Bergey's Manual of Systematic Bacteriology, Volume 4 0387950427, 9780387950426

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BERGEY’S MANUAL® OF

Systematic Bacteriology Second Edition Volume Four

The Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes

BERGEY’S MANUAL® OF

Systematic Bacteriology Second Edition Volume Four

The Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes Noel R. Krieg, James T. Staley, Daniel R. Brown, Brian P. Hedlund, Bruce J. Paster, Naomi L. Ward, Wolfgang Ludwig and William B. Whitman EDITORS, VOLUME FOUR

William B. Whitman DIRECTOR OF THE EDITORIAL OFFICE

Aidan C. Parte MANAGING EDITOR EDITORIAL BOARD Michael Goodfellow, Chairman, Peter Kämpfer, Vice Chairman,

Jongsik Chun, Paul De Vos, Fred A. Rainey and William B. Whitman WITH CONTRIBUTIONS FROM 129 COLLEAGUES

William B. Whitman Bergey’s Manual Trust Department of Microbiology 527 Biological Sciences Building University of Georgia Athens, GA 30602-2605 USA

ISBN: 978-0-387-95042-6 e-ISBN: 978-0-387-68572-4 DOI: 10.1007/978-0-387-68572-4 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010936277 © 2010, 1984–1989 Bergey’s Manual Trust Bergey’s Manual is a registered trademark of Bergey’s Manual Trust. All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper. Springer is part of Springer Science+Business Media (www.springer.com)

This volume is dedicated to our colleague Karl-Heinz Schleifer, who retired from the Board of Trustees of Bergey’s Manual as this volume was in preparation. We deeply appreciate his efforts as an editor, author and officer of the Trust. He has devoted many years to helping the Trust meet its objectives.

EDITORIAL BOARD AND TRUSTEES OF BERGEY’S MANUAL TRUST

Michael Goodfellow, Chairman Peter Kämpfer, Vice Chairman Jongsik Chun Paul De Vos Frederick Rainey William B. Whitman Don J. Brenner, Emeritus Richard W. Castenholz, Emeritus George M. Garrity, Emeritus John G. Holt, Emeritus Noel R. Krieg, Emeritus John Liston, Emeritus James W. Moulder, Emeritus R.G.E. Murray, Emeritus Karl-Heinz Schleifer, Emeritus Peter H. A. Sneath, Emeritus James T. Staley, Emeritus Joseph G. Tully, Emeritus

Preface to volume 4 of the second edition of Bergey’s Manual ® of Systematic Bacteriology

Prokaryotic systematics has remained a vibrant and exciting field of study, one of challenges and opportunities, great discoveries and gradual advances. To honor the leaders of our field, the Trust presented the 2010 Bergey’s Award in recognition of outstanding contributions to the taxonomy of prokaryotes to Antonio Ventosa. We expect that this will be the last volume to be edited by Jim Staley and Noel Krieg, both of whom served on the Trust for many years and continued to be active after their retirements. Noel contributed to Bergey’s Manual of Determinative Bacteriology, in both the 8th edition as an author and the 9th edition as an editor. Moreover, he also edited volume 1 of the 1st edition of Bergey’s Manual of Systematic Bacteriology. This was a massive achievement for this volume included the “Gram-negatives” and comprised more than one-third of that edition. In the 2nd edition of Bergey’s Manual of Systematic Bacteriology, Noel and Jim edited volume 2 along with Don Brenner. This three-part volume comprised the Proteobacteria and was also a major portion of the current edition. In the current volume, Noel edited the phylum Bacteroidetes, which is the largest phylum in this work. In addition to his passion for prokaryotic systematics, Noel is well known by his colleagues and students at Virginia Tech as a dedicated and passionate teacher. Jim Staley’s service to the Trust paralleled that of Noel’s. Jim also contributed to Bergey’s Manual of Determinative Bacteriology, in both the 8th edition as an author and the 9th edition as an editor. He edited volume 3 of the 1st edition of Bergey’s Manual of Systematic Bacteriology along with Marvin Bryant and Norbert Pfennig and volume 2 of the 2nd edition. In the current volume, he edited the phyla Acidobacteria, Chlamydiae, Dictoyoglomi, Fibrobacteres, Fusobacteria, and Gemmatimonadetes. More importantly, he coached and mentored the rest of us throughout the entire editorial process. Most recently, Jim has led the efforts of the Trust to form Bergey’s International Society for Microbial Systematics (BISMiS), whose purpose is to

promote excellent research in microbial systematics as well as enhance global communication among taxonomists who study the Bacteria and Archaea. The society will also serve internationally as an advocate for research efforts on microbial systematics and diversity. We wish Jim the best in this new adventure.

Acknowledgements The Trust is indebted to all of the contributors and reviewers, without whom this work would not be possible. The Editors are grateful for the time and effort that each has expended on behalf of the entire scientific community. We also thank the authors for their good grace in accepting comments, criticisms, and editing of their manuscripts. The Trust recognizes its enormous debt to Aidan Parte, whose enthusiasm and professionalism have made this work possible. His expertise and good judgment have been extremely valued. We also recognize the special efforts of Jean Euzéby in checking and correcting where necessary the nomenclature and etymology of every described taxon in this volume. The Trust also thanks its Springer colleagues, Editorial Director Andrea Macaluso and Production Manager Susan Westendorf. In addition, we thank Amina Ravi, our manager at our typesetters, SPi, for her work in the proofing and production of the book. We thank our current copyeditors, proofreaders and other staff, including Susan Andrews, Joanne Auger, Francis Brenner, MaryAnn Brickner, Travis Dean, Robert Gutman, Judy Leventhal and Linda Sanders, without whose hard work and attention to detail the production of this volume would be impossible. Lastly, we thank the Department of Microbiology at the University of Georgia for its assistance and encouragement in thousands of ways. William B. (Barny) Whitman

ix

Contents

Preface to volume 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . On using the Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Road map of volume 4 phyla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Taxonomic outline of volume 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix xix xxv 1 21

Phylum XIV. Bacteroidetes phyl. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Bacteroidia class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Bacteroidales ord. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Bacteroidaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Bacteroides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Acetofilamentum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Acetomicrobium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Acetothermus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus V. Anaerorhabdus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. Marinilabiliaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Marinilabilia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Anaerophaga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Alkaliflexus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family III. Rikenellaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Rikenella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Alistipes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family IV. Porphyromonadaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Porphyromonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Barnesiella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Dysgonomonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Paludibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus V. Petrimonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VI. Proteiniphilum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VII. Tannerella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family V. Prevotellaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Prevotella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Xylanibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class II. Flavobacteriia class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Flavobacteriales ord. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Flavobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Flavobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Aequorivita . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Algibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Aquimarina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus V. Arenibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VI. Bergeyella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25 25 25 25 27 41 42 44 45 49 49 51 53 54 55 56 61 62 70 71 76 77 77 78 85 86 102 105 105 106 112 155 157 158 161 165

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Genus VII. Bizionia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VIII. Capnocytophaga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IX. Cellulophaga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus X. Chryseobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XI. Cloacibacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XII. Coenonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XIII. Costertonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XIV. Croceibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XV. Dokdonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XVI. Donghaeana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XVII. Elizabethkingia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XVIII. Empedobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XIX. Epilithonimonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XX. Flaviramulus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXI. Formosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXII. Gaetbulibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXIII. Gelidibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXIV. Gillisia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXV. Gramella. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXVI. Kaistella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXVII. Kordia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXVIII. Krokinobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXIX. Lacinutrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXX. Leeuwenhoekiella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXXI. Lutibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXXII. Maribacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXXIII. Mariniflexile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXXIV. Mesonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXXV. Muricauda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXXVI. Myroides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXXVII. Nonlabens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXXVIII. Olleya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XXXIX. Ornithobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XL. Persicivirga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XLI. Polaribacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XLII. Psychroflexus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XLIII. Psychroserpens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XLIV. Riemerella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XLV. Robiginitalea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XLVI. Salegentibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XLVII. Sandarakinotalea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XLVIII. Sediminicola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XLIX. Sejongia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus L. Stenothermobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LI. Subsaxibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LII. Subsaximicrobium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LIII. Tenacibaculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LIV. Ulvibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LV. Vitellibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LVI. Wautersiella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LVII. Weeksella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LVIII. Winogradskyella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LIX. Yeosuana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LX. Zhouia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus LXI. Zobellia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

166 168 176 180 197 198 199 199 201 201 202 210 212 213 214 218 219 221 226 227 228 230 231 232 234 235 238 239 240 245 248 249 250 254 255 258 261 262 264 266 269 270 271 275 275 277 279 283 284 285 286 288 291 292 292

CONTENTS

Family II. Blattabacteriaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Blattabacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family III. Cryomorphaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Cryomorpha . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Brumimicrobium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Crocinitomix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Fluviicola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus V. Lishizhenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VI. Owenweeksia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class III. Sphingobacteriia class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Sphingobacteriales ord. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Sphingobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Sphingobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Pedobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. Chitinophagaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Chitinophaga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Terrimonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family III. Saprospiraceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Saprospira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Aureispira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Haliscomenobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Lewinella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class IV. Cytophagia class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Cytophagales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Cytophagaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Cytophaga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Adhaeribacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Arcicella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Dyadobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus V. Effluviibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VI. Emticicia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VII. Flectobacillus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VIII. Flexibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IX. Hymenobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus X. Larkinella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XI. Leadbetterella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XII. Meniscus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XIII. Microscilla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XIV. Pontibacter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XV. Runella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XVI. Spirosoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus XVII. Sporocytophaga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. Cyclobacteriaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Cyclobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Algoriphagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Aquiflexum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Belliella. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus V. Echinicola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VI. Rhodonellum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family III. Flammeovirgaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Flammeovirga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Fabibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Flexithrix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Persicobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus V. Reichenbachiella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xiii

315 315 322 323 323 326 327 327 328 330 330 331 331 339 351 351 356 358 359 361 363 366 370 370 371 371 375 377 380 387 388 389 392 397 404 405 406 408 410 412 415 418 423 423 426 433 434 437 440 442 442 447 448 450 452

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Genus VI. Roseivirga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order II. Incertae sedis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Rhodothermaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Rhodothermus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Salinibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order III. Incertae sedis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Thermonema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order IV. Incertae sedis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Toxothrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

453 457 457 458 460 465 465 467 467

Phylum XV. Spirochaetes phyl. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Spirochaetia class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Spirochaetales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Spirochaetaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Spirochaeta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Borrelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Cristispira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Treponema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. Brachyspiraceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Brachyspira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family III. Brevinemataceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Brevinema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family IV. Leptospiraceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Leptospira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Leptonema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Turneriella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hindgut spirochetes of termites and Cryptocercus punctulatus . . . . . . . . . . . . . . . .

471 471 471 473 473 484 498 501 531 531 545 545 546 546 556 558 563

Phylum XVI. Tenericutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Mollicutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Mycoplasmatales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Mycoplasmataceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Ureaplasma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. Incertae sedis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Eperythrozoon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Haemobartonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order II. Entomoplasmatales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Entomoplasmataceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Entomoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Mesoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. Spiroplasmataceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Spiroplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order III. Acholeplasmatales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Acholeplasmataceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Acholeplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. Incertae sedis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. “Candidatus Phytoplasma” gen. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . Order IV. Anaeroplasmatales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Anaeroplasmataceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Anaeroplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Asteroleplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

567 568 574 575 575 613 639 640 642 644 645 646 649 654 654 687 687 688 696 696 719 720 720 722

CONTENTS

xv

Phylum XVII. Acidobacteria phyl. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Acidobacteriia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Acidobacteriales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Acidobacteriaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Acidobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Edaphobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Terriglobus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class II. Holophagae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Holophagales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Holophagaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Holophaga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Geothrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order II. Acanthopleuribacterales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Acanthopleuribacteraceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Acanthopleuribacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

725 727 727 728 728 729 730 731 731 732 732 732 734 734 734

Phylum XVIII. “Fibrobacteres” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Fibrobacteria class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Fibrobacterales ord. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Fibrobacteraceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Fibrobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

737 739 739 739 740

Phylum XIX. “Fusobacteria” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Fusobacteriia class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Fusobacteriales ord. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Fusobacteriaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Fusobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Cetobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Ilyobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Propionigenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. Leptotrichiaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Leptotrichia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Sebaldella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Sneathia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Streptobacillus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

747 747 747 748 748 758 759 761 766 766 769 770 771

Phylum XX. Dictyoglomi phyl. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Dictyoglomia class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Dictyoglomales ord. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Dictyoglomaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Dictyoglomus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

775 776 776 776 776

Phylum XXI. Gemmatimonadetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Gemmatimonadetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Gemmatimonadales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Gemmatimonadaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Gemmatimonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

781 781 781 782 782

Phylum XXII. Lentisphaerae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Lentisphaeria class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Lentisphaerales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Lentisphaeraceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Lentisphaera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

785 787 788 788 788

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CONTENTS

Order II. Victivallales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Victivallaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Victivallis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

791 791 791

Phylum XXIII. Verrucomicrobia phyl. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Verrucomicrobiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Verrucomicrobiales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Verrucomicrobiaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Verrucomicrobium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Prosthecobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. Akkermansiaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Akkermansia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family III. Rubritaleaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Rubritalea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class II. Opitutae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Opitutales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Opitutaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Opitutus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Alterococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order II. Puniceicoccales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Puniceicoccaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Puniceicoccus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Cerasicoccus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Coraliomargarita . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Pelagicoccus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class III. Spartobacteria class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Chthoniobacterales ord. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Chthoniobacteraceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Chthoniobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. “Candidatus Xiphinematobacter”. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

795 799 802 803 803 805 809 809 812 812 817 820 820 820 821 823 824 824 825 827 829 834 836 837 837 838

Phylum XXIV. “Chlamydiae” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Chlamydiia class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Chlamydiales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Chlamydiaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Chlamydia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family II. “Candidatus Clavichlamydiaceae” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. “Candidatus Clavichlamydia”. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family III. Criblamydiaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Criblamydia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family IV. Parachlamydiaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Parachlamydia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Neochlamydia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Protochlamydia gen. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family V. “Candidatus Piscichlamydiaceae” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. “Candidatus Piscichlamydia” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family VI. Rhabdochlamydiaceae fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Rhabdochlamydia gen. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family VII. Simkaniaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Simkania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. “Candidatus Fritschea” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family VIII. Waddliaceae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Waddlia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

843 844 844 845 846 865 865 867 867 867 868 869 870 872 872 873 873 874 875 875 876 877

CONTENTS

xvii

Phylum XXV. “Planctomycetes” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Class I. Planctomycetia class. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order I. Planctomycetales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. Planctomycetaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus I. Planctomyces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus II. Blastopirellula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus III. Gemmata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus IV. Isosphaera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus V. Pirellula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VI. Rhodopirellula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VII. Schlesneria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus VIII. Singulisphaera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order II. “Candidatus Brocadiales” ord. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Family I. “Candidatus Brocadiaceae” fam. nov. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

879 879 879 880 881 895 897 900 903 906 910 913 918 918

Author index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

927

Index of scientific names of Archaea and Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

931

Contributors

Wolf-Rainer Abraham Helmholtz Center for Infection Research, 125, Chemical Microbiology, Inhoffenstrasse 7, D-38124 Braunschweig, Germany [email protected] Rudolf Amann Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany [email protected] Josefa Antón Department of Physiology, Genetics and Microbiology, University of Alicante, Apartado 99, 03080 Alicante, Spain [email protected] Mitchell F. Balish Miami University, Department of Microbiology, 80 Pearson Hall, Oxford, OH, USA [email protected] Patrik M. Bavoil Department of Microbial Pathogenesis, University of Maryland Dental School, 650 West Baltimore Street, Baltimore, MD 21201, USA [email protected] Yoshimi Benno Benno Laboratory, Center for Intellectual Property Strategies, RIKEN, Wako, Saitama 351-0198, Japan [email protected], [email protected] Jean-François Bernardet Unité de Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas cedex, France [email protected] Luise Berthe-Corti Institute for Chemistry and Biology of the Marine Environment (ICBM), University of Oldenburg, P.O. Box 2503, D-26111, Oldenburg, Germany [email protected] Joseph M. Bové 18 chemin Feyteau, 33650 La Brède, France [email protected] John P. Bowman Tasmanian Institute of Agricultural Research, Private Bag 54, University of Tasmania, Hobart, TAS 7001, Australia [email protected] Janet M. Bradbury Infectious Diseases Group, Department of Veterinary Pathology, Jordan Building, University of Liverpool, Leahurst Neston CH64 7TE, UK [email protected]

Ingrid Brettar Helmholtz-Zentrum für Infektionsforschung GmbH (HZI), Helmholtz Centre for Infection Research, Department of Vaccinology & Applied Microbiology, Inhoffenstrasse 7, D-38124 Braunschweig, Germany [email protected] John A. Breznak Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824-4320, USA [email protected] Daniel R. Brown Infectious Diseases and Pathology, Box 110880, University of Florida, Basic Sciences Building BSB3-31/BSB3-52, 1600 SW Archer Road, Gainesville, FL 32610, USA [email protected] Andreas Brune Max Planck Institute for Terrestrial Microbiology, Karl-vonFrisch-Straße, 35043 Marburg, Germany [email protected] Alke Bruns Lohmann Animal Health GmbH & Co. KG Zeppelinstrasse 2, D-27472 Cuxhaven, Germany [email protected] Brita Bruun Department of Clinical Microbiology, Hillerød Hospital, 3400 Hillerød, Denmark [email protected] Sandra Buczolits Institut fur Bakteriologie, Mykologie und Hygiene, Veterinarmedizinische Universitat, Veterinarplatz 1, A-1210 Wien, Austria [email protected] Hans-Jürgen Busse Institut für Bakteriologie, Mykologie und Hygiene, Veterinarmedizinische Universitat, Veterinarplatz 1, A-1210 Wien, Austria [email protected] Margaret K. Butler Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Building 75, St Lucia, QLD 4072, Australia [email protected] Michael J. Calcutt 201 Connaway Hall, University Missouri-Columbia, Columbia, MO, USA [email protected]

xix

xx

CONTRIBUTORS

Richard W. Castenholz Center for Ecology & Evolutionary Biology, Department of Biology, University of Oregon, Eugene, OR 97403-1210, USA [email protected] Marie Anne Chattaway Molecular Identification Services Unit, Department for Bioanalysis and Horizon Technologies, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK Jang-Cheon Cho Department of Biological Sciences, Division of Biology and Ocean Sciences, Inha University, Incheon 402-751, Republic of Korea [email protected] Richard Christen UMR 6543 CNRS – Université Nice Sophia Antipolis, Centre de Biochimie, Parc Valrose, F-06108 Nice, France [email protected] Jongsik Chun School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea [email protected] John D. Coates Department of Plant and Microbial Biology, 271 Koshland Hall, University of California, Berkeley, Berkeley, CA 94720, USA [email protected] August Coomans Zoological Institute, Department of Biology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium [email protected] Kyle C. Costa Department of Microbiology and Astrobiology Program, Box Number 357242, University of Washington, Seattle, WA 98195, USA [email protected] Robert E. Davis Molecular Plant Pathology Laboratory, USDA-Agricultural Research Service, Beltsville, MD 20705, USA [email protected] Willem M. de Vos Laboratory of Microbiology, Wageningen University, 6703 CT Wageningen, The Netherlands [email protected] Svetlana N. Dedysh S.N. Winogradsky Institute of Microbiology RAS, Prospect 60-Letya Oktyabrya 7/2, Moscow 117312, Russia [email protected] Muriel Derrien Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands [email protected] Kirstin J. Edwards Applied and Functional Genomics, Centre for Infections, Health Protection Agency, 6l Colindale Avenue, London NW9 5EQ, UK [email protected] Jean P. Euzéby Ecole Nationale Veterinaire, 23 chemin des Capelles, B.P. 87614, 31076 Toulouse cedex 3, France [email protected]

Mark Fegan Biosciences Research Division, Department of Primary Industries, Attwood, VIC 3049, Australia [email protected] Sydney M. Finegold Infectious Diseases Section (111 F), VA Medical Center West Los Angeles, Los Angeles, CA 90073, USA [email protected] Cecil W. Forsberg Department of Molecular and Cellular Biology, Science Complex, University of Guelph, Guelph, ON, Canada N1G 2W1 [email protected] John A. Fuerst School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane St Lucia, QLD 4072, Australia [email protected] Ferran Garcia-Pichel School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA [email protected] George M. Garrity Department of Microbiology, Michigan State University, 6162 Biomedical and Physical Sciences Building, East Lansing, MI 48824-4320, USA [email protected] Gail E. Gasparich Department of Biological Sciences, 8000 York Road, Towson University, Towson, MD 21252, USA [email protected] Saheer E. Gharbia Applied and Functional Genomics, Centre for Infections, Health Protection Agency, 6l Colindale Avenue, London NW9 5EQ, UK [email protected] Peter Gilbert (Deceased) School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, UK Stephen J. Giovannoni Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA [email protected] John I. Glass The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD, USA [email protected] Dawn Gundersen-Rindal Insect Biocontrol Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Research Center, Plant Sciences Institute, Beltsville, MD 20705, USA [email protected] Hafez M. Hafez Free University Berlin, Königsweg 63, 14163 Berlin, Germany [email protected] Nigel A. Harrison Research and Education Center, 3205 College Avenue, University of Florida, Fort Lauderdale, FL 33314-7799, USA [email protected]

CONTRIBUTORS

Brian P. Hedlund School of Life Sciences, University of Nevada Las Vegas, Box 4004, 4505 Maryland Parkway, Las Vegas, NV 89154-4004, USA [email protected] Peter Hirsch Institut für Allgemeine Mikrobiologie der Biozentrum, Universität Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany [email protected] Manfred G. Höfle Helmholtz-Zentrum für Infektionsforschung GmbH (HZI), Helmholtz Centre for Infection Research, Department of Vaccinology & Applied Microbiology, Inhoffenstrasse 7, D-38124 Braunschweig, Germany [email protected] Stanley C. Holt The Forsyth Institute, Boston, MA, USA [email protected] Matthias Horn Department of Microbial Ecology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria [email protected] Celia J. Hugo Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein 9300, South Africa [email protected] Roar L. Irgens 715 Lilac Drive, Mount Vernon, WA 98273-6613, USA [email protected] Elena P. Ivanova Faculty of Life and Social Science, Swinburne University of Technology, H31, P.O. Box 218, Hawthorn, VIC 3122, Australia [email protected] Peter H. Janssen Grasslands Research Centre, AgResearch, Private Bag 11008, Palmerston North 4442, New Zealand [email protected] Mike S. M. Jetten Radboud University, Institute for Water and Wetland Research, Department of Microbiology, Huygens Building Room HG02.339, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands [email protected] Karl-Erik Johansson Department of Bacteriology, Ulls Vaeg 2A-2C, National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden [email protected] Bernhard Kaltenboeck Department of Pathobiology, College of Veterinary Medicine, Auburn University, 270 Greene Hall, Auburn, AL 36849-5519, USA [email protected] Yoichi Kamagata Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido 062-8517, Japan [email protected] Srinivas Kambhampati Professor of Insect Genetics and Evolution, Department of Entomology, Kansas State University, Manhattan, KS 66506, USA [email protected]

xxi

Peter Kämpfer Institut für Angewandte Mikrobiologie, Justus-LiebigUniversität Giessen, Heinrich-Buff-Ring 26-32 (IFZ), D-35392 Giessen, Germany [email protected] Hiroaki Kasai Marine Biotechnology Institute, 3-75-1, Heita, Kamaishi, Iwate 026-0001, Japan [email protected] Sang-Jin Kim Marine Biotechnology Research Centre, Korea Ocean Research & Development Institute, Department of Marine Biotechnology, University of Science and Technology, P.O. Box 29, Ansan 425-600, Republic of Korea [email protected] Seung Bum Kim Department of Microbiology, Chungnam National University, 220 Gung-dong, Yusong, Daejon 305-764, Republic of Korea [email protected] Eija Könönen Laboratory Head, Anaerobe Reference Laboratory, Department of Bacterial and Inflammatory Diseases, National Public Health Institute (KTL), Mannerheimintie 166, FIN00300, Helsinki, Finland [email protected] Noel R. Krieg 617 Broce Drive, Blacksburg, VA 24060-2801, USA [email protected] Lee R. Krumholz Department of Botany and Microbiology and Institute for Energy and the Environment, 770 Van Vleet Oval, The University of Oklahoma, Norman, OK 73019, USA [email protected] J. Gijs Kuenen Delft University of Technology, Department of Biotechnology, Julianalaan 67, 2628 BC Delft, The Netherlands [email protected] Irina S. Kulichevskaya S.N. Winogradsky Institute of Microbiology RAS, Prospect 60-Letya Oktyabrya 7/2, Moscow 117312, Russia [email protected] Cho-chou Kuo Department of Pathobiology, University of Washington, Seattle, WA 98195, USA [email protected] Edward R. Leadbetter Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Mail Stop 52, Woods Hole, MA 02543, USA [email protected] Kuo-Chang Lee School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane St Lucia, QLD 4072, Australia [email protected] Susan Leschine University of Massachusetts Amherst, Department of Microbiology, 639 North Pleasant Street, Amherst, MA 01003, USA [email protected] Ralph A. Lewin (Deceased) Scripps Institution of Oceanography, University of California, La Jolla, CA 92093, USA

xxii

CONTRIBUTORS

Chengxu Liu 11511 Reed Hartman Hwy, Cincinnati, OH 45241, USA [email protected], [email protected] Julie M. J. Logan Molecular Identification Services, Centre for Infections, Health Protection Agency, 6l Colindale Avenue, London NW9 5EQ, UK [email protected] Wolfgang Ludwig Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4, D-85350 Freising, Germany [email protected] Heinrich Lünsdorf Helmholtz Center for Infection Research, Vaccinology Department, Inhoffenstrasse 7, D-38124 Braunschweig, Germany [email protected], [email protected] Alexandre J. Macedo Universidade Federal do Rio Grande do Sul, Faculdade de Farmácia and Centro de Biotecnologia, Porto Alegre, RS, Brazil [email protected] Rosa Margesin Institut für Mikrobiologie, Universität Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria [email protected] Meghan May Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611-0880, USA [email protected] Joanne B. Messick Comparative Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA [email protected] Valery V. Mikhailov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of Russian Academy of Sciences, Prospect 100 Let Vladivostoku 159, Vladivostok 690022, Russia [email protected] Yasuyoshi Nakagawa Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, 2-5-8, Kazusakamatari, Kisarazu, Chiba 292-0818, Japan [email protected] Jason B. Navarro School of Life Sciences, University of Nevada, Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154-4004, USA [email protected] Olga I. Nedashkovskaya Pacific Institute of Bioorganic Chemistry, of the Far-Eastern Branch of the Russian Academy of Sciences, Pr. 100 let Vladivostoku 159, Vladivostok 690022, Russia [email protected], [email protected] Harold Neimark Department of Microbiology & Immunology, Box 44, 450 Clarkson Avenue, State University of New York, College of Medicine, Brooklyn, NY 11203, USA [email protected]

Denis I. Nikitin Russian Academy of Sciences, Institute of Microbiology, Prosp. 60 let Oktyabrya, 7a, 117811 Moscow, Russia [email protected] Steven J. Norris Greer Professor and Vice Chair for Research, Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, MSB 2.120, P.O. Box 20708, Houston, TX 77030, USA [email protected] Ingar Olsen Institute of Oral Biology, Faculty of Dentistry, P.B. 1052 Blindern, N-0316 Oslo, Norway [email protected] Huub J. M. Op den Camp Radboud University, Institute for Water and Wetland Research, Department of Microbiology, Huygens Building Room HG02.340, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands [email protected] Bruce J. Paster The Forsyth Institute, 245 First Street, Cambridge, MA 021421200, USA [email protected] Bharat K. C. Patel Microbial Discovery Research Unit, School of Biomolecular and Physical Sciences, Griffith University, Nathan Campus, Kessels Road, Brisbane, QLD 4111, Australia [email protected] Caroline M. Plugge Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands [email protected] Lakshani Rajakurana Molecular Identification Services Unit, Department for Bioanalysis and Horizon Technologies, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK Merja Rautio Division of Clinical Microbiology, Huslab, Jorvi Hospital, Espoo, Finland Gundlapally S.N. Reddy Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500 007, India [email protected], [email protected] Laura B. Regassa Georgia Southern University, 202 Georgia Avenue, Statesboro, GA, USA [email protected] Joël Renaudin UMR Génomique Diversité et Pouvoir Pathogène, INRA-Université de Bordeaux 2, IBVM, 71 Avenue Edouard Bourlaux, BP 81, F-33883 Villenave d’Ornon, France [email protected] Alexander H. Rickard Department of Epidemiology, School of Public Health, University of Michigan, 1415 Washington Heights, 4647 SPH Tower, Ann Arbor, MI, USA [email protected]

CONTRIBUTORS

Janet A. Robertson Department of Medical Microbiology and, Immunology, University of Alberta, 1-49 Medical Sciences Building, Edmonton, AB, Canada T6G 2H7 [email protected], [email protected] Ramon Rosselló-Mora Institut Mediterrani d’Estudis Avançats (CSIC-UIB), C/Miquel Marque’s 21, E-07290 Esporles, Mallorca, Spain [email protected] Collette Saillard UMR 1090 Génomique Diversité Pouvoir Pathogène, INRA, Université Victor Ségalen Bordeaux 2, 71 Avenue Edouard Bourlaux BP 81, F-33883 Villenave d’Ornon, France [email protected] Mitsuo Sakamoto Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan [email protected] Bernhard Schink Lehrstuhl für Mikrobielle Ökologie, Fakultät für Biologie, Universität Konstanz, Fach M 654, D-78457 Konstanz, Germany [email protected] Heinz Schlesner Institut für Allgemeine Mikrobiologie, Christian-AlbrechtsUniversität, Am Botanischen Garten 1-9, D-24118 Kiel, Germany [email protected] Jean M. Schmidt Arizona State University, Department of Microbiology, Box 2701, Tempe, AZ 85287, USA [email protected] Ira Schwartz Department of Microbiology & Immunology, New York Medical College, Valhalla, NY 10595, USA [email protected] Haroun N. Shah Molecular Identification Services Unit, Department for Bioanalysis and Horizon Technologies, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK [email protected] Wung Yang Shieh Institute of Oceanography, National Taiwan University, Taipei, Taiwan [email protected] Sisinthy Shivaji Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500 007, India [email protected] Lindsay I. Sly Department of Microbiology and Parasitology, University of Queensland, Brisbane, QLD, Australia [email protected] Robert M. Smibert Department of Anaerobic Microbiology, Virginia Tech, Blacksburg, VA, USA Yuli Song 8700 Mason Montgomery Road, MBC DV2-5K1, 533; Mason, OH 45040-9462, USA [email protected]

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Anne M. Spain Department of Botany and Microbiology, 770 Van Vleet Oval, The University of Oklahoma, Norman, OK 73019, USA [email protected] James T. Staley Department of Microbiology, University of Washington, Seattle, WA 98195-2700, USA [email protected] Thaddeus B. Stanton Agricultural Research Service – Midwest Area, National Animal Disease Center, United States Department of Agriculture, P.O. Box 70, 1920 Dayton Ave, Building 24, Ames, IA 50010-0070, USA [email protected] Richard S. Stephens Program in Infectious Diseases and Immunity, School of Public Health, University of California at Berkeley, 235 Warren Hall, Berkeley, CA 94720, USA [email protected] Marc Strous Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany [email protected] Paula Summanen Anaerobe Laboratory, VA Medical Center West Los Angeles, 11301 Wilshire Boulevard, Building 304, Room E3-237, Los Angeles, CA 90073, USA [email protected] Makoto Suzuki Kyowa Hakko Bio Co., Ltd, 1-6-1 Ohtemachi, Chiyoda-ku, Tokyo 100-8185, Japan [email protected] Anne C. R. Tanner Department of Molecular Genetics, The Forsyth Institute, 140 The Fenway, Boston, MA 02115, USA [email protected] Séverine Tasker School of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, UK [email protected] David Taylor-Robinson 6 Vache Mews, Vache Lane, Chalfont St Giles, Buckingham HP8 4UT, UK [email protected] J. Cameron Thrash Department of Microbiology, Oregon State University, Corvallis, OR, USA [email protected] Stefanie Van Trappen BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium [email protected] Marc Vancanneyt BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium Tom T. M. Vandekerckhove BioBix: Laboratory for Bioinformatics and Computational Genomics, Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium [email protected]

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CONTRIBUTORS

Guiqing Wang Department of Pathology, New York Medical College, Valhalla, NY 10595, USA [email protected] Naomi L. Ward Department of Molecular Biology, University of Wyoming, Department 3944, 1000 E. University Ave, Laramie, WY 82071, USA [email protected] Robert Whitcomb (Deceased) Patagonia, AZ 85624, USA William B. Whitman Department of Microbiology, University of Georgia, 527 Biological Sciences Building, Cedar Street, Athens, GA 306022605, USA [email protected] David L. Williamson 4 Galahad Lane, Nesconset, NY 11767-2220, USA [email protected]

Hana Yi School of Biological Sciences, Seoul National University, NS70, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Korea [email protected] Jaewoo Yoon Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi Bunkyo-Ku, Tokyo 113-0032, Japan [email protected] Erwin G. Zoetendal Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands [email protected] Richard L. Zuerner Infectious Bacterial Diseases Research Unit, USDA-ARSNADC, P.O. Box 70, 1920 Dayton Avenue, Building 24, Ames, IA 50010, USA [email protected]

On using the Manual NOEL R. KRIEG AND GEORGE M. GARRITY

Citation The Systematics is a peer-reviewed collection of chapters, contributed by authors who were invited by the Trust to share their knowledge and expertise of specific taxa. Citations should refer to the author, the chapter title, and inclusive pages rather than to the editors.

Arrangement of the Manual As in the previous volumes of this edition, the Manual is arranged in phylogenetic groups based upon the analyses of the 16S rRNA presented in the introductory chapter “Road map of the phyla Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae and Planctomycetes”. These groups have been substantially modified since the publication of volume 1 in 2001, reflecting both the availability of more experimental data and a different method of analysis. Since volume 4 includes only the phylum Firmicutes, taxa are arranged by class, order, family, genus and species. Within each taxon, the nomenclatural type is presented first and indicated by a superscript T. Other taxa are presented in alphabetical order without consideration of degrees of relatedness.

Articles Each article dealing with a bacterial genus is presented wherever possible in a definite sequence as follows: a. Name of the genus. Accepted names are in boldface, followed by “defining publication(s)”, i.e. the authority for the name, the year of the original description, and the page on which the taxon was named and described. The superscript AL indicates that the name was included on the Approved Lists of Bacterial Names, published in January 1980. The superscript VP indicates that the name, although not on the Approved Lists of Bacterial Names, was subsequently validly published in the International Journal of Systematic and Evolutionary Microbiology (or the International Journal of Systematic Bacteriology). Names given within quotation marks have no standing in nomenclature; as of the date of preparation of the Manual they had not been validly published in the International Journal of Systematic and Evolutionary Microbiology, although they may have been “effectively published” elsewhere. Names followed by the term “nov.” are newly proposed but will not be validly published until they appear in a Validation List in the International Journal of Systematic and Evolutionary Microbiology. Their proposal in the Manual constitutes only “effective publication”, not valid publication.

b. Name of author(s). The person or persons who prepared the Bergey’s article are indicated. The address of each author can be found in the list of Contributors at the beginning of the Manual. c. Synonyms. In some instances a list of some synonyms used in the past for the same genus is given. Other synonyms can be found in the Index Bergeyana or the Supplement to the Index Bergeyana. d. Etymology of the name. Etymologies are provided as in previous editions, and many (but undoubtedly not all) errors have been corrected. It is often difficult, however, to determine why a particular name was chosen, or the nuance intended, if the details were not provided in the original publication. Those authors who propose new names are urged to consult a Greek and Latin authority before publishing in order to ensure grammatical correctness and also to ensure that the meaning of the name is as intended. e. Salient features. This is a brief resume of the salient features of the taxon. The most important characteristics are given in boldface. The DNA G+C content is given. f. Type species. The name of the type species of the genus is also indicated along with the defining publication(s). g. Further descriptive information. This portion elaborates on the various features of the genus, particularly those features having significance for systematic bacteriology. The treatment serves to acquaint the reader with the overall biology of the organisms but is not meant to be a comprehensive review. The information is normally presented in the following sequence: Colonial morphology and pigmentation Growth conditions and nutrition Physiology and metabolism Genetics, plasmids, and bacteriophages Phylogenetic treatment Antigenic structure Pathogenicity Ecology h. Enrichment and isolation. A few selected methods are presented, together with the pertinent media formulations. i. Maintenance procedures. Methods used for maintenance of stock cultures and preservation of strains are given. j. Procedures for testing special characters. This portion provides methodology for testing for unusual characteristics or performing tests of special importance.

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ON USING THE MANUAL

k. Differentiation of the genus from other genera. Those characteristics that are especially useful for distinguishing the genus from similar or related organisms are indicated here, usually in a tabular form. l. Taxonomic comments. This summarizes the available information related to taxonomic placement of the genus and indicates the justification for considering the genus a distinct taxon. Particular emphasis is given to the methods of molecular biology used to estimate the relatedness of the genus to other taxa, where such information is available. Taxonomic information regarding the arrangement and status of the various species within the genus follows. Where taxonomic controversy exists, the problems are delineated and the various alternative viewpoints are discussed. m. Further reading. A list of selected references, usually of a general nature, is given to enable the reader to gain access to additional sources of information about the genus. n. Differentiation of the species of the genus. Those characteristics that are important for distinguishing the various species within the genus are presented, usually with reference to a table summarizing the information. o. List of species of the genus. The citation of each species is given, followed in some instances by a brief list of objective synonyms. The etymology of the specific epithet is indicated. Descriptive information for the species is usually presented in tabular form, but special information may be given in the text. Because of the emphasis on tabular data, the species descriptions are usually brief. The type strain of each species is indicated, together with the collection(s) in which it can be found. (Addresses of the various culture collections are given in the article in volume 1 entitled Culture Collections: An Essential Resource for Microbiology.) The 16S rRNA gene sequence used in phylogenetic analysis and placement of the species into the taxonomic framework is given, along with the GenBank (or other database) accession number. Additional comments may be provided to point the reader to other well-characterized strains of the species and any other known DNA sequences that may be relevant. p. Species incertae sedis. The List of Species may be followed in some instances by a listing of additional species under the heading “Species Incertae sedis” or “Other organisms”, etc. The taxonomic placement or status of such species is questionable, and the reasons for the uncertainty are presented. q. References. All references given in the article are listed alphabetically at the end of the family chapter.

Tables In each article dealing with a genus, there are generally three kinds of table: (a) those that differentiate the genus from similar or related genera, (b) those that differentiate the species within the genus, and (c) those that provide additional information about the species (such information not being particularly useful for differentiation). The meanings of symbols are as follows: +, 90% or more of the strains are positive d, 11–89% of the strains are positive −, 90% or more of the strains are negative D, different reactions occur in different taxa (e.g., species of a genus or genera of a family) v, strain instability (NOT equivalent to “d”) w, weak reaction. nd, not determined or no data. nr, not reported. These symbols, and exceptions to their use, as well as the meaning of additional symbols, are given in footnotes to the tables.

Use of the Manual for determinative purposes Many chapters have keys or tables for differentiation of the various taxa contained therein. For identification of species, it is important to read both the generic and species descriptions because characteristics listed in the generic descriptions are not usually repeated in the species descriptions. The index is useful for locating the articles on unfamiliar taxa or in discovering the current classification of a particular taxon. Every bacterial name mentioned in the Manual is listed in the index. In addition, an up-to-date outline of the taxonomic framework is provided in the introductory chapter “Road map of the phyla Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae and Planctomycetes”.

Errors, comments, and suggestions As in previous volumes, the editors and authors earnestly solicit the assistance of all microbiologists in the correction of possible errors in Bergey’s Manual of Systematic Bacteriology. Comments on the presentation will also be welcomed as well as suggestions for future editions. Correspondence should be addressed to: Editorial Office Bergey’s Manual Trust Department of Microbiology University of Georgia Athens, GA 30602-2605, USA Tel: +1-706-542-4219; fax +1-706-542-6599 e-mail: [email protected]

Road map of the phyla Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes Wolfgang Ludwig, Jean Euzéby and William B. Whitman

This revised road map updates previous outlines of Garrity and Holt (2001) and Garrity et al. (2005) with the description of additional taxa and new phylogenetic analyses. While the outline/ road map seeks to be complete for all taxa validated prior to July 1, 2006, some taxa described after that date are included. The new phylogenetic trees are strict consensus trees based on various maximum-likelihood and maximum-parsimony analyses and corrected according to results obtained when applying alternative treeing methods. Multifurcations indicate that a common branching order was not significantly supported after applying alternative treeing approaches. Detailed branching orders are shown if supported by at least 50% of the “treeings” performed in addition to the maximum-likelihood approach. Given that the focus is on the higher taxonomic ranks, rather restrictive variability filters were applied. Consequently, resolution power is lost for lower levels. Of special importance, relationships within genera lack the resolution that would be obtained with genus–family level analyses. Furthermore, the type strain tree, which is available online at www.bergeys.org, is an extract of comprehensive trees comprising some thousand sequences. Thus, trees for the specific groups in subsequent chapters, which are based upon smaller datasets and include the variable sequence positions, may differ with respect to detailed topology, especially at levels of closer relationships within and between genera. In the trees shown here, branch lengths – in first instance – indicate significance and only approximate estimated number of substitutions. Starting with the second edition of Bergey’s Manual of Systematic Bacteriology, the arrangement of content follows a phylogenetic framework or road map based largely on analyses of the nucleotide sequences of the ribosomal small-subunit RNA rather than on phenotypic data (Garrity et al., 2005). Implicit in the use of the road map are the convictions that prokaryotes have a phylogeny and that phylogeny matters. However, phylogenies, like other experimentally derived hypotheses, are not static but may change whenever new data and/or improved methods of analysis become available (Ludwig and Klenk, 2005). Thus, the large increases in data since the publication of the taxonomic outlines in the preceding volumes have led to a re-evaluation of the road map. Not surprisingly, the taxonomic hierarchy has been modified or newly interpreted for a number of taxonomic units. These changes are described in the following paragraphs.

The taxonomic road map proposed in volume 1 and updated and emended in volume 2 was derived from phylogenetic and principal-component analyses of comprehensive datasets of small-subunit rRNA gene sequences. A similar approach is continued here. Since the introduction of comparative rRNA sequencing (Ludwig and Klenk, 2005; Ludwig and Schleifer, 2005), there has been a continuous debate concerning the justification and power of a single marker molecule for elucidating phylogeny and establishing taxonomy of organisms. Although generally well established in taxonomy, the polyphasic approach cannot currently be applied for sequence-based analyses due to the lack of adequate comprehensive datasets for alternative marker molecules. Even in the age of genomics, the datasets for non-rRNA markers are poor in comparison to more than 400,000 rRNA primary structures available in general and special databases (Cole et al., 2007; Pruesse et al., 2007). ­Nevertheless, the data provided by the full genome-sequencing projects allow the definiton of a small set of genes representing the conserved core of prokaryotic genomes (Cicarelli et al., 2006; Ludwig and Schleifer, 2005). Furthermore, comparative analyses of the core gene sequences globally support the smallsubunit rRNA derived view of prokaryotic evolution. Although the tree topologies reconstructed from alternative markers differ in detail, the major groups (and taxa) are verified or at least not disproved (Ludwig and Schleifer, 2005). Consequently, the structuring of this volume is based on updated and curated (http://www.arb-silva.de; Pruesse et  al., 2007) databases of ­processed small-subunit rRNA primary structures.

Data analysis The current release of the integrated small-subunit rRNA database of the SILVA project (Pruesse et al., 2007) provides the basis for these phylogenetic analyses. The tools of the arb software package (Ludwig et  al., 2004) were used for data evaluation, optimization, and phylogenetic inference. A subset of about 33,000 high-quality sequences from Bacteria was extracted from the current SILVA SSU Ref database. Among the criteria for restrictive quality analyses and data selection were coverage of at least positions 18–1509 (Escherichia coli 16S rRNA numbering), no ambiguities or missing sequence stretches, no chimeric primary structures, low deviation from overall and group-­specific consensus and conservation profiles, and good agreement of tree topologies and branch length with processed sequence 1

2 2

Road map

data. Unfortunately, only some of the type strain sequences successfully passed this restrictive quality check. The alignment of the sequences of this subset, as well as all type strain sequences initially excluded given incompleteness or lower quality, was manually evaluated and optimized. Phylogenetic treeing was first based on the high-quality dataset and performed applying phylum specific position filters (50% positional identity). The partial or lower quality type strain sequences were subsequently added using a special arb-tool allowing the optimal positioning of branches to the reference tree without admitting topology changes (Ludwig and Klenk, 2005). The consensus trees used for evaluating or modifying the taxonomic outline were based on maximum-likelihood analyses (RAXML, implemented in the arb package; Stamatakis et al., 2005) and further evaluated by maximum-parsimony and distance matrix analyses with the respective arb tools (Ludwig et al., 2004).

"Bacteroidetes"

"Spirochaetes"

"Acidobacteria"

Taxonomic interpretation The phylogenetic conclusions were used for evaluating and modifying the taxonomic outline of the phyla “Bacteroidetes”, “Spirochaetes”, Tenericutes (Mollicutes), “Acidobacteria”, “Fibrobacteres”, “Fusobacteria”, “Dictyoglomi”, Gemmatimonadetes, Lentisphaerae, “Verrucomicrobia”, “Chlamydiae”, and “Planctomycetes”. These include all the phyla not described in earlier volumes with the exception of the Actinobacteria, which will be included in the fifth and last volume of this edition. There is no particular rationale for inclusion in this volume. Although some of the phyla may be related in a kingdom or superphylum (i.e., “Chlamydiae”, Lentisphaerae, “Planctomycetes”, and “Verrucomicrobia”) (Griffiths and Gupta, 2007; Lee et  al., 2009; Pilhofer et  al., 2008; Wagner and Horn, 2006), most are unrelated to each other (Figure 1). Some are major pathogens of humans, other animals, and plants. Some are exotic and only described in the last decade. In order to ensure applicability and promote acceptance, the proposed taxonomic modifications were made following a conservative procedure. The overall organization follows the type “taxon” principle as applied in the previous volumes. Taxa defined in the outline of the preceding volumes were only unified, dissected, or transferred in the cases of strong phylogenetic support. This approach is justified by the well-known low significance of local tree topologies (also called “range of unsharpness” around the nodes; Ludwig and Klenk, 2005). Thus, many of the cases of paraphyletic taxa found were maintained in the current road map if the respective (sub)-clusters rooted closely together, even if they were separated by intervening clusters representing other taxa. While reorganization of these taxa may be warranted, it was not performed in the absence of confirmatory evidence. The names of validly published, but phylogenetically misplaced, type strains are also generally maintained. These strains are mentioned in the context of the respective phylogenetic groups. In cases of paraphyly, all concerned species or higher taxa are assigned to the respective (sub)-groups. New higher taxonomic ranks are only proposed if species or genera – previously assigned to different higher taxonomic units – are significantly unified in a monophyletic branch. Upon the recommendation of the Judicial Commission (De Vos et al., 2005), many of the names and classifications previously proposed by Cavalier-Smith (2002) are not used in this work. The classification used categories not covered by the Rules of the Code and priority and proposed types without standing in nomenclature. For these reasons, the following phylum (or

"Chlamydiae"

"Verrucomicrobia"

Lentisphaerae "Planctomycetes" "Fibrobacteres" Gemmatimonadetes

Tenericutes

"Fusobacteria" "Dictyoglomi" FIGURE 1.  Phyla Bacteroidetes, Spirochaetes, Tenericutes, Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes. While the phyla Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes may be specifically related to each other, the other phyla included in volume 4 are not related.

division) names are not used: Planctobacteria, Sphingobacteria, and Spirochaetae. Likewise, the following class names are not used: Acidobacteria, Chlamydiae, Flavobacteria, Planctomycea, and Spirochaetes. Lastly, priority for the order name Acidobacteriales is no longer attributed to Cavalier-Smith (2002).

Phylum “Bacteroidetes” In previous classifications, the phylum “Bacteroidetes” was proposed to comprise three classes, “Bacteroidia”, “Flavobacteriia”, and “Sphingobacteriia” (Garrity et al., 2005). While the analyses performed here, which were based upon many more sequences and differ-



3

Road map Bacteroidaceae "Prevotellaceae" "Porphyromonadaceae" "Bacteroidia" "Rikenellaceae" "Marinilabiliaceae" Flavobacteriaceae "Blattabacteriaceae"

"Flavobacteriia"

Cryomorphaceae Sphingobacteriaceae "Chitinophagaceae"

"Sphingobacteriia"

"Saprospiraceae" Cytophagaceae "Cytophagia"

"Cyclobacteriaceae" "Flammeovirgaceae" Balneola

Incertae sedis

"Rhodothermaceae"

FIGURE 2.  Overview of the phylum “Bacteroidetes”. This phylum contains 15 families classified within four classes.

Currently, the incertae sedis taxa Balneola and “Rhodothermaceae” are classified within the class “Cytophagia”.

ent methods, generally support this conclusion, they also justify formation of a fourth class within this phylum, the “Cytophagia” (Figure 2). This new class comprises many genera previously ­classified within the “Flexibacteraceae”, “­Flammeovirgaceae”, and Crenotrichaceae (see below). Thus, the phylum “Bacteroidetes” ­comprises at least four phylogenetic groups that are well delineated on the basis of their 16S rRNA gene sequences. In addition, two groups are affiliated with the phylum but could not be readily assigned to one of these classes. While additional evidence may warrant classification with one of the known or novel classes, these organisms were grouped within Incertae sedis of the “Cytophagia” for the present time (Figure 2).

Class “Bacterioidia” and order “Bacteroidales” The class “Bacterioidia” contains five families, all classified within the order “Bacteroidales”. These families include the four families proposed previously (Garrity et al., 2005), Bacteroidaceae, “Rikenellaceae”, “Porphyromonadaceae”, and “Prevotellaceae”, as well as a new family proposed here, “Marinilabiliaceae” (Figure 3). In addition, on the basis of the dissimilarity of its 16S rRNA gene sequence to other members of the order, Odoribacter (Bacteroides) splanchnicus may represent an additional undescribed family or a member of the “Marinilabiliaceae”. However, chemotaxonomic characteristics and analyses of the fimA gene imply a close relationship to the family “Porphyromonadaceae” (Hardham et al., 2008). Therefore, its reclassification is not proposed at this time. Lastly, the recently described marine organism, Prolixibacter bellariivorans, appears to represent a deep lineage in this class but whose affilitation with these families is ambiguous (Holmes et al., 2007).

Family Bacteroidaceae In addition to the type genus, Bacteroides, this family comprises three monospecific genera, Acetofilamentum, Acetothermus, and Anaerorhabdus, and one genus, Acetomicrobium, comprising two species. Because complete 16S rRNA gene sequences are not available for representatives of these four genera, these ­assignments are tentative. Two genera previously assigned to

this ­family have also been reassigned. As recommended by Morotomi et al. (2007), Megamonas has been transferred to the Firmicutes. Based on its rRNA gene sequence, Anaerophaga has been transferred to the new family “Marinilabiliaceae”. The genus Bacteroides comprises at least six lineages or clades. The type species, Bacteroides fragilis, together with Bacteroides acidifaciens, caccae, finegoldii, nordii, ovatus, salyersiae, thetaiotaomicron, and xylanisolvens, represent a cluster slightly separated from the other members of the genus. If supported by other evidence, each of the other lineages could be classified as new genera within this family. The other lineages are represented by Bacteroides cellulosilyticus and intestinalis; Bacteroides coprosuis and propionifaciens; Bacteroides pyogenes, suis, and tectus; Bacteroides barnesiae, coprocola, coprophilus, dorei, helcogenes, massiliensis, plebeius, salanitronis, uniformis, and vulgatus. The species Bacteroides eggerthii, gallinarum, and stercoris cannot clearly be assigned to one of the lineages. In addition to these clades within the genus, the following validly published species are probably misclassified. Bacteroides splanchnicus was recently reclassified as Odoribacter splanchnicus (Hardham et al., 2008); this genus may represent a novel member of family “Porphyromonadaceae” (see above). Bacteroides capillosus and cellulosolvens are probably members of the phylum Firmicutes. In addition, rRNA gene sequences are not available for Bacteroides capillus, forsythus, furcosus, polypragmatus, and salivosus, so their assignment is uncertain. Lastly, the family Bacteroidaceae appears to be paraphyletic, and the family “Prevotellaceae” falls within the radiation of ­Bacteroides clades. Because the members of the “Prevotellaceae” are generally closely related and the branch length to the ­Bacteroidaceae is fairly long, this conclusion is tentative. While these families were not combined at this time, this classification may warrant further investigation.

Family “Marinilabiliaceae” This family represents a group of sister but not clearly monophyletic branches within the “Bacteroidales” and comprises three genera.

4 4

Road map Bacteroides

Bacteroidaceae Prevotella Xylanibacter

"Prevotellaceae"

Dysgonomonas Paludibacter Barnesiella Tannerella

"Porphyromonadaceae" Porphyromonas Parabacteroides

Proteiniphilum Petrimonas Rikenella Alistipes

"Rikenellaceae"

Marinilabilia Anaerophaga

"Marinilabiliaceae" Odoribacter

Alkaliflexus Prolixibacter FIGURE 3.  Genera of the class “Bacteroidia”. This class comprises five families and the genus Prolixibacter, which has

not yet been assigned to a family.

The type genus, Marinilabilia, contains two species, Marinilabilia salmonicolor and agarovorans, and was previously classified within the “Rikenellaceae” (Garrity et  al., 2005). The remaining taxa include Alkaliflexus imshenetskii and Anaerophaga thermohalophila, the latter of which was formerly classified within the Bacteroidaceae (Garrity et al., 2005). The current analysis suggests that Cytophaga fermentans should be reclassified as a novel genus that is associated with this family. Lastly, rRNA analyses suggest that Odoribacter (Bacteroides) splanchnicus, which was proposed after the deadline for inclusion in this volume, may represent an additional member of the “Marinilabiliaceae”. However, chemotaxonomic characteristics and analyses of the fimA gene imply a close relationship to the family “Porphyromonadaceae” (Hardham et al., 2008), so this classification is not proposed at this time.

Family “Rikenellaceae” The family comprises the monospecific genus Rikenella microfusus and the closely related genus Alistipes. The latter genus comprises the type species Alistipes putredinis and Alistipes finegoldii, onderdonkii, and shahii. Marinilabilia, which was classified within this family by Garrity et al. (2005), is now classified within the family “Marinilabiliaceae”.

Family “Porphyromonadaceae” The genus Porphyromonas, which was formed by reclassification of various species of Bacteroides (Shah and Collins, 1988), is the type for this family. Originally, this family comprised the ­genera Porphyromonas, Dysgonomonas, and Tannerella (Garrity et  al., 2005). The genus Porphyromonas comprises five subclusters: (1) the type species Porphyromonas asaccharolytica and Porphyromonas ­circumdentaria, endodontalis, gingivicanis, and uenonis; (2) Porphyromonas cangingivalis, canoris, levii, and somerae; (3) Porphyromonas crevioricanis, gingivalis, and gulae; (4) Porphyromonas catoniae and macacae; and (5) Porphyromonas cansulci.

The genus Dysgonomonas comprises the type Dysgonomonas gadei and the closely related species Dysgonomonas capnocytophagoides and mossii. The last genus is monospecific, Tannerella forsythia. The current analyses add five other genera to this family. These include three monospecific genera represented by ­Paludibacter propionicigenes, Petrimonas sulfuriphila, and Proteiniphilum acetatigenes. Also included is the recently described genus comprising Barnesiella viscericola and intestinihominis (Morotomi et  al., 2008; Sakamoto et  al., 2007). Lastly, the genus Parabacteroides comprises the type species Parabacteroides distasonis and three closely related species Parabacteroides goldsteinii, johnsonii, and merdae (Sakamoto and Benno, 2006). This last genus was also described after the deadline for inclusion in this volume.

Family “Prevotellaceae” Although the family “Prevotellaceae” appears within the cluster of species of the family Bacteroidaceae, the genera representing the “Prevotellaceae” are well separated from the Bacteroidaceae. Therefore, both families are continued in the current classification. The genus Prevotella, which was formed by reclassification of ­various species of Bacteroides (Shah and Collins, 1990), is the type for this family. It comprises a number of phylogenetic groups, each of which may warrant reclassification into one or more genera if supported by additional evidence: (1) the type species Prevotella melaninogenica and Prevotella histolytica and veroralis; (2) Prevotella denticola and multiformis; (3) Prevotella corporis, disiens, falsenii, intermedia, nigrescens, and pallens; (4) Prevotella maculosa, oris, and salivae; (5) Prevotella bryantii, and multisaccharivorax; (6) Prevotella baroniae, buccae and dentalis; (7) Prevotella enoeca and pleuritidis; (8) Prevotella buccalis and timonensis; (9) Prevotella loescheii and shahii; (10) Prevotella brevis and ruminicola; and (11) Prevotella amnii and bivia.



Road map

The species Prevotella albensis, bergensis, copri, marshii, oralis, oulorum, paludivivens, and stercorea cannot be clearly assigned to one of the lineages. Xylanibacter oryzae is also found within the radiation of the described Prevotella clusters. Prevotella tannerae represents a more distant branch of the family. In contrast, Prevotella heparinolytica and zoogleoformans are clearly separated from the other members of this family and may warrant reclassification. Lastly, Hallella seregens is closely related to Prevotella dentalis, which has priority (Willems and Collins, 1995). Therefore, Hallella seregens is not used.

Class “Flavobacteriia” and order “Flavobacteriales” This class comprises a single order, “Flavobacteriales”, and is essentially unchanged from the original proposal of Garrity et al. (2005). The order comprises three families, Flavobacteriaceae, “Blattabacteriaceae”, and Cryomorphaceae (Figures 4 and 5) (Bowman et  al., 2003). The family “Myroidaceae” proposed by

Costertonia Flagellimonas Muricauda Maribacter Pibocella Robiginitalea Zeaxanthinibacter Zobellia Arenibacter Sediminicola Cellulophaga Dokdonia Krokinobacter Aquimarina Salinimicrobium Gillisia Psychroflexus Zunongwangia Gramella Salegentibacter Mesonia Donghaeana Persicivirga Nonlabens Sandarakinotalea Stenothermobacter Galbibacter Joostella Zhouia Coenonia Capnocytophaga Aequorivita Vitellibacter Flavobacterium Myroides Leeuwenhoekiella Leptobacterium Flaviramulus Tamlana Mariniflexile Yeosuana Gaetbulibacter Algibacter Lacinutrix Olleya Mesoflavibacter Bizionia

Flavobacteriaceae

FIGURE 4.  Genera of the class “Flavobacteriia”. This class comprises

three families. The first part of the family Flavobacteriaceae is shown here.

5

Garrity et  al. (2005) was judged to be insufficiently resolved from the Flavobacteriaceae and was not used.

Family Flavobacteriaceae This extraordinarily diverse family comprises over 70 genera. The rRNA analyses indicate the presence of many phylogenetic clusters that may warrant separation into novel families if supported by additional evidence. Many of the clusters described here are identical to those found by Bernardet and Nakagawa (2006) or include mostly taxa described after their work. Cluster (1) includes the type genus Flavobacterium and Myroides. This latter genus includes three closely related species, Myroides odoratus, odoratimimus, and pelagicus. Although the genus Flavobacterium is very diverse, the rRNA phylogeny lacks clear indication of clades that might serve as the basis for further subdivision. Species included in this genus include the type species Flavobacterium aquatile and, in alphabetical order, ­Flavobacterium antarcticum, aquidurense, branchiophilum, columnare, croceum, cucumis, daejeonense, defluvii, degerlachei, denitrificans, flevense, frigidarium, frigidimaris, frigoris, fryxellicola, gelidilacus, gillisiae, granuli, hercynium, hibernum, hydatis, johnsoniae, limicola, micromati, omnivorum, pectinovorum, psychrolimnae, psychrophilum, saccharophilum, saliperosum, segetis, soli, succinicans, suncheonense, tegetincola, terrae, terrigena, weaverense, xanthum, and xinjiangense. In addition, there are some species for which sequences are not available, including Flavobacterium acidificum, acidurans, oceanosedimentum, and thermophilum. (2) Capnocytophaga ochracea (type species), canimorsus, cynodegmi, gingivalis, granulosa, haemolytica, ochracea, and sputigena; and Coenonia anatina. Although Galbibacter mesophilus (Khan et  al., 2007c), Joostella marina (Quan et  al., 2008), and Zhouia amylolytica are associated with this cluster, this relationship is not strong. (3) Actibacter sediminis (Kim et  al., 2008a); Aestuariicola saemankumensis (Yoon et al., 2008d); Lutibacter litoralis; Lutimonas vermicola (Yang et  al., 2007); Polaribacter filamentus (type species), butkevichii, franzmannii, and glomeratus; Polaribacter dokdonensis (which forms a separate clade from the other species of this genus); Tenacibaculum maritimum (type species), adriaticum, aestuarii, aiptasiae, amylolyticum, galleicum, litopenaei, litoreum, lutimaris, mesophilum, ovolyticum, skagerrakense, and soleae. (4) Chryseobacterium gleum (type species), aquaticum, aquifrigidense, arothri, balustinum, bovis, caeni, daecheongense, daeguense, defluvii, flavum, formosense, gambrini, gregarium, haifense, hispanicum, hominis, hungaricum, indologenes, indoltheticum, jejuense, joostei, luteum, marina, molle, oranimense, pallidum, piscium, scophthalmum, shigense, soldanellicola, soli, taeanense, taichungense, taiwanense, ureilyticum, vrystaatense, and wanjuense. In addition to these species, the following taxa appear within the radiation of Chryseobacterium, including Epilithonimonas tenax, Kaistella koreensis, Sejongia antarctica (type species) and jeonii. Other taxa within this cluster include Bergeyella zoohelcum, Cloacibacterium normanense, Elizabethkingia meningoseptica (type species) and miricola, Empedobacter brevis, Ornithobacterium rhinotracheale, Riemerella anatipestifer (type species) and columbina, Wautersiella falsenii, and Weeksella virosa. (5) Arenibacter latericius (type species), certesii, echinorum, palladensis, and troitsensis; Cellulophaga algicola, baltica, and pacifica (a clade which does not include the type species); Costertonia aggregata; Flagellimonas eckloniae (Bae et  al., 2007); Maribacter

6 6

Road map

Bizionia Gelidibacter Subsaxibacter Subsaximicrobium Croceibacter Sediminibacter Psychroserpens Winogradskyella Formosa Marixanthomonas Ulvibacter Gilvibacter Aestuariicola Lutimonas Actibacter Polaribacter Tenacibaculum Lutibacter Fulvibacter Kordia Kaistella Sejongia Epilithonimonas Chryseobacterium Cloacibacterium Bergeyella Riemerella Elizabethkingia Empedobacter Wautersiella Weeksella Ornithobacterium Blattabacterium Cryomorpha Brumimicrobium Lishizhenia Fluviicola Crocinitomix Owenweeksia

Flavobacteriaceae

"Blattabacteriaceae"

Cryomorphaceae

FIGURE 5.  Genera of the class “Flavobacteriia”. This class comprises three families. The second part of the family Flavobacteriaceae and the remaining two families are shown here.

­sedimenticola (type species), aquivivus, arcticus, dokdonensis, forsetii, orientalis, polysiphoniae, and ulvicola; Muricauda ruestringensis (type species), aquimarina, lutimaris, and flavescens; Pibocella ponti (which appears within the cluster of Maribacter species); Robiginitalea biformata and myxolifaciens ; Sediminicola luteus; Zeaxanthinibacter enoshimensis (Asker et al., 2007); and Zobellia galactanivorans (type species), amurskyensis, laminariae, russellii, and uliginosa. In addition, Cellulophaga lytica (the type species of this genus) and fucicola appear as either a deep branch of this cluster (Bernardet and Nakagawa, 2006) or as an associated but independent group (this analysis). In either case, the reclassification of Cellulophaga algicola, baltica, and pacifica to a new genus would appears to be warranted. Lastly, the type strain of Pibocella ponti has been lost.

If available, this strain would be reclassified within Maribacter. For that reason, this genus is not included in the outline. (6) Algibacter lectus and mikhailovii; Flaviramulus basaltis; ­Gaetbulibacter saemankumensis and marinus; Mariniflexile gromovii and fucanivorans; Tamlana crocina (Lee, 2007); and Yeosuana ­aromativorans. (7) Croceibacter atlanticus and Sediminibacter furfurosus (Khan et al., 2007a). (8) Gelidibacter algens (type species), gilvus, mesophilus, and salicanalis; Subsaxibacter broadyi; Subsaximicrobium wynnwilliamsii (type species) and saxinquilinus. (9) Lacinutrix copepodicola, algicola, and mariniflava; and Olleya marilimosa.



7

Road map

(10) Gilvibacter sediminis (Khan et al., 2007a); Marixanthomonas ophiurae (Romanenko et  al., 2007). Ulvibacter litoralis and antarcticus. (11) Dokdonia donghaensis; Krokinobacter genikus (type species), diaphorus, eikastus, and genicus. (12) Donghaena dokdonensis; Nonlabens tegetincola; Persicivirga xylanidelens; Sandarakinotalea sediminis; and Stenothermobacter spongiae. (13) Gillisia limnaea (type species), hiemivivida, illustrilutea, mitskevichiae, myxillae, and sandarakina; Gramella echinicola (type species) and portivictoriae; Mesonia algae (type species) and mobilis; Psychroflexus torques (type species), gondwanensis, and tropicus; Salegentibacter salegens (type species), agarivorans, flavus, holothuriorum, mishustinae, salaries, and salinarum; Salinimicrobium catena, terrae, and xinjiangense (Chen et al., 2008; Lim et al., 2008); and Zunongwangia profunda (Qin et  al., 2007). Among these taxa, Salinimicrobium catena was previously classified as Salegentibacter catena (Lim et al., 2008). (14) Aequorivita antarctica (type species), crocea, lipolytica, and sublithincola and Vitellibacter vladivostokensis. In addition to these well delineated clusters, a large number of taxa were not closely associated with any of these clusters or each other. These include: Aquimarina muelleri (type species), brevivitae, intermedia, and latercula; Bizionia paragorgiae (type species), gelidisalsuginis, and saleffrena; a second clade of Bizionia species including Bizionia algoritergicola and myxarmorum; Formosa algae (type species) and agariphila; Fulvibacter tottoriensis; Kordia algicida; Leeuwenhoekiella marinoflava (type species), aequorea, and blandensis; Leptobacterium flavescens; Mesoflavibacter zeaxanthinifaciens; Psychroserpens burtonensis (type species) and mesophilus; and Winogradskyella thalassocola (type species), epiphytica, eximia, and poriferorum. The rRNA gene sequences of the following pairs of genera are closely related, which may justify combining them: Sandarakinotalea–Nonlabens; Dokdonia–Krokinobacter.

Family “Blattabacteriaceae” This family comprises Blattabacterium cuenoti, which is an endosymbiont of insects that has not been grown in pure culture.

Family Cryomorphaceae Proposed by Bowman et al. (2003) to include novel genera of cold-tolerant marine bacteria isolated from sea ice and other polar environments, this family comprises six monospecific genera: Cryomorpha ignava, Brumimicrobium glaciale, Crocinitomix catalasitica, Fluviicola taffensis, Lishizhenia caseinilytica, and Owenweeksia hongkongensis. The phylogenetic analyses conducted here suggest that this family is polyphenetic and contains three lineages that cluster together at the base of the phylogenetic tree for the Flavobacteriales. Cryomorpha and Owenweeksia each comprise one monogeneric lineage, with the remaining four genera comprising the third lineage. However, in the absence of additional evidence, these lineages were not separated at this time.

Class “Sphingobacteriia” and order “Sphingobacteriales” This class comprises a single order, the “Sphingobacteriales”. It is more circumscribed than the original proposal (2005) and excludes many taxa previously classified within the “Flexibacteraceae”. The order comprises three families: Sphingobacteriaceae, “Chitinophagaceae”, and “Saprospiraceae” (Figure 6). The family Crenotrichaceae was removed because the type genus Crenothrix was transferred to the Proteobacteria (Stoecker et al., 2006). The genus Chitinophaga then became the type for a new family within the order. Based upon their rRNA gene sequence similarities, the genera Rhodothermus and Salinibacter, which were also previously classified within the Crenotrichaceae, were transferred to the class “Cytophagia” as an order incertae sedis (see below). Similarly, Balneola, which was described after the deadline for

Sphingobacterium Pseudosphingobacterium Parapedobacter Sphingobacteriaceae

Olivibacter Pedobacter Nubsella Mucilaginibacter Chitinophaga Terrimonas Niabella Niastella

"Chitinophagaceae"

Flavisolibacter Sediminibacterium Segetibacter Saprospira Aureispira Lewinella Haliscomenobacter FIGURE 6.  Genera of the class “Sphingobacteriia”.

"Saprospiraceae"

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Road map

inclusion in this volume, was classified within the Crenotrichaceae based in part upon its similarity to Rhodothermus (Urios et al., 2006). Analyses performed here suggest that it may also be a deep lineage of the “Cytophagia”. Lastly, the sequence of the 16S rRNA gene for Toxothrix trichogenes is not available, so this genus was transferred to incertae sedis. Even with these changes, this class is not clearly monophyletic (Figure 2). The families “Chitinophagaceae” and “Saprospiraceae” may represent a sister lineage to the family Sphingobacteriaceae. However, it the absence of confirmatory evidence, the grouping of the three families into one class was retained in the current outline.

Family Sphingobacteriaceae At the time these analyses were performed, seven genera were identified within this family. The genera Sphingobacterium, Olivibacter, Parapedobacter, and Pseudosphingobacterium form one phylogenetic cluster. The genera Pedobacter, Mucilaginibacter, and Nubsella form a second cluster. In addition, the species Flexibacter canadensis represents an additional deep phylogenetic group within this family that may warrant classification as a novel genus. The genus Sphingobacterium comprises Sphingobacterium spiritivorum (type species), anhuiense, canadense, composti, daejeonense, faecium, kitahiroshimense, mizutaii, multivorum, siyangense, and thalpophilum. Interestingly, the species epithet Sphingobacterium composti was independently proposed for two different organisms by Ten et al. (2006, 2007) and Yoo et al. (2007). Because Sphingobacterium composti Ten et al. (2007) has priority, the species of Yoo et  al. (2007) warrants renaming. In addition, this genus contains Sphingobacterium antarcticum, whose rRNA gene sequence is not available. Related to the genus Sphingobacterium are the taxa Olivibacter sitiensis (type species), ginsengisoli, soli, and terrae (Ntougias et al., 2007; Wang et al., 2008), Parapedobacter koreensis (type species) and soli (Kim et al., 2007b, 2008b), and Pseudosphingobacterium domesticum (Kim et  al., 2007b; ­Vaz-Moreira et al., 2007). These genera were described after the deadline for inclusion in this volume. The second cluster is composed of Pedobacter species, which itself comprises four subclusters. The first subcluster contains Pedobacter heparinus (type species), africanus, caeni, cryoconitis, duraquae, ginsengisoli, himalayensis, metabolipauper, panaciterrrae, piscium, steynii, and westerhofensis. The second subcluster ­comprises Pedobacter insulae and koreeensis. The third sub­cluster comprises Pedobacter daechungensis, lentus, saltans, and ­terricola, which may warrant reclassification into a novel genus if ­supported by additional evidence. A fourth sub­cluster is represented by Mucilaginibacter gracilis, kameinonesis, and paludis (Pankratov et al., 2007; Urai et al., 2008). A number of species were not closely associated with any of these clusters or each other: Nubsella zeaxanthinifaciens (Asker et al., 2008); Pedobacter agri, aquatilis, composti, roseus, sandarokinus, suwonensis, and ­terrae.

Family “Chitinophagaceae” This family contains two phylogenetic clusters. The first cluster includes the genus Chitinophaga. This genus comprises Chitinophaga pinensis (type species), arvensicola, filiformis, ginsengisegetis, ginsengisoli, japonensis, sancti, skermani, and terraei. The second cluster includes six related genera with ten species: Flavisolibacter ginsengisoli and ginsengiterrae (Yoon and Im, 2007); Niabella aurantiaca and soli (Kim et  al., 2007a; Weon et  al.,

2008a); Niastella koreensis (type species) and yeongjuensis (Weon et al., 2006); Sediminibacterium salmoneum (Qu and Yuan, 2008); Segetibacter koreensis (An et  al., 2007); and Terrimonas ferruginea (type species) and lutea. Flavisolibacter, Niabella, Niastella, Sediminibacterium, and Segetibacter were described after the deadline for inclusion in this volume (Weon et al., 2006).

Family “Saprospiraceae” As originally proposed by Garrity et al. (2005), the family comprises three related genera and nine species. These include: Saprospira grandis; Haliscomenobacter hydrossis, and Lewinella cohaerens (type species), agarilytica, antarctica, lutea, marina, nigricans, and persica. Recently, the newly discovered genus Aureispira (marina and maritime) has also been classified within this family (Hosoya et al., 2006, 2007).

Class “Cytophagia” and order Cytophagales Analyses performed here of the rRNA genes indicate that many of the genera previously classified within the families “Flexibacteraceae” and “Flammeovirgaceae” are not closely related to the “Sphingobacteriia” and should be transferred to a novel class (­Figure 7). The order Cytophagales is designated the type for the new class. The genus Cytophaga is the type for the order and ­family Cytophagaceae. Because the family Cytophagaceae includes the type genera of the families “Flexibacteraceae” and Spirosomaceae, these classifications are not used.

Cytophaga Sporocytophaga Flexibacter Dyadobacter

Persicitalea Runella Emticicia Leadbetterella Spirosoma Rudanella Larkinella Arcicella Flectobacillus Hymenobacter Pontibacter Effluviibacter Adhaeribacter Microscilla Algoriphagus Aquiflexum Cyclobacterium Belliella Rhodonellum Echinicola Fabibacter Roseivirga Fulvivirga Persicobacter Reichenbachiella "Candidatus Cardinium" Flexithrix Rapidithrix Flammeovirga Perexilibacter Limibacter Sediminitomix Thermonema Balneola Rhodothermus Salinibacter

Cytophagaceae

"Cyclobacteriaceae"

"Flammeovirgaceae"

"Rhodothermaceae"

FIGURE 7.  Genera of the class “Cytophagia”. This class comprises three families and four orders incertae sedis.



Road map

Family Cytophagaceae This family comprises 19 genera distributed within seven phylogenetic clusters: the first cluster includes Cytophaga hutchinsonii (type species) and aurantiaca; the second cluster includes Sporocytophaga myxococcoides (type and only species); the third cluster includes Effluviibacter roseus; Hymenobacter roseosalivarius (type species), actinosclerus, aerophilus, chitinivorans, deserti, gelipurpurascens, norwichensis, ocellatus, psychrotolerans, rigui, soli, and xinjiangensis; and Pontibacter actiniarum (type species), akesuensis, and korlensis. In addition, Adhaeribacter aquaticus appears to represent a deep lineage in this cluster. The fourth cluster includes Arcicella aquatica and rosea; Dyadobacter fermentans (type species), alkalitolerans, beijingensis, crusticola, ginsengisoli, hamtensis, and koreensis; Emticicia ginsengisoli and oligotrophica; Flectobacillus major (type species) and lacus; Larkinella insperata; Leadbetterella byssophila; Persicitalea jodogahamensis (Yoon et al., 2007b); Rudanella lutea (Weon et al., 2008b); Runella slithyformis (type species), defluvii, limosa, and zeae; and Spirosoma linguale, panaciterrae, and rigui. The fifth cluster includes Flexibacter roseolus, elegans, and Microscilla marina. The sixth cluster comprises Flexibacter flexilis (type species). The ­seventh cluster comprises Flexibacter ruber. Cyclobacterium and Reichenbachiella, two genera previously classified with this group (Garrity et  al., 2005), have been transferred to the “Cyclobacteriaceae” and “Flammeovirgaceae”, respectively. In addition, Meniscus glaucopis is retained within the Cytophagaceae even though the sequence of its rRNA gene is not available.

Family “Cyclobacteriaceae” This family includes the genus Cyclobacterium, which was previously classified within the “Flexibacteraceae”, and five related genera: Cyclobacterium marinum (type species), amurskyense, and lianum; Aquiflexum balticum; Algoriphagus ratkowskyi (type species), alkaliphilus, antarcticus, aquimarinus, boritolerans, chordae, halophilus, locisalis, mannitolivorans, marincola, ornithinivorans, terrigena, vanfongensis, winogradskyi, and yeomjeoni. This cluster includes Chimaereicella and Hongiella species that were transferred to Algoriphagus (Nedashkovskaya et  al., 2007b); Belliella baltica; Echinicola pacifica (type species) and vietnamensis; and Rhodonellum psychrophilum represent further genera.

Family “Flammeovirgaceae” This family includes the genus Flammeovirga and at least seven related genera and one Candidatus taxon. This family comprises two phylogenetic groups which are neighbors in all trees but not clearly monophyletic. In addition, Thermonema, which was previously classified in this family (Garrity et  al., 2005), possesses only low similarity to the other genera and was reclassified to an order incertae sedis. Subsequently, it was found that this reassignment was equivocal, and analyses with more representatives of this family are ambiguous (Figure 7). For the purposes of this road map, this genus was retained in an order incertae sedis. As a result, this family comprises two phylogenetic groups: Flammeovirga aprica (type species), arenaria, kamogawensis, and yaeyamensis; Flexibacter aggregans, litoralis, and polymorphus, which appear to be misclassified; Flexithrix dorotheae; Limibacter armeniacum (Yoon et  al., 2008b); Perexilibacter aurantiacus (Yoon et  al., 2007a); Rapidithrix thailandica

9

(Srisukchayakul et  al., 2007); and Sediminitomix flava (Khan et al., 2007b). The second group comprises Fabibacter halotolerans; Fulvivirga kasyanovii (Nedashkovskaya et al., 2007a); Reichenbachiella agariperforans; Roseivirga ehrenbergii (type species), echinicomitans, seohaensis, and spongicola; and Persicobacter diffluens. In addition, Flexibacter tractuosus, which appears to be misclassified, and “Candidatus Cardinium hertigii”, a symbiont of parasitoid wasps (Zchori-Fein et al., 2004), are neighboring lineages.

Class “Cytophagia” orders incertae sedis In addition to the members of these families whose taxonomic position is relatively well defined, three deep lineages are classified within “Cytophagia” as separate orders incertae sedis. These lineages include (1) the family “Rhodothermaceae”, comprising Rhodothermus marinus and Salinibacter ruber; (2) the genus Balneola, with species Balneola vulgaris (type) and alkaliphila, which were described after the deadline for inclusion in the volume (Urios et al., 2006, 2008); and (3) Thermonema lapsum (type species) and rossianum (which may also be assigned to the “Flammeovirgaceae”). The assignment of the first two lineages to this class is ambiguous, and their reclassification may be warranted with additional evidence. Toxothrix trichogenes, for which the rRNA gene sequence is not available, is also included as incertae sedis within this class.

Phylum “Spirochaetes” As a result of the current analyses of 16S rRNA gene sequences, a single class and order are recognized within the phylum “Spirochaetes”. Members of the “Spirochaetes” possess a cellular ultrastructure unique to bacteria with internal organelles of motility, namely periplasmic flagella.

Class “Spirochaetia” and order Spirochaetales The class comprises a single order. The order Spirochaetales comprises four families that are well delineated by 16S rRNA gene sequences (Figure 8). Compared to the previous outline (Garrity et  al., 2005), the families Spirochaetaceae and Leptospiraceae are retained in the current classification. However, the genus Serpulina was judged to be a subjective synonym of Brachyspira (Ochiai et  al., 1997). As a consequence, the family “Serpulinaceae” was replaced with “Brachyspiraceae”. The genus Brevinema was also transferred from the family Spirochaetaceae to a novel family “Brevinemataceae” in recognition of the differences in 16S rRNA gene sequences. Lastly, four genera of arthropod symbionts for which no sequences are available were transferred from the Spirochaetaceae to a fifth family, incertae sedis.

Family Spirochaetaceae This family comprises four genera that are well delineated on the basis of their 16S rRNA gene sequences. Compared to previous classifications, the genus Brevinema was transferred to a new family on the basis of substantial differences in its 16S rRNA gene sequence. Likewise, the genera Clevelandina, Diplocalyx, Hollandina, and Pillotina were transferred to a family incertae sedis in the absence of rRNA gene sequences. The culture for the type species of the genus Spirochaeta, Spirochaeta plicatilis, is not available, and its rRNA gene has not

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Road map

Spirochaeta Treponema Borrelia

Spirochaetaceae

Cristispira Brevinema Brachyspira

"Brevinemataceae" "Brachyspiraceae"

Leptospira Leptonema

Leptospiraceae

Turneriella FIGURE 8.  Genera of the phylum “Spirochaetes”.

been sequenced. Of the remaining species, three are more closely related to Treponema and should probably be reclassified within that group (see below). The remaining species of the genus Spirochaeta comprise at least seven phylogenetic groups: (1) Spirochaeta africana and asiatica; (2) Spirochaeta alkalica, americana, and halophila; (3) Spirochaeta aurantia; (4) Spirochaeta bajacaliforniensis and smaragdinae; (5) Spirochaeta coccoides; (6) Spirochaeta isovalerica and litoralis; and (7) Spirochaeta thermophila. The genus Borrelia comprises three phylogenetic groups. One group contains the type species, Borrelia anserina, and the causative agents of relapsing fever, Borrelia coriaceae, crocidurae, duttonii, hermsii, hispanica, miyamotoi, parkeri, persica, recurrentis, theileri, and turicatae.. Many of these species are transmitted by soft-bodied ticks. The second group includes the causative agent of Lyme disease, Borrelia burgdorferi, and species transmitted by hard-bodied ticks, Borrelia afzelii, burgdorferi, garinii, japonica, lusitaniae, sinica, spielmanii, tanukii, turdi, and valaisiana. The third group consists solely of Borrelia turcica. In addition, sequences are not available for some named species, including Borrelia baltazardii, brasiliensis, caucasica, dugesii, graingeri, harveyi, latyschewii, mazzottii, tillae, and venezuelensis. The genus Cristispira is represented by a single species, Cristispira pectinis, which is related to Borrelia. This microorganism has been identified in the crystalline styles of oysters (Paster et al., 1996). The genus Treponema comprises three phylogenetic groups: Treponema pallidum (type species), “calligyrum”, denticola, medium, phagedenis, putidum, “refringens”, and “vincentii”; in addition, Spirochaeta zuelzerae is associated with this group. The second group comprises Treponema amylovorum, berlinense, bryantii, brennaborense, lecithinolyticum, maltophilum, parvum, pectinovorum, porcinum, saccharophilum, socranskii, and succinifaciens. The third group comprises Treponema azotonutricium and primita; in addition, Spirochaeta caldaria and stenostrepta are associated with this group. Lastly, no sequence is available for Treponema minutum, so its placement is ambiguous.

Family “Brachyspiraceae” This family comprises a single genus of closely related species: Brachyspira aalborgi (type species), alvinipulli, hyodysenteriae, innocens, intermedia, murdochii, and pilosicoli. Many of the species in this genus were previously classified in the genus Serpulina, which is not used in the current classification (Ochiai et al., 1997).

Family “Brevinemataceae” This family is represented by a single genus and species, Brevinema andersonii, isolated from rodents.

Family Leptospiraceae This family comprises the large genus Leptospira and two monospecies genera, Leptonema illini and Turneriella parva. These latter genera were previously classified within the Leptospira. However, on the basis of differences in their 16S rRNA gene sequences, they were transferred to novel genera. The genus Leptospira comprises three phylogenetic groups: (1) Leptospira interrogans (type species), alexanderi, borgpetersenii, kirschneri, noguchii, santarosai, and weilii; (2) Leptospira broomii, fainei, inadai, licerasiae, and wolffii; and (3) Leptospira biflexa, meyeri, and wolbachii.

Spirochaetales family incertae sedis This family includes four genera of symbionts of arthropod invertebrates. Although their morphologies have been described in detail (Bermudes et  al., 1988), their 16S rRNA genes have not been sequenced, and their phylogenetic placements are unknown. They are Clevelandina reticulitermitidis, Diplocalyx calotermitidis, Hollandina pterotermitidis, and Pillotina calotermitidis.

Phylum Tenericutes This phylum comprises a single class, Mollicutes, which was previously classified within the Firmicutes (Garrity et al., 2005). Elevation of these organisms to a separate phylum is justified in part by analyses of a number of conserved phylogenetic markers such as the elongation factor Tu and RNA polymerase (Ludwig and Schleifer, 2005). This classification is further supported by the presence of a wall-less cytoplasmic membrane which is a distinctive cellular structure of this group.

Class Mollicutes This class comprises four orders, Mycoplasmatales, Entomoplasmatales, Acholeplasmatales, and Anaeroplasmatales. While these orders do not agree well with the 16S rRNA gene phylogeny, efforts to reorganize the taxonomy are confounded by the presence of many human and animal pathogens within the group and the priority of some genus names that are seldom used (Brown et al., 2010). A major difficulty is the polyphyletic nature of the genus Mycoplasma, species of which are found in 13 distinct clusters distributed over three deep lineages. A fuller



Road map

discussion of the complexities of this group along with rRNA gene trees is found in the chapter on Mycoplasmatales.

Order Mycoplasmatales This order is the type for the class and comprises two families and four genera. The genera Mycoplasma and Ureaplasma are classified within the family Mycoplasmataceae. The other two genera, Eperythrozoon and Haemobartonella, contain many blood parasites that have not been cultivated. Although some of the species have been transferred to the genus Mycoplasma, the genera are classified within a family incertae sedis in recognition of the remaining uncertainties in their classification.

Family Mycoplasmataceae This family contains the genera Mycoplasma and Ureaplasma. While Ureaplasma is well defined on the basis of its rRNA gene sequence phylogeny, Mycoplasma is found in at least three deep phylogenetic lineages or groups. The first group contains the type species, Mycoplasma mycoides, which is actually more closely related to Entomoplasma, the type genus of the order Entomoplasmatales, than to most other species of Mycoplasma and Ureaplasma. A second lineage, called the “pneumoniae group”, includes the genus Ureaplasma as well as four Mycoplasma ­clusters. The third lineage, called the “hominis group”, includes the remaining eight Mycoplasma clusters. The group containing the type species includes: Mycoplasma mycoides (type species), capricolum, cottewii, putrefaciens, and yeatsii. The “hominis group” includes eight clusters of Mycoplasma ­species. (1) The “bovis” cluster comprises Mycoplasma adleri, agalactiae, bovigenitalium, bovis, californicum, caviae, columbinasale, columbinum, felifaucium, fermentans, gallinarum, iners, leopharyngis, lipofaciens, maculosum, meleagridis, opalescens, phocirhinis, primatum, simbae, and spermatophilum. (2) The “equigenitalium” cluster comprises ­Mycoplasma elephantis and equigenitalium. (3) The “hominis” cluster comprises Mycoplasma alkalescens, anseris, arginini, arthritidis, auris, buccale, canadense, cloacale, equirhinis, falconis, faucium, gateae, gypis, hominis, hyosynoviae, indiense, orale, phocicerebrale, phocidae, salivarium, spumans, and subdolum. (4) The “lipophilum” cluster comprises Mycoplasma hyopharyngis and lipophilum. (5) The “neurolyticum” cluster comprises Mycoplasma bovoculi, collis, cricetuli, conjunctivae, dispar, flocculare, hyopneumoniae, hyorhinis, iguanae, lagogenitalium, molare, neurolyticum, and ovipneumoniae. (6) The “pulmonis” cluster comprises Mycoplasma agassizii, pulmonis, and testudineum. (7) The “sualvi “ cluster comprises Mycoplasma moatsii, mobile, and sualvi. (8) The “synoviae” cluster comprises Mycoplasma alligatoris, anatis, bovirhinis, buteonis, canis, citelli, columborale, corogypsi, crocodyli, cynos, edwardii, felis, gallinaceum, gallopavonis, glycophilum, leonicaptivi, ­mustelae, oxoniensis, pullorum, sturni, synoviae, and verecundum. The “pneumoniae group” includes four clusters of Mycoplasma species and Ureaplasma. (1) The “fastidiosum” cluster comprises Mycoplasma cavipharyngis and fastidiosum. (2) The “hemotrophic” cluster comprises many species that were formerly classified within the genera Eperythrozoon and Haemobartonella (see below), including Mycoplasma coccoides, haemocanis, haemofelis, haemomuris, ovis, suis, and wenyonii. (3) The “muris” cluster comprises Mycoplasma iowae, microti, muris, and penetrans. (4) The “pneumoniae” cluster comprises Mycoplasma alvi, amphoriforme, gallisepticum, genitalium, imitans, pirum, pneumoniae, and testudinis. The genus Ureaplasma comprises Ureaplasma urealyticum (type species), canigenitalium, cati, diversum, felinum, gallorale, and parvum.

11

Mycoplasmatales family incertae sedis This family includes the genera of blood parasites Eperythrozoon and Haemobartonella. Species whose 16S rRNA genes have been sequenced are also classified within the Mycoplasma hemotrophic cluster. On the basis of their 16S rRNA gene sequences, the species of these genera are intermixed in two groups. The first group comprises Eperythrozoon coccoides (type species) and Haemobartonella canis and felis. Haemobartonella muris, which is the type species of its genus, is a deep lineage in this group. Upon reclassification to Mycoplasma, the Haemobartonella species were renamed haemocanis, haemofelis, and haemomuris, respectively, to distinguish them from previously named Mycoplasma species. The second group comprises Eperythrozoon ovis, suis, and wenyonii.

Order Entomoplasmatales This order contains two families, Entomoplasmataceae and Spiroplasmataceae. The order is paraphyletic because it includes the type species of the genus Mycoplasma, most species of which are classified in the Mycoplasmatales.

Family Entomoplasmataceae This family comprises the genera Entomoplasma and Mesoplasma. However, on the basis of their 16S rRNA gene sequences, some species of Acholeplasma appear to be misclassified within this group. The family comprises four phylogenetic lineages: (1) Entomoplasma ellychniae (type species), Mesoplasma florum (type species), and Mesoplasma chauliocola, coleopterae, corruscae, entomophilum, grammopterae, and tabanidae; (2) Mesoplasma photuris, seiffertii, and syrphidae; Entomoplasma lucivorax, luminosum, and somnilux; and Acholeplasma multilocale; (3) Mesoplasma lactucae; and (4) the group containing the type species of Mycoplasma, Mycoplasma mycoides (see above).

Family Spiroplasmataceae This family comprises the single genus Spiroplasma, which itself comprises three relatively deep phylogenetic lineages. In fact, these lineages are no more closely related to each other than to some Mycoplasma species. These lineages include (1) Spiroplasma citri (type species), chrysopicola, insolitum, melliferum, penaei, phoeniceum, poulsonii, and syrphidicola; (2) Spiroplasma alleghenense, cantharicola, chinense, corruscae, culicicola, diabroticae, diminutum, gladiatoris, helicoides, lampyridicola, leptinotarsae, lineolae, litorale, montanense, sabaudiense, turonicum, and velocicrescens; and (3) Spiroplasma ixodetis and platyhelix.

Order Acholeplasmatales and family Acholeplasmataceae This order comprises the family Acholeplasmataceae and a family incertae sedis of uncultured plant pathogens classified within “Candidatus Phytoplasma”. On the basis of their 16S rRNA gene sequences, both of these groups are well defined phylogenetically. The family Acholeplasmataceae comprises four closely related lineages that are all classified with the genus Acholeplasma: (1) Acholeplasma laidlawii (type species), equifetale, granularum, oculi, and pleciae; (2) Acholeplasma axanthum, cavigenitalium, and modicum; Mycoplasma feliminutum; (3) Acholeplasma brassicae, morum, and vituli; and (4) Acholeplasma palmae and parvum.

12 12

Road map

Order Anaeroplasmatales and family Anaeroplasmataceae

Phylum “Fibrobacteres” This phylum comprises the class “Fibrobacteria”, the order “Fibrobacterales”, the family “Fibrobacteraceae”, and the genus Fibrobacter. This genus contains two species, Fibrobacter succinogenes (type species) and intestinalis.

This order and family comprises two genera which, on the basis of 16S rRNA gene sequence similarity, are not closely related. Anaeroplasma is related to members of the order Acholeplasmatales. The second genus, Asteroleplasma, appears to represent a very deep lineage within the phylum. The genus Anaeroplasma comprises three closely related species: Anaeroplasma abactoclasticum (type species), bactoclasticum, and varium. In addition, the species Anaeroplasma intermedium has been described for which no sequence is available. Asteroleplasma anaerobium is the sole species in the genus Asteroleplasma.

Phylum “Fusobacteria” This phylum comprises a single class, “Fusobacteriia”, and order “Fusobacteriales”. Two families are currently described (Figure 10). While the family “Leptotrichiaceae” is well defined on the basis of 16S rRNA gene sequences, the family “Fusobacteriaceae” is more complicated. It comprises five genera. The genus Fusobacterium is paraphyletic and includes the lineage containing the genus Cetobacterium. The genera Ilyobacter and Propionigenium are also intermixed. If additional evidence supports these conclusions, reclassification within this family would be warranted. The phylogenetic groups within the family “Fusobacteriaceae” are (1) Fusobacterium nucleatum (type species), canifelinum, equinum, gonidiaformans, mortiferum, necrogenes, necrophorum, perfoetens, periodonticum, russii, simiae, ulcerans, and varium; and Cetobacterium ceti (type species) and somerae, representing a deeper branch; (2) Ilyobacter polytropus (type species), insuetus, and tartaricus; Propionigenium modestum (type species) and maris; and (3) Psychrilyobacter atlanticus, which was described after the deadline for inclusion in this volume, but it appears to be a deep lineage of this family (Zhao et al., 2009). The phylogenetic groups within the family “Leptotrichiaceae” are (1) Leptotrichia buccalis (type species), hofstadii, shahii, trevisanii, and wadei; (2) Leptotrichia goodfellowii; (3) Sebaldella termitidis; (4) Sneathia sanguinegens; and (5) Streptobacillus moniliformis.

Phylum “Acidobacteria” With only seven species, this phylum of mostly oligotrophic heterotrophs comprises two classes of validly published bacteria (Figure 9). However, surveys of environmental DNA indicate that this is one of the most abundant groups of bacteria in soil and many other habitats.

Class “Acidobacteriia”, order “Acidobacteriales”, and family “Acidobacteriaceae” These taxa comprise two monospecific genera, represented by Acidobacterium capsulatum and Terriglobus roseus, and Edaphobacter modestus (type species) and aggregans.

Class Holophagae, order Holophagales, family Holophagaceae, order Acanthopleuribacterales, and family Acanthopleuribacteraceae The family Holophagaceae comprises two monospecific genera, represented by Holophaga foetida and Geothrix fermentans. The family Acanthopleuribacteraceae comprises one monospecific genus, Acanthopleuribacter.

Phylum “Dictyoglomi” This phylum comprises the class “Dictyoglomia”, the order “Dictyoglomales”, the family “Dictyoglomaceae”, and the genus

Acidobacterium Edaphobacter Terriglobus Geothrix Holophaga Acanthopleuribacter

"Acidobacteriaceae"

"Acidobacteriia"

Holophagaceae

Holophagae

Acanthopleuribacteraceae

FIGURE 9.  Genera of the phylum “Acidobacteria”.

Fusobacterium Cetobacterium Ilyobacter

"Fusobacteriaceae"

Propionigenium Psychrilyobacter Leptotrichia Sebaldella Streptobacillus Sneathia FIGURE 10.  Genera of the phylum “Fusobacteria”.

"Leptotrichiaceae"



Road map

13

This phylum comprises the class Gemmatimonadetes, the order Gemmatimonadales, the family Gemmatimonadaceae, and the genus Gemmatimonas. This genus contains one species, Gemmatimonas aurantiaca.

albus, croceus, and litoralis. In addition, the genus “Fucophilus”, which has been described but whose name has never been validly published, is a member of this family. The class “Spartobacteria” comprises the order “Chthoniobacterales”, which includes the family “Chthoniobacteraceae”. This family comprises “Chthoniobacter flavus” and the nematode symbionts “Candidatus Xiphinematobacter brevicolli” (type species), “americani”, and “rivesi”.

Phylum Lentisphaerae

Phylum “Chlamydiae”

On the basis of their 16S rRNA gene sequences and other molecular markers, this phylum is related to the phyla “Verrucomicrobia”, “Chlamydiae”, and “Planctomycetes”, which form a deep group within the Bacteria. The phylum Lentisphaerae comprises the class “Lentisphaeria” and two orders. The order Lentisphaerales comprises the family “Lentisphaeraceae” and the monospecific genus Lentisphaera, the type of which is Lentisphaera araneosa. The order Victivallales comprises the family “Victivallaceae”and the monospecific genus Victivallis, the type of which is Victivallis vadensis.

On the basis of their 16S rRNA gene sequences and other molecular markers, this phylum is related to the phyla Lentisphaerae, “Planctomycetes”, and “Verrucomicrobia”, which form a deep group within the bacteria. All known members of the ­phylum “Chlamydiae” are obligate intracellular bacteria and multiply in eukaryotic hosts, including humans and other animals and protozoa. They also possess a developmental cycle that is characterized by morphologically and physiologically distinct stages. The intracellular lifestyle of chlamydiae is thus thought to be an ancient trait of this phylum (Everett et al., 1999). As a consequence of the intracellular lifestyle, no species has ever been grown in axenic culture. Because of changes to the Bacteriological Code beginning in 1997, only the species described before that time have been validly published, and many of the newer taxa are limited to Candidatus status (Labeda, 1997; ­Murray and Stackebrandt, 1995). In addition, even though some species have been cultivated in the free-living amoebae Acanthamoeba castellanii and Acanthamoeba polyphaga, they have not been deposited in two public culture collections, and thus their names have not been validly published (Heyrman et al., 2005). The phylum “Chlamydiae” comprises a single class, “Chlamydiia”, and order, Chlamydiales. The order comprises eight families of varying relatedness based upon 16S rRNA gene sequence similarities (Figure 12). The family Chlamydiaceae contains the type genus for the order. Two taxonomies are in widespread use for this family. One taxonomy assigns all species within this family to the genus Chlamydia. The second taxonomy classifies many of these species within a second genus, Chlamydophila, in recognition of their differences in a variety of molecular markers including the 16S rRNA gene and some phenotypic ­markers (Everett et al., 1999). The merits of these approaches have been discussed (Everett and Andersen, 2001; Schachter et al., 2001). While this taxonomic outline uses the taxonomy of Everett et al. (1999), the first taxonomy is used by the authors of the chapter Chlamydiaceae (Kuo and Stephens, 2010). On the basis of the taxonomy of Everett et al. (1999), the genus Chlamydia comprises Chlamydia trachomatis (type species), muridarum, and suis. The genus Chlamydophila comprises Chlamydophila psittaci (type species), abortus, caviae, felis, pecorum, and pneumoniae. The remaining families in the order are: “Candidatus Clavichlamydiaceae” comprising “Candidatus Clavichlamydia salmonicola”; “Criblamydiaceae” comprising “Criblamydia sequanensis”; Parachlamydiaceae comprising Parachlamydia acanthamoebae (type species and genus), Neochlamydia hartmannellae, and “Protochlamydia amoebophila”; “Candidatus Piscichlamydiaceae” comprising “Candidatus Piscichlamydia salmonis”; “Rhabdochlamydiaceae” comprising “Candidatus Rhabdochlamydia porcellionis” and “Candidatus Rhabdochlamydia crassificans”; Simkaniaceae comprising Simkania negevensis (type species and genus) and “Candidatus Fritschea

­ ictyoglomus. This genus contains two species, Dictyoglomus therD mophilum (type species) and turgidum.

Phylum Gemmatimonadetes

Phylum “Verrucomicrobia” On the basis of their 16S rRNA gene sequences and other molecular markers, this phylum is related to the phyla “Chlamydiae”, Lentisphaerae, and “Planctomycetes”, which form a deep group within the bacteria. “Verrucomicrobia” comprises three classes, Verrucomicrobiae, Opitutae, and “Spartobacteria” (Figure 11). Currently, the class Verrucomicrobiae comprises the order Verrucomicrobiales, which comprises the families Verrucomicrobiaceae, “Akkermansiaceae”, and “Rubritaleaceae”. The family Verrucomicrobiaceae comprises Verrucomicrobium spinosum, Prosthecobacter fusiformis (type species), debontii, dejongeii, and vanneervenii. In addition, Prosthecobacter fluviatilis, which was described after the deadline for inclusion in this volume, is a member of this family (Takeda et  al., 2008). The family “Akkermansiaceae” comprises the monospecific genus Akkermansia, the type of which is Akkermansia muciniphila. The family “Rubritaleaceae” comprises Rubritalea marina (type species), sabuli, spongiae, squalenifaciens, and tangerina. In addition to these genera, four genera were described after the deadline for inclusion in this volume. Persicirhabdus sediminis; and Roseibacillus ishigakijimensis (type species), persicicus, and ponti (Yoon et al., 2008a), are affiliated with the family “Rubritaleaceae”. The remaining two genera, Haloferula rosea (type species), harenae, helveola, phyci, rosea, and sargassicola (Yoon et al., 2008c) and Luteolibacter pohnpeiensis (type species) and algae (Yoon et  al., 2008a), appear to be members of the order Verrucomicrobiales, but their affiliation with a particular family is more ambiguous. For this reason, they have not been included in the Taxonomic Outline. The class Opitutae comprises the orders Opitutales and Puniceicoccales. The order Opitutales comprises a single family, Opitutaceae, and two monospecific genera, the type species of which are Opitutus terrae and Alterococcus agarolyticus. The order Puniceicoccales comprises a single family, Puniceicoccaceae, and four genera. The genera form two clusters. The first cluster includes three monospecific genera, the type species of which are Puniceicoccus vermicola, Cerasicoccus arenae, and Coraliomargarita akajimensis. The second cluster includes Pelagicoccus mobilis (type species),

Roseibacillus Akkermansia

"Fucophilus"

Cerasicoccus

Puniceicoccus

"Candidatus Xiphinematobacter"

Alterococcus

Pelagicoccus

Coraliomargarita

Chthoniobacter

Prosthecobacter

Verrucomicrobium

Luteolibacter

Haloferula

FIGURE 11.  Genera of the phylum “Verrucomicrobia”.

Opitutus

Rubritalea Persicirhabdus

Opitutaceae

Puniceicoccaceae

"Chthoniobacteraceae"

Verrucomicrobiaceae

"Akkermansiaceae"

"Rubritaleaceae"

Opitutae

"Spartobacteria"

Verrucomicrobiae

14 14 Road map

FIGURE 12.  Genera of the phylum “Chlamydiae”.

Waddlia

Parachlamydia

Neochlamydia

"Candidatus

"Candidatus Fritschea"

Piscichlamydia"

"Candidatus Rhabdochlamydia"

Simkania

"Protochlamydia"

"Criblamydia"

"Candidatus Clavichlamydia"

Chlamydophila

Chlamydia

"Piscichlamydiaceae"

Waddliaceae

Simkaniaceae

"Rhabdochlamydiaceae"

Parachlamydiaceae

"Criblamydiaceae"

"Clavichlamydiaceae"

Chlamydiaceae

Road map 15

16 16

Road map Planctomyces Schlesneria Singulisphaera Isosphaera Gemmata

Planctomycetales

Blastopirellula Rhodopirellula Pirellula "Candidatus Anammoxoglobus" "Candidatus Jettenia" "Candidatus Brocadia"

"Brocadiales"

"Candidatus Kuenenia" "Candidatus Scalindua" FIGURE 13.  Genera of the phylum “Planctomycetes”.

bemisiae” and “Candidatus Fritschea eriococci”; and Waddliaceae comprising Waddlia chondrophila and “Waddlia malaysiensis” (Chua et al., 2005).

Phylum “Planctomycetes” This phylum comprises a single class, “Planctomycetia”, and two orders, Planctomycetales and “Brocadiales” (Figure 13). The order Planctomycetales comprises the family Planctomycetaceae, containing eight diverse genera. The type genus is Planctomyces. However, a strain and 16S rRNA gene sequence are not available for the type species, Planctomyces bekefii, or for two other validly published species in this genus, Planctomyces guttaeformis and stranskae. Therefore, the taxonomy of this group is based upon the properties of the species that are available: Planctomyces brasiliensis, limnophilus, and maris. Most of the other genera in this family are monospecific and represented by Blastopirellula marina, Gemmata obscuriglobus, Isosphaera pallida, Pirellula staleyi (type species) and marina, Rhodopirellula baltica, Schlesneria paludicola, and Singulisphaera acidiphila. In addition to these, Zavarzinella formosa was described after the deadline for this volume but could be classified within this family (Kulichevskaya et al., 2009). The order “Brocadiales” and family “Brocadiaceae” comprises Candidatus species. They include “Candidatus Brocadia anammoxidans” and “fulgida”, “Candidatus Anammoxoglobus propionicus”, “Candidatus Jettenia asiatica”, “Candidatus Kuenenia stuttgartiensis”, and “Candidatus Scalindua brodiae”, “­sorokinii”, and “wagneri”.

References An, D.S., H.G. Lee, W.T. Im, Q.M. Liu and S.T. Lee. 2007. Segetibacter koreensis gen. nov., sp. nov., a novel member of the phylum Bacteroidetes, isolated from the soil of a ginseng field in South Korea. Int. J. Syst. Evol. Microbiol. 57: 1828–1833. Asker, D., T. Beppu and K. Ueda. 2007. Zeaxanthinibacter enoshimensis gen. nov., sp. nov., a novel zeaxanthin-producing marine bacterium of the family Flavobacteriaceae, isolated from seawater off Enoshima Island, Japan. Int. J. Syst. Evol. Microbiol. 57: 837–843. Asker, D., T. Beppu and K. Ueda. 2008. Nubsella zeaxanthinifaciens gen. nov., sp. nov., a zeaxanthin-producing bacterium of the family Sphingobacteriaceae isolated from freshwater. Int. J. Syst. Evol. Microbiol. 58: 601–606.

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Cole, J.R., B. Chai, R.J. Farris, Q. Wang, A.S. Kulam-Syed-Mohideen, D.M. McGarrell, A.M. Bandela, E. Cardenas, G.M. Garrity and J.M. Tiedje. 2007. The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data. Nucleic Acids Res. 35: D169–D172. De Vos, P., H.G. Truper and B.J. Tindall. 2005. Judicial Commission of the International Committee on Systematics of Prokaryotes Minutes. Xth International (IUMS) Congress of Bacteriology and Applied Microbiology. Int. J. Syst. Bacteriol. 55: 525–532. Everett, K.D., R.M. Bush and A.A. Andersen. 1999. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int. J. Syst. Bacteriol. 49: 415–440. Everett, K.D.E. and A.A. Andersen. 2001. Radical changes to chlamydial taxonomy are not necessary just yet: reply. Int. J. Syst. Evol. Microbiol. 51: 251–253. Garrity, G.M. and J.G. Holt. 2001. The road map to the manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 1, The Archaea and the Deeply Branching and Phototrophic Bacteria (edited by Boone, Castenholz and Garrity). Springer, New York, pp. 119–166. Garrity, G.M., J.A. Bell and T. Lilburn. 2005. The revised road map to the manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, The Proteobacteria, Part A, Introductory Essays (edited by Brenner, Krieg, Staley and Garrity). Springer, New York, pp. 159–206. Griffiths, E. and R.S. Gupta. 2007. Phylogeny and shared conserved inserts in proteins provide evidence that Verrucomicrobia are the closest known free-living relatives of chlamydiae. Microbiology 153: 2648–2654. Hardham, J.M., K.W. King, K. Dreier, J. Wong, C. Strietzel, R.R. Eversole, C. Sfintescu and R.T. Evans. 2008. Transfer of Bacteroides splanchnicus to Odoribacter gen. nov. as Odoribacter splanchnicus comb. nov., and description of Odoribacter denticanis sp. nov., isolated from the crevicular spaces of canine periodontitis patients. Int. J. Syst. Evol. Microbiol. 58: 103–109. Heyrman, J., M. Rodriguez-Diaz, J. Devos, A. Felske, N.A. Logan and P. De Vos. 2005. Bacillus arenosi sp. nov., Bacillus arvi sp. nov. and Bacillus humi sp. nov., isolated from soil. Int. J. Syst. Evol. Microbiol. 55: 111–117. Holmes, D.E., K.P. Nevin, T.L. Woodard, A.D. Peacock and D.R. Lovley. 2007. Prolixibacter bellariivorans gen. nov., sp. nov., a sugar-fermenting, psychrotolerant anaerobe of the phylum Bacteroidetes, isolated from a marine-sediment fuel cell. Int. J. Syst. Evol. Microbiol. 57: 701–707. Hosoya, S., V. Arunpairojana, C. Suwannachart, A. Kanjana-Opas and A. Yokota. 2006. Aureispira marina gen. nov., sp. nov., a gliding, arachidonic acid-containing bacterium isolated from the southern coastline of Thailand. Int. J. Syst. Evol. Microbiol. 56: 2931–2935. Hosoya, S., V. Arunpairojana, C. Suwannachart, A. Kanjana-Opas and A. Yokota. 2007. Aureispira maritima sp. nov., isolated from marine barnacle debris. Int. J. Syst. Evol. Microbiol. 57: 1948–1951. Khan, S.T., Y. Nakagawa and S. Harayama. 2007a. Sediminibacter furfurosus gen. nov., sp. nov. and Gilvibacter sediminis gen. nov., sp. nov., novel members of the family Flavobacteriaceae. Int. J. Syst. Evol. Microbiol. 57: 265–269. Khan, S.T., Y. Nakagawa and S. Harayama. 2007b. Sediminitomix flava gen. nov., sp. nov., of the phylum Bacteroidetes, isolated from marine sediment. Int. J. Syst. Evol. Microbiol. 57: 1689–1693. Khan, S.T., Y. Nakagawa and S. Harayama. 2007c. Galbibacter mesophilus gen. nov., sp. nov., a novel member of the family Flavobacteriaceae. Int. J. Syst. Evol. Microbiol. 57: 969–973. Kim, B.Y., H.Y. Weon, S.H. Yoo, S.B. Hong, S.W. Kwon, E. Stackebrandt and S.J. Go. 2007a. Niabella aurantiaca gen. nov., sp. nov., isolated from a greenhouse soil in Korea. Int. J. Syst. Evol. Microbiol. 57: 538–541.

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Kim, J.H., K.Y. Kim, Y.T. Hahm, B.S. Kim, J. Chun and C.J. Cha. 2008a. Actibacter sediminis gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from tidal flat sediment. Int. J. Syst. Evol. Microbiol. 58: 139–143. Kim, M.K., J.R. Na, D.H. Cho, N.K. Soung and D.C. Yang. 2007b. Parapedobacter koreensis gen. nov., sp. nov. Int. J. Syst. Evol. Microbiol. 57: 1336–1341. Kim, M.K., Y.A. Kim, Y.J. Kim, N.K. Soung, T.H. Yi, S.Y. Kim and D.C. Yang. 2008b. Parapedobacter soli sp. nov., isolated from soil of a ginseng field. Int. J. Syst. Evol. Microbiol. 58: 337–340. Kulichevskaya, I.S., O.I. Baulina, P.L. Bodelier, W.I. Rijpstra, J.S. Damste and S.N. Dedysh. 2009. Zavarzinella formosa gen. nov., sp. nov., a novel stalked, Gemmata-like planctomycete from a Siberian peat bog. Int. J. Syst. Evol. Microbiol. 59: 357–364. Kuo, C.C. and R.S. Stephens. 2010. Family I. Chlamydiaceae. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 4, The Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes (edited by Krieg, Staley, Brown, ­Hedlund, Paster, Ward, Ludwig and Whitman). Springer, New York, p. 845. Labeda, D.P. 1997. International Committee on Systematic Bacteriology, VIIth International Congress of Microbiology and Applied ­Bacteriology Minutes. Int. J. Syst. Bacteriol. 47: 597–600. Lee, K.C., R.I. Webb, P.H. Janssen, P. Sangwan, T. Romeo, J.T. Staley and J.A. Fuerst. 2009. Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes. BMC Microbiol. 9: 5. Lee, S.D. 2007. Tamlana crocina gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae, isolated from beach sediment in Korea. Int. J. Syst. Evol. Microbiol. 57: 764–769. Lim, J.M., C.O. Jeon, S.S. Lee, D.J. Park, L.H. Xu, C.L. Jiang and C.J. Kim. 2008. Reclassification of Salegentibacter catena Ying et  al. 2007 as Salinimicrobium catena gen. nov., comb. nov. and description of Salinimicrobium xinjiangense sp. nov., a halophilic bacterium isolated from Xinjiang province in China. Int. J. Syst. Evol. Microbiol. 58: 438–442. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Forster, I. Brettske, S. Gerber, A.W. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. Konig, T. Liss, R. Lussmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N. Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode and K.H. Schleifer. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32: 1363–1371. Ludwig, W. and H.P. Klenk. 2005. Overview: a phylogenetic backbone and taxonomic framework for procaryotic systematics. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, The Proteobacteria, Part A, Introductory Essays (edited by Brenner, Krieg, Staley and Garrity). Springer, New York, pp. 49–65. Ludwig, W. and K.H. Schleifer. 2005. Molecular phylogeny of bacteria based on comparative sequence analysis of conserved genes. In Microbial Phylogeny and Evolution, Concepts and Controversies (edited by Sapp). Oxford University Press, New York, pp. 70–98. Morotomi, M., F. Nagai and H. Sakon. 2007. Genus Megamonas should be placed in the lineage of Firmicutes; Clostridia; Clostridiales; ‘Acidaminococcaceae’; Megamonas. Int. J. Syst. Evol. Microbiol. 57: 1673–1674. Morotomi, M., F. Nagai, H. Sakon and R. Tanaka. 2008. Dialister succinatiphilus sp. nov. and Barnesiella intestinihominis sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol. 58: 2716–2720. Murray, R.G. and E. Stackebrandt. 1995. Taxonomic note: implementation of the provisional status Candidatus for incompletely described procaryotes. Int. J. Syst. Bacteriol. 45: 186–187. Nedashkovskaya, O.I., S.B. Kim, D.S. Shin, I.A. Beleneva and V.V. Mikhailov. 2007a. Fulvivirga kasyanovii gen. nov., sp. nov., a novel member of the phylum Bacteroidetes isolated from seawater in a mussel farm. Int. J. Syst. Evol. Microbiol. 57: 1046–1049.

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Nedashkovskaya, O.I., M. Vancanneyt, S.B. Kim, B. Hoste and K.S. Bae. 2007b. Algibacter mikhailovii sp. nov., a novel marine bacterium of the family Flavobacteriaceae, and emended description of the genus Algibacter. Int. J. Syst. Evol. Microbiol. 57: 2147–2150. Ntougias, S., C. Fasseas and G.I. Zervakis. 2007. Olivibacter sitiensis gen. nov., sp. nov., isolated from alkaline olive-oil mill wastes in the region of Sitia, Crete. Int. J. Syst. Evol. Microbiol. 57: 398–404. Ochiai, S., Y. Adachi and K. Mori. 1997. Unification of the genera Serpulina and Brachyspira, and proposals of Brachyspira hyodysenteriae comb. nov., Brachyspira innocens comb. nov. and Brachyspira pilosicoli comb. nov. Microbiol. Immunol. 41: 445–452. Pankratov, T.A., B.J. Tindall, W. Liesack and S.N. Dedysh. 2007. Mucilaginibacter paludis gen. nov., sp. nov. and Mucilaginibacter gracilis sp. nov., pectin-, xylan- and laminarin-degrading members of the family Sphingobacteriaceae from acidic Sphagnum peat bog. Int. J. Syst. Evol. Microbiol. 57: 2349–2354. Paster, B.J., D.A. Pelletier, F.E. Dewhirst, W.G. Weisburg, V. Fussing, L.K. Poulsen, S. Dannenberg and I. Schroeder. 1996. Phylogenetic position of the spirochetal genus Cristispira. Appl. Environ. ­Microbiol. 62: 942–946. Pilhofer, M., K. Rappl, C. Eckl, A.P. Bauer, W. Ludwig, K.H. Schleifer and G. Petroni. 2008. Characterization and evolution of cell division and cell wall synthesis genes in the bacterial phyla Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes and phylogenetic comparison with rRNA genes. J. Bacteriol. 190: 3192–3202. Pruesse, E., C. Quast, K. Knittel, B. Fuchs, W. Ludwig, J. Peplies and F.O. Glöckner. 2007. SILVA: a comprehensive online resource for quality checked and aligned rRNA sequence data compatible with ARB. Nucleic Acids Res. 35: 7188–7196. Qin, Q.L., D.L. Zhao, J. Wang, X.L. Chen, H.Y. Dang, T.G. Li, Y.Z. Zhang and P.J. Gao. 2007. Wangia profunda gen. nov., sp. nov., a novel marine bacterium of the family Flavobacteriaceae isolated from southern Okinawa Trough deep-sea sediment. FEMS Microbiol. Lett. 271: 53–58. Qu, J.H. and H.L. Yuan. 2008. Sediminibacterium salmoneum gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from sediment of a eutrophic reservoir. Int. J. Syst. Evol. Microbiol. 58: 2191–2194. Quan, Z.X., Y.P. Xiao, S.W. Roh, Y.D. Nam, H.W. Chang, K.S. Shin, S.K. Rhee, Y.H. Park and J.W. Bae. 2008. Joostella marina gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from the East Sea. Int. J. Syst. Evol. Microbiol. 58: 1388–1392. Romanenko, L.A., M. Uchino, G.M. Frolova and V.V. Mikhailov. 2007. Marixanthomonas ophiurae gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from a deep-sea brittle star. Int. J. Syst. Evol. Microbiol. 57: 457–462. Sakamoto, M. and Y. Benno. 2006. Reclassification of Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae as Parabacteroides distasonis gen. nov., comb. nov., Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov. Int. J. Syst. Evol. Microbiol. 56: 1599–1605. Sakamoto, M., P.T. Lan and Y. Benno. 2007. Barnesiella viscericola gen. nov., sp. nov., a novel member of the family Porphyromonadaceae isolated from chicken caecum. Int. J. Syst. Evol. Microbiol. 57: 342–346. Schachter, J., R.S. Stephens, P. Timms, C. Kuo, P.M. Bavoil, S. ­Birkelund, J. Boman, H. Caldwell, L.A. Campbell, M. Chernesky, G. ­Christiansen, I.N. Clarke, C. Gaydos, J.T. Grayston, T. Hackstadt, R. Hsia, B. Kaltenboeck, M. Leinonnen, D. Ojcius, G. McClarty, J. Orfila, R. Peeling, M. Puolakkainen, T.C. Quinn, R.G. Rank, J. Raulston, G.L. ­Ridgeway, P. Saikku, W.E. Stamm, D.T. Taylor-Robinson, S.P. Wang and P.B. Wyrick. 2001. Radical changes to chlamydial taxonomy are not necessary just yet. Int. J. Syst. Evol. Microbiol. 51: 249; author reply 251–253. Shah, H.N. and M.D. Collins. 1988. Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int. J. Syst. Bacteriol. 38: 128–131. Shah, H.N. and D.M. Collins. 1990. Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int. J. Syst. Bacteriol. 40: 205–208.

Srisukchayakul, P., C. Suwanachart, Y. Sangnoi, A. Kanjana-Opas, S. Hosoya, A. Yokota and V. Arunpairojana. 2007. Rapidithrix thailandica gen. nov., sp. nov., a marine gliding bacterium isolated from samples collected from the Andaman sea, along the southern coastline of Thailand. Int. J. Syst. Evol. Microbiol. 57: 2275–2279. Stamatakis, A.P., T. Ludwig and H. Meier. 2005. RAxML-II: a program for sequential, parallel & distributed inference of large phylogenetic trees. Concurrency Comput. Pract. Exp. 17: 1705–1723. Stoecker, K., B. Bendinger, B. Schoning, P.H. Nielsen, J.L. Nielsen, C. Baranyi, E.R. Toenshoff, H. Daims and M. Wagner. 2006. Cohn’s Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase. Proc. Natl. Acad. Sci. U.S.A. 103: 2363–2367. Takeda, M., A. Yoneya, Y. Miyazaki, K. Kondo, H. Makita, M. Kondoh, I. Suzuki and J. Koizumi. 2008. Prosthecobacter fluviatilis sp. nov., which lacks the bacterial tubulin btubA and btubB genes. Int. J. Syst. Evol. Microbiol. 58: 1561–1565. Ten, L.N., O.M Liu, W.T. Im, Z. Aslam and S.T. Lee. 2006. Sphingobacterium composti sp. nov., a novel DNase-producing bacterium isolated from compost. J. Microbiol. Biotechnol. 16: 1728–1733. Ten, L.N., Q.-M. Liu, W.-T. Im, Z. Aslam and S.-T. Lee. 2007. List of new names and new combinations previously effectively, but not ­validly, published. Validation List no. 116 Int. J. Syst. Evol. Microbiol 57: 1372. Urai, M., T. Aizawa, Y. Nakagawa, M. Nakajima and M. Sunairi. 2008. Mucilaginibacter kameinonensis sp., nov., isolated from garden soil. Int. J. Syst. Evol. Microbiol. 58: 2046–2050. Urios, L., H. Agogue, F. Lesongeur, E. Stackebrandt and P. Lebaron. 2006. Balneola vulgaris gen. nov., sp. nov., a member of the phylum Bacteroidetes from the north-western Mediterranean Sea. Int. J. Syst. Evol. Microbiol. 56: 1883–1887. Urios, L., L. Intertaglia, F. Lesongeur and P. Lebaron. 2008. Balneola alkaliphila sp. nov., a marine bacterium isolated from the Mediterranean Sea. Int. J. Syst. Evol. Microbiol. 58: 1288–1291. Vaz-Moreira, I., M.F. Nobre, O.C. Nunes and C.M. Manaia. 2007. ­Pseudosphingobacterium domesticum gen. nov., sp. nov., isolated from home-made compost. Int. J. Syst. Evol. Microbiol. 57: 1535–1538. Wagner, M. and M. Horn. 2006. The Planctomycetes, Verrucomicrobia, Chlamydiae and sister phyla comprise a superphylum with biotechnological and medical relevance. Curr. Opin. Biotechnol. 17: 241–249. Wang, L., L.N. Ten, H.G. Lee, W.T. Im and S.T. Lee. 2008. Olivibacter soli sp. nov., Olivibacter ginsengisoli sp. nov. and Olivibacter terrae sp. nov., from soil of a ginseng field and compost in South Korea. Int. J. Syst. Evol. Microbiol. 58: 1123–1127. Weon, H.Y., B.Y. Kim, S.H. Yoo, S.Y. Lee, S.W. Kwon, S.J. Go and E. Stackebrandt. 2006. Niastella koreensis gen. nov., sp. nov. and Niastella yeongjuensis sp. nov., novel members of the phylum Bacteroidetes, isolated from soil cultivated with Korean ginseng. Int. J. Syst. Evol. Microbiol. 56: 1777–1782. Weon, H.Y., B.Y. Kim, J.H. Joa, S.W. Kwon, W.G. Kim and B.S. Koo. 2008a. Niabella soli sp. nov., isolated from soil from Jeju Island, Korea. Int. J. Syst. Evol. Microbiol. 58: 467–469. Weon, H.Y., H.J. Noh, J.A. Son, H.B. Jang, B.Y. Kim, S.W. Kwon and E. Stackebrandt. 2008b. Rudanella lutea gen. nov., sp. nov., isolated from an air sample in Korea. Int. J. Syst. Evol. Microbiol. 58: 474–478. Willems, A. and M.D. Collins. 1995. 16S ribosomal RNA gene similarities indicate that Hallella seregens (Moore and Moore) and Mitsuokella dentalis (Haapasalo et al.) are genealogically highly related and are members of the genus Prevotella: emended description of the genus Prevotella (Shah and Collins) and description of Prevotella dentalis comb. nov. Int. J. Syst. Bacteriol. 45: 832–836. Yang, S.J., Y.J. Choo and J.C. Cho. 2007. Lutimonas vermicola gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from the marine polychaete Periserrula leucophryna. Int. J. Syst. Evol. Microbiol. 57: 1679–1684. Yoo, S.H., H.Y. Weon, H.B. Jang, B.Y. Kim, S.W. Kwon, S.J. Go and E. Stackebrandt. 2007. Sphingobacterium composti sp. nov., isolated from cotton-waste composts. Int. J. Syst. Evol. Microbiol. 57: 1590–1593.



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Yoon, J., S. Ishikawa, H. Kasai and A. Yokota. 2007a. Perexilibacter aurantiacus gen. nov., sp. nov., a novel member of the family ‘Flammeovirgaceae’ isolated from sediment. Int. J. Syst. Evol. Microbiol. 57: 964–968. Yoon, J., S. Ishikawa, H. Kasai and A. Yokota. 2007b. Persicitalea jodogahamensis gen. nov., sp. nov., a marine bacterium of the family ‘Flexibacteraceae’, isolated from seawater in Japan. Int. J. Syst. Evol. Microbiol. 57: 1014–1017. Yoon, J., Y. Matsuo, K. Adachi, M. Nozawa, S. Matsuda, H. Kasai and A. Yokota. 2008a. Description of Persicirhabdus sediminis gen. nov., sp. nov., Roseibacillus ishigakijimensis gen. nov., sp. nov., Roseibacillus ponti sp. nov., Roseibacillus persicicus sp. nov., Luteolibacter pohnpeiensis gen. nov., sp. nov. and Luteolibacter algae sp. nov., six marine members of the phylum ‘Verrucomicrobia’, and emended descriptions of the class Verrucomicrobiae, the order Verrucomicrobiales and the family Verrucomicrobiaceae. Int. J. Syst. Evol. Microbiol. 58: 998–1007. Yoon, J., Y. Matsuo, H. Kasai and A. Yokota. 2008b. Limibacter armeniacum gen. nov., sp. nov., a novel representative of the family ‘Flammeovirgaceae’ isolated from marine sediment. Int. J. Syst. Evol. Microbiol. 58: 982–986.

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Yoon, J., Y. Matsuo, A. Katsuta, J.H. Jang, S. Matsuda, K. Adachi, H. Kasai and A. Yokota. 2008c. Haloferula rosea gen. nov., sp. nov., Haloferula harenae sp. nov., Haloferula phyci sp. nov., Haloferula helveola sp. nov. and Haloferula sargassicola sp. nov., five marine representatives of the family Verrucomicrobiaceae within the phylum ‘Verrucomicrobia’. Int. J. Syst. Evol. Microbiol. 58: 2491–2500. Yoon, J.H., S.J. Kang, Y.T. Jung and T.K. Oh. 2008d. Aestuariicola saemankumensis gen. nov., sp. nov., a member of the family Flavobacteriaceae, isolated from tidal flat sediment. Int. J. Syst. Evol. Microbiol. 58: 2126–2131. Yoon, M.H. and W.T. Im. 2007. Flavisolibacter ginsengiterrae gen. nov., sp. nov. and Flavisolibacter ginsengisoli sp. nov., isolated from ginseng ­cultivating soil. Int. J. Syst. Evol. Microbiol. 57: 1834–1839. Zchori-Fein, E., S.J. Perlman, S.E. Kelly, N. Katzir and M.S. Hunter. 2004. Characterization of a ‘Bacteroidetes’ symbiont in Encarsia wasps (Hymenoptera: Aphelinidae): proposal of ‘Candidatus Cardinium hertigii’. Int. J. Syst. Evol. Microbiol. 54: 961–968. Zhao, J.S., D. Manno and J. Hawari. 2009. Psychrilyobacter atlanticus gen. nov., sp. nov., a marine member of the phylum Fusobacteria that produces H2 and degrades nitramine explosives under low temperature conditions. Int. J. Syst. Evol. Microbiol. 59 : 491–497.

Taxonomic outlines of the phyla Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), ­Acidobacteria, Fibrobacteres, Fusobacteria, ­Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes Wolfgang Ludwig, Jean Euzéby and William B. Whitman

All taxa recognized within this volume of the rank of genus and above are listed below. Within each classification, the ­nomenclatural type is listed first followed by the remaining taxa in alphabetical order. Taxa appearing on the Approved Lists are denoted by the superscript AL. Taxa that were otherwise validly published are denoted by the superscript VP. Taxa that have not been validly pub­ lished are presented in quotations. Taxa which were described after the deadline of July 1, 2006, and are therefore not included in this volume are indicated by an asterisk (*).

Phylum XIV. “Bacteroidetes” Class I. “Bacteroidia” Order I. “Bacteroidales” Family I. BacteroidaceaeAL Genus I. BacteroidesAL(T) Genus II. AcetofilamentumVP Genus III. AcetomicrobiumVP Genus IV. AcetothermusVP Genus V. AnaerorhabdusVP Family II. “Marinilabiliaceae” Genus I. MarinilabiliaVP(T) Genus II. AlkaliflexusVP Genus III. AnaerophagaVP Family III. “Rikenellaceae” Genus I. RikenellaVP(T) Genus II. AlistipesVP Family IV. “Porphyromonadaceae” Genus I. PorphyromonasVP(T) Genus II. BarnesiellaVP Genus III. DysgonomonasVP Genus IV. PaludibacterVP Genus V. ParabacteroidesVP* Genus VI. PetrimonasVP Genus VII. ProteiniphilumVP Genus VIII. Tannerella VP Family V. “Prevotellaceae” Genus I. PrevotellaVP(T) Genus II. XylanibacterVP Class II. “Flavobacteriia” Order I. “Flavobacteriales” Family I. FlavobacteriaceaeVP Genus I. FlavobacteriumAL(T) Genus II. ActibacterVP* Genus III. AequorivitaVP Genus IV. AestuariicolaVP*

Genus V. AlgibacterVP Genus VI. AquimarinaVP Genus VII. ArenibacterVP Genus VIII. BergeyellaVP Genus IX. BizioniaVP Genus X. CapnocytophagaVP Genus XI. CellulophagaVP Genus XII. ChryseobacteriumVP Genus XIII. CloacibacteriumVP Genus XIV. CoenoniaVP Genus XV. CostertoniaVP Genus XVI. CroceibacterVP Genus XVII. DokdoniaVP Genus XVIII. DonghaeanaVP Genus XIX. ElizabethkingiaVP Genus XX. EmpedobacterVP Genus XXI. EpilithonimonasVP Genus XXII. FlagellimonasVP* Genus XXIII. FlaviramulusVP Genus XXIV. FormosaVP Genus XXV. FulvibacterVP* Genus XXVI. GaetbulibacterVP Genus XXVII. GalbibacterVP* Genus XXVIII. GelidibacterVP Genus XXIX. GillisiaVP Genus XXX. GilvibacterVP* Genus XXXI. GramellaVP Genus XXXII. JoostellaVP* Genus XXXIII. KaistellaVP Genus XXXIV. KordiaVP Genus XXXV. KrokinobacterVP Genus XXXVI. LacinutrixVP Genus XXXVII. LeeuwenhoekiellaVP Genus XXXVIII. LeptobacteriumVP* Genus XXXIX. LutibacterVP 21

22

Taxonomic outlines

Genus XL. LutimonasVP* Genus XLI. MaribacterVP Genus XLII. MariniflexileVP Genus XLIII. MarixanthomonasVP* Genus XLIV. MesoflavibacterVP* Genus XLV. MesoniaVP Genus XLVI. MuricaudaVP Genus XLVII. MyroidesVP Genus XLVIII. NonlabensVP Genus XLIX. OlleyaVP Genus L. OrnithobacteriumVP Genus LI. PersicivirgaVP Genus LII. Polaribacter VP Genus LIII. PsychroflexusVP Genus LIV. PsychroserpensVP Genus LV. RiemerellaVP Genus LVI. RobiginitaleaVP Genus LVII. SalegentibacterVP Genus LVIII. SalinimicrobiumVP* Genus LIX. SandarakinotaleaVP Genus LX. SediminibacterVP* Genus LXI. SediminicolaVP Genus LXII. SejongiaVP Genus LXIII. StenothermobacterVP Genus LXIV. SubsaxibacterVP Genus LXV. SubsaximicrobiumVP Genus LXVI. TamlanaVP* Genus LXVII. TenacibaculumVP Genus LXVIII. UlvibacterVP Genus LXIX. VitellibacterVP Genus LXX. WautersiellaVP Genus LXXI. WeeksellaVP Genus LXXII. WinogradskyellaVP Genus LXXIII. YeosuanaVP Genus LXXIV. Zeaxanthinibacter VP* Genus LXXV. ZhouiaVP Genus LXXVI. ZobelliaVP Genus LXXVII. ZunongwangiaVP* Family II. “Blattabacteriaceae” Genus I. BlattabacteriumAL(T) Family III. CryomorphaceaeVP Genus I. CryomorphaVP(T) Genus II. BrumimicrobiumVP Genus III. CrocinitomixVP Genus IV. FluviicolaVP Genus V. LishizheniaVP Genus VI. OwenweeksiaVP Class III. “Sphingobacteriia” Order I. “Sphingobacteriales” Family I. SphingobacteriaceaeVP Genus I. SphingobacteriumVP(T) Genus II. MucilaginibacterVP* Genus III. NubsellaVP* Genus IV. OlivibacterVP* Genus V. ParapedobacterVP* Genus VI. PedobacterVP Genus VII. PseudosphingobacteriumVP* Family II. “Chitinophagaceae” Genus I. ChitinophagaVP(T)

Genus II. FlavisolibacterVP* Genus III. NiabellaVP* Genus IV. NiastellaVP* Genus V. SediminibacteriumVP* Genus VI. SegetibacterVP* Genus VII. TerrimonasVP Family III. “Saprospiraceae” Genus I. Saprospira AL(T) Genus II. AureispiraVP Genus III. HaliscomenobacterAL Genus IV. LewinellaVP Class IV. “Cytophagia” Order I. CytophagalesAL(T) Family I. Cytophagaceae AL Genus I. Cytophaga AL(T) Genus II. AdhaeribacterVP Genus III. ArcicellaVP Genus IV. DyadobacterVP Genus V. EffluviibacterVP Genus VI. EmticiciaVP Genus VII. FlectobacillusAL Genus VIII. Flexibacter AL Genus IX. HymenobacterVP Genus X. LarkinellaVP Genus XI. LeadbetterellaVP Genus XII. MeniscusAL Genus XIII. Microscilla AL Genus XIV. PersicitaleaVP* Genus XV. PontibacterVP Genus XVI. Rudanella VP* Genus XVII. RunellaAL Genus XVIII. SpirosomaAL Genus XIX. SporocytophagaAL Family II. “Cyclobacteriaceae” Genus I. CyclobacteriumVP(T) Genus II. AlgoriphagusVP Genus III. AquiflexumVP Genus IV. BelliellaVP Genus V. EchinicolaVP Genus VI. RhodonellumVP Family III. “Flammeovirgaceae” Genus I. FlammeovirgaVP(T) Genus II. FabibacterVP Genus III. FlexithrixAL Genus IV. FulvivirgaVP* Genus V. LimibacterVP* Genus VI. PerexilibacterVP* Genus VII. PersicobacterVP Genus VIII. RapidithrixVP* Genus IX. ReichenbachiellaVP Genus X. RoseivirgaVP Genus XI. SediminitomixVP* Order II. Incertae sedis Family I. “Rhodothermaceae” Genus I. RhodothermusVP(T) Genus II. SalinibacterVP Order III. Incertae sedis Genus I. BalneolaVP* Order IV. Incertae sedis

23

Taxonomic outlines

Genus I. ThermonemaVP Order V. Incertae sedis Genus I. ToxothrixAL Phylum XV. “Spirochaetes” Class I. “Spirochaetia” Order I. SpirochaetalesAL(T) Family I. SpirochaetaceaeAL Genus I. SpirochaetaAL(T) Genus II. BorreliaAL Genus III. CristispiraAL Genus IV. TreponemaAL Family II. “Brachyspiraceae” Genus I. BrachyspiraVP(T) Family III. “Brevinemataceae” Genus I. BrevinemaVP(T) Family IV. LeptospiraceaeAL Genus I. LeptospiraAL(T) Genus II. LeptonemaVP Genus III. TurneriellaVP Family V. Incertae sedis Genus I. ClevelandinaVP Genus II. DiplocalyxVP Genus III. HollandinaVP Genus IV. PillotinaVP Phylum XVI. TenericutesVP Class I. Mollicutes AL Order I. Mycoplasmatales AL(T) Family I. MycoplasmataceaeAL Genus I. MycoplasmaAL(T) Genus II. UreaplasmaVP Family II. Incertae sedis Genus I. EperythrozoonAL Genus II. HaemobartonellaAL Order II. EntomoplasmatalesVP Family I. EntomoplasmataceaeVP Genus I. EntomoplasmaVP(T) Genus II. MesoplasmaVP Family II. SpiroplasmataceaeVP Genus I. SpiroplasmaAL(T) Order III. AcholeplasmatalesVP Family I. AcholeplasmataceaeAL Genus I. AcholeplasmaAL(T) Family II. Incertae sedis Genus I. “Candidatus Phytoplasma” Order IV. AnaeroplasmatalesVP Family I. AnaeroplasmataceaeVP Genus I. AnaeroplasmaAL(T) Genus II. AsteroleplasmaVP Phylum XVII. “Acidobacteria” Class I. “Acidobacteriia” Order I. “Acidobacteriales”(T) Family I. “Acidobacteriaceae” Genus I. AcidobacteriumVP(T) Genus II. EdaphobacterVP Genus III. TerriglobusVP Class II. HolophagaeVP Order I. HolophagalesVP(T) Family I. HolophagaceaeVP Genus I. HolophagaVP(T)

Genus II. GeothrixVP Order II. AcanthopleuribacteralesVP Family I. AcanthopleuribacteraceaeVP Genus I. AcanthopleuribacterVP(T) Phylum XVIII. “Fibrobacteres” Class I. “Fibrobacteria” Order I. “Fibrobacterales”(T) Family I. “Fibrobacteraceae” Genus I. FibrobacterVP(T) Phylum XIX. “Fusobacteria” Class I. “Fusobacteriia” Order I. “Fusobacteriales”(T) Family I. “Fusobacteriaceae” Genus I. FusobacteriumAL(T) Genus II. CetobacteriumVP Genus III. IlyobacterVP Genus IV. PropionigeniumVP Genus V. PsychrilyobacterVP* Family II. “Leptotrichiaceae” Genus I. LeptotrichiaAL(T) Genus II. SebaldellaVP Genus III. SneathiaVP Genus IV. StreptobacillusAL Phylum XX. “Dictyoglomi” Class I. “Dictyoglomia” Order I. “Dictyoglomales”(T) Family I. “Dictyoglomaceae” Genus I. DictyoglomusVP(T) Phylum XXI. Gemmatimonadetes Class I. GemmatimonadetesVP Order I. GemmatimonadalesVP(T) Family I. GemmatimonadaceaeVP Genus I. GemmatimonasVP(T) Phylum XXII. Lentisphaerae Class I. “Lentisphaeria” Order I. LentisphaeralesVP(T) Family I. “Lentisphaeraceae” Genus I. LentisphaeraVP(T) Order II. VictivallalesVP Family I. “Victivallaceae” Genus I. VictivallisVP(T) Phylum XXIII. “Verrucomicrobia” Class I. VerrucomicrobiaeVP Order I. VerrucomicrobialesVP(T) Family I. VerrucomicrobiaceaeVP Genus I. VerrucomicrobiumVP(T) Genus II. ProsthecobacterVP Family II. “Akkermansiaceae” Genus I. AkkermansiaVP(T) Family III. “Rubritaleaceae” Genus I. RubritaleaVP(T) Genus II. PersicirhabdusVP* Genus III. RoseibacillusVP* VP Class II. Opitutae Order I. OpitutalesVP(T) Family I. OpitutaceaeVP Genus I. OpitutusVP(T) Genus II. AlterococcusVP

24

Taxonomic outlines

Order II. PuniceicoccalesVP Family I. PuniceicoccaceaeVP Genus I. PuniceicoccusVP(T) Genus II. CerasicoccusVP Genus III. CoraliomargaritaVP Genus IV. PelagicoccusVP Class III. “Spartobacteria” Order I. “Chthoniobacterales”(T) Family I. “Chthoniobacteraceae” Genus I. “Chthoniobacter”(T) Genus II. “Candidatus Xiphinema­ tobacter” Phylum XXIV. “Chlamydiae” Class I. “Chlamydiia” Order I. Chlamydiales AL(T) Family I. Chlamydiaceae AL Genus I. Chlamydia AL(T) Genus II. ChlamydophilaVP Family II. “Clavichlamydiaceae” Genus I. “Candidatus Clavichlamydia” Family III. “Criblamydiaceae” Genus I. “Criblamydia”(T) Family IV. ParachlamydiaceaeVP Genus I. ParachlamydiaVP(T) Genus II. NeochlamydiaVP Genus III. “Protochlamydia” Family V. “Piscichlamydiaceae” Genus I. “Candidatus Piscichlamydia”(T) Family VI. “Rhabdochlamydiaceae” Genus I. “Candidatus Rhabdo­ chlamydia”VP(T)

Family VII. SimkaniaceaeVP Genus I. SimkaniaVP(T) Genus II. “Candidatus Fritschea”VP Family VIII. WaddliaceaeVP Genus I. WaddliaVP(T) Phylum XXV. “Planctomycetes” Class I. “Planctomycetia” Order I. PlanctomycetalesVP(T) Family I. PlanctomycetaceaeVP Genus I. PlanctomycesAL(T) Genus II. BlastopirellulaVP Genus III. GemmataVP Genus IV. IsosphaeraVP Genus V. PirellulaVP Genus VI. RhodopirellulaVP Genus VII. SchlesneriaVP Genus VIII. SingulisphaeraVP Order II. “Brocadiales” Family I. “Brocadiaceae” Genus I. “Candidatus Brocadia”VP(T) Genus II. “Candidatus Anammoxo­ globus” Genus III. “Candidatus Jettenia” Genus IV. “Candidatus Kuenenia” Genus V. “Candidatus Scalindua”

Acknowledgements Jean-François Bernardet, Brian Hedlund, Noel Krieg, Olga Nedashkovskaya, Bruce Paster, Bernhard Schink, Jim Staley and Naomi Ward provided valuable advice concerning the prepara­ tion of this outline.

Phylum XIV. Bacteroidetes phyl. nov. Noel R. Krieg, Wolfgang Ludwig, Jean Euzéby and William B. Whitman Bac.te.ro.i.de′tes. N.L. fem. pl. n. Bacteroidales type order of the phylum; N.L. fem. pl. n. Bacteroidetes the phylum of Bacteroidales.

The phylum Bacteroidetes is a phenotypically diverse group of Gram-stain-negative rods that do not form endospores. They are circumscribed for this volume on the basis of phylogenetic analysis of 16S rRNA gene sequences. The phylum contains four classes, Bacteroidia, Cytophagia, Flavobacteriia, and Sphin-

gobacteria. In addition, the genera Rhodothermus, Salinibacter, and Thermonema appear to represent deep groups of the phylum that can not be readily assigned to any of the four classes. Type order: Bacteroidales ord. nov.

Class I. Bacteroidia class. nov. Noel R. Krieg Bac.te.ro.i.di¢a. N.L. masc. n. Bacteroides type genus of the type order Bacteroidales; suff. -ia ending proposed by Gibbons and Murray and by Stackebrandt et al. to denote a class; N.L. neut. pl. n. Bacteroidia the Bacteroidales class. The class presently contains one order, Bacteroidales. The description of the class is the same as that given for the order.

Type order: Bacteroidales ord. nov.

Order I. Bacteroidales ord. nov. Noel R. Krieg Bac.te.ro.i.da¢les. N.L. masc. n. Bacteroides type genus of the order; suff. -ales ending to denote an order; N.L. fem. pl. n. Bacteroidales the Bacteroides order. The order presently includes the families Bacteroidaceae, Marinilabiliaceae, Porphyromonadaceae, Prevotellaceae, and Rikenellaceae. Straight, fusiform or thin rods and coccobacilli that stain Gramnegative. Nonsporeforming. Mostly anaerobic, although some are facultatively anaerobic, and saccharolytic, although proteins and other substrates may be utilized. Nonmotile or motile by gliding.

Type genus: Bacteroides Castellani and Chalmers 1919, 959AL.

Reference Castellani, A. and A.J. Chalmers. 1919. Manual of Tropical Medicine, 3rd edn. Williams Wood and Co., New York, pp. 959–960.

Family I. Bacteroidaceae Pribam 1933, 10AL The Editorial Board Bac.te.ro.i.da.ce¢a.e. N.L. masc. n. Bacteroides type genus of the family; suff. -aceae ending to denote a family; N.L. fem. pl. n. Bacteroidaceae the Bacteroides family. The family Bacteroidaceae was circumscribed for this volume on the basis of phylogenetic analysis of 16S rRNA gene sequences; the family contains the genera Acetofilamentum, Acetomicrobium, Acetothermus, Anaerorhabdus, and Bacteroides. The genera Anaerophaga and Megamonas, which were previously classified with this family,

have been transferred to the family Marinilabiliaceae and the Firmicutes, respectively (Morotomi et al., 2007). Straight rods that stain Gram-negative. Anaerobic. Nonsporeforming. Some characteristics that differentiate the genera are given in Table 1. Type genus: Bacteroides Castellani and Chalmers 1919, 959AL.

25

a

Bacteroides 0.5–2.0 × 1.6–12 − + + Succinate and acetate; trace to moderate amounts of isobutyrate and isovalerate Major constituents of the normal human colonic flora; also found in smaller numbers in the female genital tract; not common in the mouth or upper respiratory tract 39–49

Symbols: +, >85% positive; −, 0–15% positive.

DNA G+C content (mol%)

Isolated from

Cell size (mm) Motility Saccharolytic Growth occurs below 50°C Major products of glucose fermentation

Characteristic

~47

Secondary sewage sludge

0.18–0.3 × 2–8 − + + Acetate and H2 (in a molar ratio of 1:2) and CO2

Acetofilamentum

TABLE 1.  Some characteristics that differentiate the genera of the family Bacteroidaceae  a

47

0.6–0.8 × 2–7 + + + Acetate and H2 or acetate, lactate, ethanol, and H2 Sewage sludge

Acetomicrobium

~62

Thermophilically fermenting sewage sludge

0.5 × 5–8 − + − Acetate and H2 in a molar ratio of 1:1:2.

Acetothermus

34

Infected appendix, lung abscesses and abdominal abscesses; infrequently from human and pig feces

0.3–1.5 × 1–3 − − + Acetate and lactate

Anaerorhabdus

26 FAMILY i. Bacteroidaceae

27

Genus I. Bacteroides

Genus I. Bacteroides Castellani and Chalmers 1919, 959AL emend. Shah and Collins 1989, 85 Yuli Song, Chengxu Liu and Sydney M. Finegold Bac.te.ro.i¢des. N.L. n. bacter rod; L. suff. -oides (from Gr. suff. -eides, from Gr. n. eidos that which is seen, form, shape, figure), resembling, similar; N.L. masc. n. Bacteroides rodlike.

morphologies and were biochemically and physiologically extremely heterogeneous. Therefore, Shah and Collins (1989) proposed that this genus should be restricted to Bacteroides fragilis and closely related organisms. The application of molecular biology techniques such as DNA–DNA hybridization and 16S rRNA gene sequencing has done much to clarify the inter- and intrageneric structure of Bacteroides. 16S rRNA gene sequence analysis indicates that the genus Bacteroides is equivalent to the Bacteroides cluster within the Bacteroides subgroup of the “­Bacteroidetes” (previously referred as the phylum Cytophaga–Flavobacteria–Bacteroides) (Paster et al., 1994; Woese, 1987). The currently described species that conform to the emended description of the genus Bacteroides based on biochemical, chemical, and genetic criteria include the members of the “Bacteroides fragilis group” [not a formal taxonomic group, but the ten species conforming to the 1989 proposal (Shah and Collins) to restrict the genus] and several other later described species (Figure  14). Other Bacteroides species with validly published names that do not conform to the emended generic description require f­ urther study (Table 5).

Rod-shaped cells with rounded ends. Gram-stain-negative. Cells are fairly uniform if smears are prepared from young cultures on blood agar. Nonmotile. Anaerobic. Colonies are 1–3 mm in diameter, smooth, white to gray, and nonhemolytic on blood agar. Chemo-organotrophic. Saccharolytic. Weakly proteolytic. Most species grow in the presence of 20% bile, but are not always stimulated. Esculin is usually hydrolyzed. Nitrate is not reduced to nitrite. Indole variable. Major fermentation products are succinate and acetate. Trace to moderate amounts of isobutyrate and isovalerate may be produced. Predominant cellular fatty acid is C15:0 anteiso. DNA G+C content (mol%): 39–49. Type species: Bacteroides fragilis (Veillon and Zuber 1898) ­Castellani and Chalmers 1919, 959AL.

Further descriptive information As defined in Bergey’s Manual of Systematic Bacteriology, 1st edition, the genus Bacteroides comprised more than 60 species (Holdeman et al., 1984). These species exhibited a variety of cellular

Bacteroides coprocol a JCM 12979T (AB200224)

100

Bacteroides plebeius JCM 12973T (AB200217)

94 99 100 100

Bacteroides massiliensis CCUG 48901T (AY126616) Bacteroides vulgatus ATCC 8482T (M58762)

Bacteroides helcogenes JCM 6297T (AB200227) Bacteroides pyogenes JCM 6294T (AB200229) Bacteroides tectu s JCM 10003T (AB200228)

100

Prevotella heparinolytica ATCC 35895T (L16487) Prevotella zoogleoforman s ATCC 33285T (L16488)

Bacteroides uniformi s ATCC 8492T (L16486) Bacteroides stercori s ATCC 43183T (X83953) Bacteroides eggerthii DSM 20697T (AB050107) 59

Bacteroides finegoldi i JCM 13345T (AB222699) Bacteroides thetaiotaomicro n ATCC 29148T (L16489) Bacteroides ovatus NCTC 11153T (L16484)

69

Bacteroides acidifacien s JCM 10556T (AB021156) Bacteroides cacca e ATCC 43185T (X83951)

82 81

Bacteroides nordii ATCC BAA-998T (AY608697) Bacteroides salyersiae ATCC BAA-997T (AY608696)

77

Bacteroides fragilis ATCC 25285T (L11656) Bacteroides intestinalis JCM 13265T (AB214328) Bacteroides coprosuis CCUG 50528T (AF319778)

100

Bacteroides distasonis ATCC 8503T (M86695) 57

97

Bacteroides goldsteinii CCUG 48944T (AY974070) Bacteroides merdae ATCC 43184T (X83954) Bacteroides forsythus FDC 338T (L16495) Bacteroides splanchnicus NCTC 10825T (L16496)

100

Bacteroides capillosus ATCC 29799T (AY136666) Bacteroides cellulosolvens ATCC 35603T (L35517) Bacteroides ureolyticus ATCC 33387T (L04321)

1%

FIGURE 14.  Phylogenetic tree based on 16S rRNA gene sequence comparisons (>1300 aligned bases) showing the phylogenetic relationships of Bacteroides species. The tree, constructed using the neighbor-joining method, was based on a comparison of approximately 1400 nt. Bootstrap values, expressed as percentages of 1000 replications, are given at branching points. Bar = 1% sequence divergence.

28

FAMILY i. Bacteroidaceae

Cell size (µm) Growth in 20% bile Production of:   Indole   Catalase Starch hydrolyzed Gelatin digested G6PDH and 6PGDH Acid produced from:   Arabinose   Cellobiose   Xylan   Glucose   Glycogen   Inositol   Lactose   Maltose   Mannitol   Mannose   Melezitose   Melibiose   Raffinose   Rhamnose   Ribose   Salicin   Sucrose   Trehalose   Xylose Enzyme activity: d   a-Galactosidase   b-Galactosidase   a-Glucosidase   b-Glucosidase   a-Arabinosidase   b-Glucuronidase  N-Acety1  b-glucosaminidase  Glutamic acid   decarboxylase   a-Fucosidase   Alkaline phosphatase   Arginine arylamidase   Leucine arylamidase  Glutamyl glutamic   acid arylamidase   Glycine arylamidase  Leucyl glycine   arylamidase   Alanine arylamidase Major metabolic end product(s)e

0.8–1.3 × 1.6–8.0 +

0.8–1.3 × 1.4–1.6 × 0.8–1.0 × 0.8–3.0 × 1.3–1.9 × 1.6–4.2 × 0.4–1.0 × 1.6–8.0 2.5–12 4.0 0.5–1.5 1.6–5.0 0.8–1.2 1.0–6.0 + + + + + + +

0.8 × 1.5–4.5 +

11. B. helcogenes

10. B. goldsteinii

9. B. finegoldii

8. B. eggerthii

7. B. dorei

6. B. distasonis

5. B. coprosuis

4. B. coprocola

3. B. caccae

2. B. acidifaciens

Characteristic

1. B. fragilis

TABLE 2.  Descriptive characteristics of different Bacteroides speciesa,b

0.9–1.5 × 0.5–0.6 × 1.2–10 0.8–4.0 + w

− + + w +

v nr d nr +

− d w w +

+ − nr − nr

− − + − nr

− d + w +

− nr nr nr nr

+ − w − +

− nr nr − nr

− nr nr nr nr

− − + − nr

− d − + + − + + − + − w + − − − + − +

nr nr nr nr nr nr nr nr nr nr nr nr nr nr nr nr nr nr nr

+ d − + w nr nr nr nr nr d + + d + d + + +

− + nr + nr nr + + − + − nr + + nr + + − +

− − nr + nr nr nr + nr nr nr nr nr nr nr nr nr nr −

d + − + − − + + − + + + + d d + + + +

+ − nr + nr nr + + − + − nr + + nr − + − +

+ d + + + − + + − + − − − w − − − − +

+ + nr + nr nr + + − + − nr + + nr + nr − +

− + − + nr nr nr nr nr + nr nr + + nr − + − +

− + + + + nr + + nr + − + + nr nr + + − +

+ + + + − − +

nr nr nr nr nr nr nr

+ + w w + − +

+ + + + + − +

w + + + − − +

+ + + + + − +

+ + + + + + +

+ + + + + − +

+ + + + + − +

+ + + − − − +

nr nr nr nr nr + nr

+

nr

w

+

nr

+

+

+

+

−

v

+ + + + +

nr nr nr nr nr

+ + + + +

+ + − − +

+ + + − +

− + + + +

+ + + + +

− + − − +

− + − − +

− + + + +

nr nr − − nr

− +

nr nr

− +

− +

− +

+ +

+ +

− +

− +

+ +

− nr

+ A, S, p, pa (ib, iv, l)

nr A, S

+ A, S, p, (iv)

+ nr

+ A, S, p

+ A, S, p (pa, ib, iv, l)

+ nr

+ A, S, p (pa, ib, iv, l)

+ nr

+ A, S, p (iv, f)

nr A, S (p, iv)

Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive; +−, usually positive, sometimes negative; w, weak reaction; nr, not reported. b Esculin is usually hydrolyzed and nitrate is not reduced to nitrite. c Type strain is negative. d Enzyme activity tested using API kits. e Fermentation products: A, acetic acid; B, butyric acid; P, propionic acid; PA, phenylacetic acid; S, succinic acid; ib, isobutyric acid; iv, isovaleric acid; f, formic acid; l, lactic acid. Capital letters indicate >1 meq/100 ml of broth; small letters, 99% of clinical isolates of Bacteroides include metronidazole, chloramphenicol, and carbapenems; however, strains of the Bacteroides fragilis group with resistance to imipenem and metronidazole are also encountered. Agents active against 95–99% of Bacteroides fragilis isolates include the b-lactam/b-lactamase inhibitor combinations. Bacteroides fragilis group species other than Bacteroides fragilis are more likely to be resistant to b-lactam/b-lactamase inhibitor combinations than Bacteroides fragilis. The level of chloramphenicol susceptibility remains quite high, whereas almost uniform resistance to aminoglycosides is observed and resistance to quinolones is common. Several multicenter surveys have documented an alarming gradual increase in resistance rates of Bacteroides species worldwide (Snydman et al., 2002). Multiresistant Bacteroides fragilis group strains, some of which are capable of transferring resistance genes, have been isolated in several countries. Although the rate of Bacteroides fragilis resistance to carbapenems or b-lactam/b-lactamase inhibitor combination drugs remains low, some investigators have noted a gradual increase in minimum inhibitory concentrations. Agents such as clindamycin and some cephalosporins, which have traditionally been considered good choices against Bacteroides fragilis group strains, are losing activity against these clinically important pathogens.

The ­classical mechanisms of resistance to b-lactams are: (a) production of b-lactamases; (b) alteration of penicillin-binding proteins; and (c) changes in outer membrane permeability to b-lactams. Resistance to clindamycin is mediated by modification of the ribosome. Tetracycline resistance is ­mediated by both tetracycline efflux and ribosomal protection. 5-Nitroimidazole resistance appears to be caused by a ­combination of decreased antibiotic uptake and decreased nitroreductase activity (Fang et  al., 2002). Two main mechanisms – alteration of target enzymes (gyrase) caused by chromosomal mutations in encoding genes and reduced intracellular accumulation due to increased efflux of the drug – are associated with quinolone resistance in the Bacteroides fragilis group (Oh and Edlund, 2003). Natural habitats and clinical significance.  Members of the “Bacteroides fragilis group” are major constituents of the normal human colonic flora and are also found in smaller numbers in the female genital tract, but are not common in the mouth or upper respiratory tract. The “Bacteroides fragilis group” makes up one-third of all clinical isolates of anaerobes. Bacteroides fragilis is the most frequently encountered, with Bacteroides thetaiotaomicron second. They are recovered from most intra-abdominal infections and may occur in infections at other sites. Bacteroides fragilis strains producing a potent zinc-dependent metalloprotease or enterotoxin with a variety of pathological effects on intestinal mucosal cells have been identified. Enterotoxin-producing Bacteroides fragilis strains have been isolated from the intestinal tracts of young farm animals and small children with diarrhea and in cases of extra-abdominal infections including bacteremia, but they have also been isolated from fecal samples from healthy children and adults (Sears et al., 1995). To date, three types of the enterotoxin, each having different virulence and geographical distribution, have been characterized. Other members of the Bacteroides fragilis group are present less often in infection.

Enrichment and isolation procedures A complex medium containing peptone, yeast extract, vitamin K1, and hemin is recommended for isolation of most species. The use of selective media along with nonselective media will increase yield and save time in terms of isolation of Bacteroides. Fresh or pre-reduced media (commercially available from Anaerobe Systems) are recommended, as both increase the initial isolation efficiency. The recommended minimum medium setup for culture of clinical specimens includes: (a) a nonselective, enriched, Brucella base sheep blood agar plate supplemented with vitamin K1 and hemin; and (b) a BBE agar plate for specimens from below the diaphragm for the selection and presumptive identification of the “Bacteroides fragilis group” (and Bilophila spp.). When indicated, a phenylethyl alcohol-sheep blood agar plate to prevent overgrowth by aerobic Gram-stain-negative rods and swarming of some clostridia may also be used. After inoculation, the anaerobic plates should be placed immediately in an anaerobic environment containing at least 5% carbon dioxide. Carbon dioxide is required for optimal growth of most of the saccharolytic species. The plates are examined after incubation for 48 h at 35 or 37°C.

31

Genus I. Bacteroides

and generally produce acetic and succinic acids as the major end products of glucose metabolism, in contrast to asaccharolytic species in the genus Porphyromonas and moderately saccharolytic species in Prevotella. Bacteroides species can also be readily distinguished from Porphyromonas species by not being pigmented and containing predominantly 12-methyl-tetradecanoic acid (C15:0 anteiso) as the long-chain fatty acid, in contrast to pigmented Porphyromonas species, which contain mainly 13-methyl-tetradecanoic acid (C15:0 iso). All Bacteroides species examined so far possess the enzymes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), which differs from other genera. Furthermore, the dibasic amino acid of their cell wall peptidoglycan is mesodiaminopimelic acid. The principal respiratory quinones are menaquinones with 10 or 11 isoprene units or both. Sphingolipids are produced (Shah and Collins, 1983). Unfortunately, these features have not been tested in many of the recently described Bacteroides species. Characteristics that differentiate Bacteroides from related taxa within the phylum “Bacteroidetes” are presented in Table 3.

Maintenance procedures The following procedures are those of Jousimies-Somer et al. (2002). Cultures of Bacteroides species can be maintained by regularly subculturing. A young, actively growing culture can be put into stock from liquid or solid media by freezing at −70°C or lyophilizing in a well-buffered medium containing no fermentable carbohydrate. Supplemented (vitamin K1 and hemin) thioglycolate medium or chopped meat broth incubated for 24–48 h, depending on the growth rate of the isolate, can be used to prepare stock cultures. Broth culture (0.5 ml) is added to an equal volume of sterile 20% skim milk or 15% glycerol prepared in an unbreakable screw-capped vial. For toxin-­producing organisms, storage under anaerobic conditions at −70°C after a minimal number of subcultures may be important.

Differentiation of the genus Bacteroides from other genera Bacteroides can be differentiated from other known related genera by 16S rRNA gene sequence analysis because the members form a distinct cluster within the Bacteroides subgroup with mean interspecies similarities of approximately 93% (Paster et al., 1994). The stability of this cluster has also been verified by bootstrap analysis with a confidence level of 100%. Species of the genus Bacteroides can also be distinguished from species of other closely related genera by bile resistance, cellular fatty acid content, fermentation, and pigmentation (Table 3). Members of the genus can be distinguished from those of the most closely related genera Prevotella and Porphyromonas by their resistance to 20% bile. In addition, Bacteroides species are highly ­fermentative

Taxonomic comments The taxonomy of the genus Bacteroides has been in a state of great change in recent years. On the basis of 16S rRNA gene sequence analysis, Bacteroides forms the “CFB group” with Cytophaga and Flavobacterium (Woese, 1987). The “CFB group” is divided into the Cytophaga, Flavobacterium, and Bacteroides subgroups. The Bacteroides subgroup is further divided into the Prevotella cluster, Porphyromonas cluster, Bacteroides cluster, and two new unnamed clusters. Accordingly, it is now generally

Prevotella

Alistipes d

Tannerella

Pigment production Growth in 20% bile Susceptibility to: e   Vancomycin (5 mg)   Kanamycin (1 mg)   Colistin (10 mg) Catalase production Indole production Nitrate reduction G6PDH and 6PGDH dehydrogenase production Proteolytic activity Carbohydrate fermentation Major metabolic end product(s)f Major cellular fatty acids DNA G+C content (mol%) Type species

Porphyromonas c

Characteristic

Bacteroides b

TABLE 3.  Descriptive characteristics of Bacteroides and related generaa

− +

+− −

+/− −

+− +−

− −

R R R − +/− − + − + A, S C15:0 anteiso 40–48 B. fragilis

S/R R R − +/− − − +/− −+ A, B C15:0 iso 40–55 P. asaccharolytica

R S/R S/R − +/− − − +/− + A, S C15:0 anteiso 39–60 P. melaninogenica

R R R −+ +− − − + +− S C15:0 iso 55–58 A. putredinis

R S S − − − − + − A, S, PA C15:0 anteiso 44–48 T. forsythia

Symbols: +, positive; −, negative; −+, usually negative, sometimes positive; +/−, positive or negative; +−, usually positive, sometimes negative. Bacteroides sensu stricto. c Unlike other Porphyromonas spp., P. catoniae does not produce pigment and is moderately saccharolytic. d Unlike other Alistipes spp., A. putredinis does not produce pigment, is susceptible to bile, catalase-positive, and nonsaccharolytic. e Special potency antimicrobial identification disks; R, resistant; S, susceptible; S/R, either sensitive or resistant. f A, acetic acid; B, butyric acid; PA, phenylacetic acid; S, succinic acid. a

b

32

FAMILY i. Bacteroidaceae

recognized that the majority of the original Bacteroides species fall into three genera: Prevotella (bile-sensitive, moderately saccharolytic, pigmented and nonpigmented species), Porphyromonas (bile-sensitive, pigmented, asaccharolytic species), and Bacteroides (bile-resistant, nonpigmented, saccharolytic species) (Shah and Collins, 1988, 1989, 1990). Several other genera have been described subsequently for those clearly unrelated Bacteroides taxa that do not conform to these three major groups (e.g., Alistipes, Anaerorhabdus, Dichelobacter, Fibrobacter, ­Megamonas,

­Mitsuokella, Rikenella, Sebaldella, Tannerella, and ­Tissierella) (Table 4). However, a large number of taxa still remain unclassified and several, such as Bacteroides capillosus, Bacteroides cellulosolvens, Bacteroides ureolyticus, and Bacteroides splanchnicus, are retained in Bacteroides until valid new genera are proposed. The genus Bacteroides now contains mainly the species that were formerly described as the “Bacteroides fragilis group” (including Bacteroides eggerthii), as well as several later described species. It is evident from the phylogenetic data (Figure 14) that three

TABLE 4.  Taxonomy changes in the genus Bacteroides

Previous name

References

Current name

Reference(s)

Holdeman and Moore (1970) van Steenbergen et al. (1984) Coykendall et al. (1980) Johnson and Holdeman (1983) Slots and Genco (1980) Love et al. (1987)

Porphyromonas asaccharolytica Porphyromonas endodontalis Porphyromonas gingivalis Porphyromonas levii Porphyromonas macacae Porphyromonas macacae

Shah and Collins (1988) Shah and Collins (1988) Shah and Collins (1988) Shah et al. (1995) Love (1995) Love (1995)

Holdeman and Johnson (1977) Bryant et al. (1958) Holdeman and Johnson (1982) Kornman and Holt (1981) Shah and Collins (1981) Shah and Collins (1981) Johnson and Holdeman (1983) Shah and Collins (1981)

Prevotella bivia Prevotella brevis Prevotella buccae Prevotella buccae Prevotella buccae Prevotella buccalis Prevotella corporis Prevotella denticola

B. disiens B. heparinolyticus B. intermedius B. loescheii

Holdeman and Johnson (1977) Okuda et al. (1985) Holdeman and Moore (1970) Holdeman and Johnson (1982)

Prevotella disiens Prevotella heparinolytica Prevotella intermedia Prevotella loescheii

  B. melaninogenicus

Oliver and Wherry (1921) emend. Roy and Kelly (1939) Loesche et al. (1964) Holdeman and Johnson (1982) Shah et al. (1985) Bryant et al. (1958) Watabe et al. (1983)

Prevotella melaninogenica

Weinberg et al. (1937) emend. Cato et al. (1982)

Prevotella zoogleoformans

Shah and Collins (1990) Avgustin et al. (1997) Shah and Collins (1990) Shah and Collins (1990) Shah and Collins (1990) Shah and Collins (1990) Shah and Collins (1990) Shah and Collins (1990) emend. Wu et al. (1992) Shah and Collins (1990) Shah and Collins (1990) Shah and Collins (1990) Shah and Collins (1990) emend. Wu et al. (1992) Shah and Collins (1990) emend. Wu et al. (1992) Shah and Collins (1990) Shah and Collins (1990) Shah and Collins (1990) Shah and Collins (1990) Shah and Collins (1990) emend. Wu et al. (1992) Shah and Collins (1990) emend. Moore et al. (1994)

Hamlin and Hungate (1956) Tanner et al. (1986) Veillon and Zuber (1898) Tanner et al. (1981) Harrison and Hansen (1963) Kaneuchi and Mitsuoka (1978) Mitsuoka et al. (1974) Beveridge (1941) Prévot et al. (1956) Olitsky and Gates (1921) Tissier (1908) Weinberg et al. (1937) Hungate (1950) Sebald (1962)

Ruminobacter amylophilus Tannerella forsythia Anaerorhabdus furcosa Campylobacter gracilis Megamonas hypermegale Rikenella microfusus Mitsuokella multacida Dichelobacter nodosus Capnocytophaga ochracea Dialister pneumosintes Tissierella praeacuta Alistipes putredinis Fibrobacter succinogenes Sebaldella termitidis

Species reclassified into the genus Porphyromonas:   B. asaccharolyticus   B. endodontalis   B. gingivalis   B. levii   B. macacae   B. salivosus Species reclassified into the genus Prevotella:   B. bivius   B. ruminicola subsp. brevis   B. buccae   B. capillus   B. pentosaceus   B. buccalis   B. corporis   B. denticola        

         

B. oralis B. oris B. oulorum B. ruminicola subsp. ruminicola B. veroralis

  B. zoogleoformans Species reclassified into other genera:   B. amylophilus   B. forsythus   B. furcosus   B. gracilis   B. hypermegas   B. microfusus   B. multiacidus   B. nodosus   B. ochraceus   B. pneumosintes   B. praeacutus   B. putredinis   B. succinogenes   B. termitidis

Prevotella oralis Prevotella oris Prevotella oulorum Prevotella ruminicola Prevotella veroralis

Stackebrandt and Hippe (1986) Sakamoto et al. (2002) Shah and Collins (1986) Vandamme et al. (1995) Shah and Collins (1982b) Collins et al. (1985) Shah and Collins (1982a) Dewhirst et al. (1990) Leadbetter et al. (1979) Moore and Moore (1994) Collins and Shah (1986b) Rautio et al. (2003) Montgomery et al. (1988) Collins and Shah (1986a)

Genus I. Bacteroides

bile-resistant “Bacteroides fragilis group” species – Bacteroides ­distasonis, Bacteroides merdae, and the recently described Bacteroides goldsteinii – cluster close to the bile-sensitive Tannerella forsythia (formerly Bacteroides forsythus) and display a loose affinity with the first subcluster (the genus Porphyromonas), suggesting that a novel genus should be established to accommodate these three species. Sakamoto and Benno (2006) created the genus Parabacteroides to encompass three species that were previously classified in Bacteroides, viz., Parabacteroides distasonis (previously Bacteroides distasonis), Parabacteroides goldsteinii (previously Bacteroides goldsteinii), and Parabacteroides merdae (previously Bacteroides merdae). A fourth species, Parabacteroides johnsonii, was subsequently added (Sakamoto et  al., 2007). The description of Parabacteroides is as follows (Sakamoto and Benno, 2006). Rods (0.8–1.6 × 1.2–12 mm). Nonmotile. Non-spore-forming. Gram-stain-negative. Obligately anaerobic. Colonies on Eggerth–Gagnon (EG) agar plates are 1–2 mm in diameter, gray to off-white gray, circular, entire, slightly convex, and smooth. Saccharolytic. Major end products are acetic and succinic acids;

33

lower levels of other acids may be produced. Growth occurs in the presence of 20% bile. Esculin is hydrolyzed. Indole is not produced. G6PDH, 6PGDH, malate dehydrogenase, and glutamate dehydrogenase are present, but not a-fucosidase. The principal respiratory quinones are menaquinones MK-9 and MK-10. Both nonhydroxylated and 3-hydroxylated longchain fatty acids are present. The nonhydroxylated acids are predominantly of the saturated straight chain and anteisomethyl branched chain types. The DNA G+C content is 43–46 mol%. The type species is Parabacteroides distasonis (Eggerth and Gagnon, 1933) Sakamoto and Benno 2006, 1602VP. Interestingly, the bile-sensitive oral species Prevotella heparinolytica and Prevotella zoogleoformans fall, phylogenetically, within the Bacteroides cluster. The Subcommittee on Gram-negative Rods of the International Committee on Systematics of Prokaryotes recommended designating them Bacteroides heparinolytica and Bacteroides zoogleoformans, respectively, but studies other than 16S rRNA gene sequence analysis should be performed before the status of these taxa is finalized.

List of species of the genus Bacteroides 1. Bacteroides fragilis (Veillon and Zuber 1898) Castellani and Chalmers 1919, 959AL [Bacillus fragilis Veillon and Zuber 1898, 536; Fusiformis fragilis (Veillon and Zuber 1898) Topley and Wilson 1929, 393; Ristella fragilis (Veillon and Zuber 1898) Prévot 1938; Bacteroides fragilis subsp. fragilis (Veillon and Zuber 1898) Castellani and Chalmers 1919, 959] fra¢gi.lis. L. masc. adj. fragilis fragile (relating to the brittle colonies that may form under some culture conditions). Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly or in pairs and have rounded ends. Vacuoles are often present, particularly in broth media containing a fermentable carbohydrate. Surface colonies on blood agar are 1–3 mm in diameter, circular, entire, low convex, and translucent to semi-opaque. They often have an internal structure of concentric rings when reviewed by obliquely transmitted light. In general, strains produce no hemolysis on blood agar, although a few strains may be slightly hemolytic, particularly in the area of confluent growth. Growth is often enhanced by 20% bile. Growth may occur at 25 or 45°C. Most strains grow at pH 8.5, but they grow more slowly and less luxuriantly than at pH 7.0. Cells can survive exposure to air for at least 6–8 h. Hemin is either required for growth or markedly stimulates growth. The major fatty acids are C15:0 anteiso, C15:0 iso, C17:0 iso 3-OH, and C16:0. Strains have been isolated from various types of human clinical specimens or conditions including appendicitis, peritonitis, endocarditis, bacteremia, perirectal abscess, infected pilonidal cyst, postsurgical wound infections, and lesions of the urogenital tract; occasionally isolated from the mouth and vagina. It is the major obligately anaerobic, Gram-stainnegative bacterium isolated from various infections. Although Bacteroides fragilis strains show little phenotypic variability, the species comprises genetically heterogeneous strains (Johnson, 1978). A DNA–DNA hybridization study

by Johnson and Ault (1978) led to the distinction of two DNA homology groups, I and II, with about 80% of strains isolated in clinical studies assigned to homology group I. Similarly, two genotypically distinct Bacteroides fragilis groups have been identified on the basis of ribotyping, restriction fragment length polymorphism analysis, PCR-generated fingerprinting, insertion sequence content, 16S rRNA gene sequence alignments, multilocus enzyme electrophoresis, and genetic marker sequence analysis (Gutacker et  al., 2000, 2002). One group is characterized by the presence of the cfiA gene (encoding a metallo-b-lactamase of Ambler’s class B) and the absence of the cepA gene (encoding a b-lactamase of Ambler’s class A). The second group is characterized by the absence of the cfiA gene and of the associated insertion sequences, the frequent presence of the cepA gene, and a higher genetic heterogeneity (Podglajen et al., 1995; Ruimy et al., 1996). By including strains obtained from Johnson (1978) in their 16S rRNA gene sequence comparison, Ruimy et al. (1996) showed that the two groups described above could be related to DNA homology groups II and I, respectively. However, data obtained so far are still insufficient to clarify definitively whether the two groups may be considered as two distinct genospecies that have ­diverged recently, or if they represent two Bacteroides fragilis groups not yet separated at the species level, but evolving in this direction. Putative virulence factors of Bacteroides fragilis include attachment mechanisms, relative aerotolerance, extracellular enzyme production, and resistance to complementmediated killing and phagocytosis. A polysaccharide capsule contributes to this resistance and resistance to T-cell activity (Patrick, 2002). An enterotoxin termed Bacteroides fragilis toxin, or BFT, is a recognized virulence factor. BFT has been characterized as a 20-kDa zinc-dependent metalloprotease (Moncrief et al., 1995) that mediates the cleavage of E-cadherin, resulting in an altered morphology of certain human intestinal carcinoma cell lines (particularly

34

FAMILY i. Bacteroidaceae

HT29/C1cells), fluid accumulation in ligated lamb ileal loops, and intestinal epithelial cell proliferation. It has been reported that the bft gene is contained in a 6-kb pathogenicity island termed the Bacteroides fragilis pathogenicity island or BfPAI (Franco et al., 1999). The genomes of the Bacteroides fragilis type strain, NCTC 9343T, and a clinical strain, YCH46, have been sequenced. The genome of Bacteroides fragilis NCTC 9343T contains a single circular chromosome of 5,205,140 bp that is ­predicted to encode 4274 genes and a plasmid, pBF9343. It shows considerable variation, even within the same strain, and “invertable promoters” appear to regulate much of this variance in many surface-exposed and secreted proteins (Cerdeno-Tarraga et al., 2005). The complete genome sequence has revealed an unusual breadth (in number and in effect) of DNA inversion events that potentially control expression of many different components, including surface and secreted components, regulatory molecules, and restriction-modification proteins. This may be related to its niche as a commensal and opportunistic pathogen, because the resulting diversity in surface structures could increase both immune evasion and the ability to colonize novel sites. Source: human clinical specimens. DNA G+C content (mol%): 41–44 (HPLC). Type strain: ATCC 25285, CCUG 4856, CIP 77.16, DSM 2151, JCM 11019, LMG 10263, NCTC 9343. Sequence accession no. (16S rRNA gene): AB050106, X83935, M11656; genome sequences of strain NCTC 9343T, NC_003228, CR626927; genome sequences of strain YCH46, NC_006347, AP006841. 2. Bacteroides acidifaciens Miyamoto and Itoh 2000, 148VP a.ci.di¢fa.ci.ens. N.L. n. acidum (from L. adj. acidus sour) acid; L. v. facio produce; N.L. part. adj. acidifaciens acid-producing. Characteristics are as described for the genus and as ­given in Table 2, with the following additional characteristics. After 48 h incubation, surface colonies on EG agar (­Mitsuoka et al., 1965) are 1–3 mm in diameter, circular, entire, raised, convex, smooth, and grayish. The specific character of this species is reduction of the pH of pre-reduced anaerobically sterilized peptone-yeast extract (PY) broth with Fildes’ digest (PYF) (Fildes et al., 1936) broth without carbohydrate. Source: mouse cecum. DNA G+C content (mol%): 39.4–42.4 (HPLC). Type strain: strain A40, JCM 10556. Sequence accession no. (16S rRNA gene): AB021164. 3. Bacteroides caccae Johnson, Moore and Moore 1986, 499VP cac¢cae. Gr. n. kakkê feces; N.L. gen. n. caccae of feces, referring to the source of isolate. Characteristics are as described for the genus and as ­ iven in Table 2, with the following additional characterg istics. Cells of the type strain from peptone-yeast-glucose (PYG) broth cultures occur singly or in pairs; cells may ­appear vacuolated or beaded in strains from broth cultures in media with a fermentable carbohydrate. Surface colonies on supplemented brain heart infusion (BHI) blood

agar plates (Jousimies-Somer et  al., 2002) incubated for 48 h are 0.5–1 mm in diameter, circular, entire, convex, gray, translucent, shiny, and smooth. Rabbit blood may be slightly hemolyzed. Strains grow equally well at 30 and 37°C, but less well at 25 and 45°C. The type strain reduces neutral red and does not produce H2S. Pyruvate is converted to acetate. Lactate and threonine are not utilized. The ­major fatty acids are C15:0 anteiso, C15:0 iso, C17:0 iso 3-OH, and C16:0. Source: human feces and blood. DNA G+C content (mol%): 40–42 (HPLC). Type strain: ATCC 43185, CCUG 38735, CIP 104201, JCM 9498, NCTC 13051, VPI 3452A. Sequence accession no. (16S rRNA gene): X83951. Additional remarks: Previously referred to as “3542A” DNA homology group (Johnson, 1978; Johnson and Ault, 1978). 4. Bacteroides coprocola Kitahara, Sakamoto, Ike, Sakata and Benno 2005, 2146VP co.pro.co¢la. Gr. n. kopros feces; L. suff. -cola (from L. n. incola) inhabitant; N.L. n. coprocola inhabitant of feces. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Surface colonies on EG blood agar incubated for 48 h are 1.0 to approximately 3.0 mm in diameter, disc shaped, and grayish white. The major fatty acids are C15:0 anteiso, C16:0 3-OH, C16:0, C17:0 iso 3-OH, and C18:1 w9c. Source: feces of healthy humans. DNA G+C content (mol%): 42.4 (HPLC). Type strain: strain M16, DSM 17136, JCM 12979. Sequence accession no. (16S rRNA gene): AB200224. 5. Bacteroides coprosuis Whitehead, Cotta, Collins, Falsen and Lawson 2005, 2517VP co.pro.su¢is. Gr. n. kopros feces; L. gen. n. suis of a pig; N.L. gen. n. coprosuis of pig feces, from which the organism was isolated. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells usually occur singly, but occasionally in pairs in PYG. Colonies grown on BHI agar plates after 48 h incubation are cream-colored, circular, convex, entire, opaque, and reach a diameter of 1 mm. Strains grow at 25–37°C, but not at 42 or 45°C, with an optimum of 37°C. The major fatty acids are C15:0 anteiso and C17:0 iso 3-OH. Significant amounts of C17:0 iso and C15:0 iso are also present. Source: swine feces. DNA G+C content (mol%): 36.4 (HPLC). Type strain: PC139, CCUG 50528, NRRL B-41113. Sequence accession no. (16S rRNA gene): AJ514258, AF319778. 6. Bacteroides distasonis Eggerth and Gagnon 1933, 403AL [Ristella distasonis (Eggerth and Gagnon 1933) Prévot 1938, 291; Bacteroides fragilis subsp. distasonis (Eggerth and Gagnon 1933) Holdeman and Moore 1970, 35] dis.ta.so¢nis. N.L. gen. masc. n. distasonis of Distaso, named after A. Distaso, a Romanian bacteriologist. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics.

Genus I. Bacteroides

In PYG, cells usually occur singly, but occasionally in pairs. Colonies on sheep-blood agar are pinpoint to 0.5 mm in diameter, circular, entire, convex, translucent to opaque, gray-white, soft, smooth, and a-hemolytic (sheep blood). Hemin is required or is highly stimulatory for growth. Resazurin, but not neutral red, is reduced. Propionate is not formed from lactate or threonine. Neither pyruvate nor gluconate is converted to products other than those found after growth in PY. The major fatty acids are C15:0 anteiso, C15:0 iso, C17:0 iso 3-OH, and C16:0. Isolated primarily from human feces, where it is one of the most common species; also isolated from human clinical specimens. This species has been recently reclassified as Parabacteroides distasonis Sakamoto and Benno (2006); see Taxonomic comments, above). Source: human feces, human clinical specimens. DNA G+C content (mol%): 43–45 (HPLC). Type strain: ATCC 8503, CCUG 4941, CIP 104284, DSM 20701, JCM 5825, NCTC 11152. Sequence accession no. (16S rRNA gene): M86695. 7. Bacteroides dorei Bakir, Sakamoto, Kitahara, Matsumoto and Benno 2006c, 1642VP do.re¢i. N.L. gen. masc. n. dorei of Doré, in honor of the French microbiologist Joel Doré, in recognition of his many contributions to intestinal (gut) microbiology. Cells are Gram-stain-negative rods, anaerobic, nonmotile, and non-spore-forming. Typical cells are 1.6–4.2 × 0.8–1.2 mm and occur singly. Colonies on EG agar plates after 48 h incubation at 37°C under 100% CO2 are circular, whitish, raised, and convex, and attain a diameter of 2.0 mm. Optimum temperature for growth is 37°C. Growth occurs in the presence of bile. Esculin is not hydrolyzed. Nitrate is not reduced. No activity is detected for urease and gelatin. Acid is produced from glucose, sucrose, xylose, rhamnose, lactose, maltose, arabinose, mannose, and raffinose. Acid is not produced from cellobiose, salicin, trehalose, mannitol, glycerol, melezitose, or sorbitol. Positive reactions are obtained using API rapid ID 32A for a-fucosidase, a-galactosidase, b-galactosidase, 6-phospho-b-galactosidase, a-glucosidase, b-glucosidase, a-arabinosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, glutamic acid, decarboxylase, alkaline phosphatase, arginine arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, leucine arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, and serine arylamidase. Negative reactions are obtained for arginine dihydrolase, and proline arylamidase. Major fatty acids are C15:0 anteiso (26–32%), C17:0 iso 3-OH (17–19%) and C18:1 w9c (9–12%). Source: human feces. DNA G+C content (mol%): 43 (HPLC). Type strain: 175, JCM 13471, DSM 17855 [strain 219 (JCM 13472) is included in this species]. Sequence accession no. (16S rRNA gene): AB242142. 8. Bacteroides eggerthii Holdeman and Moore 1974, 260AL eg.gerth¢i.i. N.L. gen. masc. n. eggerthii of Eggerth, named after Arnold H. Eggerth, an American bacteriologist.

35

Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. In PYG broth, cells are pleomorphic rods, ranging from coccoid to large rods with vacuoles or swellings, occurring singly or in pairs. Colonies on blood agar are punctiform, circular, entire convex, translucent, gray-white, shiny, smooth, and nonhemolytic. Hemin markedly stimulates growth, but is not required. Without hemin, malate and lactate are produced; with hemin, succinate and acetate are produced. Vitamin B12 is required for production of propionate from succinate. The optimum temperature for growth is 37°C. There is good growth at 30 and 45°C, moderate growth at 25°C. Neutral red is reduced. The major fatty acids are C15:0 anteiso, C15:0 iso, C17:0 iso 3-OH, and C16:0. Source: human feces and occasionally from clinical specimens. DNA G+C content (mol%): 44–46 (HPLC). Type strain: ATCC 27754, CCUG 9559, CIP 104285, DSM 20697, NCTC 11155. Sequence accession no. (16S rRNA gene): AB050107, L16485. 9. Bacteroides finegoldii Bakir, Kitahara, Sakamoto, Matsumoto and Benno 2006b, 934VP fine.gold¢i.i. N.L. gen. masc. n. finegoldii of Finegold, in honor of Sydney M. Finegold, a contemporary researcher in anaerobic bacteriology and infectious diseases. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly. Surface colonies on EG blood agar plates after 48 h are 1–2 mm in diameter, circular, translucentwhitish, raised, and convex. The major fatty acids are C15:0 anteiso and C17:0 iso 3-OH. Source: feces of healthy humans. DNA G+C content (mol%): 42.4–43 (HPLC). Type strain: strain 199, JCM 13345, DSM 17565. Sequence accession no. (16S rRNA gene): AB222699. 10. Bacteroides goldsteinii Song, Liu, Lee, Bolanos, Vaisanen and Finegold 2006, 499VP (Effective publication: Song, Liu, Lee, Bolanos, Vaisanen and Finegold 2005a, 4526.) gold.stein¢i.i. N.L. gen. masc. n. goldsteinii of Goldstein, in honor of an infectious disease clinician who has done much work with anaerobes, Ellie J.C. Goldstein. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly, occasionally in pairs. Colonies on Brucella blood agar plates at 48 h are gray, circular, convex, entire, opaque, and attain a diameter of 1–2 mm. The major fatty acids are C15:0 anteiso and C17:0 iso 3-OH. Significant amounts of C18:1w9c and C17:0 anteiso 3-OH are also present. Habitat is probably the human gut. This species has been recently reclassified as Parabacteroides goldsteinii (Sakamoto and Benno, 2006; see Taxonomic comments, above). Source: human clinical specimens of intestinal origin. DNA G+C content (mol%): 43 (HPLC). Type strain: WAL 12034, CCUG 48944, ATCC BAA-1180. Sequence accession no. (16S rRNA gene): AY974070.

36

FAMILY i. Bacteroidaceae

11. Bacteroides helcogenes Benno, Watabe and Mitsuoka 1983a, 896VP (Effective publication: Benno, Watabe and Mitsuoka 1983b, 404.) hel.co.ge¢nes. Gr. n. helkos abscess; N.L. suff. -genes (from Gr. v. gennaô to produce), producing; N.L. adj. helcogenes abscess-producing. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells are single or in pairs. After 48 h of incubation on EG agar, surface colonies are 1.0–2.0 mm in diameter, circular with entire edges, convex, translucent to opaque, gray to graywhite, shiny, and smooth. Most strains do not grow in peptone-yeast-Fildes-glucose (PYFG) broth containing 20% bile. The major fatty acids are C15:0 anteiso and C17:0 iso 3-OH. Source: swine abscesses and feces. DNA G+C content (mol%): 45.3–46.3 (HPLC). Type strain: strain P 36-108, ATCC 35417, CCUG 15421, DSM 20613, JCM 6297. Sequence accession no. (16S rRNA gene): AB200227. 12. Bacteroides intestinalis Bakir, Kitahara, Sakamoto, Matsumoto and Benno 2006a, 153VP in.tes.ti.na¢lis. L. n. intestinum gut, intestine; L. masc. suff. -alis suffix denoting pertaining to; N.L. masc. adj. intestinalis pertaining to the intestine. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly. Surface colonies on EG blood agar plates after 2 d are 1–3 mm in diameter, circular, translucentwhitish, raised, and convex. The major fatty acids are C15:0 anteiso and C17:0 iso 3-OH. Source: feces of healthy humans. DNA G+C content (mol%): 44 (HPLC). Type strain: strain 341, DSM 17393, JCM 13265. Sequence accession no. (16S rRNA gene): AB214328. 13. Bacteroides massiliensis Fenner, Roux, Mallet and Raoult 2005, 55VP mas.si¢li.en.sis. L. masc. adj. massiliensis of Massilia, the ancient Roman name for Marseille, France, where the type strain was isolated. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells usually occur singly. Surface colonies on sheep blood agar plates after 2 d are 1–2 mm in diameter, circular, whitegrayish, translucent, raised, and convex. No hemolysis on sheep blood agar. Optimum growth temperature is 37°C, but growth is observed at 25–42°C. The major fatty acid is C15:0 anteiso. Source: human blood culture. DNA G+C content (mol%): 49 (HPLC). Type strain: strain B84634, CCUG 48901, CIP 107942, JCM 13223. Sequence accession no. (16S rRNA gene): AY126616, AB200226. 14. Bacteroides merdae Johnson, Moore and Moore 1986, 499VP mer¢dae. L. gen. n. merdae of feces, referring to the source of the isolate.

Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly or in pairs or short chains. Surface colonies on BHI blood agar plates supplemented with vitamin K and hemin are 0.5–1.0 mm in diameter, circular to slightly irregular, entire, convex, white, shiny, and smooth. Rabbit blood is slightly hemolyzed. The type strain reduces neutral red and resazurin and produces hydrogen sulfide after incubation for 5 d. Pyruvate is converted to acetate and propionate. Lactate, threonine, and gluconate are not utilized. The major fatty acids are C15:0 anteiso, C15:0 iso, C17:0 iso 3-OH, and C16:0. This species has been recently reclassified as Parabacteroides merdae (Sakamoto and Benno, 2006; see Taxonomic comments, above). Source: human feces and occasionally clinical specimens. DNA G+C content (mol%): 43–46 (HPLC). Type strain: strain ATCC 43184, CCUG 38734, CIP 104202, JCM 9497, NCTC 13052, VPI T4-1. Sequence accession no. (16S rRNA gene): AY169416, X83954, U50416. Additional remarks: Previously referred to as “T4-1” DNA homology group (Johnson, 1978; Johnson and Ault, 1978). 15. Bacteroides nordii Song, Liu, McTeague and Finegold 2005b, 983VP (Effective publication: Song, Liu, McTeague and Finegold 2004, 5569.) nor.di.i. N.L. gen. masc. n. nordii of Nord, to honor Carl Erik Nord, who has contributed much to our knowledge of anaerobic bacteriology and intestinal bacteriology. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly or in pairs. Colonies on Brucella blood agar plates at 48 h are gray, circular, convex, entire, opaque, and attain a diameter of 1–2 mm. The major fatty acids are C15:0 anteiso, C15:0 iso, C16:0 3-OH, and C18:1 w9c. Probably of intestinal origin. Source: clinical sources such as peritoneal fluid, appendix tissue, and intra-abdominal abscesses. DNA G+C content (mol%): 41.4 (HPLC). Type strain: strain WAL 11050, ATCC BAA-998, CCUG 48943. Sequence accession no. (16S rRNA gene): AY608697. 16. Bacteroides ovatus Eggerth and Gagnon 1933, 405AL [Pasteurella ovata Eggerth and Gagnon 1933) Prévot 1938, 292; Bacteroides fragilis subsp. ovatus (Eggerth and Gagnon 1933) Holdeman and Moore 1970, 35] o.va¢tus. L. masc. adj. ovatus ovate, egg-shaped (relating to the cellular shape). Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells are oval, occurring singly, but occasionally in pairs. Capsules are detected in some strains. Surface colonies on blood agar are 0.5–1.0 mm, circular, entire, convex, pale buff, semi-opaque, and may have a mottled appearance. There is no hemolysis of sheep blood. Hemin is required or is highly stimulatory for growth. The major fatty acids are C15:0 anteiso, C17:0 iso 3-OH, C18:1 w9c, and C15:0 iso.

Genus I. Bacteroides

Source: human feces and occasionally human clinical specimens. DNA G+C content (mol%): 39–43 (HPLC). Type strain: ATCC 8483, BCRC 10623, CCUG 4943, CIP 103756, DSM 1896, JCM 5824, NCTC 11153. Sequence accession no. (16S rRNA gene): AB050108, L16484, X83952. 17. Bacteroides plebeius Kitahara, Sakamoto, Ike, Sakata and Benno 2005, 2146VP ple.bei¢us. L. masc. adj. plebeius common, of low class. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Colonies grown on EG blood agar plates are 1–3 mm in diameter, disc-shaped, grayish-white, and translucent. The major fatty acids are C15:0 anteiso, C17:0 iso 3-OH, C16:0 3-OH, C18:1 w9c, and C16:0. Source: feces of healthy humans. DNA G+C content (mol%): 42.4–43.9 (HPLC). Type strain: strain M12, DSM 17135, JCM 12973. Sequence accession no. (16S rRNA gene): AB200217– AB200222. 18. Bacteroides pyogenes Benno, Watabe and Mitsuoka 1983a, 896VP (Effective publication: Benno, Watabe and Mitsuoka 1983b, 402.) py.o¢ge.nes. Gr. n. pyum pus; N.L. suff. -genes (from Gr. v. gennaô to produce) producing; N.L. adj. pyogenes pus-producing. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly or occasionally in pairs with longer rods. After 2 d of incubation on EG agar, surface colonies are 0.5–1.5 mm in diameter, circular with entire edges, low convex, translucent to opaque, cream to light gray, shiny, and smooth. Usually, no hemolysis occurs on blood agar. Strains do not grow well in PYFG–20% bile broth. Some strains hydrolyze esculin. Source: swine abscess and feces. DNA G+C content (mol%): 46.1–47.6 (HPLC). Type strain: strain P 39-88, ATCC 35418, CCUG 15419, DSM 20611, JCM 6294. Sequence accession no. (16S rRNA gene): AB200229. 19. Bacteroides salyersiae corrig. Song, Liu, McTeague and Finegold 2005b, 983VP (Effective publication: Song, Liu, McTeague and Finegold 2004, 5569.) [Bacteroides salyersae (sic) Song, Liu, McTeague and Finegold 2004, 5569] sal¢yer.si.ae. N.L. gen. fem. n. salyersiae of Salyers, named after Abigail Salyers, an American bacteriologist who has contributed so much to our knowledge of intestinal bacteriology and anaerobic bacteriology in general. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly, occasionally in pairs. Colonies on Brucella blood agar plates at 24 h are gray, circular, convex, entire, opaque, and attain a diameter of 1–2 mm. The major fatty acids are C15:0 anteiso, C15:0 iso, C16:0 3-OH, C16:0, and C18:1 w9c. Probably of intestinal origin. Source: clinical sources such as peritoneal fluid, appendix tissue, and intra-abdominal abscesses.

37

DNA G+C content (mol%): 42.0 (HPLC). Type strain: strain WAL 10018, ATCC BAA-997, CCUG 48945. Sequence accession no. (16S rRNA gene): AY608696. 20. Bacteroides stercoris Johnson, Moore and Moore 1986, 501VP ster¢co.ris. L. n. stercus feces; L. gen. n. stercoris of feces, referring to source of isolate. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly and in pairs. Vacuolated cells are seen sometimes in cultures in broths that contain a fermentable carbohydrate. Surface colonies on supplemented BHI blood agar with rabbit blood are 0.5–1.0 mm in diameter, circular, entire, convex, transparent to translucent, shiny, smooth, and b-hemolytic. Resazurin is reduced, neutral red is not reduced. Pyruvate is converted to acetate. Lactate, threonine, and gluconate are not used. The major fatty acids are C15:0 anteiso, C15:0 iso, C17:0 iso 3-OH, and C16:0. Source: human feces and occasionally human clinical specimens. DNA G+C content (mol%): 43–47 (HPLC). Type strain: ATCC 43183, CCUG 38733, CIP 104203, JCM 9496, NCTC 13053, VPI B5-21. Sequence accession no. (16S rRNA gene): X83953, U50417. Additional remarks: previously referred to as the “subsp. a” DNA homology group (Johnson, 1978; Johnson and Ault, 1978). 21. Bacteroides suis Benno, Watabe and Mitsuoka 1983a, 896VP (Effective publication: Benno, Watabe and Mitsuoka 1983b, 403.) su¢is. L. n. sus swine; L. gen. n. suis of the pig. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells are short rods, single or in pairs; longer rods occur only occasionally. After 2 d of incubation on EG agar, surface colonies are 0.5–1.5 mm in diameter, circular with entire edge, low convex, translucent to opaque, gray to gray-white, shiny, and smooth. Slight greening or indefinite hemolysis often apparent on blood agar plates. Either no growth or markedly inhibited and delayed growth occurs in PYFG broth containing 20% bile. Source: swine abscess and feces. DNA G+C content (mol%): 42.3–45.7 (HPLC). Type strain: P 38024, ATCC 35419, DSM 20612, CCUG 15420, JCM 6292. Sequence accession no. (16S rRNA gene): DQ497991. 22. Bacteroides tectus corrig. Love, Johnson, Jones, Bailey and Calverley 1986, 126VP [Bacteroides tectum (sic) Love, Johnson, Jones, Bailey and Calverley 1986, 126] tec¢tus. L. masc. adj. tectus (from L. v. tego) secret, concealed, hidden, referring to difficulty in species identification. Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells occur singly, in pairs, and in short chains. On sheep blood agar, surface colonies after 48 h are 2–3 mm in diameter, circular, entire, dome shaped, grayish, and opaque. Source: subcutaneous fight wound abscesses from cats and dogs, pyothorax of cats, and mouths of normal cats.

38

FAMILY i. Bacteroidaceae

DNA G+C content (mol%): 47–48 (HPLC). Type strain: strain 160, ATCC 43331, CCUG 25929, JCM 10003, NCTC 11853. Sequence accession no. (16S rRNA gene): AB200228. 23. Bacteroides thetaiotaomicron (Distaso 1912) Castellani and Chalmers 1919, 960AL [Bacillus thetaiotaomicron Distaso 1912, 444; Sphaerocillus thetaiotaomicron (Distaso 1912) Prévot 1938, 300; Bacteroides fragilis subsp. thetaiotaomicron (Distaso 1912) Holdeman and Moore 1970, 35] the.ta.i.o.ta.o¢mi.cron. N.L. n. thetaiotaomicron a combination of the Greek letters theta, iota and omicron (relating to the morphology of vacuolated forms). Characteristics are as described for the genus and as ­ iven in Table 2, with the following additional characterisg tics. Cells are pleomorphic and occur singly or in pairs. Cells from blood agar plates or PY broth are smaller and more homogeneous in size and shape than cells from media with a fermentable carbohydrate. Capsules are present in some strains. Surface colonies on blood agar are punctiform, circular, entire, convex, semiopaque, whitish, soft, and shiny. Sheep blood is not hemolyzed. Hemin is either required or is highly stimulatory for growth. The major fatty acids are C15:0 anteiso, C17:0 iso 3-OH, C18:1 w9c, and C16:0 3-OH. The genome of Bacteroides thetaiotaomicron VPI-5482 has been sequenced. The genome of Bacteroides fragilis NCTC 9343T contains a single circular chromosome of 6,260,361 bp. The genome has large expansions of many paralogous groups of genes that encode products essential to the organism’s ability to successfully compete in the human colonic microbiota. Most notable of these is the organism’s abundant machinery for utilizing a large variety of complex polysaccharides as a source of carbon and energy. The proteome also reveals the organism’s extensive ability to adapt and regulate expression of its genes in response to the changing ecosystem. These factors suggest a highly flexible and adaptable organism that is exquisitely equipped to dominate in its challenging and competitive niche (Xu et al., 2003). Source: human feces, frequently found in human clinical specimens. DNA G+C content (mol%): 40–43 (HPLC). Type strain: ATCC 29148, CCUG 10774, CIP 104206, DSM 2079, JCM 5827, NCTC 10582, VPI 5482. Sequence accession no. (16S rRNA gene): M58763, AB050109, L16489; genome sequence for strain VPI 5482, NC_004663, AE015928. 24. Bacteroides uniformis Eggerth and Gagnon 1933, 400AL u.ni.form¢is. L. masc. adj. uniformis having only one form, uniform.

Characteristics are as described for the genus and as ­ iven in Table 2, with the following additional characterg istics. Cells occur singly or in pairs; an occasional filament may be seen. Vacuoles that do not greatly swell the cell are often present in cells grown in media containing a fermentable carbohydrate. Surface colonies on blood agar are 0.5–2.0 mm in diameter, circular, entire, low convex, grayto-white, and translucent to slightly opaque. There usually is no hemolysis of blood agar, but some strains may produce a slight greening of the agar. The optimum temperature for growth is 35–37°C. Growth may occur at 25 or 45°C. The major fatty acids are C15:0 anteiso, C15:0 iso, C17:0 iso 3-OH, and C16:0. Strains of Bacteroides uniformis and Bacteroides thetaiotaomicron are very similar phenotypically, but they differ in their DNA G+C content and are not related by DNA– DNA hybridization analysis. In general, strains of Bacteroides uniformis do not grow as well in 20% bile as strains of Bacteroides thetaiotaomicron. Source: part of human and swine fecal flora; also isolated from various human clinical specimens. DNA G+C content (mol%): 45–48 (HPLC). Type strain: ATCC 8492, CCUG 4942, CIP 103695, DSM 6597, JCM 5828, NCTC 13054. Sequence accession no. (16S rRNA gene): L16486, AB050110. 25. Bacteroides vulgatus Eggerth and Gagnon 1933, 401AL [Pasteurella vulgatus (Eggerth and Gagnon, 1933) Prévot 1938, 292; Bacteroides fragilis subsp. vulgatus (Eggerth and Gagnon 1933) Holdeman and Moore 1970, 35] vul.ga¢tus. L. masc. adj. vulgatus common (referring to the frequent occurrence of the species in fecal flora). Characteristics are as described for the genus and as given in Table 2, with the following additional characteristics. Cells may be pleomorphic with swellings or vacuoles, but less so than strains of Bacteroides fragilis. In broth cultures, cells usually occur singly, occasionally in pairs or short chains. Capsules are detected in some strains. Surface colonies on blood agar are 1–2 mm in diameter, circular, entire, convex, and semi-opaque. Sheep blood is not hemolyzed. Hemin is required or is highly stimulatory for growth. The major fatty acids are C15:0 anteiso, C15:0 iso, C17:0 iso 3-OH, and C16:0. Source: human feces, occasionally isolated from human infections. DNA G+C content (mol%): 40–42 (HPLC). Type strain: ATCC 8482, BCRC (formerly CCRC) 12903, CCUG 4940, CIP 103714, DSM 1447, IFO (now NBRC) 14291, JCM 5826, LMG 7956, LMG 17767, NCTC 11154. Sequence accession no. (16S rRNA gene): AJ867050, AB050111.

Species incertae sedis 1. Bacteroides capillosus (Tissier 1908) Kelly 1957, 433AL [Bacillus capillosus Tissier 1908, 193; Ristella capillosa (Tissier 1908) Prévot 1938, 292] ca.pil.lo¢sus. L. masc. adj. capillosus very hairy. Comparative 16S rRNA gene sequence analysis demonstrates that Bacteroides capillosus is phylogenetically far

removed from the genus Bacteroides and, in fact, has a closer affinity to clostridia in the Firmicutes and is closely related genealogically to the type strain of Clostridium orbiscindens in Clostridium cluster IV. Cells are straight or curved rods, 0.7–1.1 × 1.6–7.0 mm, occurring singly, in pairs, or in short chains after 24 h ­incubation in

39

Genus I. Bacteroides

glucose broth. Vacuoles, swellings, and filaments with tapered ends are observed. Surface colonies are minute to 1 mm, circular, entire, convex, translucent, and smooth. Growth of most strains is enhanced by hemin, rumen fluid, or Tween 80. Strains are generally nonfermentative unless Tween 80 is added to the medium, in which case they may be slightly fermentative. Good growth occurs at 37 and 45°C, slight growth at 30°C, and no growth at 25°C. Other characteristics are given in Table 5. Source: cysts and wounds, human mouth, human infant and adult feces, intestinal tracts of dogs, mice, and termites, and sewage sludge. DNA G+C content (mol%): 60 (HPLC). Type strain: ATCC 29799, CCUG 15402. Sequence accession no. (16S rRNA gene): AY136666.

2. Bacteroides cellulosolvens Murray, Sowden and Colvin 1984, 186VP cell.u.lo.sol¢vens. N.L. n. cellulosum cellulose; L. v. solvere to dissolve; N.L. part. adj. cellulosolvens cellulose dissolving, so named because of its ability to ferment cellulosic substrates. Comparative 16S rRNA gene sequence analysis has demonstrated that Bacteroides cellulosolvens is phylogenetically far removed from the genus Bacteroides and, in fact, has a closer affinity to the clostridia of the Firmicutes and is closely related genealogically to the type strain of Acetivibrio cellulolyticus in Clostridium cluster III (Lin et al., 1994). Cells are straight, rod-shaped, approximately 0.8 × 6 mm, and occur singly. They are nonmotile and no flagella are detected by staining or electron microscopy. Non-­spore-forming. Bright

3. B. coagulans

4. B. galacturonicus

5. B. pectinophilus

6. B. polypragmatus

7. B. splanchnicus

8. B. xylanolyticus

Growth in 20% bile Esculin hydrolyzed Indole produced Nitrate reduced Catalase produced Starch hydrolyzed Gelatin digested Acid produced from:   Arabinose   Cellobiose   Xylan   Sorbitol   Fructose   Glucose   Glycogen   Inositol   Lactose   Maltose   Mannitol   Mannose   Melezitose   Melibiose   Raffinose   Rhamnose   Ribose   Salicin   Sucrose   Trehalose   Xylose Fermentation ­productsc

2. B. cellulosolvens

Characteristic

1. B. capillosus

TABLE 5.  Characteristics of misplaced Bacteroides speciesa,b

− + − − nr − w

− nr − − − − −

nr − + − nr nr +

+ − nr nr − − −

+ − nr nr − nr +

nr + + − − + −

+ + + − nr − d

nr nr nr nr nr nr nr

− − − − − w − − − − − − − − − − − − − − − s, a (l, f, p)

− − − − − − − − − − − − − − − − − − − − − H2, CO2, a, ethanol, ld

− − − − − − − − − − − − − − − − − − − − − a (f, s, l)

− − − nr d − nr nr − − nr − nr nr nr − − nr − nr − A, F (l, ethanol)e

− − − nr − − nr nr − − nr − nr nr nr − − nr − nr − A, F (l)e

+ + nr nr + + + − + + + + + + + + + + − + + H2, CO2, ethanol, a (b)

+ − nr nr d + − − + − − + − d − − − − − − − S, A, P (b, ib, iv, l)

+ + + nr nr + nr nr nr nr nr + nr nr nr nr nr nr nr nr + H2, CO2, ethanol, a

a Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); –, 0–15% positive; w, weak reaction; nr, not reported. Fermentation ­products: A, acetic acid; B, butyric acid; F, formic acid; ib, isobutyric acid; iv, isobutyric acid; l, lactic acid; p, propionic acid; S, succinic acid. Upper-case letters indicate >1 meq/100 ml of broth; lower-case letters, 85% positive; –, 0–15% positive.

a

Genus IV. Acetothermus Dietrich, Weiss and Winter 1988a, 328VP (Effective publication: Dietrich, Weiss and Winter 1988c, 179.) The Editorial Board A.ce¢to.ther¢mus. L.n. acetum vinegar; Gr. adj. thermos hot; N.L. masc. n. Acetothermus a thermophilic microorganism producing acetic acid.

Straight rods 0.5 × 5–8 mm, occurring singly or in pairs. Gramstain-negative. Nonsporeforming. Nonmotile. Chemoorganotrophic. Anaerobic, having a strictly fermentative type of metabolism. Optimum temperature, 58°C; no growth occurs below 50°C or above 60°C. Optimum pH, 7-8. Yeast extract, peptone, and vitamin B12 are required. Only glucose and fructose are used as energy sources. Glucose is fermented by glycolysis to acetate, CO2, and H2 in a molar ratio of 1:1:2. Increasing H2 partial pressures inhibit growth, but elevated acetate concentrations do not affect growth. Isolated from thermophilically fermenting sewage sludge. DNA G+ C content (mol%): ~62. Type species: Acetothermus paucivorans Dietrich, Weiss and Winter 1988a, 328VP (Effective publication: Dietrich, Weiss and Winter 1988c, 179.).

Further descriptive information In the absence of vitamin B12 the organisms form long filaments 0.5 mm in diameter and up to 50 mm long. Optimal growth occurs in a medium containing 0.2% yeast extract and 0.2% peptone. Growth is directly proportional to the concentration of vitamin B12 up to 1 mg/l. NaCl is not required; >1 g/l is inhibitory. The cell-wall peptidoglycan contains ornithine as the diamino acid, unlike most other Gram-stain-negative bacteria, which usually contain diaminopimelic acid as the diamino acid. The peptidoglycan structure corresponds to the directly crosslinked A1b type described by Schleifer and Kandler (1972).

Glucose and fructose are used as energy sources. The following carbon sources are not used: arabinose, mannose, ribose, xylose, galactose, mannitol, sorbitol, sucrose, maltose, lactose, melibiose, raffinose, starch, cellulose, glycerol, Casamino acids, meat extract, glycogen, pyruvate, glycine, alanine, glutamate, formate, and methanol.

Enrichment and isolation procedures Enrichment and isolation procedures from sewage sludge digesting at 60°C were done using the medium described for isolation of Acetomicrobium faecale (see footnote in the chapter on Acetomicrobium for formula) modified by the omission of acetate.

Taxonomic comments No significant DNA–DNA hybridization occurs between Acetothermus and Acetomicrobium (Dietrich et  al., 1988c). The taxonomic placement of Acetothermus as a member of the family Bacteroidaceae is not certain because of the lack of rRNA gene sequence data.

Differentiation of the genus Acetothermus from other genera Characteristics differentiating Acetothermus from Acetofilamentum, Acetivibrio, and Acetomicrobium are listed in Table 6 in the chapter on Acetofilamentum.

List of species of the genus Acetothermus 1. Acetothermus paucivorans Dietrich, Weiss and Winter 1988a, 328VP (Effective publication: Dietrich, Weiss and ­Winter 1988c, 179.) pau.ci.vo¢rans. L. masc. adj. paucus few, little; L. v. vorare to eat, swallow; L. part. adj. vorans eating, swallowing; N.L. part. adj. paucivorans utilizing only a very restricted number of the supplied substrates.

The characteristics are as described for the genus. Source: thermophilically fermenting sewage sludge. DNA G+C content (mol%): 62 (Tm). Type strain: TN, DSM 20768. (Note: this strain is no longer listed in the DSMZ catalog.) Sequence accession no.: not reported.

Genus V. Anaerorhabdus

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Genus V. Anaerorhabdus Shah and Collins 1986, 573VP (Effective publication: Shah and Collins 1986, 86.) Haroun N. Shah An.ae.ro.rhab¢dus. Gr. pref. an not; Gr. n. aer air: anaero (not living) in air; Gr. fem. n. rhabdos rod; N.L. fem. n. Anaerohabdus rod-shaped bacterium not living in air.

Short rods. Nonspore-forming. Nonmotile. Gram-negative. Anaerobic. Nonsaccharolytic, although a few carbohydrates may be fermented weakly. Acetic and lactic acids are the major metabolic end products in peptone-yeast extract-glucose broth (PYG). Glucose-6-phosphate dehydrogenase is produced, but 6-phosphogluconate dehydrogenase, malate dehydrogenase, and glutamate dehydrogenase are absent. Sphingolipids and menaquinones are not produced. The nonhydroxylated longchain fatty acids are primarily of the straight-chain saturated and monounsaturated types. Methyl branched fatty acids are either absent or present in small amounts. Isolated from infected appendix, lung abscesses, and abdominal abscesses. Infrequently isolated from human and pig feces. DNA G+C content (mol%): 34. Type species: Anaerorhabdus furcosa (Veillon and Zuber 1898) Shah and Collins 1986, 573VP [Effective publication: Anaerorhabdus furcosus (sic) (Veillon and Zuber 1898) Shah and Collins 1986, 86.] [Bacillus furcosus Veillon and Zuber 1898, 541; Fusiformis furcosus (Veillon and Zuber 1898) Topley and Wilson 1929, 302; Bacteroides furcosus (Veillon and Zuber 1898) Hauduroy, Ehringer, Urbain, Guillot and Magrou 1937, 61AL; Ristella furcosa (Veillon and Zuber 1898) Prévot 1938, 291].

Enrichment and isolation procedures Anaerorhabdus isolates grow on blood agar producing small colonies (0.5 mm in diameter) but is improved by culturing on fastidious anaerobic agar. Growth is improved by the addition of rumen fluid.

Differentiation of the genus Anaerorhabdus from other genera Anaerorhabdus differs from Bacteroides fragilis and related species in having a significantly lower DNA base composition, in being nonfermentative or weakly fermentative, and in lacking 6-phosphogluconate dehydrogenase, malate dehydrogenase, and glutamate dehydrogenase. Anaerorhabdus primarily synthesizes fatty acids of the straight-chain saturated and monounsaturated type, and methyl-branched acids are present in only small amounts. In contrast, the fatty acids of Bacteroides fragilis and related species are mainly of the straight-chain saturated, anteiso-, and iso-methyl branched chain types, with monounsaturated acids either absent or present in only trace amounts. Moreover, Anaerorhabdus lacks menaquinones and sphingolipids, whereas Bacteroides fragilis and related species synthesize menaquinones and sphingolipids.

List of species of the genus Anaerorhabdus 1. Anaerorhabdus furcosa (Veillon and Zuber 1898) Shah and Collins 1986, 573 [Effective publication: Anaerorhabdus furcosus (sic) (Veillon and Zuber 1898) Shah and Collins 1986, 86.] [Bacillus furcosus Veillon and Zuber 1898, 541; Fusiformis furcosus (Veillon and Zuber 1898) Topley and Wilson 1929, 302; Bacteroides furcosus (Veillon and Zuber 1898) Hauduroy, Ehringer, Urbain, Guillot and Magrou 1937, 61AL; Ristella furcosa (Veillon and Zuber 1898) Prévot 1938, 291]. fur.co¢sa. L. fem. adj. furcosa forked (pertaining to cell shape). (Note: the original epithet furcosus was corrected by Euzéby and Boemare, 2000.) The description is the one given for the genus, plus the following additional features. Pleomorphic rods, 0.3–1.5 × 1–3 mm, occurring singly, in pairs, or short chains. Some cells appear to be forked or Y-shaped. Colonies on blood agar are 0.5 mm in diameter, circular, entire, low convex, translucent, colorless to gray, smooth, and shiny. Glucose

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broth cultures are turbid with smooth sediment; growth is stimulated by rumen fluid. Optimum growth temperature, 30–37°C; slight growth occurs at 25°C but none at 45°C. Acetic and lactic acids are the major metabolic end products in PYG broth; trace amounts of formic and succinic acids may also be produced. Most strains hydrolyze esculin. Phosphate positive. Indole, acetylmethylcarbinol, urease, and H2S are not produced. Gelatin liquefaction is usually negative although a few strains may be weakly positive. Starch and hippurate are not hydrolyzed. Nitrate is not reduced. The cell walls do not contains diaminopimelic acid, heptose, or ketodeoxyoctonic acid (KDO). Non-hydroxylated long chain fatty acids are primarily of the straight-chain saturated and mono-unsaturated types with hexadecanoic, octadecanoic, and octadecenoic acids predominating. DNA G+C content (mol%): 34 (Tm). Type strain: ATCC 25662, VPI 3253. Sequence accession no. (16S rRNA gene): none available.

Bakir, M.A., M. Kitahara, M. Sakamoto, M. Matsumoto and Y. Benno. 2006b. Bacteroides finegoldii sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol. 56: 931–935. Bakir, M.A., M. Sakamoto, M. Kitahara, M. Matsumoto and Y. Benno. 2006c. Bacteroides dorei sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol. 56: 1639–1643. Benno, Y., J. Watabe and T. Mitsuoka. 1983a. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 12. Int. J. Syst. Bacteriol. 33: 896–897.

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FAMILY i. Bacteroidaceae

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Genus V. Anaerorhabdus homology groupings for Bacteroides fragilis and other saccharolytic Bacteroides species. Int. J. Syst. Bacteriol. 28: 257–268. Johnson, J.L. and L.V. Holdeman. 1983. Bacteroides intermedius comb. nov. and descriptions of Bacteroides corporis sp. nov. and Bacteroides levii sp. nov. Int. J. Syst. Bacteriol. 33: 15–25. Johnson, J.L., W.E.C. Moore and L.V.H. Moore. 1986. Bacteroides caccae sp. nov., Bacteroides-merdae sp. nov., and Bacteroides stercoris sp. nov. isolated from human feces. Int. J. Syst. Bacteriol. 36: 499–501. Jousimies-Somer, H.R., P. Summanen, D.M. Citron, E.J. Baron, H.M. Wexler and S.M. Finegold. 2002. Wadsworth-KTL anaerobic bacteriology manual. Star Publishing Company, Belmont, CA. Kaneuchi, C. and T. Mitsuoka. 1978. Bacteroides microfusus, a new species from intestines of calves, chickens, and Japanese quails. Int. J. Syst. Bacteriol. 28: 478–481. Kelly, C.D. 1957. Genus I. Bacteroides Castellani and Chalmers 1919. In Bergey’s Manual of Determinative Bacteriology, 7th edn (edited by Breed, Murray and Smith). Williams & Wilkins, Baltimore, pp. 424–436. Kitahara, M., M. Sakamoto, M. Ike, S. Sakata and Y. Benno. 2005. Bacteroides plebeius sp. nov. and Bacteroides coprocola sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol. 55: 2143–2147. Kornman, K.S. and S.C. Holt. 1981. Physiological and ultrastructural characterization of a new Bacteroides species (Bacteroides capillus) isolated from severe localized periodontitis. J. Periodont. Res. 16: 542–555. Leadbetter, E.R., S.C. Holt and S.S. Socransky. 1979. Capnocytophaga: new genus of Gram-negative gliding bacteria. 1. General characteristics, taxonomic considerations and significance. Arch. Microbiol. 122: 9–16. Lin, C., J.W. Urbance and D.A. Stahl. 1994. Acetivibrio cellulolyticus and Bacteroides cellulosolvens are members of the greater clostridial assemblage. FEMS Microbiol. Lett. 124: 151–155. Loesche, W.J., S.S. Socransky and R.J. Gibbons. 1964. Bacteroides oralis, proposed new species isolated from the oral cavity of man. J. Bacteriol. 88: 1329–1337. Love, D.N., J.L. Johnson, R.F. Jones, M. Bailey and A. Calverley. 1986. Bacteroides tectum sp. nov. and characteristics of other nonpigmented Bacteroides isolates from soft-tissue infections from cats and dogs. Int. J. Syst. Bacteriol. 36: 123–128. Love, D.N., J.L. Johnson, R.F. Jones and A. Calverley. 1987. Bacteroides salivosus sp. nov., an asaccharolytic, black-pigmented species from cats. Int. J. Syst. Bacteriol. 37: 307–309. Love, D.N. 1995. Porphyromonas macacae comb. nov., a consequence of Bacteroides macacae being a senior synonym of Porphyromonas salivosa. Int. J. Syst. Bacteriol. 45: 90–92. Mitsuoka, T., T. Sega and S. Yamamoto. 1965. [Improved methodology of qualitative and quantitative analysis of the intestinal flora of man and animals]. Zentralbl. Bakteriol. [Orig.] 195: 455–469. Mitsuoka, T., A. Terada, K. Watanabe and K. Uchida. 1974. Bacteroides multiacidus, a new species from feces of humans and pigs. Int. J. Syst. Bacteriol. 24: 35–41. Miyamoto, Y. and K. Itoh. 2000. Bacteroides acidifaciens sp. nov., isolated from the caecum of mice. Int. J. Syst. Evol. Microbiol. 50: 145–148. Moncrief, J.S., R. Obiso, Jr., L.A. Barroso, J.J. Kling, R.L. Wright, R.L. Van Tassell, D.M. Lyerly and T.D. Wilkins. 1995. The enterotoxin of Bacteroides fragilis is a metalloprotease. Infect. Immun. 63: 175–181. Montgomery, L., B. Flesher and D. Stahl. 1988. Transfer of Bacteroides succinogenes (Hungate) to Fibrobacter gen. nov. as Fibrobacter succinogenes comb. nov. and description of Fibrobacter intestinalis sp. nov. Int. J. Syst. Bacteriol. 38: 430–435. Moore, L.V.H., J.L. Johnson and W.E.C. Moore. 1994. Descriptions of Prevotella tannerae sp. nov. and Prevotella enoeca sp. nov. from the human gingival crevice and emendation of the description of Prevotella zoogleoformans. Int. J. Syst. Bacteriol. 44: 599–602. Moore, L.V.H. and W.E.C. Moore. 1994. Oribaculum catoniae gen. nov., sp. nov., Catonella morbi gen. nov., sp. nov., Hallella seregens gen. nov., sp. nov., Johnsonella ignava gen. nov., sp. nov., and Dialister pneumosintes gen. nov., comb. nov., nom. rev., anaerobic Gram-negative bacilli from the human gingival crevice. Int. J. Syst. Bacteriol. 44: 187–192.

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Morotomi, M., F. Nagai and H. Sakon. 2007. Genus Megamonas should be placed in the lineage of Firmicutes; Clostridia; Clostridiales; ‘Acidaminococcaceae’; Megamonas. Int. J. Syst. Evol. Microbiol. 57: 1673–1674. Murray, W.D., L.C. Sowden and J.R. Colvin. 1984. Bacteroides cellulosolvens sp. nov., a cellulolytic species from sewage sludge. Int. J. Syst. Bacteriol. 34: 185–187. Oh, H. and C. Edlund. 2003. Mechanism of quinolone resistance in anaerobic bacteria. Clin. Microbiol. Infect. 9: 512–517. Okuda, K., T. Kato, J. Shiozu, I. Takazoe and T. Nakamura. 1985. Bacteroides heparinolyticus sp. nov. isolated from humans with periodontitis. Int. J. Syst. Bacteriol. 35: 438–442. Olitsky, P.K. and F.L. Gates. 1921. Experimental studies of the naso-pharyngeal secretions from influenza patients. J. Exp. Med. 33: 713–729. Oliver, W.W. and W.B. Wherry. 1921. Notes on some bacterial parasites of the human mucous membranesq. J. Infect. Dis. 28: 341–344. Paster, B.J., F.E. Dewhirst, I. Olsen and G.J. Fraser. 1994. Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria. J. Bacteriol. 176: 725–732. Patel, G.B. and C. Breuil. 1981. Isolation and characterization of Bacteroides polypragmatus sp. nov., an isolate which produces carbon dioxide, hydrogen and acetic acid during growth on various organic substrates. In Advances in Biotechnology (edited by Moo-Young and Robinson). Pergamon Press, Toronto, pp. 291–296. Patel, G.B. and C. Breuil. 1982. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 8. Int. J. Syst. Bacteriol. 32: 266–268. Patrick, S. 2002. Bacteroides. In Molecular Medical Microbiology (edited by Sussman). Academic Press, London, pp. 1921–1948. Podglajen, I., J. Breuil, I. Casin and E. Collatz. 1995. Genotypic identification of two groups within the species Bacteroides fragilis by ribotyping and by analysis of PCR-generated fragment patterns and insertion sequence content. J. Bacteriol. 177: 5270–5275. Prévot, A.R. 1938. Etudes de systematique bacterienne. III. Invalidite du genre Bacteroides Castellani et Chalmers demembrement et reclassification. Ann. Inst. Pasteur 20: 285–307. Prévot, A.R., P. Ardieux, L. Joubert and F. De Cadore. 1956. Recherches sur Fusiformis nucleatus (Knorr) et son pouvoir pathogène pour l’homme et les animaux. Ann. Inst. Pasteur (Paris) 91: 787–798. Pribram, E. 1933. Klassification der Schizomyceten. F. Deuticke, Leipzig, pp. 1–143. Rautio, M., E. Eerola, M.L. Vaisanen-Tunkelrott, D. Molitoris, P. Lawson, M.D. Collins and H. Jousimies-Somer. 2003. Reclassification of Bacteroides putredinis (Weinberg et al. 1937) in a new genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp. nov., from human sources. Syst. Appl. Microbiol. 26: 182–188. Roy, T.E. and C.D. Kelly. 1939. Genus VIII. Bacteroides Castellani and Charmers. In Bergey’s Manual of Determinative Bacteriology, 5th edn (edited by Bergey, Breed, Murray and Hitchens). Williams & Wilkins, Baltimore, pp. 569–570. Ruimy, R., I. Podglajen, J. Breuil, R. Christen and E. Collatz. 1996. A recent fixation of cfiA genes in a monophyletic cluster of Bacteroides fragilis is correlated with the presence of multiple insertion elements. J. Bacteriol. 178: 1914–1918. Sakamoto, M., M. Suzuki, M. Umeda, I. Ishikawa and Y. Benno. 2002. Reclassification of Bacteroides forsythus (Tanner et al. 1986) as Tannerella forsythensis corrig., gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 52: 841–849. Sakamoto, M. and Y. Benno. 2006. Reclassification of Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae as Parabacteroides distasonis gen. nov., comb. nov., Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov. Int. J. Syst. Evol. Microbiol. 56: 1599–1605. Sakamoto, M., M. Kitahara and Y. Benno. 2007. Parabacteroides johnsonii sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol. 57: 293–296. Schleifer, K.H. and O. Kandler. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36: 407–477.

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FAMILY i. Bacteroidaceae

Scholten-Koerselman, I., F. Houwaard, P. Janssen and A.J.B. Zehnder. 1986. Bacteroides xylanolyticus sp. nov., a xylanolytic bacterium from methane producing cattle manure. Antonie van Leeuwenhoek 52: 543–554. Scholten-Koerselman, I., F. Houwaard, P. Janssen and A.J.B. Zehnder. 1988. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 24. Int. J. Syst. Bacteriol. 38: 136–137. Sears, C.L., L.L. Myers, A. Lazenby and R.L. Van Tassell. 1995. Enterotoxigenic Bacteroides fragilis. Clin. Infect. Dis. 20 Suppl 2: S142–S148. Sebald, M. 1962. Étude sur les bactéries anaérobies gram-négatives asporulées. Thèses de l’Université Paris, Imprimerie Barnéoud S.A., Laval, France. Shah, H.N. and M. Collins. 1981. Bacteroides buccalis, sp. nov., Bacteroides denticola, sp. nov., and Bacteroides pentosaceus, sp. nov., new species of the genus Bacteroides from the oral cavity. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I Abt. Orig. C. 2: 235–241. Shah, H.N. and M.D. Collins. 1982a. Reclassification of Bacteroides multiacidus (Mitsuoka, Terada, Watanabe and Uchida) in a new genus Mitsuokella, as Mitsuokella multiacidus comb. nov. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I Abt. Orig. C. 3: 491–494. Shah, H.N. and M.D. Collins. 1982b. Reclassification of Bacteroides hypermegas (Harrison and Hansen) in a new genus Megamonas, as Megamonas hypermegas comb. nov. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. C3: 394–398. Shah, H.N. and M.D. Collins. 1983. Genus Bacteroides. A chemotaxonomical perspective. J. Appl. Bacteriol. 55: 403–416. Shah, H.N., M.D. Collins, J. Watabe and T. Mitsuoka. 1985. Bacteroides oulorum sp. nov., a nonpigmented saccharolytic species from the oral cavity. Int. J. Syst. Bacteriol. 35: 193–197. Shah, H.N. and M.D. Collins. 1986. Reclassification of Bacteroides furcosus Veillon and Zuber (Hauduroy, Ehringer, Urbain, Guillot and Magrou) in a new genus Anaerorhabdus, as Anaerorhabdus furcosus comb. nov. Syst. Appl. Microbiol. 8: 86–88. Shah, H.N. and M.D. Collins. 1986. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 22. Int. J. Syst. Bacteriol. 36: 573–576. Shah, H.N. and M.D. Collins. 1988. Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int. J. Syst. Bacteriol. 38: 128–131. Shah, H.N. and M.D. Collins. 1989. Proposal to restrict the genus Bacteroides (Castellani and Chalmers) to Bacteroides fragilis and closely related species. Int. J. Syst. Bacteriol. 39: 85–87. Shah, H.N. and D.M. Collins. 1990. Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int. J. Syst. Bacteriol. 40: 205–208. Shah, H.N., M.D. Collins, I. Olsen, B.J. Paster and F.E. Dewhirst. 1995. Reclassification of Bacteroides levii (Holdeman, Cato, and Moore) in the genus Porphyromonas, as Porphyromonas levii comb. nov. Int. J. Syst. Bacteriol. 45: 586–588. Slots, J. and R.J. Genco. 1980. Bacteroides melaninogenicus subsp. macacae: new subspecies from monkey periodontopathic indigenous microflora. Int. J. Syst. Bacteriol. 30: 82–85. Snydman, D.R., N.V. Jacobus, L.A. McDermott, R. Ruthazer, E.J. Goldstein, S.M. Finegold, L.J. Harrell, D.W. Hecht, S.G. Jenkins, C. Pierson, R. Venezia, J. Rihs and S.L. Gorbach. 2002. National survey on the susceptibility of Bacteroides fragilis Group: report and analysis of trends for 1997–2000. Clin. Infect. Dis. 35: S126–134. Song, Y., C. Liu, J. Lee, M. Bolanos, M.L. Vaisanen and S.M. Finegold. 2005a. “Bacteroides goldsteinii sp. nov.” isolated from clinical specimens of human intestinal origin. J. Clin. Microbiol. 43: 4522–4527. Song, Y., C. Liu, J. Lee, M. Bolaños, M.-L. Vaisanen and S.M. Finegold. 2006. In List of new names and new combinations previously effectively, but not validly, published. List no. 108. Int. J. Syst. Evol. Microbiol. 56: 499–500. Song, Y.L., C.X. Liu, M. McTeague and S.M. Finegold. 2004. “Bacteroides nordii” sp. nov. and “Bacteroides salyersae” sp. nov. isolated from clinical specimens of human intestinal origin. J. Clin. Microbiol. 42: 5565–5570.

Song, Y.L., C.X. Liu, M. McTeague and S.M. Finegold. 2005b. In Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 103. Int. J. Syst. Evol. Microbiol. 55: 983–985. Soutschek, E., J. Winter, F. Schindler and O. Kandler. 1984. Acetomicrobium flavidum, gen. nov., sp. nov., a thermophilic, anaerobic bacterium from sewage-sludge, forming acetate, CO2 and H2 from glucose. Syst. Appl. Microbiol. 5: 377–390. Soutschek, E., J. Winter, F. Schindler and O. Kandler. 1985. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no.17. Int. J. Syst. Bacteriol. 35: 223–225. Stackebrandt, E. and H. Hippe. 1986. Transfer of Bacteroides amylophilus to a new genus Ruminobacter gen. nov., nom. rev. as Ruminobacter amylophilus comb. nov. Syst. Appl. Microbiol. 8: 204–207. Tanner, A.C.R., S. Badger, C.H. Lai, M.A. Listgarten, R.A. Visconti and S.S. Socransky. 1981. Wolinella gen-nov, Wolinella succinogenes (Vibrio succinogenes Wolin et  al.) comb. nov., and description of Bacteroides gracilis sp. nov., Wolinella recta sp. nov., Campylobacter concisus sp. nov., and Eikenella corrodens from humans with periodontal disease. Int. J. Syst. Bacteriol. 31: 432–445. Tanner, A.C.R., M.A. Listgarten, J.L. Ebersole and M.N. Strezempko. 1986. Bacteroides forsythus sp. nov., a slow-growing, fusiform Bacteroides sp. from the human oral cavity. Int. J. Syst. Bacteriol. 36: 213–221. Tissier, H. 1908. Recherches sur la flore intestinale normale des enfants agés d’un an à cinq ans. Ann. Inst. Pasteur (Paris) 22: 189–208. Topley, W.W.C. and G.S. Wilson. 1929. The Principles of Bacteriology and Immunity, vol. 1. Edward Arnold, London. Van Steenbergen, T.J.M., A.J. Van Winkelhoff, D. Mayrand, D. Grenier and J. De Graaff. 1984. Bacteroides endodontalis sp. nov., an asaccharolytic black-pigmented bacteriodes species from infected dental root canals. Int. J. Syst. Bacteriol. 34: 118–120. Vandamme, P., M.I. Daneshvar, F.E. Dewhirst, B.J. Paster, K. Kersters, H. Goossens and C.W. Moss. 1995. Chemotaxonomic analyses of Bacteroides gracilis and Bacteroides ureolyticus and reclassification of B. gracilis as Campylobacter gracilis comb. nov. Int. J. Syst. Bacteriol. 45: 145–152. Veillon, A. and A. Zuber. 1898. Recherches sur quelques microbes strictement anaérobies et leur rôle en pathologie. Arch. Med. Exp. 10: 517–545. Watabe, J., Y. Benno and T. Mitsuoka. 1983. Taxonomic study of Bacteroides oralis and related organisms and proposal of Bacteroides veroralis sp. nov. Int. J. Syst. Bacteriol. 33: 57–64. Weinberg, M., R. Nativelle and A.R. Prévot. 1937. Les microbes anaérobies. Masson et Cie, Paris. Werner, H., G. Rintelen and H. Kunstek-Santos. 1975. A new butyric acid-producing Bacteroides species: B. splanchnicus n. sp. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I 231: 133–144. Wexler, H.M. and S.M. Finegold. 1998. Current susceptibility patterns of anaerobic bacteria. Yonsei Med. J. 39: 495–501. Whitehead, T.R., M.A. Cotta, M.D. Collins, E. Falsen and P.A. Lawson. 2005. Bacteroides coprosuis sp. nov., isolated from swine-manure storage pits. Int. J. Syst. Evol. Microbiol. 55: 2515–2518. Winter, J., E. Braun and H.P. Zabel. 1987. Acetomicrobium faecalis spec. nov., a strictly anaerobic bacterium from sewage sludge, producing ethanol from pentoses. Syst. Appl. Microbiol. 9: 71–76. Winter, J., E. Braun and H.P. Zabel. 1988. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 24. Int. J. Syst. Bacteriol. 38: 136–137. Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev. 51: 221–271. Wolin, E.A., M.G. Wolin and R.S. Wolfe. 1963. Formation of methane by bacterial extracts. J. Biol. Chem. 238: 2882–2886. Wu, C.C., J.L. Johnson, W.E.C. Moore and L.V.H. Moore. 1992. Emended descriptions of Prevotella denticola, Prevotella loescheii, Prevotella veroralis, and Prevotella melaninogenica. Int. J. Syst. Bacteriol. 42: 536–541. Xu, J., M.K. Bjursell, J. Himrod, S. Deng, L.K. Carmichael, H.C. Chiang, L.V. Hooper and J.I. Gordon. 2003. A genomic view of the human–Bacteroides thetaiotaomicron symbiosis. Science 299: 2074–2076.

49

Genus I. Marinilabilia

Family II. Marinilabiliaceae fam. nov. Wolfgang Ludwig, Jean Euzéby and William B. Whitman Ma.ri.ni.la.bi.li.a.ce′ae. N.L. fem. n. Marinilabilia type genus of the family; suff. -aceae ending to denote family; N.L. fem. pl. n. Marinilabiliaceae the Marinilabilia family. The family Marinilabiliaceae was circumscribed for this volume on the basis of phylogenetic analysis of 16S rRNA gene sequences and includes the type genus Marinilabilia, Alkaliflexus, and Anaerophaga. All genera are composed of slender, flexible rods. Except for Anaerophaga, which does not grow on

solid surfaces in culture, cells are motile by gliding. Cells stain Gram-negative. Saccharolytic and require NaCl for growth. Some characteristics that differentiate the three genera are given in Table 8. Type genus: Marinilabilia Nakagawa and Yamasato 1996, 600VP.

TABLE 8.  Some characteristics that differentiate the genera of the family Marinilabiliaceae a

Characteristic Motile by gliding Can grow aerobically Major products of glucose fermentation Growth above 50°C Isolated from DNA G+C content (mol%)

Marinilabilia

Alkaliflexus

Anaerophaga

+ + Acetate, succinate, propionate, lactate, formate and H2 − Marine mud containing decayed algae 37–41

+ − Propionate, acetate, succinate and formate − Mud from an alkaline soda lake

nt − Propionate, acetate, and succinate

44

+ Oil contaminated sediment in an oil separation tank 41

nt, Not tested.

a

Genus I. Marinilabilia Nakagawa and Yamasato 1996, 600VP Makoto Suzuki Ma.ri.ni.la.bil′i.a. L. adj. marinus marine, pertaining to the sea; L. adj. labilis gliding; N.L. fem. n. Marinilabilia marine gliding organisms.

Short to very long flexible rods, 0.3–0.5 × 2–50 mm. Resting stage is absent. Motile by gliding. Gram-negative. Colonies are pink to salmon in color. Chemo-organotrophic. Facultatively anaerobic, having both respiratory and fermentative types of metabolism. Catalase-positive. Decompose several kinds of biomacromolecules. Marine organisms requiring elevated salt concentrations. Optimum temperature: 28–37°C. Optimum pH: around 7. The major polyamine is spermidine. The major respiratory quinone is MK-7. DNA G+C content (mol%): 41 (HPLC). Type species: Marinilabilia salmonicolor (Veldkamp 1961) Nakagawa and Yamasato 1996, 600VP emend. Suzuki, Nakagawa, Harayama and Yamamoto 1999, 1555 (Cytophaga salmonicolor Veldkamp 1961, 339).

Further descriptive information Phylogenetic analysis based on 16S rRNA gene sequences indicates that the genus Marinilabilia belongs to the family “Marinilabiaceae”. The phylogenetic relationships of the genus Marinilabilia and related organisms are shown in Figure 15. The nearest neighbor is the genus Anaerophaga and the similarities between the species of Marinilabilia and Anaerophaga are 90.3–92.4%. Two misclassified facultatively anaerobic Cytophaga species, Cytophaga fermantans and Cytophaga xylanolytica, are more distantly related to members of the genus Marinilabilia. There is 86.8–89.6% similarity between these two organisms and members of the genus Marinilabilia. Cells of Marinilabilia salmonicolor are slender rods with rounded or slightly tapered ends. They are extremely flexible and motile

by gliding. The cells usually vary from 2 to 30 mm in length, but occasionally, very long elements of up to 50 mm long can be seen. These long cells occur even in young cultures and often show flexing and gliding movements. The mean length of cells grown aerobically on agar plates is usually greater than that of cells from stab or anaerobic liquid cultures. In the stationary phase, spherical cells are always found. They vary in diameter from 1.0 to 3.5 mm. Spermidine is the dominant cellular polyamine when cells are cultivated in marine broth (Hosoya and Hamana, 2004). Strains of the genus Marinilabilia can grow well anaerobically in glucose-mineral medium supplemented with a vitamin mixture (Veldkamp, 1961). Under anaerobic conditions, growth is poor when bicarbonate is omitted from the medium. Under aerobic ­conditions, Marinilabilia strains can grow well when the medium contains 0.1% (w/v) yeast extract, corn steep liquor, or nutrient broth. Products of fermentation are acetic acid, succinic acid, propionic acid, lactic acid, formic acid, CO2, H2, and trace amounts of ethanol (Veldkamp, 1961). Marinilabilia strains inhabit coastal marine sediments. They have been isolated from marine mud containing decayed algae. One biovar can actively degrade agar.

Enrichment and isolation procedures Organisms of this group have been recognized as incidentally occurring organisms in enrichment cultures of green sulfur bacteria (Veldkamp, 1961). They were originally enriched by using anaerobic cultures in which agar was the carbon and

50

Family II. Marinilabiliaceae

energy source. The anaerobic enrichment cultures were incubated in the dark at 30°C for 3–5 d. Anaerobic plate cultures were used for the isolation of these organisms. The medium used for these cultures contained (in g/l): NaCl, 30; KH2PO4, 1; NH4Cl, 1; MgCl2·6H2O, 0.5; CaCl2, 0.04; NaHCO3, 5; Na2S·9H2O, 0.1; ferric citrate, 0.03; agar, 5; yeast extract, 0.3; pH 7. The medium was supplemented with a trace element mixture (Veldkamp, 1961).

Maintenance procedures Cultures are maintained as paraffin-covered stab cultures at 4°C; transfers are made monthly. Cultures can be kept in broth containing 10% glycerol at −80°C or in the gas phase of liquid nitrogen. They can be preserved by a liquid-drying method using 75% bovine serum in marine broth as a protective reagent.

Differentiation of the genus Marinilabilia from other genera Marinilabilia can be differentiated from related organisms by its relation to oxygen, salt requirement, some physiological characters, and cell mass color. Differential features are given in Table 9.

39 59

100

100

Marinilabilia salmonicolor Anaerophaga thermohalophila Cytophaga fermentans Alkaliflexus imshenetskii Rikenella microfusus Alistipes finegoldii Bacteroides fragilis

0.02

FIGURE 15.  16S rRNA gene sequence-based phylogenetic tree of the genus Marinilabilia and related organisms. The tree was constructed by the neighbor-joining method. The numbers at nodes show the percentage bootstrap value of 500 replicates. Bar = evolutionary distance of 0.02 (Knuc).

Marinilabilia can be differentiated from Anaerophaga, Alkaliflexus, Alistipes, and Rikenella by its relation to oxygen. It can also be differentiated from Cytophaga fermentans by its cell mass color and from Cytophaga xylanolytica by its salt requirement.

Taxonomic comments There are two known isolates of the genus Marinilabilia. Strain NBRC 15948T (=ATCC 19041T), the type strain of Marinilabilia salmonicolor, was isolated from marine mud off the coast of California. The second strain, NBRC 14957 (=ATCC 19043), which was isolated from the same environment, has the same characteristics as the type strain and, in addition, is able to degrade agar. These two strains were originally reported as Cytophaga salmonicolor and “Cytophaga salmonicolor var. agarovorans” by Veldkamp (1961). Reichenbach (1989) described Cytophaga agarovorans to accommodate “Cytophaga salmonicolor var. agarovorans”, based on its remarkable agar-degrading ability and higher DNA G+C content. Later, Nakagawa and Yamasato (1996) included these two organisms in the genus Marinilabilia as Marinilabilia salmonicolor and Marinilabilia agarovorans. Phylogenetic analysis of these two organisms using 16S rRNA gene and gyrB sequences indicated their close relatedness (Nakagawa and Yamasato, 1996; Suzuki et al., 1999). In particular, the gyrB sequences of strains NBRC 15948T and NBRC 14957 are identical, as determined for a 1.2 kb region. DNA–DNA hybridization also confirmed their close relatedness: over 70% DNA relatedness was found between the two strains (Suzuki et  al., 1999). Reichenbach (1989) stated that there was a small percentage difference in the G+C content of the DNA between Marinilabilia agarovorans and Marinilabilia salmonicolor, namely, 41 vs. 37 mol%, respectively (buoyant density method). A second determination of the G+C content of Marinilabilia strains was performed by the HPLC method (Suzuki et al., 1999) and indicated that the G+C content was almost the same for the two strains; by this method,

0.3 × 4–8 − Orange to red

0.3–0.4 × 4–10 + Pink

0.5–0.7 × 2–10 + Yellow

0.4 × 3–24 + Orange to salmon

+

−

−

+

−

−

−

− −

+ −

+ −

− −

− +

+ −

+ −

+ + 37–41

− + 42

+ + 44

+ + 39

w − 46

trace nd 55–58

nd − 60–61

Symbols: +, >85% positive; −, 0–15% positive; w, weak reaction; nd, not determined.

a

Rikenella

Cytophaga xylanolytica

0.3–0.5 × 2–20 + Pink to salmon

Alistipes

Cytophaga fermentans

Relation to oxygen  Facultative   anaerobe   Strict anaerobe  Aerotolerant   anaerobe Catalase Salt requirement DNA G+C content (mol%)

Alkaliflexus

Cell size, mm Gliding motility Cell mass color

Anaerophaga

Characteristic

Marinilabilia

TABLE 9.  Differential characteristics of Marinilabilia and related organismsa

0.3–0.5 × 0.9–3.0 0.2–0.3 × 0.3–1.5 − − Gray, brown to black Gray

Genus II. Anaerophaga

the DNA G+C contents of strains NBRC 14957 (Marinilabilia agarovorans) and NBRC 15948T (Marinilabilia salmonicolor) were 41.2 and 41.5 mol%, respectively. All of the above information indicates that these two strains belong to a single species. Since Marinilabilia salmonicolor was first described as Cytophaga salmon-

51

icolor, Marinilabilia salmonicolor is a senior subjective synonym of Marinilabilia agarovorans. However, the agar-degrading ability of strain NBRC 14957 is a prominent biochemical characteristic; therefore, this strain is considered as Marinilabilia salmonicolor biovar Agarovorans.

List of species of the genus Marinilabilia 1. Marinilabilia salmonicolor  (Veldkamp 1961) Nakagawa and Yamasato 1996, 600VP emend. Suzuki, Nakagawa, Harayama and Yamamoto 1999, 1555 (Cytophaga salmonicolor Veldkamp 1961, 339) sal.mo.ni¢co.lor. L. masc. n. salmo -onis salmon; L. masc. n. color color; N.L. adj. salmonicolor intended to mean salmoncolored. Slender flexible cylindrical rods with rounded or slightly tapering end, 0.3–0.5 × 2–50 µm, usually around 10–20 mm in length. Resting stages are not observed. Motile by gliding. Gram-negative. The color of the cell mass is salmon to pink. Chemo-organotrophic. Facultative anaerobe. Good growth is obtained in a normal aerobic atmosphere. Marine strains require elevated salt concentrations. Peptones, ­Casamino

acids, yeast extract, ammonium, and nitrate are suitable nitrogen sources. Arabinose, xylose, glucose, galactose, mannose, fructose, sucrose, lactose, maltose, cellobiose, trehalose, raffinose, inulin, and starch are fermented. Gelatin is liquefied slowly. Catalase is produced. The optimum temperature is 28–37°C. The optimum pH is around 7. The major respiratory quinone is MK-7. The major polyamine is spermidine. Source: marine mud containing decayed algae. DNA G+C content (mol%): 41.2–41.5 (HPLC). Type strain: ATCC 19041, CIP 104809, DSM 6480, LMG 1346, NBRC 15948. Note: strain NBRC 14957 can degrade agar and is biovar Agarovorans.

Genus II. Anaerophaga Denger, Warthmann, Ludwig and Schink 2002, 177VP Bernhard Schink An.a.e.ro.pha′ga. an Gr. pref. an not; Gr. n. aer aeros air; Gr. v. phagein to devour, to eat up; N.L. fem. n. Anaerophaga an anaerobic eater.

Slender flexible rods with rounded ends. Strictly anaerobic. Chemoorganotrophic. Nonphotosynthetic. Have a fermentative type of metabolism, using organic compounds as substrates. Inorganic electron acceptors are not used. Media containing a reductant, e.g., cysteine, are necessary for growth. Catalase-negative. Isolated from oil-contaminated sediment in an oil separation tank. Type species: Anaerophaga thermohalophila Denger, Warthmann, Ludwig and Schink 2002, 177VP. DNA G+C content (mol%): 42.

Further descriptive information The only described species so far is Anaerophaga thermohalophila. The strains Fru22T and Glc12 were isolated as possible agents for tenside (surfactant) production in microbially improved oil recovery (MIOR). Therefore, the organism was enriched and selected under conditions of elevated temperature (50°C) and increased salinity (7.5%, w/v). The isolated strains ferment hexoses and pentoses to equal molar amounts of acetate, propionate, and succinate (Denger and Schink, 1995; Denger et al., 2002), according to the following equations: 3 C6H12O6 → 2 C2H3O2− + 2 C3H5O2− + 2 C4H4O42− + 8 H+ + 2 H2O, or 9 C5H10O5 → 5 C2H3O2− + 5 C3H5O2− + 5 C4H4O42− + 20 H+ + 5 H2O No CO2 is released in this type of mixed-acid fermentation. Upon prolonged exposure of fully grown cultures to daylight, an orange-red pigment was produced, which could be extracted from cell pellets by acetone or hexane, indicating

that it was a lipophilic component. Absorption spectra of these extracts showed maxima at 488 and 518 nm and a further shoulder around wavelength 460 nm, as is typical of carotenoids (Reichenbach et  al., 1974). Carotenoids are known to be produced by several representatives of aerobic gliding bacteria, including Flexibacter sp., Cytophaga sp., and several myxobacteria (Reichenbach and Dworkin, 1981). Strain Fru22T is the first strict anaerobe producing such pigments, although the moderately oxygen-tolerant anaerobe Cytophaga xylanolytica also produces carotenoids (Haack and Breznak, 1993). When pigmented cell material after centrifugation was treated with 10% KOH, the color turned dark-red to brownish, similar to the flexirubins of Cytophaga, Sporocytophaga, and Flexibacter spp. (Achenbach et  al., 1978; Reichenbach et  al., 1974). Unfortunately, these strains do not grow on agar surfaces, even if the medium is poured and stored in an oxygen-free glove box under a N2/CO2 atmosphere (90/10, v/v). Thus, whether gliding motility occurs could not be determined. Spore-like structures or spheres are observed in ageing cultures. Surface-active compounds were produced by strain Fru22T optimally at the end of exponential growth (Denger and Schink, 1995). This tenside efficiently stabilized hexadecane/ water emulsions. Production was enhanced in the presence of hexadeca ne, which provides a lipophilic surface in the culture. The surface-active compound(s) was associated partly with the cells and cell surfaces, but was also released into the culture medium.

52

Family II. Marinilabiliaceae

Enrichment and isolation procedures Anaerophaga thermohalophila strain Fru22 and a similar strain, Glc12, were enriched originally from blackish-oily sedimentary residues in an oil separation tank near Hannover, Germany. The mineral salts medium for enrichment and cultivation was bicarbonate-buffered (50 mM), cysteine-reduced (1 mM), and contained, together with other minerals, 75 g of NaCl and 4.0 g of MgCl2·7H2O per liter (Denger and Schink, 1995). The pH was 6.7–6.8. During the enrichment, the medium received a few drops (50 ml per 25 ml of medium) of hexadecane to provide a lipophilic boundary layer. Subcultures were inoculated with oily drops from the surface of the preculture. All details concerning cultivation and physiological characterization have been described before (Denger and Schink, 1995). Growth rates and yields were best at 20–70 g of total salt per liter. At 80 g of salt per liter, growth was partly inhibited, and there was no growth at 1300 aligned bases) showing the phylogenetic relationship of

the Alistipes species to related taxa. The tree was constructed using the neighbor-joining method, following distance analysis of aligned sequences. Bootstrap analysis was used, with 1000 repetitions.

(Rautio et al., 2003a; Song et al., 2006) as well as from farm soil (Cato et al., 1979) indicates that the habitat is the human and animal gut.

Enrichment and isolation procedures Alistipes strains can be isolated on solid culture media appropriate for anaerobes, including nonselective Brucella blood agar enriched with vitamin K1 and hemin, and fastidious anaerobe agar (FAA; Lab M). In addition, pigment-producing, bile-resistant species are also isolated from selective KVLB and BBE agar plates (Rautio et al., 1997). Growth in liquid media is especially poor. Tween 80 (0.5%) has been used to enhance growth of Alistipes putredinis (­Holdeman et  al., 1984). Of various supplements (bile, formate-fumarate, hemin, horse serum, pyruvate, sodium bicarbonate, and Tween) tested for enhancement of growth of pigment-producing Alistipes species in thioglycolate broth

medium, bile and horse serum showed a weak stimulation (Rautio et al., 1997).

Maintenance procedures For long term storage, young (2–3 d) cultures are transferred into vials containing sterilized 20% skim milk and kept frozen at –70°C. Twenty-year-old strains have been revived successfully from stocks by scraping frozen bacterial suspension onto fresh or prereduced Brucella blood agar and incubating the plates in anaerobic jars or in an anaerobic chamber for 3–5 d before subcultivation for verifying the purity of strains.

Methods for characterization tests In general, the anaerobic methods described in the WadsworthKTL Anaerobic Bacteriology Manual (Jousimies-Somer et  al., 2002) are suitable for the study of members of this genus. The spot indole test may give false-negative results, therefore, the

58

FAMILY iii. Rikenellaceae

TABLE 11.  Characteristics differentiating Alistipes from related generaa

Characteristic

Alistipes b

Growth in air and CO2 – Gram reaction – Pigment production + Growth in 20% bile + Susceptibility to:e   Vancomycin (5 mg) R   Kanamycin (1 mg) R   Colistin (10 mg) R Catalase production – Indole production + Nitrate reduction – Proteolytic activity + Carbohydrate fermentation + Major metabolic end product(s)f S Major cellular fatty acid C15:0 iso DNA G+C content (mol%) 55–58 Type species A. putredinis

Bacteroides c

Porphyromonas d

Prevotella

Rikenella

Tannerella

– – – +

– – + –

– – D –

– – – +

– – – –

R R R – D – – + A, S C15:0 anteiso 40–48 B. fragilis

D R R – D – D – A, B C15:0 iso 40–55 P. asaccharolytica

R D D – D – D + A, S C15:0 anteiso 39–60 P. melaninogenica

R S R – – – D – P, S C15:0 iso 60–61 R. microfusus

R S S – – – + – A, B, IV, P, PA C15:0 anteiso 44–48 T. forsythia

Symbols: +, positive; –, negative; D, different reactions in different taxa (species). Unlike other Alistipes spp., Alistipes putredinis does not produce pigment, and is susceptible to bile, catalase positive, and asaccharolytic. c Bacteroides sensu stricto. d Unlike other Porphyromonas spp., Porphyromonas catoniae does not produce pigment, and is moderately saccharolytic. e Special potency antimicrobial identification disks. Symbols: R, resistant; S, susceptible. f Symbols: A, acetic acid; B, butyric acid; IV, isovaleric acid; P, propionic acid; PA, phenylacetic acid; S, succinic acid. a

b

tube indole test is recommended. Commercial API ZYM and API rapid ID 32 A test kits (bioMérieux) and individual Rosco diagnostic tablets (Rosco) can be useful for examining biochemical characteristics. A heavy inoculum from young (2–3 d) cultures should be used for biochemical testing to avoid poor reproducibility of reactions. The enzyme profiles ­generated by the API ZYM test kit (bioMérieux) proved to be most useful in distinguishing the Alistipes species from each other. For ­demonstration of carbohydrate fermentation, prereduced, anaerobically sterilized (PRAS) peptone-yeast-sugar broth tubes, with or without additional supplements, are used, but results can be affected by poor growth of Alistipes strains in liquid media.

Differentiation of the genus Alistipes from other genera Characteristics that differentiate Alistipes from related taxa within the phylum Bacteroidetes (previously referred to as the

“Cytophaga–Flavobacteria–Bacteroides” phylum) are presented in Table 11.

Taxonomic comments The genus Alistipes currently includes four validly published species, Alistipes putredinis and Alistipes finegoldii (Rautio et  al., 2003a, b), and Alistipes onderdonkii and Alistipes shahii (Song et al., 2006). Phylogenetic analyses of the 16S rRNA gene sequence reveal further heterogeneity within the genus, since occasional strains that are phenotypically similar to but phylogenetically diverse from Alistipes species described in the current literature have been reported (Song et al., 2005, 2006). Also, preliminary results on bile-resistant, pigment-producing Alistipes-like organisms in ongoing studies by the authors (unpublished) revealed (among approximately 150 strains examined) several groups having a 16S rRNA gene sequence divergence of more than 3% compared to any known Alistipes species.

List of species of the genus Alistipes 1. Alistipes putredinis (Weinberg, Nativelle and Prévot 1937) Rautio, Eerola, Väisänen-Tunkelrott, Molitoris, Lawson, Collins and Jousimies-Somer 2003b, 1701 (Effective publication: Rautio, Eerola, Väisänen-Tunkelrott, Molitoris, Lawson, Collins and Jousimies-Somer 2003a, 186.) (Bacillus putredinis Weinberg, Nativelle and Prévot 1937, 755; Ristella putredinis Prévot 1938, 291; Bacteroides putredinis Kelly 1957, 420) put.re¢di.nis. L. n. putredo -inis rottenness, putridity; L. gen. n. putredinis of putridity. The description is based on previous literature (Cato et  al., 1979; Holdeman et  al., 1984; Rautio et  al., 2003a; Song et al., 2006). Surface colonies on supplemented Brucella sheep blood agar after incubation for 4 d are pinpoint to 0.5 × mm in diameter, circular, entire or slightly irregular, low convex, translucent, gray, dull, and smooth. Colonies

do not produce pigment. No growth occurs in 20% bile. Although only traces of acid products can be detected in a 6-d-old PYG broth (pH > 6), in a 24-h-old meat chopped carbohydrate broth, the main acid detected is succinate. Catalase and indole are produced. The type strain, examined with the API ZYM and API rapid ID 32 A test kits, is positive for alkaline and acid phosphatases, esterase, esterase lipase, naphthol-AS-BI-phosphohydrolase, a-glucosidase, leucyl glycine, alanine, and serine arylamidases, glutamic decarboxylase, and indole. Some characteristics of the species are listed in Table 12. DNA G+C content (mol%): 55 (HPLC). Type strain: ATCC 29800, CCUG 45780, CIP 104286, DSM 17216. Sequence accession no. (16S rRNA gene): L16497.

+ + + – + S a, p, (iv, l) C15:0 iso 57 CCUG 46020 Human feces, appendix tissue

– – + – – S a, iv, p C15:0 iso 55 ATCC 29800 Human and animal feces, appendicitis and, related infections, intraabdominal and, perianal infections, sheep foot rot, farm soil

b

a

S a, p, (iv, l) C15:0 iso 56 CCUG 48946 Human feces, appendix tissue, abdominal abscess, urine

d – + – +

b + + –

Alistipes onderdonkii Straight rods, 0.3–0.9 × 0.5–3 mm, singly or in pairs 0.5–0.8 mm, circular, entire, opaque

Alistipes finegoldii Straight rods, 0.2 × 0.8–2 mm, singly, long filaments occur 0.3–1.0 mm, circular, entire, translucent or opaque b + + –

Alistipes putredinis

Straight or slightly curved rods, 0.3–0.5 × 0.9–3 mm, singly or in pairs Pinpoint to 0.5 mm, circular, slightly irregular, low convex, translucent – – – +

Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); -, 0–15% positive. Based on the reactions generated by the API ZYM test kit. c Symbols: a, acetic acid; iv, isovaleric acid; l, lactic acid; p, propionic acid; S, major amount of succinic acid.

Hemolysis Pigment production Growth in 20% bile Catalase production Enzyme activities: b   a-Chymotrypsinase   a-Fucosidase   a-Glucosidase   b-Glucosidase Glucose fermentation Metabolic end products:   Major   Minor (trace) amountsc Major cellular fatty acid DNA G+C content (mol%) Type strain Isolation sit

Colony morphology

Cell morphology

Characteristic

TABLE 12.  Characteristics differentiating species of the genus Alistipesa.

Alistipes shahii

S a, iv C15:0 iso 58 CCUG 48947 Human feces, appendix tissue, intraabdominal fluid

d + + + +

b + + –

Straight rods, 0.5–0.8 × 0.6–4 mm, singly or in pairs 0.5–1 mm, circular, entire, opaque

Genus II. Alistipes 59

60

FAMILY iii. Rikenellaceae

2. Alistipes finegoldii Rautio, Eerola, Väisänen-Tunkelrott, Molitoris, Lawson, Collins and Jousimies-Somer 2003b, 1701VP (Effective publication: Rautio, Eerola, VäisänenTunkelrott, Molitoris, Lawson, Collins and Jousimies-Somer 2003a, 186.) fine.gold¢i.i. N.L. gen. masc. n. finegoldii of Finegold; named after Sydney M. Finegold, an American contemporary researcher and clinician in recognition of his contribution to anaerobic bacteriology and infectious diseases. The description is based on previous literature (Rautio et  al., 2003a, 1997; Song et  al., 2006). Surface colonies on supplemented Brucella sheep blood agar after 4 d of incubation are pinpoint to 1.0 mm in diameter, circular, entire, raised, gray, translucent or opaque, and (weakly) b-­ hemolytic. Colonies are light brown to brown. No fluorescence is observed under long-wave UV light (365 nm), but the colonies appear black. Esculin hydrolysis differs among strains. Acid is produced from glucose. The strains, examined with the API ZYM and API rapid ID 32 A test kits , are positive for alkaline and acid phosphatases, esterase, esterase lipase, a-chymotrypsin, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, N-acetyl-bglucosaminidase, a-fucosidase, leucyl glycine, alanine, and glutamyl glutamic arylamidases, and indole. Some characteristics of the species are listed in Table 12. DNA G+C content (mol%): 57 (HPLC). Type strain: AHN 2437, CCUG 46020, CIP 107999, DSM 17242. Sequence accession no. (16S rRNA gene): AJ518874. 3. Alistipes onderdonkii Song, Könönen, Rautio, Liu, Bryk, Eerola and Finegold 2006, 1988VP on.der.don¢ki.i. N.L. gen. masc. n. onderdonkii of Onderdonk; named after Andrew B. Onderdonk, a contemporary American microbiologist, for his contribution to increased knowledge about the intestinal microbiota and anaerobic bacteria. The description is based on the investigation of 15 strains (Song et al., 2006). Cells are 0.2–0.5 mm × 0.5–3 mm. ­Surface colonies on supplemented Brucella sheep blood agar after 4 d are pinpoint to 0.8 mm in diameter, circular, entire, convex, opaque, gray, and (weakly) b-hemolytic. Colonies are light brown to brown. No fluorescence is observed under

References Cato, E.P., L.V. Holdeman and W.E.C. Moore. 1979. Proposal of neotype strains for seven non-saccharolytic Bacteroides species. Int. J. Syst. Bacteriol. 29: 427–434. Collins, M.D., H.N. Shah and T. Mitsuoka. 1985a. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 18. Int. J. Syst. Bacteriol. 35: 375–376. Collins, M.D., H.N. Shah and T. Mitsuoka. 1985b. Reclassification of Bacteroides microfusus (Kaneuchi and Mitsuoka) in a new genus Rikenella, as Rikenella microfusus comb. nov. Syst. Appl. Microbiol. 6: 79–81. Eggerth, A.H. and B.H. Gagnon. 1933. The Bacteroides of human feces. J. Bacteriol. 25: 389–413. Holdeman, L.V., R.W. Kelly and W.E.C. Moore. 1984. Genus I. Bacteroides. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 604–631. Jousimies-Somer, H.R., P. Summanen, D.M. Citron, E.J. Baron, H.M. Wexler and S.M. Finegold. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual. Star Publishing Company, Belmont, CA.

long-wave UV light (365 nm), but the colonies appear black. Grows in the presence of 20% bile. Indole positive. Catalase negative. Esculin hydrolysis differs among strains. Acid is produced from glucose. When tested by the API rapid ID 32 A system (bioMeriéux), mannose and raffinose are fermented. The strains, examined with the API ZYM and API rapid ID 32A test kits, are positive for alkaline and acid phosphatases, esterase, esterase lipase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, N-acetyl-bglucosaminidase, leucyl glycine, alanine, and glutamyl glutamic arylamidases, and indole. Some characteristics of the species are listed in Table 12. DNA G+C content (mol%): 56 (HPLC). Type strain: WAL 8169, ATCC BAA-1178, CCUG 48946. Sequence accession no. (16S rRNA gene): AY974071. 4. Alistipes shahii Song, Könönen, Rautio, Liu, Bryk, Eerola and Finegold 2006, 1999VP sha¢hi.i. N.L. gen. masc. n. shahii of Shah; to honor Haroun N. Shah, a contemporary British microbiologist for his contributions to anaerobic bacteriology. The description is based on the investigation of six strains (Song et al., 2006). Cells are 0.1–0.2 mm × 0.6–4 mm. Surface colonies on supplemented Brucella sheep blood agar after 4 d are pinpoint to 1.0 mm in diameter, circular, entire, convex, opaque, gray, and (weakly) b-hemolytic. Colonies are light brown to brown. No fluorescence is observed under long-wave UV light (365 nm), but the colonies appear black. Grows in the presence of 20% bile. Indole positive. Catalase negative. Esculin is hydrolyzed. Acid is produced from glucose. When tested by the API rapid ID 32 A system (bioMeriéux), mannose and raffinose are fermented. The strains, examined with the API ZYM and API rapid ID 32 A test kits (bioMeriéux), are positive for alkaline and acid phosphatases, esterase, esterase lipase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetylb-glucosaminidase, a-fucosidase, leucyl glycine, alanine, and glutamyl glutamic arylamidases, and indole. Some characteristics of the species are listed in Table 12. DNA G+C content (mol%): 58 (HPLC). Type strain: WAL 8301, ATCC BAA-1179, CCUG 48947. Sequence accession no. (16S rRNA gene): AY974072.

Kaneuchi, C. and T. Mitsuoka. 1978. Bacteroides microfusus, a new species from intestines of calves, chickens, and Japanese quails. Int. J. Syst. Bacteriol. 28: 478–481. Kelly, C.D. 1957. Genus I. Bacteroides Castellani and Chalmers 1919. In Bergey’s Manual of Determinative Bacteriology, 7th edn (edited by Breed, Murray and Smith). Williams & Wilkins, Baltimore, pp. 424–436. Mitsuoka, T., T. Sega and S. Yamamoto. 1965. Improved methodology of qualitative and quantitative analysis of the intestinal flora of man and animals. Zentralbl. Bakteriol. [Orig.] 195: 455–469. Ohkuma, M., S. Noda, Y. Hongoh and T. Kudo. 2002. Diverse bacteria related to the bacteroides subgroup of the CFB phylum within the gut symbiotic communities of various termites. Biosci. Biotechnol. Biochem. 66: 78–84. Paster, B.J., F.E. Dewhirst, I. Olsen and G.J. Fraser. 1994. Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria. J. Bacteriol. 176: 725–732. Prévot, A.R. 1938. Etudes de systematique bacterienne. III. Invalidite du genre Bacteroides Castellani et Chalmers demembrement et reclassification. Ann. Inst. Pasteur 20: 285–307.



61

Genus II. Alistipes

Rautio, M., M. Lonnroth, H. Saxen, R. Nikku, M.L. Vaisanen, S.M. Finegold and H. Jousimies-Somer. 1997. Characteristics of an unusual anaerobic pigmented gram-negative rod isolated from normal and inflamed appendices. Clin. Infect. Dis. 25 Suppl 2: S107–S110. Rautio, M., H. Saxen, A. Siitonen, R. Nikku and H. Jousimies-Somer. 2000. Bacteriology of histopathologically defined appendicitis in children. Pediatr. Infect. Dis. J. 19: 1078–1083. Rautio, M., E. Eerola, M.L. Vaisanen-Tunkelrott, D. Molitoris, P. Lawson, M.D. Collins and H. Jousimies-Somer. 2003a. Reclassification of Bacteroides putredinis (Weinberg et  al., 1937) in a new genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp. nov., from human sources. Syst. Appl. Microbiol. 26: 182–188. Rautio, M., E. Eerola, M.L. Väisänen-Tunkelrott, D. Molitoris, P. ­Lawson, M.D. Collins and H.R. Jousimies-Somer. 2003b. In Valida-

tion of the publication of new names and new combinations previously effectively published outside the IJSEM. List no. 94. Int. J. Syst. Evol. Microbiol. 53: 1701–1702. Song, Y., C. Liu, M. Bolanos, J. Lee, M. McTeague and S.M. Finegold. 2005. Evaluation of 16S rRNA sequencing and reevaluation of a short biochemical scheme for identification of clinically significant Bacteroides species. J. Clin. Microbiol. 43: 1531–1537. Song, Y., E. Kononen, M. Rautio, C. Liu, A. Bryk, E. Eerola and S.M. Finegold. 2006. Alistipes onderdonkii sp. nov. and Alistipes shahii sp. nov., of human origin. Int. J. Syst. Evol. Microbiol. 56: 1985–1990. Weinberg, M., R. Nativelle and A.R. Prévot. 1937. Les microbes anaérobies. Masson et Cie, Paris. Willis, A.T. 1960. Anaerobic Bacteriology in Clinical Medicine. Butterworths, London.

Family IV. Porphyromonadaceae fam. nov. Noel R. Krieg Por.phy.ro.mo.na.da.ce′a.e. N.L. fem. n. Porphyromonas type genus of the family; suff. -aceae ending to denote a family; N.L. fem. pl. n. Porphyromonadaceae the Porphyromonas family.

The family Porphyromonadaceae is a phenotypically diverse group of genera that was circumscribed for this volume on the basis of phylogenetic analysis of 16S rRNA gene sequences. The family contains the genera Porphyromonas (type genus), Barnesiella, Dysgonomonas, Paludibacter, Petrimonas, Proteiniphilum, and Tannerella. In addition, Parabacteroides, which was described after the deadline for this volume

should be ­classified within this family (Sakamoto and Benno, 2006; Sakamoto et  al., 2007a). All are nonmotile rods that stain Gram-negative. Except as noted below, strict anaerobes and saccharolytic. Some characteristics that differentiate the genera are given in Table 13. Type genus: Porphyromonas Shah and Collins 1988, 129VP emend. Willems and Collins 1995, 580.

TABLE 13.  Some characteristics that differentiate the genera of the family Porpyromonadaceae a

Characteristic

Porphyromonas

Cell shape

Short rods or coccobacilli

Rods

Coccobacilli to short rods

−

−

− −

−; Some species are weakly positive Butyric and acetic acids; propionic, isovaleric, isobutyric, and phenylacetic acids may also be produced MK-9, MK-10

Growth in the presence of bile Can grow aerobically N-Acetylglucosamine required for growth Saccharolytic

Products of glucose fermentation:

Predominant menaquinone Isolated from:

DNA G+C content (mol%)

Oral infections and various other clinical specimens of human and animal origin 44–55

Barnesiella

Dysgonomonas

Paludibacter

Proteiniphilum

Tannerella

Rods

Rods

Fusiform cells

+

Rods with ends usually round to slightly tapered −

nt

−

−

− −

+ −

− −

− −

− −

− +, except bite wound isolates

+

+

+

+

−

+

Acetic and succinic acids

Propionic, lactic, and succinic acids

Acetic and propionic acids; succinic acid is a minor product

Acetic acid and H2

na

Acetic, butyric, isovaleric, propionic, and phenylacetic acids; smaller amounts of isobutyric and succinic acids may be produced

MK-11, MK-12 Chicken cecum

nt

MK-8

MK-8

nt

MK-10, MK-11

Human clinical specimens and stools

Rice plant residue (rice straw) collected from irrigated rice-field soil

Oilfield well head

UASB reactor treating brewery wastewater

38

39

41

47–49

Human subgingival, gingival, and periodontal pockets, in dental root canals, and around infected dental implants 44–48

52

Symbols: +, >85% positive; -, 0–15% positive; w, weak reaction; na, not available; nt, not tested.

a

Petrimonas

62

Family III. Porphyromonadaceae

Genus I. Porphyromonas Shah and Collins 1988, 129VP emend. Willems and Collins 1995, 580. Paula Summanen and Sydney M. Finegold Por.phy.ro.mo′nas. Gr. adj. porphyreos purple; Gr. n. monas unit; N.L. fem. n. Porphyromonas porphyrin cell.

Short rods or coccobacilli, 0.3–1 × 0.8–3.5 mm. Gram-negative, non-sporeforming, and nonmotile. Obligately anaerobic. Generally cells form brown to black colonies on blood agar due to protoheme production. Most species are asaccharolytic: growth is not significantly affected by carbohydrates but is enhanced by protein hydrolysates such as proteose peptone or yeast extract. Major fermentation products are usually n-butyric acid and acetic acid; propionic, isovaleric, isobutyric, and phenylacetic acid may also be produced. The major cellular fatty acid is 13-methyltetradecanoic acid (C15:0 iso). Indole is produced by most strains. Nitrate is not reduced to nitrite. Esculin is not hydrolyzed. Most species do not hydrolyze starch. Isolated from oral infections and various other clinical specimens of human and animal origin. DNA G+C content (mol%): 40–55 (Tm). Type species: Porphyromonas asaccharolytica (Holdeman and Moore 1970) Shah and Collins 1988, 128VP [Bacteroides asaccharolyticus (Holdeman and Moore 1970) Finegold and Barnes 1977, 390; Bacteroides melaninogenicus subsp. asaccharolyticus Holdeman and Moore 1970, 33].

Further descriptive information Cell morphology.  Most cells in broth cultures are small (0.3– 1.0  ×  0.8–3.5  mm); however, occasionally longer cells and filaments (³5  mm) may be formed. Cells from growth on a solid medium are commonly shorter and can appear spherical. Cell-wall composition.  The cell-wall peptidoglycan contains lysine as the diamino acid. 2-Keto-3-deoxyoctulosonic acid is absent. The principal respiratory quinones are unsaturated menaquinones with 9 or 10 isoprene units. Both nonhydroxylated and 3-hydroxylated fatty acids are present. The nonhydroxylated fatty acids are predominantly methyl-branched-chain fatty acids. The predominant fatty acid is C15:0 iso; a few species contain comparable amounts of C15:0 iso and C15:0 anteiso acids. The 3-hydroxylated fatty acids are generally straight-chain saturated fatty acids. Information on the fatty acid content and cell wall composition can be found in Collins et al. (1994) and Brondz and Olsen (1991). Colony morphology.  Colonies on blood agar plates are usually round, entire, smooth (occasionally rough), shiny, convex, and 0.5–3 mm in diameter. The colonies of all but one species (Porphyromonas catoniae) are pigmented. The black pigmentation of Porphyromonas is caused by the accumulation of hemin used as an iron source for bacterial growth. The species vary in the degree and rapidity of pigment production depending primarily on the type of blood used in the growth medium. Laked rabbit blood agar is considered the most reliable medium for detecting the pigment. The pigmentation ranges from tan to black and may take several days to develop. Growth conditions.  The optimum temperature for growth is 37°C. Porphyromonas species favor a slightly alkaline environmental pH and 100% humidity. Hemin and vitamin K1 are either required or greatly stimulate the growth of most species.

Although some species are weakly saccharolytic, their growth is not significantly affected by carbohydrates. Nitrogenous substances, such as proteose peptone, trypticase, and yeast extract markedly enhance growth. Metabolism and metabolic pathways.  Malate dehydrogenase and glutamate dehydrogenase are present; glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are absent from most species. Proteolytic activity is variable. The strains have a limited ability to ferment amino acids such as aspartate and asparagine. Genetics.  Porphyromonas forms a distinct phylogenetic group, and 16S rRNA gene sequencing can be reliably used to differentiate Porphyromonas from other genera and from each other, with the exception of Porphyromonas asaccharolytica and Porphyromonas uenonis, where DNA–DNA reassociation studies are required to distinguish these two species genetically. The genome of Porphyromonas gingivalis has been sequenced and studied by the Forsyth Institute and The Institute for Genomic Research (TIGR) (Nelson et al., 2003). The genome size of this species was determined to be 2176  kb from XbaI restriction enzyme digests and 2250 kb from SpeI digests. Information and a schematic representation of the Porphyromonas gingivalis W83 genome can be found at www.tigr.org. Comparative analysis of the whole-genome sequence with other available complete genome sequences confirmed the close relationship between the phylum Bacteroidetes [Cytophaga–Flavobacteria–Bacteroides (CFB)] and the green sulfur bacteria. Within the phylum “Bacteroidetes” the genomes of Bacteroides thetaiotaomicron and Bacteroides fragilis were most similar to that of Porphyromonas gingivalis. The genome analysis revealed a range of virulence determinants that relate to the novel biology of this bacterium. It also revealed that Porphyromonas gingivalis can metabolize a range of amino acids and generate end products that are toxic to the human host. Antibiotic susceptibility.  Porphyromonas species are generally very susceptible to most of the antimicrobial agents commonly used for treatment of anaerobic infections, such as amoxicillin-clavulanate, piperacillin-tazobactam, ampicillin-sulbactam, imipenem, cephalosporins, and metronidazole. b-Lactamase production has been described in Porphyromonas asaccharolytica (Aldridge et al., 2001), Porphyromonas catoniae (Kononen et al., 1996), Porphyromonas somerae (Summanen et  al., 2005), and Porphyromonas uenonis (Finegold et  al., 2004). The frequency of b-lactamase production has been reported at approximately 20%. Animal-derived Porphyromonas species are more often b-lactamase producers than those derived from humans. Occasional resistance to clindamycin and ciprofloxacin may occur in Porphyromonas asaccharolytica and Porphyromonas somerae; also, ciprofloxacin resistance of Porphyromonas gingivalis has been reported (Lakhssassi et al., 2005). Pathogenicity.  Some species are considered true pathogens and are associated with human or animal infections. In particular, Porphyromonas gingivalis is a major causative agent in the



63

Genus I. Porphyromonas

i­nitiation and progression of severe forms of periodontal disease. Porphyromonas gingivalis possesses a multitude of cell-surface associated and extracellular activities that contribute to its virulence potential. Several of these factors are adhesins that interact with other bacteria, epithelial cells, and extracellular matrix proteins. Secreted or cell-bound enzymes, toxins, and hemolysins play a significant role in the spread of the organism through tissue, in tissue destruction, and in evasion of host defenses.

Maintenance procedures Isolates can be put into stock from broth or plate cultures. Freezing at –70°C or lyophilization of young cultures grown in a well-buffered liquid or solid medium is satisfactory for storage of Porphyromonas species. Storage of lyophilized cultures at 4°C is recommended. Even with the best storage conditions, only a portion of the original cell population survives. Therefore, a large inoculum in a supportive medium and minimal exposure to oxygen are recommended for the recovery of viable cultures from stored material. To maintain stock strains in the laboratory, it is advisable to transfer them weekly in chopped meat medium or other suitable medium that does not contain a fermentable carbohydrate.

Ecology.  Several of the members of Porphyromonas are indigenous bacterial flora in the oral cavity of humans and animals. Many species are also found in the urogenital and intestinal tracts. Porphyromonas species have been isolated from oral infections, and from many infections throughout the body, e.g., blood, amniotic fluid, umbilical cord, pleural empyema, peritoneal and pelvic abscesses, inflamed endometrium, and other infected tissues. Porphyromonas species of animal origin have been encountered in humans with animal bite infections.

Differentiation of the genus Porphyromonas from other genera The pigmented Porphyromonas species can be differentiated from other anaerobic, Gram-stain-negative genera with relative ease (Table 14). The special-potency antibiotic disks (vancomycin, 5 mg; kanamycin, 1000 mg; and colistin, 10 mg) can be used to separate the Gram-stain-negative genera: Porphyromonas species are generally sensitive to vancomycin and resistant to kanamycin and colistin, whereas the other Gram-stain-negative genera are resistant to vancomycin and vary in their resistance to kanamycin and colistin. A distinctive feature of the porphyromonads is that the predominant fatty acid is 13-methyltetradecanoic acid (C15:0 iso); by contrast, Bacteroides and Prevotella contain 12-methyl tetradecanoic acid (C15:0 anteiso) as their major cellular fatty acid. Bacteroides and Prevotella also produce a simpler metabolic end product profile of mainly acetic and succinic acids (Table 14).

Enrichment and isolation procedures A complex medium containing peptone, yeast extract, vitamin K1, and hemin, and supplemented with 5% blood is recommended for isolation of Porphyromonas from body sites. Fresh or prereduced media (commercially available from Anaerobe Systems, Morgan Hill, California) are recommended, as they increase isolation efficacy. For determination of pigmentation, rabbit blood (laked) is preferable to blood from other animals. Kanamycin-vancomycin laked-blood agar with reduced vancomycin concentration (2 mg/ ml) may be used as a selective medium when Porphyromonas species are sought (Jousimies-Somer et al. 2002). Formulas for basal media are given in Jousimies-Somer et al. (2002). Inoculated media should immediately be placed in an anaerobic environment, such as an anaerobic pouch, jar, or chamber. If anaerobic systems utilizing palladium catalysts are used, the anaerobic gas mixture must contain hydrogen to enable the palladium to reduce oxygen to water. The plates may be examined after a 48-h incubation. However, a total incubation period of at least 7 d is recommended, because not all Porphyromonas species may be detected with shorter incubation times.

Taxonomic comments The taxonomy of pigmented Gram-negative bacilli has changed greatly since the first edition of Bergey’s Manual of Systematic Bacteriology. The genus Porphyromonas was created in 1988 (Shah and Collins, 1988), and since then some species

TABLE 14.  Differentiation of the genus Porphyromonas from other anaerobic Gram-negative rods a

Characteristic Susceptibility to:   Vancomycin (5 mg)   Kanamycin (1000 mg)   Colistin (10 mg) Pigment Growth in 20% bile Proteolytic activity Major metabolic end products from PYGf Major long-chain fatty acids DNA G+C (mol%) e

Porphyromonas b

Alistipes c

Bacteroides d

Prevotella

Rikenella

Tannerella

  S R R + D A, B, iV C15:0 iso 40–55

  R R R + + + S

  R R R + A, S C15:0 anteiso 40–48

  R R D D D A, S C15:0 anteiso 39–60

  R S R + P, S C15:0 iso 60–61

  R S S + A, S, PA C15:0 anteiso 44–48

C15:0 iso 55–58

+, 90% or more of the strains are positive; -, 10% or more of the strains are negative; D, different reaction in different species. Porphyromonas catoniae does not produce pigment, is vancomycin-resistant, and fermentative. c Alistipes putredinis does not produce pigment, is susceptible to bile, and nonfermentative. d Bacteroides sensu stricto. e Special potency antimicrobial identification disks; R, resistant; S, susceptible; D, differs among species. f A, acetic acid; B, butyric acid; iV, isovaleric acid; P, propionic acid; PA, phenylacetic acid; S, succinic acid. a

b

64

Family III. Porphyromonadaceae

previously included in the genus Bacteroides (Porphyromonas gingivalis, Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas levii, and Porphyromonas macacae) were reclassified as Porphyromonas species, and several new species described. Porphyromonas now includes 16 validly published species. Porphyromonas salivosa is a later heterotypic synonym of Porphyromonas macacae (Love, 1995). The porphyromonads form a natural, but deep, phylogenetic group (Figure 17). The species exhibit levels of 16S rRNA sequence divergence of up to 15%. Phylogenetically, the closest related species to Porphyromonas are found in the genera Bacteroides and Prevotella (approx. 82–89% similarity).

Differentiation of species of the genus Porphyromonas With the exception of Porphyromonas catoniae, Porphyromonas species form a phenotypically homogeneous group: they are pigmented, sensitive to the special-potency vancomycin disk, and produce butyric acid as the major metabolic end product. Porphyromonas catoniae differs in all these aspects. The characteristics useful in differentiating the species are given in Table 15. Numerous PCR-based identification or detection systems have been described for Porphyromonas gingivalis and Porphyromonas endodontalis (de Lillo et al., 2004; Fouad et al., 2002; Gomes et  al., 2005; Jervoe-Storm et  al., 2005; Kuboniwa et  al., 2004; Kumar et al., 2003; Noguchi et al., 2005; Seol et al., 2006).

List of species of the genus Porphyromonas 1. Porphyromonas asaccharolytica (Holdeman and Moore 1970) Shah and Collins 1988, 129VP (Bacteroides asaccharolyticus (Holdeman and Moore 1970) Finegold and Barnes 1977, 390AL; Bacteroides melaninogenicus subsp. asaccharolyticus Holdeman and Moore 1970, 33) a.sac.cha.ro.ly′ti.cus. Gr. pref. a not; Gr. n. sakchâr sugar; N.L. fem. adj. lytica (from Gr. fem. adj. lutikê) able to loosen, able to dissolve; N.L. fem. adj. asaccharolytica not digesting sugar. The description is from Shah and Collins (1988) and Holdeman et  al. (1984). Proteolytic activity is weak, but gelatin liquefaction is positive and fibrinolytic activity is present. Starch is not hydrolyzed. Growth is stimulated by 0.5% NaCl. Cellular and colonial morphology and other characteristics are as described for the genus and as given in Table 15. Porphyromonas asaccharolytica can be distinguished from Porphyromonas uenonis by phenotypic tests (Table 15); however, DNA–DNA reassociation studies are required to distinguish these two species genetically. Susceptible to piperacillin-tazobactam, ampicillin-sulbactam, imipenem, meropenem, trovafloxacin, and metronidazole. Most strains (³90%) are susceptible to cefoxitin, ciprofloxacin, and clindamycin. b-Lactamase production has been described in Porphyromonas asaccharolytica at the rate of 21% (Aldridge et al., 2001). Source: various human clinical infections. DNA G+C content (mol%): 52–54 (Tm) Type strain: ATCC 25260, CCUG 7834, DSM 20707, JCM 6326, LMG 13178, L16490, VPI 4198. Sequence accession no. (16S rRNA gene): L16490. 2. Porphyromonas cangingivalis Collins, Love, Karjalainen, Kanervo, Forsblom, Willems, Stubbs, Sarkiala, Bailey, Wigney and Jousimies-Somer 1994, 676VP can.gin.gi.val′is. L. n. canis dog; L. n. gingiva gum; L. fem. suff. -alis suffix denoting pertaining to; N.L. fem. adj. cangingivalis pertaining to the gums of dogs. The description is from Collins et al. (1994). In cookedmeat carbohydrate medium and on sheep blood agar plates cells are 0.3–0.6 × 0.8–1.5 mm and occur singly and in clumps; occasionally filaments up to 16 mm long are observed. Some strains indent agar, exhibit peripheral flattening, and have a roughened and dry surface appearance on sheep blood agar. On egg yolk agar, colonies are yellow or orange. After ­incubation of 5 d, the pH range in media containing

c­ arbohydrates generally is 6.3–6.5. Ammonia is produced in cooked meat medium. Neither lactate nor threonine is converted to propionate, and pyruvate is not utilized. Other characteristics are as described for the genus and as given in Table 15. Some strains are susceptible to penicillin, amoxicillin, carbenicillin, and erythromycin; 35% of the strains produce b-lactamase. Source: diseased or healthy periodontal pockets of dogs with naturally occurring periodontitis. DNA G+C content (mol%): 55 (Tm). Type strain: ATCC 700135, CCUG 47700, NCTC 12856, VPB 4874, X76259. Sequence accession no. (16S rRNA gene): X76259. 3. Porphyromonas canoris Love, Karjalainen, Kanervo, Forsblom, Willems, Stubbs, Sarkiala, Bailey, Wigney and Jousimies-Somer 1994, 207VP can′or.is. L. n. canis dog; L. gen. n. oris of the mouth; N.L. gen. n. canoris of a dog’s mouth. The description is from Love et al. (1994). In cooked-meat carbohydrate medium and on sheep blood agar plates cells are 0.3–0.6  ×  0.8–1.5  mm and occur singly and in clumps; occasionally filaments up to 16  mm long are observed. On sheep blood agar, colonies at 48 h are circular and rough, and have an orange pigmentation. After incubation of 5 d, the pH range in media containing carbohydrates is 6.3–6.5. Ammonia is produced in cooked meat medium. Lactate is converted to propionate, but pyruvate is not utilized and threonine is not converted to propionate. Other characteristics are as described for the genus and as given in Table 15. Strains are susceptible to penicillin, amoxicillin, carbenicillin, and erythromycin. Source: subgingival pockets of dogs with naturally occurring periodontitis. DNA G+C content (mol%): 49–51 (Tm). Type strain: CCUG 36550, CIP 104881, JCM 11138, NCTC 12835, VPB 4878. Sequence accession no. (16S rRNA gene): X76261. 4. Porphyromonas cansulci Collins, Love, Karjalainen, Kanervo, Forsblom, Willems, Stubbs, Sarkiala, Bailey, Wigney and Jousimies-Somer 1994, 678VP can.sul′ci. L. n. canis dog; L. gen. n. sulci of a furrow; L. gen. n. cansulci of a dog’s furrow, referring to the habitat in the mouths of dogs.



Genus I. Porphyromonas

65

Bacteroides cellulosolvens ATCC 35603T (L35517) Capnocytophaga gingivalis ATCC 33624T (L14639) Coenonia anatina LMG 14382T (Y17612) Alistipes putredenis ATCC 29800T (L16497) Rikenella microfusus ATCC 29728T (L16498) Bacteroides splanchnicus NCTC 10825T (L16496) Tannerella forsythia JCM 10827T (L16495) Porphyromonas uenonis ATCC BAA-906T (AY570514) Porphyromonas asaccharolytica ATCC 25260T (L16490) Porphyromonas endodontalis ATCC 35406T (L16491) Porphyromonas gingivicanis ATCC 55562T (DQ677835) Porphyromonas circumdentaria NCTC 12469T (L26102) Porphyromonas catoniae ATCC 51270T (X82823) Porphyromonas somerae ATCC BAA-1230T (AY968205) Porphyromonas levii ATCC 29147T (L16493) Porphyromonas cangingivalis VPB 4874T (X76259) Porphyromonas canoris NCTC 12835T (X76261) Porphyromonas macacae ATCC 33141T (L16494) Porphyromonas salivosa NCTC 11632T (L26103) Porphyromonas crevioricanis ATCC 55563T (DQ677836) Porphyromonas cansulci VPB 4875T (X76260) Porphyromonas gulae ATCC 51700T (AF208290) Porphyromonas gingivalis ATCC 33277T (L16492) Parabacteriodes distasonis ATCC 8503T (M86695) Parabacteroides merdae ATCC 43184T (X83954) Dysgonomonas capnocytophagoides CCUG 17996T (U41355) Dysgonomonas mossii CCUG 43457T (AJ319867) Dysgonomonas gadei CCUG 42882T (Y18530) Paludibacter proprionicigenes JCM 13257T (AB078842) Proteiniphilum acetatigenes JCM 12891T (AY742226) Bacteroides fragilis ATCC 25285T (M11656) Bacteroides caccae ATCC 43185T (X83951) Bacteroides acidofaciens A40T (AB021164) Bacteroides ovatus NCTC 11153T(L16484) Bacteroides salyersiae ATCC BAA-997T (AY608696) Bacteroides thetaiotaomicron ATCC 29148T (L16489) Bacteroides nordii ATCC BAA-998T (AY608697) Bacteroides stercoris ATCC 43183T (X83953) Bacteroides eggerthii NCTC 11155T (L16485) Bacteroides uniformis ATCC 8492T (L16486) Bacteroides tectus JCM 10003T (AB200228) Bacteroides pyogenes JCM 6294T (AB200229) Bacteroides helcogenes JCM 6297T (AB200227) [Bacteroides] heparinolytica ATCC 35895T (L16487) [Bacteroides] zoogleoformans ATCC 33285T (L16488) Bacteroides vulgatus ATCC 8482T (M58762) Bacteroides massiliensisT (AY126616) Bacteroides plebeius M12T (AB200217) Bacteroides coprocola M16T (AB200224) Bacteroides coprosuis CCUG 50528T (AF319778) Prevotella tannerae ATCC 51259T (AJ005634) Prevotella brevis ATCC 19188T (AJ011682) Prevotella ruminicola ATCC 19189T (L16482) Prevotella bryantii DSM 11371T (AJ006457) Prevotella albensis DSM 11370T (AJ011683) Prevotella corporis ATCC 33547T (L16465) Prevotella disiens ATCC 29426T (L16483) Prevotella nigrescens ATCC 33563T (L16471) Prevotella intermedia ATCC 25611T (L16468) Prevotella pallens AHN 10371T (Y13105) Prevotella bivia ATCC 33574T (L16475) Prevotella melaninogenica ATCC 25845T (L16469) Prevotella veroralis ATCC 33779T (L16473) Prevotella oulora ATCC 43324T (L16472) Prevotella salivae JCM 12084T (AB108826) Prevotella oris ATCC 33573T (L16474) Prevotella multiformis JCM 12541T (AB182483) Prevotella denticola ATCC 33185T (L16466) Prevotella baroniae E9.33T (AY840553) Prevotella buccae ATCC 33690T (L16478) Prevotella multisaccharivorax JCM 12954T (AB200414) Prevotella bergensis W3326T (AY350613) Prevotella buccalis ATCC 35310T (L16476) Prevotella enoeca ATCC 51261T (AJ005635) Prevotella oralis ATCC 33269T (L16480) Prevotella marshii E9.34T (AF481227) Prevotella shahii JCM 12083T (AB108825) Prevotella loescheii ATCC 15930T (L16481) FIGURE 17.  Unrooted tree showing the phylogenetic position of Porphyromonas within the Bacteroides subgroup of the Bacteroidetes (Cytophaga–

Flavobacter–Bacteroides) phylum. The tree was constructed by the maximum-parsimony method and is based on a comparison of approximately 1400 nt. Bootstrap values, expressed as a percentage of 1000 replications, are given at the branching points. The scale bar indicates 1% sequence divergence. Courtesy of Paul A. Lawson.

66

Family III. Porphyromonadaceae

P. cansulci

P. catoniae

P. circumdentaria

P. crevioricanis

P. endodontalis

P. gingivalis

P. gingivicanis

P. gulae

P. levii

P. macacaee

P. somerae

P. uenonis

Metabolic end ­products from PYGd

P. canoris

Pigment production Fluorescence Hemagglutinin activity Indole Catalase Lipase Preformed enzyme activity: b   a-Fucosidase   a-Galactosidase   b-Galactosidase N-Acetyl-bglucosaminidase   Chymotrypsin   Trypsin Fermentation of: c   Glucose   Lactose   Maltose Glucose-6phosphate and 6-phosphogluconate dehydrogenases present Major long-chain fatty acids

P. cangingivalis

Characteristic

P. asaccharolytica

TABLE 15.  Differentiation of the species of the genus Porphyromonas a

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

-

+

+

-

+

+

+

-

+

-

d

-

d

+

-

-

-

-

na

-

+

-

+

-

+

-

-

na

na

+ -

+ + -

+ + -

+ + -

-

+ + -

+ -

+ -

+ -

+ + -

+ + -

-

+ + +−

-

+ -

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

+ -

-

+

-

+ d +

-

-

-

-f

-

+

+

+ -f

+

-

-

-

+

-

+

-

-

-

+

-

+

+

+

+

d

  -

+   -

+   -

  -

d d   + + +

+   -

  -

  -

+   -

  -

na +   -

+   w w w

+ +   w w -

+   w w w

  -

-

+

+

-

+

-

-

-

-

-

na

d

d

na

na

C15:0 iso

C15:0 iso

C15:0 iso

C15:0 iso

C15:0 iso

C15:0 iso

C15:0 iso

C15:0 iso

na

A, P, ib, B, IV, s

A, p, ib, B, IV

A, P, ib, b, IV, s

A, p, ib, B, IV, s, pa

A, P, ib, B, IV, s

A, P, ib, B, IV, s, pa

A, p, ib, B, IV, s

C15:0 C15:0 iso, iso C15:0 anteiso A, P, a, P.iv, A, P, ib, B, l, S ib, b, IV, S, IV, s, pa pa

C15:0 C15:0 C15:0 iso, iso iso, C15:0 C15:0 anteiso anteiso A, P, A, P, A, P, A, P, ib, B, ib, B, ib, B, ib, B, IV, s, IV, s IV, s, IV, s pa pa

C15:0 iso

A, P, ib, B, IV, s

a +, 90% or more of the strains are positive; –, 10% or more of the strains are negative; d, 11–89% of the strains are positive; w, weak positive reaction; na, data not available. b Reaction by API ZYM System or Rosco Diagnostic tablets. Reactivity in these systems is not always identical (see footnote f). c Fermentation of most other carbohydrates have been reported negative. d Upper-case letters indicate major metabolic products from peptone-yeast-glucose (PYG), lower-case letters indicate minor products, and parentheses indicate a variable reaction for the following acids: A, acetic; P, propionic; IB, isobutyric; B, butyric; IV, isovaleric; V, valeric; L, lactic; S, succinic; PA, phenylacetic. e Porphyromonas salivosa is a later heterotypic synonym. The cat biovar (Porphyromonas salivosa) is lipase-positive, does not ferment sorbitol, and may fluoresce under UV light. f Negative by the API ZYM System; positive with the Rosco o-nitrophenyl-b-d-galactopyranoside test.

The description is from Collins et al. (1994). On egg yolk agar, colonies may be yellow or orange. After incubation of 5 d, the pH range in media containing carbohydrates is 6.3– 6.5. Ammonia is produced in cooked-meat medium. Neither lactate nor threonine is converted to propionate and pyruvate is not utilized. Other characteristics are as described for the genus and as given in Table 15. Strains are susceptible to penicillin, amoxicillin, carbenicillin, and erythromycin. b-Lactamase producing strains have not been detected. Source: periodontal pockets of dogs with naturally occurring periodontitis.

DNA G+C content (mol%): 49–51 (Tm). Type strain: CCUG 47702, NCTC 12858, VPB 4875. Sequence accession no. (16S rRNA gene): X76260. Taxonomic note: Strain ATCC 55563T of Porphyromonas crevioricanis Hirasawa and Takada 1994, 640VP exhibits 99.9% rRNA gene sequence homology with Porphyromonas cansulci 12858T. The taxonomic standing of these two species remains to be determined. 5. Porphyromonas catoniae (Moore and Moore 1994) Willems and Collins 1995, 581VP (Oribaculum catoniae Moore and Moore 1994, 189VP)



Genus I. Porphyromonas

ca.to′ni.ae. N.L. gen. fem. n. catoniae of Cato, named in honor of Elizabeth P. Cato, an American microbiologist. The description is from Willems and Collins (1995) and Moore and Moore (1994). Cells grown in PYG broth are 0.6  ×  0.8–1.7  mm and occur in pairs and short chains. Cells grown in media containing fermentable carbohydrate may be highly vacuolated. Surface colonies on blood agar plates incubated for 2 d are 0.2–2 mm in diameter, circular, entire, flat to low convex, and transparent. Most strains are not hemolytic on rabbit blood agar; an occasional strain may be beta-hemolytic. No colonies with a dark pigment are produced. Saccharolytic. Abundant growth occurs in peptone-yeast extract or PYG broth. Broth cultures are turbid with a smooth to fine granular sediment. The pH range of PYG broth cultures is 5.0–5.5. Gelatin is hydrolyzed. Starch is hydrolyzed by most strains; some strains hydrolyze milk and meat. H2S is produced by some strains. The major cellular fatty acids are C15:0 iso and C15:0 anteiso; substantial amounts of C13:0 iso are also present. Sensitive to clindamycin and chloramphenicol. Two of 85 strains were resistant to erythromycin, one strain to tetracycline, and six strains to penicillin. b-Lactamase producing strains have been described (Kononen et al., 1996). Source: gingival crevices of humans with gingivitis or periodontitis and from humans with healthy gingivae. DNA G+C content (mol%): 49 (Tm). Type strain: ATCC 51270, CCUG 41358, NCTC 13056, VPI N3B-3. Sequence accession no. (16S rRNA gene): X82823. 6. Porphyromonas circumdentaria Love, Bailey, Collings and Briscoe 1992, 435VP cir.cum.den.ta′ri.a. L. adv. and prep. circum around, about; L. fem. adj. dentaria pertaining to teeth; N.L. fem. adj. circumdentaria referring to the isolation of the organism from the vicinity of the teeth. The description is from Love et al. (1992). In cooked-meat carbohydrate medium and on sheep blood agar plates cells are 0.3–0.6  ×  0.8–1.5  mm and occur singly, in clumps, and occasionally as filaments up to 10 mm long. On sheep blood agar, surface colonies at 72 h are 1–2 mm in diameter, and greenish brown. Both colony size and pigment formation are enhanced by growth in the presence of Staphylococcus epidermidis. After incubation for 5 d, the pH range in media containing carbohydrates generally is 6.3–6.5. Ammonia is produced in cooked meat medium. Gelatin is hydrolyzed. Neither lactate nor threonine is converted to propionate, and pyruvate is not utilized. Other characteristics are as described for the genus and as given in Table 15. Source: soft tissue infections (abscesses and empyemas), gingival margins, and subgingival plaque of felines. DNA G+C content (mol%): 40–42 (Tm). Type strain: ATCC 51356, CCUG 41934, JCM 13864, NCTC 12469, VPB 3329. Sequence accession no. (16S rRNA gene): L26102. 7. Porphyromonas crevioricanis Hirasawa and Takada 1994, 640VP cre.vi.o.ri.ca¢nis. N.L. gen. n. crevi (sic) of a crevice; L. gen. n. oris of the mouth; L. gen. n. canis of a dog; N.L. gen. n. crevioricanis of the crevice of a dog’s mouth.

67

The description is from Hirasawa and Takada (1994). Cellular and colonial morphology and other characteristics are as described for the genus and as given in Table 15. Susceptible to penicillin, amoxicillin, sulbenicillin, and erythromycin. Source: gingival crevicular fluid obtained from beagles. DNA G+C content (mol%): 44–45 (Tm). Type strain: NUM 402, ATCC 55563. Sequence accession no. (16S rRNA gene): DQ677836. Taxonomic note: Strain NCTC 12858T of Porphyromonas cansulci Collins et al. 1994, 678VP exhibits 99.9% rRNA gene sequence homology with Porphyromonas crevioricanis ATCC 55563T. The taxonomic standing of these two species remains to be determined. 8. Porphyromonas endodontalis (van Steenbergen, VanWinkelhoff, Mayrand, Grenier and de Graaff 1984) Shah and Collins 1988, 129VP (Bacteroides endodontalis van Steenbergen, VanWinkelhoff, Mayrand, Grenier and de Graaff 1984, 119VP) en.do.don′ta.lis. Gr. adv. endon within; Gr. n. odous -ontos tooth; L. fem. suff. -alis suffix denoting pertaining to; N.L. fem. adj. endodontalis pertaining to the inside of a tooth, within teeth. The description is from Shah and Collins (1988) van Steenbergen et  al. (1984), and Holdeman et  al. (1984). Protoheme is the major porphyrin produced, but protoporphyrin is also present. Most strains produce colonies that adhere strongly to the agar plates, and growth in liquid media is slow. Gelatin is hydrolyzed. No proteolytic or collagenolytic activity is detected. Arginine is hydrolyzed; starch is not hydrolyzed. H2S is produced. Cellular and colonial morphology and other characteristics are as described for the genus and as given in Table 15. Specific primers based on 16S rRNA sequencing have been described for culture-independent identification of Porphyromonas endodontalis (de Lillo et al., 2004). Similarly, the use of PCR amplification of 16S rDNA and the downstream intergenic spacer region (ISR) for culture-independent detection of periodontal pathogens revealed that Porphyromonas endodontalis was as strongly associated with periodontitis as Porphyromonas gingivalis (Kumar et al., 2003). Source: infected dental root canals, periodontal pockets, and other oral sites. DNA G+C content (mol%): 49–51 (Tm). Type strain: HG370, ATCC 35406, NCTC 13058. Sequence accession no. (16S rRNA gene): L16491. 9. Porphyromonas gingivalis (Coykendall, Kaczmarek and Slots 1980) Shah and Collins 1988, 129VP (Bacteroides gingivalis Coykendall, Kaczmarek and Slots 1980, 559VP) gin.gi.val′is. L. n. gingiva gum; L. fem. suff. -alis suffix denoting pertaining to; N.L. fem. adj. gingivalis pertaining to the gums, gingival. The description is from Shah and Collins (1988) and Holdeman et al. (1984). Protoheme is the major porphyrin produced, but traces of protoporphyrin also occur. Several amino acids, such as aspartate, arginine, cystine, histidine, serine, tryptophan, leucine, methionine, phenylalanine, and isoleucine, are utilized. Proteases are present. Starch is not hydrolyzed. Cellular and colonial morphology and other characteristics are as described for the genus and as given in Table 15.

68

Family III. Porphyromonadaceae

Porphyromonas gingivalis possesses a multitude of cell-surfaceassociated and extracellular activities, such as adhesins, secreted or cell-bound enzymes, toxins, and hemolysins that contribute to its virulence potential. Differences in the virulence exist, but the mechanisms underlying these differences are not yet fully understood. Multilocus sequence typing of Porphyromonas gingivalis strains from different geographic origins showed high genetic diversity of the species (Enersen et al., 2006). Porphyromonas gingivalis is generally very susceptible to most of the antimicrobial agents commonly used for treatment of anaerobic infections, such as amoxicillinclavulanate, piperacillin-tazobactam, ampicillin-sulbactam, imipenem, cephalosporins, and metronidazole. Ciprofloxacin resistance of Porphyromonas gingivalis has been reported (Lakhssassi et al., 2005). Porphyromonas gingivalis is considered a major periodontal pathogen and reported to be a cause of extraoral infections, such as lung abscesses and pulmonary infections. It has also been suggested that Porphyromonas gingivalis may contribute to the development of atheromas in cardiovascular disease. Source: infected dental root canals, periodontal pockets, and other oral sites. DNA G+C content (mol%): 46–48 (Tm). Type strain: 2561, ATCC 33277, CCUG 25893, CCUG 25928, CIP 103683, DSM 20709, JCM 12257, NCTC 11834. Sequence accession no. (16S rRNA gene): L16492. 10. Porphyromonas gingivicanis Hirasawa and Takada 1994, 639VP gin.gi.vi.ca′nis. L. fem. n. gingiva gum; L. gen. n. canis of a dog; N.L. gen. n. gingivicanis of the gums of a dog. The description is from Hirasawa and Takada (1994). Cellular and colonial morphology and other characteristics are as described for the genus and as given in Table 15. Susceptible to penicillin, amoxicillin, sulbenicillin, and erythromycin. Source: gingival crevicular fluid obtained from beagles. DNA G+C content (mol%): 41–42 (Tm). Type strain: NUM 301, ATCC 55562. Sequence accession no. (16S rRNA gene): DQ677835. 11. Porphyromonas gulae Fournier, Mouton, Lapierre, Kato, Okuda and Menard. 2001, 1187VP gu¢lae. L. n. gula [animal] mouth; L. gen. n. gulae from the animal mouth, referring to its isolation from subgingival plaque of various animal hosts. The description is from Fournier et  al. (2001). Porphyromonas gulae encompasses the “animal Porphyromonas gingivalis”. Cellular and colonial morphology and other characteristics are as described for the genus and as given in Table 15. Twenty strains were susceptible to amoxicillin and amoxycillin/clavulanate. Source: subgingival plaque samples of various mammals, including cat, dog, coyote, wolf, bear, and non-human primate, such as squirrel monkey and spider monkey. Porphyromonas gulae is the most prominent species of the genus Porphyromonas to be found in the oral cavity of mammals. DNA G+C content (mol%): 51 (Tm). Type strain: Loup 1, ATCC 51700, CCUG 47701, NCTC 13180. Sequence accession no. (16S rRNA gene): AF208290.

12. Porphyromonas levii (Johnson and Holdeman 1983) Shah, Collins, Olsen, Paster and Dewhirst 1995, 586VP (Bacteroides levii Johnson and Holdeman 1983, 15VP) lev′i.i. N.L. gen. masc. n. levii of Lev, named after Meir Lev, the American-English microbiologist, who first isolated this organism. The description is from Shah et  al. (1995), Holdeman et al. (1984), and Summanen et al. (2005). Cellular and colonial morphology and other characteristics are as described for the genus and as given in Table 15. Protoheme is the major porphyrin produced, but traces of protoporphyrin also occur. Succinate stimulates growth and can replace the requirement for heme. Few sugars, such as glucose and lactose, are weakly fermented. Glucose broth cultures are turbid with smooth sediment and a final pH of 5.5. Most other commonly occurring sugars, such as arabinose, cellobiose, melezitose, melibiose, raffinose, rhamnose, ribose, salicin, sucrose, trehalose, and xylose, are not fermented. Some amino acids, such as asparagine, tryptophan, phenylalanine, and glutamine, are utilized. Proteases are present. Starch is not hydrolyzed. Porphyromonas levii is genetically distant from Porphyromonas somerae; however, these two species cannot readily be differentiated by phenotypic tests. Although both species contain major C15:0 iso, C15:0 anteiso acids, the cluster analysis of the cellular fatty acid profiles (Euclidian distance of principal components accounting for the greatest variance of the organisms) differentiates the two species. Source: bovine rumen, cattle horn abscess, bovine summer mastitis, and bovine necrotizing vulvovaginitis. DNA G+C content (mol%): 46–48 (Tm). Type strain: LEV, ATCC 29147, CCUG 21027, CCUG 34320, HAMBI 467, NCTC 11028, VPI 10450, VPI 3300. Sequence accession no. (16S rRNA gene): L16493. 13. Porphyromonas macacae (Slots and Genco 1980) Love 1995, 91VP [Bacteroides melaninogenicus subsp. macacae Slots and Genco 1980, 84VP; Bacteroides macacae (Slots and Genco 1980) Coykendall, Kaczmarek and Slots 1980, 563VP; Porphyromonas salivosa Love, Bailey, Collings and Briscoe 1992, 438VP] ma.ca′cae. N.L. fem. n. Macaca genus name of the macaque; N.L. gen. n. macacae of the macaque. The description is from Love et al. (1995) and Holdeman et  al. (1984). After 6 d on blood agar plates, colonies are 0.1–0.2 mm, entire, dome shaped, and creamy brown. After 9  d in the presence of Staphylococcus epidermidis, however, the colonies are 1.0–1.5  mm in diameter, entire, umbonate with a central depression. As incubation progresses, the surfaces of colonies may become wrinkled with multiple central depressions and peripheral ridging. Gelatin is hydrolyzed. Lipase is not produced. Other characteristics are as described for the genus and as given in Table 15. Porphyromonas macacae ATCC 33141 can be distinguished by phenotypic criteria from cat strains, suggesting that cat and monkey biovars exist. (See discussion of Porphyromonas salivosa under Taxonomic note, below.) Source: oral cavities, subcutaneous abscesses, and pyothoraxes of animals, including cats and monkeys. Important pathogen in animal bite infections in humans.



Genus I. Porphyromonas

DNA G+C content (mol%): 43–44 (Tm). Type strain: 7728-L6C, Slots’ strain 7728-L6C, ATCC 33141, CCUG 47703, DSM 20710, NCTC 13100. Sequence accession no. (16S rRNA gene): L16494. Taxonomic note: Strain NCTC 11632T of Porphyromonas salivosa (Love et  al., 1987) Love et  al., 1992, 438VP exhibits 99.3% rRNA gene sequence homology (Paster et  al., 1994) and a mean level of DNA–DNA hybridization of 81% (Love, 1995) with Porphyromonas macacae ATCC 33141T; therefore, the species Porphyromonas salivosa was included in the species Porphyromonas macacae by Love (1995). However, Porphyromonas macacae ATCC 33141T can be distinguished by phenotypic criteria from cat strains (Porphyromonas salivosa), suggesting that cat and monkey biovars of Porphyromonas macacae exist. The members of the cat biovar have different colonial morphologies on blood agar: the colonies are 0.5–1.5  mm in diameter, circular, entire, dome-shaped, and brown– black at 72 h on blood agar plates. Furthermore, unlike Porphyromonas macacae ATCC 33141T, the cat biovars produce different whole-cell protein and proteinase profiles on SDS-PAGE gels, and are lipase-positive and sorbitolnegative. 14. Porphyromonas salivosa (Love, Johnson, Jones, and Calverley 1987) Love, Bailey, Collings and Briscoe 1992, 438VP = Porphyromonas macacae (senior heterotypic synonym) (Bacteroides salivosus Love, Johnson, Jones and Calverley 1987, 308) sal.i.vo¢sa. L. fem. adj. salivosa resembling saliva, slimy. The description is from Love et  al. (1995), Love et  al. (1992), and Love et  al. (1987). After 72  h on blood agar plates, colonies are 0.5–1.5 mm, entire, dome shaped, and brown–black. Gelatin is hydrolyzed. Lipase is produced. Porphyromonas salivosa NCTC 11632T can be distinguished by phenotypic criteria from monkey strains (Porphyromonas macacae), suggesting that cat and monkey biovars exist. (See Taxonomic note, above, under Porphyromonas macacae.) DNA G+C content (mol%): 42–44 (Tm). Type strain: ATCC 49407, NCTC 11632, VPB 157, CCUG 33478. Sequence accession no. (16S rRNA gene): L26103. 15. Porphyromonas somerae Summanen, Durmaz, Vaisanen, Liu, Molitoris, Eerola, Helander and Finegold 2006, 925VP (Effective publication: Summanen, Durmaz, Vaisanen, Liu, Molitoris, Eerola, Helander and Finegold 2005, 4458.) so′mer.ae. N.L. gen. fem. n. somerae, of Somer, named in honor of the late Finnish microbiologist Hannele Jousimies-Somer. The description is from Summanen et al. (2005). Colonies incubated on blood agar for 2 d often exhibit a “patchy” growth pattern, with larger colonies surrounded by smaller colonies; they are circular, entire, and convex. The colonies on laked rabbit blood agar are white–yellow to tan; after 4 d of incubation, the colonies are pigmented (light brown to dark brown) and show no or occasionally weak red fluorescence under long-wave UV light. Weakly saccharolytic, the pH of glucose, lactose, and maltose cultures is 5.3–5.4 after 5  d of incubation. Most other commonly occurring

69

sugars, such as arabinose, cellobiose, melezitose, melibiose, raffinose, rhamnose, ribose, salicin, sucrose, trehalose, and xylose are not fermented. b-lactamase is produced by 21% of the strains. Some strains are resistant to clindamycin. Other characteristics are as described for the genus and as given in Table 15. Porphyromonas somerae is genetically distant from Porphyromonas levii; however, these two species cannot readily be differentiated by phenotypic tests. Although both species contain major C15:0 iso, C15:0 anteiso acids, the cluster analysis of the cellular fatty acid profiles (Euclidian distance of principal components accounting for the greatest variance of the organisms) differentiates the two species. Source: various clinical specimens of non-oral origin, mainly from chronic foot infections of diabetics or other patients with vascular insufficiency. DNA G+C content (mol%): 47.8 (Tm). Type strain: WAL 6690, ATCC BAA-1230, CCUG 51464. Sequence accession no. (16S rRNA gene): AY968205. 16. Porphyromonas uenonis Finegold, Vaisanen, Rautio, Eerola, Summanen, Molitoris, Song, Liu and Jousimies-Somer 2005, 547VP (Effective publication: Finegold, Vaisanen, Rautio, Eerola, Summanen, Molitoris, Song, Liu and Jousimies-Somer 2004, 5301.) ue.no¢nis. N.L. gen. masc. n. uenonis of Ueno, in honor of the late Japanese microbiologist Kazue Ueno. The description is from Finegold et al. (2004). Growth is stimulated by 5% horse serum and similar additives. Cellular and colonial morphology and other characteristics are as described for the genus and as given in Table 15. Porphyromonas uenonis can be distinguished from ­Porphyromonas asaccharolytica by phenotypic tests (Table 15); however, DNA–DNA reassociation studies are required to distinguish these two species genetically. Susceptible to most antimicrobial agents. Some strains produce b-lactamase. Source: part of a mixed flora in various infections, which apparently have their origin in the intestinal tract. The habitat is probably the human gut. DNA G+C content (mol%): 52.5 (Tm). Type strain: WAL 9902, ATCC BAA-906, CCUG 48615. Sequence accession no. (16S rRNA gene): AY570514.

Other organisms Porphyromonas bennonis, a novel species isolated from human clinical specimens, was recently published in IJSEM ­(Summanen et al., 2009). DNA G+C content (mol%): 58 (Tm). Type strain: WAL 1926C, ATCC BAA-1629, CCUG 55979. Sequence accession no. (16S rRNA gene): EU414673. The 16S rRNA gene sequence of an organism called “Porphyromonas canis” was published in GenBank with accession number AB0034799. “Porphyromonas canis sp. nov. isolated from dog”. Unpublished. Strain ATCC 55562T of Porphyromonas gingivicanis Hirasawa and Takada 1994, 640VP exhibits 99.9% rRNA gene sequence homology with “Porphyromonas canis”

70

Family III. Porphyromonadaceae

Taxonomic note: Hardham, Dreier, Wong, Sfintescu and Evans (2005) mentioned an organism named “Porphyromonas denticanis”. They indicated that Porphyromonas salivosa, “Porphyromonas denticanis” (a novel species), and Porphyromonas gulae were the most frequently isolated black-pigmented anaerobic bacteria associated with canine periodontitis. However, no valid description of “Porphyromonas denticanis” can be found in the literature, and there are no GenBank entries for it.

JCM 10100, suggesting that Porphyromonas canis is a later synonym of Porphyromonas gingivicanis. No valid description of the species “Porphyromonas canis” can be found in the literature. However, the following information is available. DNA G+C content (mol%): 52.5 (Tm). Type strain: JCM 10100. Sequence accession no. (16S rRNA gene): AB034799.

Genus II. Barnesiella Sakamoto, Lan and Benno 2007b, 344VP The Editorial Board Bar.ne.si.el′la. N.L. dim. fem. n. Barnesiella named after the British microbiologist Ella M. Barnes, who ­contributed much to knowledge of intestinal bacteriology and to anaerobic bacteriology in general.

Rods (0.8–1.6  ×  1.7–11  mm). Nonsporeforming. Nonmotile. Gram-negative. Obligately anaerobic. On Eggerth–Gagnon agar, colonies are 1–2 mm in diameter, gray to off-white–gray, circular, entire, slightly convex, and smooth. Saccharolytic, with a strictly fermentative type of metabolism. Acetic and succinic acids are the main fermentation products. Growth is inhibited on a medium containing 20% bile. Esculin is hydrolyzed. Indole-negative. The predominant menaquinones are MK-11 and MK-12. Isolated from the chicken cecum. DNA G+C content (mol%): 52. Type species: Barnesiella viscericola Sakamoto, Lan and Benno 2007b, 345VP.

had menaquinones 8, 9, or 10 (except Tannerella, which has MK-10 and MK-11). Phylogenetic analysis has shown that these strains represent a new genus in the family.

Enrichment and isolation procedures Barnesiella strains were isolated from the chicken cecum under strictly anaerobic conditions by the method described by Lan et  al. (2002). They were maintained on Eggerth– Gagnon (EG) agar (Merck) supplemented with 5% (v/v) horse blood, with incubation for 2  d at 37°C in an atmosphere of 100% CO2.

Differentiation of the genus Barnesiella from other closely related genera

Further descriptive information

Some characteristics differentiating the genus Barnesiella from other genera are shown in Table 16.

Phylogenetic analyses of chicken cecal microbiota, based on 16S rRNA gene clone library analyses, have revealed a large number of novel phylotypes (Lan et al., 2002; Zhu et al., 2002). Using a special anaerobic culture technique, Sakamoto et  al. (2007b) obtained unusual strains. Like Porphyromonas strains, these obligately anaerobic, nonsporeforming, nonmotile, Gram-negative rods were inhibited by 20% bile. However, their major menaquinones (MK-11 and MK-12) differed from those of most other genera of the family Porphyromonadaceae, which

Taxonomic comments Analyses of the 16S rRNA gene sequences of two cecal isolates that failed to grow in the presence of 20% bile indicated that the strains were related to Parabacteroides distasonis (86% sequence similarity). The two strains exhibited 100% 16S rRNA gene sequence similarity with each other.

Major menaquinones Brown to black colonies on blood agar DNA G+C content (mol%)

Dysgonmonas

Paludibacter

Parabacteroides

Porphyomonas

Proteiniphilum

Tannerella

Characteristic Growth in presence of 20% bile Saccharolytic Major end products of glucose fermentation

Barnsiella

TABLE 16.  Some characteristics differentiating the genus Barnesiella from other related generaa,b

-

+

-

+

-

-

-

+ + Acetic, succinic Propionic, acids lactic, succinic acids MK-10, MK-11 nr

+ + Acetic, propi- Acetic, succinic onic acids acids

na

na

na

MK-8

MK-9, MK-10

MK-9, MK-10

nr

MK-10, MK-11

-

-

-

-

+

-

-

52

38–39

39

43–46

40–55

47

44–48

Symbols: +, >85% positive; -, 0–15% positive; na, not applicable; nr, not reported. Data taken from Chen and Dong (2005); Hofstad et al. (2000); Lawson et al. (2002a); Sakamoto and Benno (2006); Sakamoto et al. (2002); Ueki et al. (2006); Sakamoto et al. (2007b).

a

b



Genus III. Dysgonomonas

71

List of species of the genus Barnesiella 1. Barnesiella viscericola Sakamoto, Lan and Benno 2007b, 345VP vis.ce.ri′co.la. L. n. viscus, visceris intestine; L. suff. n. -cola (from L. n. incola) inhabitant; N.L. fem. n. viscericola inhabitant of the intestine. The characteristics are as given for the genus, with the following additional features. Urease and catalase-negative. Gelatin is digested. Acid is produced from d-cellobiose, glucose, maltose, d-mannose, and sucrose, but not from l-arabinose, glycerol, lactose, d-mannitol, d-melezitose, d-raffinose, l-rhamnose, salicin, d-sorbitol, d-trehalose, or d-xylose. Using the Rapid ID 32A tests, all strains are positive for a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, glutamic acid

decarboxylase, a-fucosidase, alkaline phosphatase, leucylglycine arylamidase, and alanine arylamidase. Raffinose is fermented. All of the other tests with the Rapid ID 32A system give negative results. The major end products are acetic acid and succinic acid; lower levels of other acids may be produced. Both non-hydroxylated and 3-hydroxylated long-chain fatty acids are present. The major cellular fatty acids are C15:0 anteiso and C15:0 iso. The predominant respiratory quinones are MK-11 (65–66%) and MK-12 (21–24%). MK-10 is present as a minor menaquinone (10–11%). Source: the chicken cecum. DNA G+C content (mol%): 52 (HPLC). Type strain: C46, DSM 18177, JCM 13660. Sequence accession no. (16S rRNA gene): AB267809.

Genus III. Dysgonomonas Hofstad, Olsen, Eribe, Falsen, Collins and Lawson 2000, 2194VP Ingar Olsen Dys.go.no.mo′nas. Gr. pref. dys- with notion of hard, bad, unlucky; Gr. n. gonos that which is begotten, reproduction; Gr. fem. n. monas a monad, unit; N.L. fem. n. Dysgonomonas intended to mean a weakly growing monad.

Coccobacilli to short rods. Nonmotile. Gram-negative. Facultatively anaerobic. Colonies are 1–2 mm in diameter, nonadherent, entire, gray–white, smooth, and nonhemolytic and have a slight aromatic odor. Growth is not observed on MacConkey agar. Requires X factor for growth. May be catalase-positive or -negative. Oxidase-negative. Glucose is fermented, producing acid but no gas. Alkaline phosphatase is generated but not arginine dihydrolase. Nitrate is not reduced. H2S and acetoin are not produced. Esculin may not be hydrolyzed; gelatin and urea are not hydrolyzed. Indole may be produced. Long-chain cellular fatty acids include straight-chain saturated, anteiso- and iso-methyl branched and 3-hydroxy types. Isolated from human clinical specimens and stools. DNA G+C content (mol%): 38 (Tm). Type species: Dysgonomonas gadei Hofstad, Olsen, Eribe, Falsen and Lawson 2000, 2194VP.

Further descriptive information Dysgonomonas capnocytophagoides (Hofstad et al., 2000) and Dysgonomonas mossii (Lawson et al., 2002a, b) are members of the CDC DF (dysgonic fermenter)-3 group (Daneshvar et al., 1991; Wallace et al., 1989). Dysgonomonas gadei (Hofstad et al., 2000) was isolated at the Gade Institute, Bergen, Norway. Comparative 16S rRNA sequence analysis indicates that Dysgonomonas is a distinct genus in Family III. “Porphyromonadaceae” in the phylum “Bacteroidetes”. See Taxonomic comments for details. Cells can be coccobacilli to short rods (Figures 18 and 19). Nitrate is not reduced and the oxidase reaction is negative. The catalase reaction has been reported as negative (Koneman et  al., 1997) and as negative or positive (Hofstad et  al., 2000). The organisms produce acid by fermentation of glucose (Hofstad et  al., 2000; Koneman et  al., 1997), xylose and maltose; most strains produce acid from sucrose and lactose,

FIGURE 18.  Scanning electron microscopy of cells from colonies of Dysgonomonas gadei CCUG 42886T (a), Dysgonomonas mossii CCUG 43457T (b), and Dysgonomonas capnocytophagoides CCUG 17996T (c). Cells were cultured anaerobically for 48 h at 37°C on human blood agar supplemented with hemin and vitamin K.

72

Family III. Porphyromonadaceae

FIGURE 19.  Transmission electron microscopy of Dysgonomonas gadei CCUG 42886T (a), Dysgonomonas mossii CCUG 43457T (b), and Dysgonomonas capnocytophagoides CCUG 17996T (c). Cells were cultured anaerobically for 48 h at 37°C on human blood agar supplemented with hemin and vitamin K. OM, outer membrane; IM, inner cytoplasmic membrane; G, granule; SLPS, scale-like protrusion.

but not from mannitol (Koneman et al., 1997). Esculin may be hydrolyzed (Hofstad et  al., 2000; Koneman et  al., 1997). Furthermore, alkaline phosphatase is produced but not arginine dihydrolase. H2S and acetoin are not produced. Indole may be generated (Hofstad et al., 2000). Isolates of Dysgonomonas species are rare (Koneman et  al., 1997; Martínez-Sánchez et al., 1998). They have been recovered from clinical sources such as blood, wounds, urine, peritoneal fluid, umbilicus, stools, and gallbladder. Asymptomatic carriers have been found. The first report on these organisms was made by Wagner et al. (1988), who made multiple isolations in pure culture from the stools of an elderly woman with common variable hypogammaglobulinemia of long standing. DF-3 was also found in stool specimens by Gill et  al. (1991) and Grob et  al. (1999); several of the patients suffered from prolonged diarrhea. In another study with immunocompromised patients or patients with severe underlying disease, including HIV or inflammatory bowel disease, DF-3 was isolated from stool specimens during a 1-year period (Blum et al., 1992); the clinical spectrum associated with DF-3 ranged from asymptomatic carrier state to symptomatic with chronic diarrhea. Heiner et al. (1992) described an association of enteric DF-3 infection with HIV coinfection and common variable hypogammaglobulinemia. Further, Aronson and Zbick (1988) isolated DF-3 from a 24-year-old man with relapse of acute lymphocytic leukemia, and Grob et al. (1999) detected DF-3 in bacteremia from a patient with acute myelocytic leukemia during aplasia. The former patient became granulocytopenic during intensive chemotherapy and DF-3 was isolated from blood cultures. Recently, a case of Dysgonomonas capnocytophagoides in blood culture from a severely neutropenic patient treated for acute myeloid leukemia was reported (Hansen et al., 2005). A soft-tissue abscess in a diabetic patient and a postoperative urinary tract infection in an 81-year-old man yielded DF-3, and in the latter case, DF-3 was recovered together with Escherichia coli (Bangsborg et al., 1990; Schonheyder et al., 1991). DF-3 has also been isolated together with Candida albicans, Candida glabrata, Staphylococcus aureus, and enterococci (Lawson et  al., 2002a). Melhus (1997) recovered DF-3 from a decubitous ulcer of a subfebrile patient with diarrhea. Disk diffusion and broth dilution have been used to assess antimicrobial susceptibility of DF-3 isolates (Aronson and Zbick, 1988; Blum et al., 1992; Gill et al., 1991; Heiner et al., 1992; Wagner et  al., 1988). The DF-3 strains were resistant to penicillin,

ampicillin, ampicillin-sulbactam, aztreonam, aminoglycosides, cephalosporins (including cephalotin, cefoxitin, ceftriaxone, cefoperazone, and ceftazidime), erythromycin, ciprofloxacin, and vancomycin (Koneman et al., 1997). They are usually susceptible to trimethoprim-sulfamethoxazole and chloramphenicol, and variably susceptible to piperacillin, clindamycin, tetracycline, and imipenem. Dysgonomonas gadei was sensitive to metronidazole, clindamycin, doxycycline, imipenem, meropenem, and trimethoprim/sulfamethoxazole (Hofstad et al., 2000). The organism was resistant to cefoxitin and other cephalosporins (cefotaxime, cefpirome, ceftazidime, ceftriaxone, cefuroxime, and cephalotin), aminoglycosides (gentamicin, netilmicin, sulfadiazine), fluoroquinolones (ciprofloxacin, oxafloxacin), vancomycin, and teicoplanin. Similarly, Dysgonomonas capnocytophagoides was susceptible to ampicillin, tetracycline, chloramphenicol, clindamycin, and trimethoprim-sulfamethoxazole, while it was resistant to penicillin, cephalosporins, meropenem, aminoglycosides, and ciprofloxacin (Hansen et al., 2005).

Enrichment and isolation procedures Culture from stool specimens is best performed on cefoperazonevancomycin-amphotericin blood agar incubated at 35°C in 5–7% CO2 (Koneman et al., 1997). Growth is relatively slow with pinpoint colonies visible after 24 h. After 48–72 h the colonies turn gray-white, smooth, and are nonhemolytic. A sweet odor may be produced by the organism on agar media (Bernard et al., 1991; Blum et al., 1992; Gill et al., 1991; Wagner et al., 1988).

Taxonomic comments In 2000, Hofstad et al. isolated an organism from a human gallbladder that resembled CDC Group DF-3 organisms and named it Dysgonomonas gadei, thereby creating a new genus Dysgonomonas. The authors simultaneously reclassified the organisms previously designated CDC group DF-3 as Dysgonomonas capnocytophagoides. Lawson et al. (2002a, b) described another Dysgonomonas species, Dysgonomonas mossii, from human clinical specimens. The CDC Group DF-3 has been considered closely related to Capnocytophaga species. After whole-cell protein ­electrophoresis, a separate position was occupied by a DF-3 strain when compared to well-characterized reference strains representing seven Capnocytophaga species (Vandamme et al., 1996). The whole-cell protein pattern of Dysgonomonas gadei was separate from those of two DF-3 strains and reference strains of Capnocytophaga, ­Bacteroides,



Genus III. Dysgonomonas

and Prevotella (Hofstad et al., 2000). The nearest correlation was seen with Bacteroides uniformis at approximately 58%. The cell walls of DF-3 strains had large amounts (24%) of anteiso-branched-chain fatty acid (C15:0 anteiso), moderate amounts of saturated iso-branched-chain acids (C14:0 iso and C15:0 iso), and small to moderate amounts of both branchedand straight-chain hydroxy acids (C15:0 3-OH, C16:0 iso 3-OH, C16:0 3-OH, and C17:0 iso 3-OH) (Wallace et al., 1989). The dominating cellular fatty acid content of Dysgonomonas gadei CCUG 42882T is C15:0 anteiso, C16:0, C14:0 iso, and C16:0 iso 3-OH, which is similar to that of Dysgonomonas capnocytophagoides CCUG 17996T and CCUG 42515 (Hofstad et al., 2000). For comparison, Moore et al. (1994) found that C15:0 anteiso, C15:0 iso, C17:0 iso 3-OH, and C16:0 are the major cellular fatty acids in Bacteroides and Prevotella, and C15:0 iso the major cellular fatty acid of Porphyromonas. Bernard et  al. (1991) detected C15:0 iso and C17:0 iso 3-OH as the major cellular fatty acids in Capnocytophaga. The overall fatty acid composition of DF-3 organisms and Capnocytophaga species is clearly different from that of CDC group DF-3-like organisms (Daneshvar et  al., 1991). The isoprenoid

73

quinone content of four group DF-3-like strains was similar with ubiquinone-9 (Q-9) and Q-10 as the major quinone, whereas two other group DF-3-like strains had Q-7 as their major quinones, with smaller amounts of Q-8 and Q-9. Notably, CDC group DF-3 strains F9489 and G4990 did not contain quinones. Bernard et al. (1991) found that CDC group DF-3 organisms produced significant quantities of propionic acid as metabolic product. Also lactic and succinic acids were detected, but the quantities varied among strains. Comparative 16S rRNA sequence analysis has indicated that CDC group DF-3 is phylogenetically related to but different from Bacteroides, Porphyromonas, Prevotella, and kindred organisms (Paster et al., 1994; Vandamme et al., 1996). Actually, Dysgonomonas gadei and Dysgonomonas capnocytophagoides formed a distinct phylogenic cluster within the Bacteroides–Prevotella–Porphyromonas group (Hofstad et al., 2000). Sequence analysis showed that Dysgonomonas mossii differed from Dysgonomonas gadei (91.7%) and Dysgonomonas capnocytophagoides (94.3%) (Lawson et  al., 2002a). Other taxa were related to these species (Figure 20) but showed much lower levels of sequence ­similarity, including

FIGURE 20.  Phylogenetic tree of Dysgonomonas species and related organisms. (Reproduced with permission from Lawson et al., 2002a. Syst. Appl. Microbiol. 25: 194–197.)

74

Family III. Porphyromonadaceae

Bacteroides (85–87%), Porphyromonas (84–88%), and Prevotella (79–85%) and the misclassified strict anaerobic species Bacteroides distasonis (87%), Bacteroides forsythus (88%), Bacteroides merdae (89%), and Bacteroides splanchnicus (83%) (Hofstad et  al., 2000; Lawson et al., 2002a). Comparative sequence 16S rRNA analysis unequivocally demonstrated three strains in a hitherto unrecognized subline within the Dysgonomonas clade (Lawson et al., 2002a) Divergence values of >5% with Dysgonomonas capnocytophagoides and Dysgonomonas gadei indicated that they may merit classification as a distinct species. Dewhirst et al. (1999) found that strain ASF 519 of Bacteroides distasonis fell into an unnamed genus containing Bacteroides distasonis, Bacteroides merdae, Bacteroides forsythus, and CDC group DF-3. Capnocytophaga

species were clearly distinct from Dysgonomonas (Hofstad et al., 2000).

TABLE 17.  Salient characteristics of the species of the genus Dysgon-

TABLE 18.  Fermentation profiles of species of the genus Dysgonomonas a

Differentiation of the species of the genus Dysgonomonas Similarities and differences between the three Dysgonomonas spp. are listed in Tables 17–19. Contrary to the other species Dysgonomonas gadei produces catalase. Dysgonomonas gadei can further be distinguished from Dysgonomonas capnocytophagoides and Dysgonomonas mossii by the absence of b-glucuronidase production by the latter two. Unlike the other two species, Dysgonomonas capnocytophagoides ferments trehalose but does not produce N-acetyl-bglucosaminidase and a-fucosidase. Dysgonomonas mossii does not generate glutamyl glutamic acid arylamidase.

Gray–white

Slight Coccobacilli   + + + + + + + + + nd -

Slight Slight Coccobacilli Coccobacilli and short rods and short rods     + + + + + + nd + nd + + d d + nd -

+ + nd + + -

Adonitol, dulcitol l-Arabinose, lactose, d-mannose, sucrose, d-xylose d-Arabitol, l-arabitol Cellobiose, fructose, salicin Erythritol, glycogen Inositol, d-mannitol Maltose Melibiose, melezitose Raffinose l-Rhamnose d-Ribose d-Sorbitol Starch Trehalose

D. mossii

Gray–white

D. capnocytophagoides

Gray–white

Acid produced from

D. gadei

Growth on blood agar:   CO2 required   at 25°C   at 35–37°C   at 42–43°C   Microaerophilic   Anaerobic Growth on MacConkey agar Catalase Indole Acetoin Esculin hydrolysis Gelatin hydrolysis H2S production Nitrate reduction Oxidase Resistant to ox bile Starch hydrolysis Urea hydrolysis

D. mossii

Colony pigmentation Aromatic odor Cells

D. capnocytophagoides

Characteristic

D. gadei

omonas a

+

+

nd +

nd + nd + + + w + +

nd nd + + + nd nd nd -

nd + nd w + nd d w nd nd nd +

a Symbols: +, >85% positive; -, 0–15% positive; w, weak reaction; nd, not determined.

a Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); -, 0–15% positive; nd, not determined.

List of species of the genus Dysgonomonas 1. Dysgonomonas gadei Hofstad, Olsen, Eribe, Falsen, Collins and Lawson 2000, 2194VP ga′de.i. N.L. gen. masc. n. gadei of the Gade Institute, Bergen, Norway, where the organism was first isolated. The following description is according to Hofstad et  al. (2000). Nonmotile, Gram-negative coccobacilli that grow relatively slowly on blood agar. After 48  h of aerobic ­incubation at 35°C in a CO2-enriched atmosphere, the col-

onies are 1–2  mm in diameter, nonadherent, entire, gray– white, smooth, and nonhemolytic, with a slight aromatic odor. After incubation for a few more d the colonies become coalesced, butyrous, and a-hemolytic. Growth can be seen at 25°C but not at 43°C. Growth occurs under microaerophilic and strictly anaerobic conditions. No growth occurs on MacConkey agar but does occur on nutrient agar around X and XV discs, suggesting a growth dependence for heme. Catalase-positive and oxidase-negative. No nitrate reduction



75

Genus III. Dysgonomonas

TABLE 19.  Enzymic profiles of species of the genus Dysgonomonas a,b

Enzyme a-Arabinosidase Acid phosphatase, phosphoamidase a-Fucosidase a-Galactosidase, a-glucosidase, alanine, arylamidase, alkaline phosphatase, b-glucosidase, leucyl glycine arylamidase a-Mannosidase Arginine arylamidase, arginine, dihydrolase, glutamic acid, decarboxylase, glycine arylamidase, histidine arylamidase, leucine arylamidase, proline arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, serine arylamidase, tyrosine arylamidase b-Galactosidase b-Galactosidase 6-phosphate b-Glucuronidase Chymotrypsin Cystine arylamidase, lipase C-14, valine arylamidase Esterase C-4 Ester lipase C-8 Glutamyl glutamic acid arylamidase Lysine decarboxylase, ornithine decarboxylase N-Acetyl-b-glucosaminidase Trypsin Urease

D. gadei

D. capnocytophagoides

D. mossii

+ + + +

+ + +

d nd + +

w -

-

w -

w +a + w + nd + + -

+ + d + nd

+ w nd nd nd nd nd + nd nd

Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); -, 0–15% positive; nd, not determined. Positive with API ID32A and rapid ID32E, negative with API ZYM.

a

b

or production of H2S or acetoin. Esculin is hydrolyzed, but gelatin and urea are not. Indole-positive. Resistant to ox bile. Glucose is fermented with production of acid but no gas. Acid is also produced from l-arabinose, cellobiose, fructose, lactose, d-mannose, melezitose, melibiose, raffinose, l-rhamnose, d-ribose (weak reaction), salicin, starch, sucrose, trehalose, and xylose. Positive reactions occur for N-acetyl-b-glucosaminidase, acid phosphatase, alanine arylamidase, alkaline phosphatase, a-arabinosidase, ester lipase C8 (weak), a-galactosidase, b-galactosidase (weak reaction), a-glucosidase, b-glucosidase, glutamyl glutamic acid arylamidase, a-mannosidase (weak), a-fucosidase, chymotrypsin, alanine arylamidase, leucyl glycine arylamidase, phosphoamidase, and trypsin. Positive and negative reactions are summarized in Tables 17–19. Source: an infected human gallbladder, but the habitat is unknown. DNA G+C content (mol%): not determined. Type strain: ATCC BAA-286, CCUG 42882, CIP 106420. Sequence accession no. (16S rRNA gene): Y18530. 2. Dysgonomonas capnocytophagoides Hofstad, Olsen, Eribe, Falsen, Collins and Lawson 2000, 2194VP (CDC Group DF-3, Wallace, Hollis, Weaver and Moss 1989, 735) cap.no.cy.to.pha.goi′des. N.L. n. Capnocytophaga a genus of CO2-requiring bacteria; L. suff. -oides (from Gr. suff. -eides, from Gr. n. eidos that which is seen, form, shape, figure) resembling, similar; N.L. adj. capnocytophagoides like Capnocytophaga, referring to some properties shared between these organisms. The following description is based on results obtained by Wallace et  al. (1989) and Hofstad et  al. (2000). Nonmotile, Gram-negative coccobacilli to short rods. Colonies are 1–2 mm in diameter, nonadherent, entire, gray–white, smooth, and non-hemolytic with a slight aromatic odor

after 48 h of aerobic incubation on blood agar at 35°C in a CO2-enriched atmosphere (7.5%). No growth on MacConkey agar. Facultatively anaerobic. Catalase- and oxidasenegative. Does not produce H2S or acetoin. Esculin may be hydrolyzed, but gelatin and urea are not. Indole may be produced. The organism is resistant to ox bile and does not reduce nitrate. Major products from glucose fermentation are propionic, lactic, and succinic acids. Glucose fermentation does not produce gas. Acid is produced from l-arabinose, lactose, maltose, d-mannose, melibiose, raffinose, sucrose, and d-xylose. Positive reactions occur for acid phosphatase, alanine arylamidase, alkaline phosphatase, a-arabinosidase, a-galactosidase, b-galactosidase, b-galactosidase 6-phosphate, a-glucosidase, b-glucosidase, glutamyl glutamic acid arylamidase, leucyl glycine arylamidase, and phosphoamidase. Esterase C-4 production is variable between strains. Positive and negative reactions are listed in Tables 17–19. Source: human clinical specimens, but the habitat is unknown. DNA G+C content (mol%): 38 (HPLC). Type strain: CCUG 17996, CIP 107043, LMG 11519. Sequence accession no. (16S rRNA gene): U41355. 3. Dysgonomonas mossii Lawson, Falsen, Inganäs, Weyant and Collins 2002a, 1915VP (Effective publication: Lawson, Falsen, Inganäs, Weyant and Collins 2002a, 194.) moss′i.i. N.L. gen. masc. n. mossii of Moss, to honor Claude Wayne Moss, an American microbiologist who has contributed much to microbial taxonomy. The following description is according to Lawson et  al. (2002a). Nonmotile, Gram-negative coccobacilli to short rods. After anaerobic incubation for 48 h at 37°C on blood agar, colonies are 1–2 mm in diameter, nonadherent, entire, gray–white, smooth, and nonhemolytic, and produce a

76

Family III. Porphyromonadaceae

slightly aromatic odor. Growth occurs at 25°C but not at 42°C. No growth occurs on MacConkey agar. The organisms grow on nutrient agar around X and XV disks, indicating that they have a requirement for heme. Catalase- and oxidase-negative. Nitrate is not reduced to nitrite. Indole is produced, but not acetoin. The organisms are resistant to ox bile. Esculin and starch are hydrolyzed, but gelatin and urea are not. Glucose is fermented with production of acid but no gas. Acid is produced (conventional methods) from l-arabinose, cellobiose, fructose, inositol (weak reaction), lactose, mannitol (weak reaction), maltose, l-rhamnose (weak reaction), salicin, sucrose, trehalose, and d-xylose. With API rapid ID

32A, acid is produced from mannose, and d-raffinose may be fermented. Positive reactions are obtained for N-acetyl-bglucosaminidase, alanine arylamidase, alkaline phosphatase, a-galactosidase, b-galactosidase, b-galactosidase 6-phosphate (weak), a-glucosidase, b-glucosidase, leucyl glycine arylamidase, a-mannosidase (weak reaction), and a-fucosidase. Arabinosidase may be produced. Positive and negative reactions are summarized in Tables 17–19. Source: clinical sources, but the habitat is not known. DNA G+C content (mol%): 38.5 (HPLC). Type strain: CDC F9489, CCUG 43457, CIP 107079. Sequence accession no. (16S rRNA gene): AJ319867.

Genus IV. Paludibacter Ueki, Akasaka, Suzuki and Ueki 2006, 43VP The Editorial Board Pa.lu.di.bac′ter. L. n. palus -udis a swamp, marsh; N.L. masc. n. bacter a rod; N.L. masc. n. Paludibacter rod living in swamps.

Rods (0.5–0.6 mm × 1.3–1.7 mm), with the ends usually round to slightly tapered. Nonmotile. Nonsporeforming. Gram-negative. Strictly anaerobic. Chemo-organotrophic. Optimum growth temperature, 30°C. No growth occurs at 37°C. Oxidase and catalase-negative. Nitrate is not reduced. Various sugars are fermented, and acetate and propionate are the major fermentation end products with succinate as a minor product. Major cellular fatty acids are C15:0 anteiso, C15:0, and C17:0 anteiso 3-OH. The major respiratory quinone is MK-8(H4). DNA G+C content (mol%): 39.3 (HPLC). Type species: Paludibacter propionicigenes Ueki, Akasaka, Suzuki and Ueki 2006, 43VP.

Further descriptive information Paludibacter propionicigenes was isolated from a rice plant residue (rice straw) sample collected from irrigated rice-field soil in the Shonai Branch of the Yamagata Agricultural Experimental Station (Fujishima-machi, Yamagata, Japan) during the flooding period of the field. Cells can be cultivated at 30°C under an atmosphere of 95% N2/5% CO2 in peptone-yeast (PY) broth supplemented with 1% glucose (PYG broth). Colonies on PY4S* agar are grayish white, translucent, circular with a smooth surface, and 1.0–1.5 mm in diameter after 48 h. Propionate and acetate are produced from

fermentation of glucose at a ratio of 2:1. The predominant cellular fatty acids are C15:0 anteiso (30.8%), C15:0 (19.0%), C17:0 anteiso 3-OH (17.9%), and C17:0 iso 3-OH (6.2%).

Enrichment, isolation, and maintenance procedures The type strain was isolated using the anaerobic roll tube method. PY4S agar can be used for maintenance of the strain on agar slants.

Differentiation of the genus Paludibacter from related genera Paludibacter propionicigenes is a strict anaerobe isolated from plant residue, whereas Dysgonomonas capnocytophagoides and Dysgonomonas mossii are facultative anaerobes isolated from human clinical specimens. Paludibacter propionicigenes cannot grow at 37°C and is inhibited by bile, whereas Parabacteroides merdae, Dysgonomonas capnocytophagoides, and Dysgonomonas mossii can grow at 37°C and are bile tolerant.

Taxonomic comments By analysis of 16S rRNA gene sequences, the closest neighboring species of Paludibacter propionicigenes are Dysgonomonas capnocytophagoides (90.9% similarity), Dysgonomonas mossii (89.8%), and Parabacteroides merdae (88.7%).

List of species of the genus Paludibacter 1. Paludibacter propionicigenes Ueki, Akasaka, Suzuki and Ueki 2006, 43VP pro.pi.on.i.ci′ge.nes. N.L. n. acidum propionicum propionic acid; N.L. suff. -genes (from Gr. v. gennaô to produce) producing; N.L. part. adj. propionicigenes producing propionic acid. The characteristics are as described for the genus, with the following additional features. Elongated cells are occasionally formed both singly and in chains of the short cells. Spherical cells sometimes develop after storage of slant cultures at 4°C. Growth occurs at pH 5.0–7.6 (optimum, 6.6) and at 15–35°C. Growth at 33°C is much slower than at 30°C.

*PY4S agar is peptone-yeast extract (PY) broth supplemented with (g/l): glucose, 0.25; cellobiose, 0.25; maltose, 0.25; soluble starch, 0.25; and agar, 15.0.

The NaCl concentration range for growth is 0–0.5% in PYG medium. The following compounds are used for growth and acid production: arabinose, cellobiose, fructose, galactose, glucose, mannose, maltose, melibiose, glycogen, soluble starch, and xylose. The following compounds are not used: cellulose, dulcitol, ethanol, fumarate, glycerol, inositol, lactate, lactose, malate, mannitol, melezitose, pyruvate, rhamnose, raffinose, ribose, salicin, sorbitol, sorbose, succinate, sucrose, trehalose, and xylan. Esculin is hydrolyzed, but gelatin and urea are not. H2S and indole are not produced. No growth occurs in the presence of 0.01% bile salts. Source: rice plant residue in anoxic rice-field soil in Japan. DNA G+C content (mol%): 39.3 (HPLC). Type strain: WB4, DSM 17365, JCM 13257. Sequence accession no. (16S rRNA gene): AB078842.



Genus VI. Proteiniphilum

77

Genus V. Petrimonas Grabowski, Tindall, Bardin, Blanchet and Jeanthon 2005, 1118VP The Editorial Board Pe.tri.mo′nas. L. fem. n. petra rock, stone; L. fem. n. monas a unit, monad; N.L. fem. n. Petrimonas stone monad.

Straight rods (0.7–1 × 1.5–2.0 mm) during exponential growth; some longer cells (0.5 × 4.0 mm) may be observed in old cultures. Nonsporeforming. Gram-stain-negative. Chemo-organotrophic. Mesophilic. Strictly anaerobic, having a fermentative type of metabolism. End products of glucose fermentation are acetate, H2, and CO2. Carbohydrates and some organic acids are fermented. Tryptone is required for growth. The predominant menaquinone is MK-8, with smaller amounts of MK-7 and MK-9. The major polar lipids are phosphatidylethanolamine, an unidentified phospholipid, two unidentified aminophospholipids, three unidentified phosphoglycolipids, a glycolipid, an aminolipid, and two additional uncharacterized lipids. The fatty acids include straight chain and branched fatty acids; in addition, 2-OH and 3-OH fatty acids are present. The major cellular fatty acids are C15:0 anteiso, C13:0 anteiso, C15:0 iso, and C15:0. The major hydroxy fatty acids are C3-OH-16:0 iso, C3-OH17:0 iso, and C2-OH 17:0; about one-third of each of all three appear to be amide-linked. Isolated from a biodegraded oil reservoir. DNA G+C content (mol%): 40.8 (HPLC). Type species: Petrimonas sulfuriphila Grabowski, Tindall, Bardin, Blanchet and Jeanthon 2005, 1119VP.

Further descriptive information To date, Petrimonas sulfuriphila is the only member of the phylum Bacteroidetes to be isolated from a producing well of a biodegraded oil reservoir.

Enrichment and isolation procedures Enrichment was performed in nitrate broth medium (Difco). Isolation of single colonies was accomplished using the anaerobic roll-tube technique (Hungate, 1969) with nitrate broth solidified with 2% (w/v) purified agar (Difco).

Differentiation of the genus Petrimonas from other genera The predominance of MK-8 as menaquinone is a feature that distinguishes the novel isolate from all its phylogenetic relatives, including Tannerella (MK-10, MK-11), Bacteroides (MK-10, MK-11), Parabacteroides distasonis (MK-10); Parabacteroides merdae (MK-9, MK10), and Porphyromonas (MK-9, MK-10).

Taxonomic comments According to 16S rRNA gene sequence analysis by Grabowski et al. (2005), the closest cultivated relatives of the type strain of Petrimonas sulfuriphila are Tannerella forsythia (88% similarity) and Parabacteroides merdae (87% similarity). However, very high rRNA gene sequence similarities (99.6%) were found between Petrimonas sulfuriphila and certain environmental clones retrieved from a dechlorinating consortium (GenBank accession no. AJ488088) and the bovine rumen (AB003390).

List of species of the genus Petrimonas 1. Petrimonas sulfuriphila Grabowski, Tindall, Bardin, Blanchet and Jeanthon 2005, 1119VP sul.fu.ri.phi′la. L. n. sulfur sulfur; Gr. adj. philos -ê -on loving; N.L. fem. adj. sulfuriphila sulfur-loving, indicating that sulfur stimulates growth. The characteristics are as described for the genus, with the following additional features. Cells occur singly or in pairs. The temperature range for growth at pH 7.2 is 15–40°C; optimum, 37–40°C. Growth occurs in the presence of 0–4% NaCl; optimum, 0%. Yeast extract and elemental sulfur stimulate growth. The following substrates support growth when yeast extract, tryptone, and elemental sulfur are present:

glucose, arabinose, galactose, maltose, mannose, rhamnose, lactose, ribose, fructose, sucrose, lactate, mannitol, glycerol, and cellobiose. Fumarate, pyruvate, and Casamino acids support weak growth. Acetate, formate, butyrate, propionate, methanol, peptone, ethanol, propanol, butanol, toluene, sorbose, and cellulose are not used. Elemental sulfur is reduced to sulfide. Nitrate is reduced to NH4+. Source: isolated from an oilfield well head in the Western Canadian Sedimentary Basin (Canada). DNA G+C content (mol%): 40.8 (HPLC). Type strain: BN3, DSM 16547, JCM 12565. Sequence accession no. (16S rRNA gene): AY570690.

Genus VI. Proteiniphilum Chen and Dong 2005, 2259VP The Editorial Board Pro.tei′ni.phi.lum. N.L. neut. n. proteinum protein; Gr. adj. philos -ê -on loving; N.L. neut. n. Proteiniphilum protein-loving.

Rods 0.6–0.9  ×  1.9–2.2  mm. Motile by means of lateral flagella. Nonsporeforming. Gram-stain-negative. Obligately anaerobic. Growth occurs at 20–45°C. Oxidase and catalasenegative. Chemo-organotrophic. Proteolytic. Yeast extract and peptone can be used as energy sources. Nonsaccharolytic. Carbohydrates, alcohols, and organic acids (except pyruvate) are not used. Gelatin is not hydrolyzed. Not resis-

tant to 20% bile. The major fermentation products from peptone-yeast extract (PY) medium are acetic acid and propionic acid. Nitrate is not reduced. Cellular fatty acids mainly consist of iso-branched fatty acids, predominantly C15:0 anteiso. DNA G+C content (mol %): 46.6–48.9 (Tm). Type species: Proteiniphilum acetatigenes Chen and Dong, 2005, 2261VP.

78

Family III. Porphyromonadaceae

Further descriptive information During studies of syntrophic propionate-degrading bacteria from methanogenic environments, Chen and Dong (2005) reported the isolation of mixed cultures consisting of three different organisms that, in combination, were capable of degrading propionate to acetate and methane. The three organisms were: a syntrophic, propionate-degrading bacterium; an organism resembling Methanobacterium formicicum; and a rod-shaped bacterium which did not consume propionate or synthesize methane but did accelerate the degradation of propionate by the triculture. Chen and Dong classified the latter organism as the representative of a new genus, Proteiniphilum, and a new species, Proteiniphilum acetatigenes. Colonies of Proteiniphilum acetatigenes on (PY) agar are circular, slightly convex, white, translucent, and reach 1.5 mm in diameter after 3  d at 37°C. Optimum growth occurs at 37°C. The pH range for growth is 6.0–9.7; optimum is 7.5–8.0. In addition to yeast extract and peptone, pyruvate, glycine and l-arginine can be used as carbon and energy sources. Tryptone, l-serine, l-threonine, and l-alanine support weak growth. Pyruvate is converted to acetic acid and CO2.

Enrichment and isolation procedures Proteiniphilum was isolated from methanogenic propionate-degrading mixtures obtained from the granule sludge of an upflow anaerobic sludge blanket reactor for treatment of brewery wastewater. Isolation was accomplished by initial serial dilution in anaerobic

PY medium followed by selection of colonies on anaerobic roll tubes. Repeated subculturing of colonies in PY broth followed by colony isolation is performed until purity is assured.

Maintenance procedures Routine cultivation can be accomplished in pre-reduced PY broth in anaerobic tubes under an atmosphere of 100% oxygen-free N2 with incubation at 37°C.

Differentiation of the genus Proteiniphilum from related genera The rod shape of Proteiniphilum differs from the coccobacillary shape of Dysgonomonas species; moreover, unlike Proteiniphilum, Dysgonomonas is nonmotile, facultatively anaerobic, ferments glucose, and has a higher DNA G+C content (47–49 vs 38 mol%). The motility of Proteiniphilum and failure to form succinic acid as a major end product from glucose differentiate Proteiniphilum from Parabacteroides distasonis, Parabacteroides merdae, and Tannerella forsythia. Unlike Tannerella forsythia, Proteiniphilum does not produce phenylacetic acid and butyric acid from glucose.

Taxonomic comments Analysis of 16S rRNA gene sequences indicates that the nearest neighbors of the type strain are Dysgonomonas species (89.6–90.6% sequence similarity). The type strain is more distantly related to Parabacteroides (85–87% sequence similarity), Porphyromonas (84– 88%), Prevotella (79–85%), and Tannerella forsythia (89.3%).

List of species of the genus Proteiniphilum 1. Proteiniphilum acetatigenes Chen and Dong 2005, 2261VP a.ce′ta.ti.gen.es. N.L. acetas -atis acetate; N.L. suff. -genes (from Gr. v. gennaô to produce) producing; N.L. part. adj. acetatigenes acetate-producing. The characteristics are as described for the genus, with the following additional features. Milk is not curdled. Starch and esculin are hydrolyzed. Indole is not produced. Urease, lecithinase and lipase are not produced. Methyl red and Voges– Proskauer tests are negative. H2S is not produced from peptone or thiosulfate. NH3 is produced from yeast extract, peptone and l-arginine. Acetic acid is the main product from fermentation of yeast extract, peptone, pyruvate and l-arginine; propionic acid is also produced. The following substrates are not used: adonitol, amygdalin, l-arabinose, l-aspartate, butanedioic acid, cellobiose, cellulose, citrate, l-cysteine, dulcitol, erythritol, esculin, ethanol, d-fructose, fumarate,

d-galactose, gluconate, d-glucose, l-glutamine, glycogen, hippurate, l-histidine, b-hydroxybutyric acid, inositol, inulin, l-isoleucine, d-lactose, l-leucine, l-lysine, malate, malonate, d-maltose, mannitol, mannose, melibiose, methanol, l-methionine, phenylacetic acid, l-phenylalanine, l-proline, 1-propanol, raffinose, rhamnose, ribitol, ribose, salicin, d-sorbitol, sorbose, succinate, sucrose, starch, trehalose, tryptophan, l-tyrosine, l-valine, xylan, and xylose. The major cellular fatty acids are C15:0 anteiso (46.21%), C15:0 (8.90%), C17:0 iso 3-OH (5.93%), and C17:0 anteiso (5.15%). Source: the type strain was isolated from the granule sludge of an Upflow Anaerobic Sludge Blanket (UASB) reactor treating brewery wastewater. DNA G+C content (mol%): 46.6–48.9 (Tm). Type strain: TB107, AS 1.5024, JCM 12891. Sequence accession no. (16S rRNA gene): AY742226.

Genus VII. Tannerella Sakamoto, Suzuki, Umeda, Ishikawa and Benno 2002, 848VP Mitsuo Sakamoto, Anne C. R. Tanner and Yoshimi Benno Tan.ne.rel′la. L. dim. suffix -ella; N.L. fem. dim. n. Tannerella named after the American microbiologist Anne C. R. Tanner, for her contributions to research on periodontal disease.

Fusiform cells, generally 0.3–0.5  ×  1–30  mm. Gram-negative. Nonmotile. Obligately anaerobic. N-acetylmuramic acid (NAM) is required for growth (some strains do not require NAM). Growth is inhibited in the presence of 20% bile. The major end products are acetic acid, butyric acid, isovaleric acid, propionic acid, and phenylacetic acid; smaller amounts of isobutyric

acid and succinic acid may be produced. Esculin is hydrolyzed. Indole variable. Trypsin activity is positive. Glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malate dehydrogenase, and glutamate dehydro­ genase are present. The principal respiratory quinones are menaquinones MK-10 and MK-11. Both nonhydroxylated and



Genus VII. Tannerella

3-hydroxylated long-chain fatty acids are present. The nonhydroxylated acids are predominantly of the saturated straightchain and anteiso methyl-branched-chain types. The ratio of C15:0 anteiso to C15:0 iso is high (>20). Isolated originally and principally from human oral cavity. Tannerella forsythia has been associated with periodontal disease, root canal infections, and peri-implantitis. DNA G+C content (mol%): 44–48. Type species: Tannerella forsythia corrig. (Tanner, Listgarten, Ebersole and Strzempko 1986) Sakamoto, Suzuki, Umeda, Ishikawa and Benno 2002, 848VP (Bacteroides forsythus Tanner, Listgarten, Ebersole and Strzempko 1986, 216). Sequence accession no. (16S rRNA gene): L16495.

Further descriptive information The genus Tannerella is a member of the Bacteroides subgroup of the Cytophaga–Flexibacter–Bacteroides (CFB) phylum (Paster et al., 1994) that is referred to as the phylum Bacteroidetes in this edition of the Manual. In the absence of NAM, cells of Tannerella forsythia are often pleomorphic and occur as rods with tapered (fusiform) or rounded ends, long filaments, and spheroids. The spheroids frequently occur in the center of cells and enlarge the diameter of the cell to approximately 5 mm. The spheroids are observed free from the rod forms (Tanner et al., 1979); this cell morphology has been attributed to the presence of a weakened cell wall (Wyss, 1989). In the presence of NAM, cells are regular with rounded or tapered ends (Braham and Moncla, 1992). In cell cross-sections stained with uranyl acetate and lead citrate and observed by electron microscopy, the outer membrane consists of an outer layer that is approximately 4 nm thick, an inner layer that is 1 nm thick, and a 2 nm electron-lucent space in between. The inner membrane consists of two electron-dense layers, each approximately 2 nm wide, with a 2 nm intervening electron-lucent layer. The inner and outer membranes are separated by a moderately electron-dense zone that is about 16 nm wide and appears to lack a distinct peptidoglycan layer. A regular structured external layer surrounds the outer membrane and consists of subunits, which in some cross-sections resemble adjacent arches that are 10  nm wide and 10  nm high. These subunits are separated from the outer membrane by a 12 nm electron-lucent zone. Electron-dense radial connections appear to extend from the subunits to the outer surface of the outer membrane. Negative staining of tangential sections through the cell periphery indicates that the subunits in the outermost layer are packed in an orthogonal array (Tanner et al., 1986). The predominant cellular fatty acids are C15:0 anteiso and C16:0 3-OH. In contrast, Bacteroides and Parabacteroides species possess significant levels of C15:0 anteiso and C17:0 iso 3-OH. Moreover, the ratio of C15:0 anteiso to C15:0 iso in whole-cell methanolysates of Tannerella forsythia is very much higher than those of Bacteroides and Parabacteroides species (Sakamoto and Benno, 2002, 2006): the ratios range from 22.8 to 95.2% in Tannerella forsythia strains but only from 1.9 to 10.3% in Bacteroides and Parabacteroides species. This is an important feature that differentiates Tannerella forsythia from Bacteroides and Parabacteroides species. Colonies are pale, speckled pink, circular, entire, and slightly convex on Trypticase soy agar supplemented with 5% (v/v) sheep blood (TSBA) and a NAM disk (Braham and Moncla, 1992). The colonies are generally small with a diameter of

79

0.5–2 mm after 7–10 d incubation at 37°C. Tannerella forsythia is a strict anaerobe and requires CO2 and H2 in the atmosphere for growth on agar surfaces. Growth on agar is stimulated by the presence of Fusobacterium nucleatum (and strains of Streptococcus sanguinis, Bacteroides fragilis, Campylobacter rectus, Prevotella intermedia, and Veillonella parvula) in a satellite pattern (Tanner et al., 1986). The growth of Tannerella forsythia strains is inhibited on bacteroides bile esculin agar. In contrast, the growth of Bacteroides and Parabacteroides species is not inhibited on the media containing 20% bile. In addition, Tannerella forsythia requires exogenous NAM as a growth factor (Wyss, 1989). These characteristics are important features that differentiate Tannerella forsythia from the Bacteroides fragilis group and the genus Parabacteroides. Braham and Moncla (1992) reported that Tannerella forsythia strains isolated from subgingival plaque samples from monkeys (Macaca fascicularis) also require NAM for growth. However, Tannerella forsythia strains recovered from cat and dog bite wound infections in humans do not require NAM for growth (Hudspeth et al., 1999). These results suggest that there are host-specific biotypes within the species of Tannerella forsythia. Tannerella forsythia does not react in the API 20A or API 20E test series but does react in other resting cells tests, including API ZYM, API AN-IDENT (Tanner et al., 1985, 1986), and fluorogenic substrate tests (Maiden et al., 1996). Tannerella forsythia strains give positive results for a-glucosidase, b-glucosidase, a-fucosidase, and b-glucuronidase. In the absence of NAM there is no detectable pH decrease in media supplemented with carbohydrates (Tanner et al., 1985, 1986). These results suggest that Tannerella forsythia may grow too poorly in that medium to ferment the carbohydrate, that cells may have trouble transporting the sugars across their membranes for use in the glycolytic pathway, or may overproduce methyl glyoxyl, thereby leading to toxicity and growth inhibition (Maiden et al., 2004). The genome-sequencing project of Tannerella forsythia ATCC 43037T has been completed by The Institute for Genomic Research (TIGR). A total of 3034 open reading frames are predicted from the genomic sequence of 3,405,543  bp in length based on the annotation provided by the Oral Pathogen Sequences Databases (oralgen, http://www.oralgen.lanl.gov). The preliminary genomic annotations as well as a variety of bioinformatics tools useful for studying the Tannerella forsythia genome are available at both the oralgen and Bioinformatics Resource for Oral Pathogens (brop, http://www.brop.org) (Chen et al., 2005) project web sites. The minimum inhibitory concentrations (MICs) of antimicrobial agents have been reported by Takemoto et al. (1997). Tannerella forsythia strains are most sensitive to clindamycin and metronidazole. Tannerella forsythia shows a comparatively lower susceptibility to ciprofloxacin. Most strains are sensitive to penicillin G, ampicillin, amoxycillin, tetracycline, doxycycline, and erythromycin. Tannerella forsythia has been associated with advanced forms of periodontal disease, including severe and refractory periodontitis. Unlike other periodontopathic bacteria, such as Porphyromonas gingivalis and Prevotella intermedia, Tannerella forsythia is difficult to culture, and its prevalence in periodontal disease may be underestimated. Tannerella forsythia is synergistic with Porphyromonas gingivalis, and the presence of both accelerates progression of the disease (Yoneda et  al., 2001). Pathogenic

80

Family III. Porphyromonadaceae

factors include a trypsin-like protease (PrtH) (Saito et  al., 1997) and a variety of glycosidases, such as a-D-glucosidase, N-acetyl-b-D-glucosaminidase (Hughes et al., 2003), and sialidase (Ishikura et al., 2003). Tan et al. (2001) determined the prevalence and the association of the prtH gene of Tannerella forsythia in adult periodontitis and periodontally healthy patients. Among the 86 diseased sites examined, 73 (85%) were colonized by Tannerella forsythia with the prtH genotype. In sites of the periodontally healthy, only 7 of 73 (10%) possessed Tannerella forsythia with the prtH genotype. These findings suggest a strong association of the prtH gene of Tannerella forsythia with adult periodontitis. The outer surface of Tannerella forsythia is covered by an S-layer (surface layer) composed of two protein subunits (200 and 210 kDa) that mediate adherence and invasiveness (Sabet et  al., 2003). Although mice immunized with purified S-layer and Tannerella forsythia whole cells did not develop any abscesses when challenged with viable Tannerella forsythia cells, unimmunized mice developed abscesses (Sabet et al., 2003). Other virulence factors include the cell-surface-associated protein (BspA) that mediates adherence to fibronectin (an extracellular matrix component), and fibrinogen (a clotting factor) (Sharma et al., 1998). Honma et  al. (2001) constructed a BspA-defective mutant of Tannerella forsythia that showed a reduced ability to adhere to fibronectin and fibrinogen compared with the wildtype strain (ATCC 43037T). Sharma et al. (2005) demonstrated alveolar bone loss in mice infected with the Tannerella forsythia wild-type strain, whereas this effect was impaired when the BspA mutant was used. Huang et al. (2003) investigated the distribution of Tannerella forsythia genotypes in a Japanese periodontitis population, and also the relationship between different genotypes and the periodontal status, using the arbitrarily primed polymerase chain reaction (AP-PCR) method. Tannerella forsythia ATCC 43037T and 137 clinical bacterial isolates were separated into 11 distinct AP-PCR genotypes. The majority of Tannerella forsythia strains examined belonged to AP-PCR genotypes I (including ATCC 43037T), II, III, and IV (accounting for 39.7, 20.6, 10.3, and 10.3%, respectively). The strains of Types I and III were mainly isolated from chronic periodontitis subjects, whereas the strains of Types II and IV were mainly isolated from aggressive periodontitis subjects. A Tannerella phylotype (oral clone BU063: GenBank accession no. for 16S rRNA gene is AY008308) was associated with periodontal health, in contrast to Tannerella forsythia strains that were associated with periodontal disease (Kumar et  al., 2003; Leys et al., 2002). Tannerella forsythia is found in the subgingival, gingival, and periodontal pockets, in dental root canals (Conrads et al., 1997), and around infected dental implants (Tanner et  al., 1997) of humans. Tannerella forsythia has been also found in monkey (Macaca fascicularis) subgingival plaque samples ­(Braham and Moncla, 1992). In addition, Tannerella forsythia strains have been recovered from cat- and dog-bite wound infections in humans (Hudspeth et  al., 1999). Tannerella forsythia has also been detected in atheromatous plaques (Haraszthy et al., 2000) and in buccal epithelial cells (Rudney et al., 2005) of humans. From clone libraries of 16S rRNA gene sequences from termite guts, phylotypes related to Tannerella forsythia have been found (Hongoh et al., 2005, 2003).

Enrichment and isolation procedures Isolation and identification of Tannerella species has been described (Braham and Moncla, 1992; Tanner et  al., 1998; Umeda et al., 1996). Rapid presumptive identification of clinical isolates of Tannerella forsythia is based on the following eight criteria (Braham and Moncla, 1992): (1) positive activity for a-glucosidase, (2) positive activity for b-glucosidase, (3) positive activity for sialidase, as it has been reported that sialidase activity is useful for identification of Tannerella forsythia (Moncla et  al., 1990), (4) positive activity for trypsin-like enzyme, (5) negative indole production, (6) requirement for NAM, (7) colonial morphology, and (8) Gram stain morphology from blood agar medium deficient in NAM. The trypsin-like activity is measured by the benzoyl-DL-arginine-naphthylamide (BANA) test (Loesche et al., 1990). BANA-positive non-blackpigmented bacteria are subcultured with the plates with or without the NAM disk. Subsequently, bacterial colonies that grow only on the plates with NAM are screened as Tannerella forsythia. Finally, five filter paper fluorescent spot tests [for indole (Sutter and Carter, 1972), a-glucosidase, b-glucosidase, sialidase, and trypsin-like enzyme] are used for identification of Tannerella forsythia (Braham and Moncla, 1992). For the trypsin-like enzyme assay, N-a-carbobenzoxy-l-arginine-7amino-4-methylcoumarin hydrochloride (CAMM), fluorogenic substrate, is used. An agar-free broth medium for Tannerella forsythia has been developed by Wyss (1989). This medium consists of BHI broth supplemented with fetal calf serum (FCS) and NAM. Some strains grew without the addition of FCS.

Maintenance procedures The organism can be maintained by transfer on the same media used for isolation. Recommended procedures for long-term preservation are lyophilization, freezing at –80°C, or storage in liquid nitrogen. Cryoprotective agents such as 10% glycerol or DMSO should be added to cultures before freezing, and heavy cell concentrations should be used.

Differentiation of the genus Tannerella from other genera Differential characteristics of the genus Tannerella and some related taxa are shown in Table 20. Although Tannerella forsythia is phylogenetically related to Parabacteroides species, the ratio of C15:0 anteiso to C15:0 iso in whole-cell methanolysates of Tannerella forsythia is different from those of Parabacteroides species. While the ratios of C15:0 anteiso to C15:0 iso range from 22.8 to 95.2 in Tannerella forsythia strains (Sakamoto et  al., 2002), those of Parabacteroides species range from 3.1 to 10.3. In addition, although the major menaquinones of Tannerella forsythia are MK-10 and MK-11, the major menaquinones of Parabacteroides species are MK-9 and MK-10 (Sakamoto and Benno, 2006).

Taxonomic comments As shown in Figure 21, Tannerella exhibits a close phylogenetic association with the genus Parabacteroides (Sakamoto and Benno, 2006). The name Bacteroides forsythus was published by Tanner et al. (1986) but did not appear on a Validation List in the IJSEM.



81

Genus VII. Tannerella

TABLE 20.  Differential characteristics of the genus Tannerella and some related taxaa

Characteristic

Tannerella

Parabacteroides

Bacteroides

Dysgonomonas

Paludibacter

Porphyromonas

+b +

+ -

+ D

+ + D

nt

-c

D

nt

D D + NF A, B, IV, P, PA

D + F A, S

D D D F A, S

D D D F P, L, S

+ F A, P

D D D D MF A, S

+ NF A, P

Presence of:   G6PDH   6PGDH Proteolytic activity Major cellular fatty acids

  + + + C15:0 anteiso

  + + C15:0 anteiso

  + + C15:0 anteiso

  nt nt + C15:0 anteiso

22.8–95.2

3.1–10.3

1.9–8.2

  nt nt nt C15:0 anteiso, C15:0 and C17:0 anteiso-3-OH 28

  D C15:0 anteiso

Ratio of C15:0 anteiso to C15:0 iso Predominant menaquinones

  nt nt D C14:0 iso, C15:0 anteiso and C16:0 iso 3-OH 6.0–8.8

D D +d NFe A, B, IV, P, PA, S   D D D C15:0 isof 20%) are also present, with lower levels of MK-8 (25%) with lower levels of MK-9 (1%), MK-10 (5%) and MK-11 (95%) except Pedobacter saltans, which shows >10% difference. DNA G+C content (mol%): 36–45. Type species: Pedobacter heparinus (Payza and Korn 1956) Steyn, Segers, Vancanneyt, Sandra, Kersters and Joubert 1998, 175VP; (Flavobacterium heparinum Payza and Korn 1956, 854; Cytophaga heparina Christensen 1980, 474; Sphingobacterium heparinum Takeuchi and Yokota 1992, 465–482).

for a listing of the 22 species since published. However, only the type species Pedobacter heparinus has been studied in detail. Systematic investigations do not exist. In this edition of the Manual, the genus Pedobacter is included in the phylum Bacteroidetes, class Sphingobacteriia, order Sphingobacteriales, and family Sphingobacteriaceae. Cell morphology.  Cells of Pedobacter are rod shaped (0.5–0.9 mm wide) with rounded or slightly tapering ends. The length of the rods varies from short (0.5–1.0 mm) to long (up to 6–10 mm) Figure 58. Filament formation has been observed with Pedobacter caeni, and occasionally with Pedobacter heparinus. No flagella or pili have been observed. Cell-wall composition.  The cell wall of Pedobacter species is Gram-stain-negative and there is a need to study its

Further descriptive information At the time of writing, nine species of Pedobacter had been described; five of them since 2003. See the Editorial note, below,

Figure 58.  Phase-contrast photomicrograph of Pedobacter heparinus DSM 2366, grown in nutrient broth for 24 h at 30 °C. Bar = 10 mm.

340

Family I. Sphingobacteriaceae

­ ltrastructural characteristics. It possesses sphingolipids, but u little is known with respect to other lipids that may be present. Pedobacter cryoconitis produces an extracellular mucous polysaccharide capsule (Margesin et  al., 2003). This could be an adaptative feature, as it is generally observed in psychrophilic microorganisms (Gounot, 1999). Psychrophilic species of Pedobacter, namely Pedobacter cryoconitis and Pedobacter himalayensis, also produce mucus (Margesin et al., 2003; Shivaji et al., 2005), and colonies of mesophilic Pedobacter caeni (Vanparys et  al., 2005) are slimy. In these species, the chemical nature of the mucus and the slime is yet to be determined. All species of Pedobacter contain MK-7 as the major menaquinone (68–90%) (Kwon et al., 2007; Shivaji et al., 2005; Steyn et al., 1998), however, the composition of the menaquinones varies from species to species. In Pedobacter himalayensis (Shivaji et  al., 2005), the menaquinones present are MK-7 (68%), MK-7 (H2) (5%), MK-8 (6%), MK-8 (H2) (13%), and MK-9 (H2) (8%). In Pedobacter suwonensis (Kwon et al., 2007), MK-7 constitutes 90.5% of the total, with MK-9 (5.6%), and MK-8 (3.9%) constituting less than 10% together. The predominance of MK-7 in the genus Pedobacter is a characteristic feature of the family Sphingobacteriaceae and is a discriminating chemotaxonomic feature that differentiates Pedobacter from Cytophaga (Reichenbach, 1989). The cellular fatty acid composition of the various species of Pedobacter has been studied as a chemotaxonomic marker, and the predominant fatty acids are C15:0 iso, C15:0 iso 2-OH, C15:0 iso 3-OH, C16:0, C16:1 w7c, C17:1iso w9c, and C17:0 iso 3-OH (Dees et al., 1985; Hwang et al., 2006; Kwon et al., 2007; Shivaji et al., 2005; Steyn et al., 1998; Takeuchi and Yokota, 1992; Vanparys et al., 2005, 1983; Yabuuchi and Moss, 1982). In eight of the species, namely Pedobacter suwonensis, Pedobacter himalayensis, Pedobacter cryoconitis, Pedobacter heparinus, Pedobacter africanus, Pedobacter piscium, Pedobacter saltans, and Pedobacter caeni, all of the above seven fatty acids are present, but in Pedobacter roseus, C15:0 iso 3-OH, C16:0, and C17:1 iso w9c are absent and C16:0 iso 3-OH is unique to the species (Hwang et al., 2006). Quantitatively, the predominant fatty acid is C15:0 iso (15–35%), followed by C16:1 w7c (20–31%) (Kwon et al., 2007; Margesin et al., 2003; Shivaji et al., 2005). As a group, the branched-chain fatty acids, (C15:0 iso, C15:0 iso 2-OH, C15:0 iso 3-OH, C17:1 iso w9c, and C17:0 iso 3-OH), constitute 70–80% of the total fatty acids. The unsaturated fatty acid C16:1 w7c is also present in substantial quantities (20–30%) and the saturated fatty acid C16:0 constitutes 3–9% of the total cellular fatty acid content (Shivaji et  al., 2005; Steyn et al., 1998). The other fatty acids that are observed in a few of the species of Pedobacter in trace amounts or 85% positive; −, 0–15% positive; D, different reactions occur in different taxa (species of a genus); w, weak reaction; nd, not determined.

nd Aerobic

nd Aerobic

Rings, coils, vibroids or S-shaped cells Resting stage Relationship to O2

0.9–1.7 × 0.5–0.75 × 0.5–0.5 × 2.8–4.1 2.5–3.0 2–10 − + −

Rod

Cell shape

Cell size (mm)

Adhaeribacter

Characteristic

Flectobacillus

TABLE 86.  Descriptive and differential characteristics of members of the family Cytophagaceae

372 Family I. Cytophagaceae

373

Genus I. Cytophaga

­ aking it very difficult to differentiate the genus Cytophaga from m other related genera only by phenotypic characteristics. When additional strains are isolated, other chemotaxonomic, physiologic, and biochemical characteristics of the genus Cytophaga will be found.

Taxonomic comments The genus Cytophaga was first described by Winogradsky (1929) for aerobic, cellulolytic, soil bacteria with probable gliding motility. Stanier (1940, 1941, 1942, 1947) investigated Gramstain-negative bacteria that could move by gliding and degrade cellulose, agar, and/or chitin, and suggested that the gliding motility and the ability to degrade biomacromolecules were indispensable characters for the genus. These generic concepts were accepted in principle in the 1st edition of this Manual by Reichenbach (1989c); however, he stated that the genus is “a rather heterogeneous assembly of organisms which certainly do not belong to one single genus”. In addition, several taxonomic investigations described in the reviews by Callies and Mannheim (1978), Christensen (1977), Hayes (1977), Oyaizu and Komagata (1981), and Shewan and McMeekin (1983) revealed considerable overlapping of phenotypic and chemotaxonomic characteristics between members of the genera Cytophaga and Flavobacterium. Only gliding motility could be regarded as a distinguishing feature for the two genera, but this feature is sometimes not easy to discern (Henrichsen, 1972; McMeekin and Shewan, 1978; Perry, 1973). Accordingly the two genera were called the Flavobacterium-Cytophaga complex. These developments are described in detail in the 1st edition of this Manual (Reichenbach, 1989c). Of the 23 validly published Cytophaga species, 20 were recognized in the 1st edition of this Manual. 16S rRNA sequencing analysis showed great biological diversity within the genus Cytophaga (Nakagawa and Yamasato, 1993). They also showed that Cytophaga species were characterized by either MK-6 (Cytophaga aquatilis, Cytophaga aurantiaca, Cytophaga ­columnaris, Cytophaga flevensis, Cytophaga johnsonae, Cytophaga latercula, Cytophaga lytica,

Cytophaga marina, Cytophaga marinoflava, Cytophaga pectinovora, Cytophaga psychrophila, Cytophaga saccharophila, Cytophaga succinicans) or MK-7 (Cytophaga agarovorans, Cytophaga aprica, Cytophaga arvensicola, Cytophaga diffluens, Cytophaga fermentans, Cytophaga heparina, Cytophaga hutchinsonii, Cytophaga salmonicolor). The types of major menaquinones correlated well with the phylogenetic relationships. Some of the species possessing MK-6 were closely related to Flavobacterium aquatile, the type species of the genus Flavobacterium. The understanding and acceptance of the diversity of the genus have led to several reclassifications and descriptions of new genera as summarized in Table 87 and described below. Nakagawa and Yamasato (1996) emended the genus Cytophaga to contain only two species, Cytophaga hutchinsonii and Cytophaga aurantiaca. These are the only two species that are, based on 16S rRNA sequencing analysis, justifiably members of the genus Cytophaga. Cytophaga agarovorans and Cytophaga salmonicolor, which are facultatively anaerobic, usually pink­colored marine organisms, formed a distinct cluster in the class Bacteroides and were transferred in the new genus Marinilabilia (Nakagawa and Yamasato, 1996). Later, gyrB sequence analyses and DNA–DNA hybridization studies showed that Marinilabilia salmonicolor (basonym Cytophaga salmonicolor) and Marinilabilia agarovorans (basonym, Cytophaga agarovorans) should be unified in a single species, and the name Marinilabilia salmonicolor biovar. agarovorans was proposed for it (Suzuki et al., 1999). Eight species possessing MK-6 – Cytophaga aquatilis, Cytophaga columnaris (synonym, Flexibacter columnaris), Cytophaga flevensis, Cytophaga johnsonae, Cytophaga pectinovora, Cytophaga psychrophila (synonym, Flexibacter psychrophilus), Cytophaga saccharophila, and Cytophaga succinicans – were closely related to the genus Flavobacterium and therefore transferred to the genus Flavobacterium. The family Flavobacteriaceae was emended to accommodate MK-6 organisms (Bernardet et al., 1996, 2002). Accordingly, the genus Flavobacterium now includes both gliding-motile and nonmotile organisms. This means that gliding motility is not a significant taxonomic characteristic for differentiation of genera.

TABLE 87.  Other validly published Cytophaga species showing their proposed assignment to new or different genera

Validly published species C. agarovorans C. aprica C. aquatilis C. arvensicola C columnaris C. diffluens C. fermentans C. flevensis C. heparina C. johnsonae C. latercula C. lytica C. marina C. marinoflava C. pectinovora C. psychrophila C. saccharophila C. salmonicolor C. succinicans C. uliginosa

Revised classification Marinilabilia salmonicolor biovar. agarovorans Flammeovirga aprica Flavobacterium hydatis Chitinophaga arvensicola Flavobacterium columnare Persicobacter diffluens Uncertain (probably belonging to the class Bacteroides) Flavobacterium flevense Pedobacter heparinus Flavobacterium johnsoniae Aquimarina latercula Cellulophaga lytica Tenacibaculum maritimum Leeuwenhoekiella marinoflava Flavobacterium pectinovorum Flavobacterium psychrophilum Flavobacterium saccharophilum Marinilabilia salmonicolor Flavobacterium succinicans Zobellia uliginosa

References Nakagawa and Yamasato (1996); Suzuki et al. (1999) Nakagawa et al. (1997) Bernardet et al. (1996) Kämpfer et al. (2006) Bernardet et al. (1996) Nakagawa et al. (1997) Bernardet et al. (1996) Takeuchi and Yokota (1992); Steyn et al. (1998) Bernardet et al. (1996) Nedashkovskaya et al. (2005b); Nedashkovskaya et al. (2006) Johansen et al. (1999) Suzuki et al. (2001) Nedashkovskaya et al. (2005d) Bernardet et al. (1996) Bernardet et al. (1996) Bernardet et al. (1996) Nakagawa and Yamasato (1996); Suzuki et al. (1999) Bernardet et al. (1996) Bowman (2000); Barbeyron et al. (2001)

374

Family I. Cytophagaceae

Two marine ­agarolytic organisms, Cytophaga aprica and Cytophaga diffluens, were transferred to the new genera Flammeovirga and Persicobacter, respectively (Nakagawa et al., 1997). A heparindegrading species, Cytophaga heparina was initially transferred to the genus Sphingobacterium, because it contained sphingolipids (Takeuchi and Yokota, 1992), and then was reclassified in the new genus Pedobacter as Pedobacter heparinum, together with other heparinase-producing organisms (Steyn et al., 1998). Cytophaga lytica and Cytophaga uliginosa – marine species possessing MK-6 – were classified in the new genus Cellulophaga (Bowman, 2000; Johansen et al., 1999); later, Cellulophaga uliginosa was transferred to the new genus Zobellia (Barbeyron et al., 2001). A marine fish pathogen, Cytophaga marina (heterotypic synonym, Flexibacter maritimus) was reclassified as Tenacibaculum maritimum (Suzuki et  al., 2001). Cytophaga marinoflava was assigned to the genus Leeuwenhoekiella (Nedashkovskaya et al., 2005d). The genus Stanierella was established to accommodate Cytophaga latercula; however, this new genus was eventually subsumed into another genus, Aquimarina, proposed in the same paper ­(Nedashkovskaya et al., 2005b, 2006). Cytophaga arvensicola, together with three other misclassified Flexibacter species – Flexibacter filiformis, Flexibacter japonensis, and Flexibacter sancti – was classified in the genus Chitinophaga (Kämpfer et al., 2006). At present, two of 23 validated species of the genus Cytophaga – Cytophaga fermentans and Cytophaga xylanolytica – remain to be reclassified. Cytophaga fermentans is a marine facultative ­anaerobe that possesses MK-7

and was closely related to the class Bacteroides based on the 16S rRNA sequence analysis (Nakagawa and Yamasato, 1993, 1996). Cytophaga xylanolytica is anaerobic gliding bacterium degrading xylan (Haack and Breznak, 1993). Only three partial 16S rRNA sequences (lengths, 287, 290, 350 bases, respectively) of this species are currently available; however, they show that Cytophaga xylanolytica is related to the class Bacteroides. Thus, Cytophaga fermentans and Cytophaga xylanolytica should not be included in the genus Cytophaga.

Differentiation of species of the genus Cytophaga Table 88 lists characteristics that distinguish Cytophaga species from one another. Table 88.  Characteristics differentiating the species of the genus

Cytophaga  a,b Characteristic

C. hutchinsonii

C. aurantiaca

Optimum temperature (°C) Color of cell mass Flexirubin reaction DNA G+C content (mol%)

30 Bright yellow + 39–40

20–25 Bright orange − 37–42

a Symbols: +, 90% or more of strains are positive; −, 10% or less of strains are positive.

Data from Reichenbach (1989c) and Bergey’s Manual of Determinative Bacteriology, 9th edition.

b

List of species of the genus Cytophaga 1. Cytophaga hutchinsonii Winogradsky 1929, 578AL hut.chin.so¢ni.i. N.L. masc. gen. n. hutchinsonii of Hutchinson, named in honor of English microbiologist, H. B. Hutchinson. The characteristics are as described for the genus with the following additional features taken from Bergey’s Manual of Determinative Bacteriology (9th edition) and the 1st edition of this Manual. Cells are flexible rods, 0.3–0.5 mm wide and 2–10 mm long or longer. The cell mass is bright yellow. Optimum growth temperature is 30°C. Oxidase-positive. Catalasenegative or very weakly positive. Cellulose (filter paper) and carboxymethyl cellulose are degraded, but not agar, chitin, pectin, or starch. Peptones, Casamino acids, several amino acids including aspartic and glutamic acids, NO3−, and NH4+ serve as sole nitrogen source. Cellulose, cellobiose, and glucose are used, but arabinose, fructose, galactose, mannitol, mannose, sodium pyruvate, and xylose are not. Growth does not occur in seawater media. A flexirubin type pigment is present. Source: unclear but assumed to be from soil (Reichenbach, 1989c). DNA G+C content (mol%): 39 (Bd) to 40 (Tm). Type strain: ATCC 33406, CIP 103989, DSM 1761, JCM 20678, LMG 10844, NBRC 15051, NCIMB 9469.

Sequence accession no. (complete genome): NC008255; (16S rRNA gene): M58768.

2. Cytophaga aurantiaca (ex Winogradsky 1929) Reichenbach 1989c, 2036VP (Cytophaga aurantiaca Winogradsky 1929, 597) au.ran.ti¢a.ca. N.L. fem. adj. aurantiaca orange colored. The characteristics are as described for the genus with the following additional features taken from Bergey’s Manual of Determinative Bacteriology (9th edition) and the 1st edition of this Manual. Cells are flexible rods, 0.3–0.4 mm wide and 2–8 mm long or longer. The cell mass is bright orange. Optimum growth temperature is 20–25°C. Oxidase-positive. Catalasenegative. Cellulose (filter paper) and carboxymethyl cellulose are degraded, but not agar, casein, or starch. Peptones, NO3−, and NH4+ serve as nitrogen sources. Cellulose, cellobiose, and glucose are used. Growth does not occur in seawater media. A flexirubin type pigment is absent. It is suggested that the type strain of Cytophaga aurantiaca is identical with Bortels’s strain Cytophaga aurantiaca 51, which is isolated from soil of the swampy edge of a pond in Germany (Bortels, 1956; Reichenbach, 1989c). DNA G+C content (mol%): 37–42 (Bd). Type strain: ATCC 12208, DSM 3654, JCM 8511, NBRC 16043, NCIMB 8628. Sequence accession no. (16S rRNA gene): D12658.

Other species No species listed below should be included in the genus Cytophaga, because they are not closely related to the type species of the genus based on 16S rRNA sequence analysis.

CP000383,

1. Cytophaga fermentans Bachmann 1955, 549AL fer.men¢tans. L. part. adj. fermentans fermenting.

Genus II. Adhaeribacter

The descriptions include characteristics described in Bergey’s Manual of Determinative Bacteriology (9th edition) and the 1st edition of this Manual. Cells are flexible rods, 0.3–0.7 mm wide and 8–50 mm long or longer. Motile by gliding. Nonsporeforming. Resting stages are absent. Gramstain-negative. The cell mass from aerobic cultures is bright ­yellow on peptone media, and from anaerobic cultures, it is unpigmented on minimal media. Colonies are thin spreading films with yellow blobs in shallow craters. Facultatively anaerobic. Optimum growth temperature is 30°C. Agar and starch, but not alginate, cellulose (filter paper), or chitin are degraded. Peptones, asparagine, glutamine, and NH4+ serve as nitrogen sources, but not NO3− or urea. Arabinose, cellobiose, fructose, glucose, lactose, maltose, mannitol, mannose, raffinose, starch, sucrose, and xylose are used, but acetate, agar, alginate, cellulose, chitin, dulcitol, ethanol, galactose, glycerol, rhamnose, trehalose, and sorbose are not. Glucose is fermented with production of acetate, propionate, and succinate. Growth does not occur in seawater media. Nitrate is not reduced. Marine organism, requiring elevated salt concentrations. This species is related to the class Bacteroides based on the 16S rRNA sequence analysis (Nakagawa and Yamasato, 1996). DNA G+C content (mol%): 39 (Bd) to 42 (Tm). Type strain: ATCC 19072, CIP 104805, DSM 9555, JCM 21142, NBRC 15936. Sequence accession no. (16S rRNA gene): M58766. 2. Cytophaga xylanolytica Haack and Breznak 1993, 14VP xy.lan.o.ly¢ti.ca. N.L. n. xylanum xylan (a xylose-containing heteropolysaccharides in plant cell walls); N.L. fem. adj. lytica (from Gr. fem. adj. lutikê), able to loosen, able to dissolve; N.L. fem. adj. xylanolytica xylan-dissolving.

375

The characteristics are as described by Haack and Breznak (1993). Cells are flexible rods, 0.4 mm wide and 3–24 mm long or longer. Motile by gliding. ­Nonsporeforming. ­Resting stages are absent. Gram-stain-negative. The cell mass is orange to salmon due to the presence of carotenoids. Aeroduric anaerobe. No growth occurs under conventional aerobic conditions with or without CO2 enrichment of the atmosphere. Growth occurs at 19–37°C; optimum is 30–32°C. Growth occurs at pH 6.1–8.7; optimum 7.2–8.2. Oxidase-negative. Catalase weakly positive. Sulfide is used as the sole sulfur source. Peptones, glutamine, and NH4+ serve as nitrogen sources, but not NO3−, N2, or glutamate. Xylan, laminarin, lichenin, cellobiose, lactose, glucose, galactose, mannose, xylose, and arabinose are fermented as energy sources for growth, but not cellulose, carboxymethyl cellulose, arabinogalactan, mannan, chitin, N-acetylglucosamine, sorbose, ribose, or rhamnose. Acetate, propionate, and succinate are the major products of xylan, xylose, and glucose fermentation. NO3− and SO42− are not used as terminal electron acceptors during anaerobic growth. O2 is not used as terminal electron acceptor (type strain). Growth occurs in the presence of up to 3% NaCl. Marine organism, requiring elevated salt concentrations. A flexirubin type pigment and a sulfonolipid are present. The major cellular fatty acids are C15:0 anteiso, C15:0, and C15:0 3-OH. Analysis of partial 16S rRNA sequences indicates a close relationship between this species and the class Bacteroides. DNA G+C content (mol%): 45.5 (Bd). Type strain: XM3, ATCC 51429, DSM 6779. Sequence accession no. (three partial 16S rRNA in lengths 287, 290, 350 bases): AH001617.

Genus II. Adhaeribacter Rickard, Stead, O’May, Lindsay, Banner, Handley and Gilbert 2005, 827VP Peter Gilbert* and Alexander H. Rickard Ad.haer¢i.bac.ter. L. v. adhaereo, adhaere to adhere to, to stick fast; N.L. masc. n. bacter from Gr. n. baktron rod; N.L. masc. n. Adhaeribacter sticky rod.

Rods, about 0.9–1.7 by 2.8–4.1 mm. In batch culture, mean size is dependent upon the phase of growth. During exponential growth, the rods occur singly, in aggregates, and in chains of up to approximately 4 cells in length. In the stationary phase, the cells occur singly. Cells are Gram-stain-negative, non-sporulating, nonflagellated, and nonmotile. Growth occurs in liquid and on solid interfaces. Strictly aerobic. Chemoheterotrophic. Temperature range for growth, 4–37°C; optimum, 30°C. Limited NaCl tolerance; growth occurs between 0–4.0% (w/v) NaCl. Habitat: within freshwater, multi-species biofilm communities developed on a stainless-steel surface exposed to potable water at a shear rate of 305/s. DNA G+C content (mol%): 40.0. Type species: Adhaeribacter aquaticus Rickard, Stead, O’May, Lindsay, Banner, Handley and Gilbert 2005, 827VP.

*Deceased.

Further descriptive information Based on 16S rRNA analysis, Adhaeribacter represents a separate and distinct lineage within the family Cytophagaceae of the Cytophaga–Flavobacterium–Bacteroides group (Figure 67). Colonies are pink, round, and mucoid on R2A medium (Reasoner and Geldreich, 1985) and are not easily emulsified in sterile water or phosphate buffered saline. Growth does not occur on peptone water agar or other media commonly used for the isolation of freshwater bacteria. The growth rate on agar is improved in a moist atmosphere. Cells grown in liquid R2A do not pellet tightly when centrifuged at 85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive; w, weakly positive; nd, data not reported or not applicable.

a

Data taken from Nikitin et al. (2004), Larkin and Borrall (1984a), Gosink et al. (1998), Chelius and Triplett (2000), Chelius et al. (2002), Raj and Maloy (1990), Xin et al. (2004), and Staley and Konopka (1984).

b

0.02 Cytophaga aurantiaca NCIMB 8628T (D12658)

100 64

Cytophaga hutchinsonii ATCC 33406T (M58768) Sporocytophaga myxococcoides DSM 11118T (AJ310654)

84

Roseivirga ehrenbergi i KMM 6017T (AY608410)

98

Reichenbachia agariperforans KMM 3525T (AB05891) Aquiflexum balticum BA160T (AJ744861)

92

85

Cyclobacterium marinum DSM 745T (AY533665)

78

Belliella baltica BA134T (AJ564643)

100

Hongiella mannitolivorans IMSNU 14012T (AY264838) Algoriphagus winogradskyi LMG 21969T (AJ575263)

99 10 100

Hongiella halophil a IMSNU 14013T (AY264839) Flexibacter flexilis ATCC 23079T (M62794) Flectobacillus major ATCC 29496T (M62787)

56 100

Arcicella aquatica N O- 502T (AJ535729) Dyadobacter fermentans NS114T (AF137029) Runella zeae NS12T (AF137381)

100

Runella slithyformi s ATCC 29530T (M62786)

FIGURE 70.  Unrooted neighbor-joining dendrogram of the phylogenetic relationships between Arcicella aquatica and type species of closely related genera based on a distance matrix analysis of the 16S rRNA gene sequences. Accession numbers are given in parentheses. Bootstrap percentages are indicated at tree branching points and the scale bar represents substitutions per nucleotide.

Taxonomic comments The 16S rRNA gene sequence similarity between the type strain of Arcicella aquatica and the most similar published sequence of an organism with a validly published name, Flectobacillus major, is 93.52% (Nikitin et al., 2004) (Figure 70). This

similarity value, and the differences in the polar lipids, the cellular fatty acids, and biochemical and physiological characteristics between Flectobacillus major and the type strain of Arcicella aquatica, together with the differences in the mol% G+C content of their DNA, all support the separate generic status of Arcicella.

380

Family I. Cytophagaceae

List of species of the genus Arcicella 1. Arcicella aquatica Nikitin, Strömpl, Oranskaya and Abraham 2004, 683VP (Arcocella aquatica Nikitin, Oranskaya, Pitryuk, Chernykh and Lysenko 1994, 152) a.qua.ti¢ca. L. fem. adj. aquatica aquatic. The characteristics are as given for the genus with the following additional features. Main ­phospholipids are 2-N-(2¢-hydroxyisopentadecanoyl)amino-3-hydroxy­ isoheptadeca-4(E)-ene-1-(2¢¢-aminoethyl)-phosphate, 2-N-(2¢hydro­x yisopentadecanoyl)amino-3-hydroxyoctadeca-4 (E)-ene-1-(2¢¢-aminoethyl)-phosphate, 1-pentadecanoyl-2hexadecenoylphosphatidyl-2¢-ethylamine, 1,2-bis-hexadecen­ oylphosphatidyl-2¢-ethylamine and 1-hexadecanoyl-2hexadecenoylphosphatidyl-2¢-ethylamine. Cells ­display high activities of alkaline phosphatase, leucine, and valine arylamidase, cystine arylamidase, trypsin, acid ­phosphatase, naph-

thol-AS-BI-phosphohydrolase, b-galactosidase, a-­glucosidase, catalase and N-acetyl-b-glucosaminidase, weak activities of esterase lipase (C8), a-chymotrypsin, a-­galactosidase, b-glucosidase, and a-fucosidase, and no activities of lipase (C14), a-glucuronidase, or a-mannosidase. Growth occurs on glucose, fructose, lactose, maltose, rhamnose, galactose, arabinose, ribose, sucrose, cellobiose, and inulin; some growth occurs on mannitol, sorbitol, acetate, and aspartate; no growth occurs on sorbose, dulcitol, ethanol, methanol, formate, propionate, pyruvate, monomethylamine, citrate, oxalate, succinate, malate, or amino acids, with the exception of aspartate. DNA G+C content (mol%): 34 (Tm). Type strain: NO-502, CIP 107990, LMG 21963. Sequence accession no. (16S rRNA gene): AJ535729.

Genus IV. Dyadobacter Chelius and Triplett 2000, 755VP, emend. Reddy and Garcia-Pichel 2005, 1298VP Gundlapally S. N. Reddy and Ferran Garcia-Pichel Dy.a.do.bac¢ ter. G. n. dyas two in number, pair; N.L. masc. n. bacter from the Gr. n. baktron rod or staff; N.L. masc. n. Dyadobacter rod or staff occurring in pairs.

Rods, 0.75–2 mm in length, in straight to curved arrangements, occurring in pairs (Figure 71) in young cultures and forming chains of coccoid cells in old cultures. Nonmotile. Nonsporeforming. Gram-stain-negative. Aerobic, chemoorganoheterotrophs, unable to hydrolyze cellulose or starch or gelatin. Oxidase- and catalase-positive. Cells produce a non-diffusible, yellow, flexirubin-like pigment. The major fatty acids are C15:0 iso, C16:1 w5c and C16:1 w7c. DNA G+C content (mol%): 44–49. Type species: Dyadobacter fermentans Chelius and Triplett 2000, 756VP.

FIGURE 71.  Scanning electron micrograph showing the arrangement

of cells of Dyadobacter.

Further descriptive information The genus belongs to the family Cytophagaceae in the order Cytophagales of the phylum Bacteriodetes, and presently contains six species, Dyadobacter fermentans (Chelius and Triplett, 2000), Dyadobacter crusticola (Reddy and Garcia-Pichel, 2005), Dyadobacter hamtensis (Chaturvedi et al., 2005), Dyadobacter ginsengisoli (Liu et al., 2006), Dyadobacter beijingensis (Dong et al., 2007), and Dyadobacter koreensis (Baik et al., 2007a). Dyadobacter species grown in trypticase soy agar (TSA) or R2A medium1 contain C15:0 iso (19–30%), C16:1 w5c (8–20%), and C16:1 w7c (14–40%) as major fatty acids, with 20–60% of all fatty acids being the unsaturated fatty acids [C16:1 w5c and C16:1 w7c]. The minor fatty acids common to all strains are C16:0 and C16:0 3-OH. Inasmuch as C16:1 w5c is present as a major fatty acid only in the species of the genus Dyadobacter, it can be readily used as a signature molecule to differentiate the genus Dyadobacter from other genera of the Cytophagaceae. Fatty acid profiles can also be used to differentiate among Dyadobacter species. C17:0 iso 3-OH, for example, is absent from Dyadobacter crusticola but the most abundant fatty acid in the other five species. C14:0 is absent from Dyadobacter fermentans and Dyadobacter ginsengisoli but present in the other four, as is C15:1 iso. Similarly, C15:0 iso 3-OH is absent in Dyadobacter hamtensis but present in all the other strains, and more significantly the fatty acid C15:0 iso 2-OH is present only in Dyadobacter beijingensis and not in others. All the species of Dyadobacter are yellow due to a non-diffusible flexirubin-like pigment with absorption maxima at around 428, 452, and 476 nm (Figure 72; wavelengths of peak absorption may vary slightly among species) and with a characteristic bathochromic shift (from yellow to orange and then to red) upon reaction with alkali that distinguishes them from carotenoids (Reddy and Garcia-Pichel, 2005; Weeks, 1981). These flexirubin-like pigments are not very common among bacteria and are therefore of considerable chemotaxomic value.

Genus IV. Dyadobacter

381

not yet detected ­Dyadobacter (Nagy  et  al., 2005; Reddy and ­Garcia-Pichel, 2006; Smith et al., 2004), indicating that they are not a major heterotrophic component of these communities; thus, their role remains to be clarified as well. Dyadobacter hamtensis was isolated from melt water from a Himalayan glacier and Dyadobacter ­koreensis was isolated from fresh water. By contrast, Dyadobacter ginsengisoli and Dyadobacter beijingensis were isolated from soil and rhizosphere-associated soil, respectively. The ecological role of all the above species too was not directly studied. FIGURE 72.  A typical absorption spectrum of flexirubin-like pigment

of Dyadobacter crusticola in ethanol (solid line) and alkaline ethanol (dotted line).

The six species of the genus Dyadobacter grow well on R2A solid medium at room temperature, forming visible colonies in 24–48 h. However, their physiological adaptation to extreme conditions of temperature, pH, and salinity varies widely. For instance, the type strains of Dyadobacter fermentans, Dyadobacter hamtensis, and Dyadobacter beijingensis can grow from 4 to 37°C, with an optimum growth temperature of 28°C and pH 7. Dyadobacter crusticola, Dyadobacter ginsengisoli, and Dyadobacter koreensis, by contrast, can grow from 4 to 30°C, pH 6–8, and can tolerate a salt concentration of 1%, with a temperature optimum of 25°C and pH 7. Among the six species, only Dyadobacter crusticola, Dyadobacter ginsengisoli, and Dyadobacter koreensis, can grow at 5°C, albeit slowly, and thus can be deemed psychrotolerant. Dyadobacter hamtensis is clearly halotolerant, growing in NaCl concentration up to 11.6%, whereas Dyadobacter koreensis can tolerate a pH of 11, indicating that it is alkali tolerant. All the strains can grow on various rich organic media such as nutrient agar, TSA, and R2A, and type strains of Dyadobacter fermentans and Dyadobacter hamtensis can grow on Ayer’s agar and peptone water as well (other species were not tested for growth on above two media). As shown in Table 91, differences in the metabolic versatility of the six species are substantial, ranging from the rather restricted capabilities of Dyadobacter crusticola to the wide array of carbon sources used by Dyadobacter hamtensis. So far, there seems to be no unifying theme to the ecological habitat or role of Dyadobacter species. Dyadobacter could be a genus of adventitious, generalist bacteria, but, with only a few species isolated, it is probably too early to infer such a pattern. The six species originate from disparate habitats and show widely different carbon use profiles and even divergent physiological ranges for growth Table 91. The single available strain of Dyadobacter fermentans was isolated from internal tissue of Zea mays (i.e., surface-sterilized plant stems), but because the authors could demonstrate neither a beneficial nor a detrimental effect of the bacterium on the plants, its true habitat and role remain unclear. Interestingly, putative members of Dyadobacter have been isolated from surface-sterilized soil nematode cysts on plates of rich media (Nour et al., 2003). Dyadobacter crusticola was isolated from biological soil crusts (BSCs; Garcia-Pichel, 2002) where it is thought that cyanobacterial exudates serve as a source of carbon and nitrogen, and exopolysaccharide production contributes to crusting of the soil. However, extensive surveys of soil crust environments using cultivation-independent, DNA-based methods have

Enrichment and isolation procedures Species of Dyadobacter have been isolated by directly plating on agar-solidified, relatively rich media (such as R2A or BG11-PGY) and incubated at ambient temperature in the dark for up to 15 d. Their yellow pigmentation and the typical cell shape and cell aggregation patterns that are readily observable under the compound microscope in a wet or dry mount, provide an easy system for preliminary identification on enrichment cultures. Repeated streaking of single colonies usually yields axenic, clonal strains with a few iterations. Inocula used successfully include surface sterilized maize stems, surface sterilized soil nematode cysts, biological soil crusts from arid lands, glacier melt waters, soil, rhizosphere-associated soil, and fresh water.

Maintenance procedures Stock cultures of Dyadobacter species can be maintained under aerobic conditions on a suitable medium such as R2A or 10× PGY or nutrient agar by monthly transfer onto fresh plates. Cultures also can be stored as glycerol stocks (18% glycerol) at −80°C. However, the best method of storing for many years is lyophilization and freezing at −80°C, which they tolerate with high recovery.

Procedures for testing special characters Colonies of Dyadobacter are colored yellow due to the presence of flexirubin-like pigment, and the presence of flexirubin, a characteristic feature of the genus, can be tested directly (and differentiated from carotenoid pigments) by adding a drop of 20% KOH onto colonies of Dyadobacter. This will cause change in the color of the pigment from yellow to orange and then to red (Reddy and Garcia-Pichel, 2005; Weeks, 1981). This alkali-driven shift can be also carried out on ethanolic extracts and quantified spectroscopically (Figure 72). Carotenoid-like absorption maxima at 428, 452, and 478 nm broaden upon addition of 1% KOH (final concentration) and an additional peak appears around 330 nm.

Differentiation of the genus Dyadobacter from other genera of the family Cytophagaceae The phylogenetically closest genera Runella and Spirosoma (­Figure 73) can be differentiated from Dyadobacter on the basis of pigment, cell morphology, and its variation during the growth cycle as well as fatty acid composition. For instance, unlike Dyadobacter, Runella has salmon colored colonies, no flexirubin, no cell morphology variation during the growth cycle, and lacks the fatty acid C16:1 w5c. Spirosoma has no cell morphology variation during the growth cycle, degrades gelatin and starch, contains the fatty acids C16:1 and C17:0 iso, and lacks C15:0 iso, C16:1 w5c, and C16:1 w7c. Within the Cytophagaceae, variable cell morphology is

382

Family I. Cytophagaceae

Source Maximum salt tolerance (as NaCl; w/v) pH Growth at 5°C Maximum growth temperature (°C) Lipase Phosphatase Esculin hydrolysis Nitrate reduction Lysine decarboxylase Oxidation/fermentation: Glucose Sucrose Acid from: d-Glucose Sucrose d-Fructose d-Maltose d-Arabinose, d-xylose Carbon compounds utilized: Sodium acetate d-Adonitol d-Arabinose l-Arabinose Cellulose, dextran Citric acid Dulcitol d-Fructose Fumarate d-Galactose Glycerol meso-Inositol Inulin Lactic acid Malonate d-Mannitol d-Mannose l-Melibiose l-Rhamnose, d-rhamnose d-Ribose l-Sorbose d-Sorbitol Tartrate d-Xylose Glycine l-Alanine, l-asparagine, l-aspartic acid, l-cysteine, l-glutamic acid, l-histidine, l-isoleucine, l-leucine, l-phenylalanine, l-proline, l-serine, l-threonine. l-tyrosine, l-valine l-Arginine, l-glutamine, l-lysine, l-methionine, l-tryptophan

Plant (Zea mays) 1.5 6–8 − 37 − − + − +

Soil crusts 1.0 6–8 + 30 + + + − −

Glacier water 11.0 6–8 − 37 − + − − −

+ +

− −

+ + − + +

Soil

D. koreensis (WPCB159T)g

D. beijingensis (A54T)f

D. ginsengisoli (Gsoil 043)e

D. hamtensis (HHS 11T)d

D. crusticola (C183-8T)c

Characteristic

D. fermentans (NS-114T)b

TABLE 91.  Characteristics that differentiate the species of the genus Dyadobactera

Freshwater

1.0 5.5–8.5 + 30 − nd − + nd

Rhizosphereassociated 1.5 6–8 + 35 nd nd + − −

+ −

− −

+ −

− −

− − + − −

+ − − − +

− − nd nd −

+ + + nd −

nd nd nd nd nd

+ − + + − − w + + + w + + − + + + + + − + w + + + −

− − − − − − + − − − − + + + − + − + − + − + − + − −

+ + − + + + − − + + + − + + + − − − − − + − + − + +

− + − + − − − + − + − − + − − − + + + − − − − + − nd

− + − + nd + + + nd + − + + − nd + + + − − − − − + nd nd

nd − + + nd nd − + nd + − − − nd nd − + + w − − − + nd nd

+

−

+

−

nd

nd

1.0 5–9.011 + 30 − + + − −

(Continued)

383

Genus IV. Dyadobacter

a

D. hamtensis (HHS 11T)d

D. ginsengisoli (Gsoil 043)e

D. beijingensis (A54T)f

D. koreensis (WPCB159T)g

Sensitivity to antibiotics (mg/disc): Amoxycillin (30) Ampicillin (25), chloramphenicol (30), streptomycin (25) Ciprofloxacin (30) Colistin (10) Erythromycin (15) Gentamicin (10) Kanamycin (30) Lincomycin (20) Novobiocin (30) Rifampin (25) Roxithromycin (30) Tetracycline (100) Trimethoprim (25) Vancomycin (10) DNA G+C content (mol%) 16S rRNA gene sequence similarity: D. fermentans D. crusticola D. hamtensis D. ginsengisoli D. beijingensis D. koreensis Composition of cellular fatty acids: C14:0 C15:1 iso C15:0 iso C15:0 anteiso C16:1 w5c C16:1 w7c C16:0 C18:0 C18:1 C15:0 iso 2-OH C15:0 iso 3-OH C16:0 iso 3-OH C17:0 iso 3-OH Unknown

D. crusticola (C183-8T)c

Characteristic

D. fermentans (NS-114T)b

TABLE 91.  (Continued)

nd R

nd R

R S

nd R

S S

R S

S R R R R nd R R R S R S 48

R R R S nd nd S S R S R R 48

S S R R S R R S S S S R 49

nd nd nd nd R nd nd R nd R nd nd 48

nd nd S S S S nd R S R nd R 49.2

R nd nd S nd R S S nd R nd S 44

100 95.9 95.2 96.4 97.4 95.2

95.9 100 95.7 96.0 94.5 95.2

95.2 95.7 100 96.4 94.4 98.0

96.4 96.0 96.4 100 96.4 96.6

97.4 94.5 94.4 96.4 100 94.6

95.2 95.2 98.0 96.6 94.6 100

− − 22.85 − 14.28 28.57 5.71 − − − 2.9 2.85 22.85 1.3

0.7 0.5 28.24 − 15.5 39.4 13.6 1.9 0.34 − 2.4 0.5 − 0.3

1.23 3.7 24.69 − 19.75 14.81 8.64 − 2.46 − − 2.46 22.22 −

− − 23.0 − 7.9 44.6 11.0 − − − 3.9 2.3 7.6 −

0.6 0.5 19.2 1.0 10.3 17.5 2.9 − − 23.4 2.6 3.1 12.4 2.1

0.7 − 24.2 − 11.0 34.8 9.4 − − − 2.8 2.5 9.5 −

Symbols: +, positive; −, negative or absent; w, weak; R, resistant; S, sensitive; nd, not done.

b

Data from Chelius and Triplett (2000).

Data from Reddy and Garcia-Pichel (2005).

c

Data from Chaturvedi et al. (2005).

d

Data from Liu et al. (2006).

e

Data from Dong et al. (2007).

f

Data from Baik et al. (2007a).

g

a hallmark of Dyadobacter, but it is also a trait of Flectobacillus. The latter genus, however, is pink-pigmented, not yellow, and does not contain flexirubin. The genus Dyadobacter can further be differentiated from Flectobacillus on the basis of fatty acid composition: members of Dyadobacter contain C16:1 w5c and C16:1 w7c as major fatty acids and lack C17:0 iso (Table 92). Besides

Dyadobacter, five other genera contain flexirubin-like pigments. However, compared to other genera, Dyadobacter is in the only one that contains the fatty acid C16:1 w5c and varies in morphology and aggregation during its growth cycle (Table 92). Other genus-specific characteristics that differentiate Dyadobacter from closely related genera are listed in Table 92.

384

Family I. Cytophagaceae Dyadobacter koreensi s WPCB 159 T

100 38

Dyadobacter hamtensi s HHS 11

38

T

Dyadobacter ginsengisoli Gsoil 04 3T (AB245369) Dyadobacter crusticol a CP183- 8T (AJ821885)

100

Dyadobacter beijingensis A54T (DQ335125)

99 63 54

Dyadobacter fermentans NS11 4T (AF137029) Runella slithyformi s LMG 11500T Spirosoma lingual e ATCC 23276T Leadbetterella byssophila 4M15T (AY854022)

30

Cytophaga hutchinsoni i ATCC 33406T

83

Flectobacillus majo r ATCC 29496T Microscilla furvescen s ATCC 23129T

94

25

Flexibacter tractuosu s ATCC 23168T

52 81 25

Marinicola seohaensis SW-152T (AY739663) 59

Reichenbachiella agariperforans KMM 3525T Persicobacter diffluen s ATCC 23140T Flavobacterium johnsoniae DSM 425T

46 70

Myroides odoratus ATCC 4651T

100 29 28

Cellulophaga lytica ATCC 23178T Cytophaga fermentans ATCC 19072T

29

Chryseobacterium indologene s ATCC 29897T

66

Weeksella virosa ATCC 43766T

71 100

Bacteroides fragilis Bfr81T (X83943) 74

Sphingobacterium multivoru m OM-A8T (AB020205)

95

Pedobacter heparinu s ATCC 13125T 100

Flexibacter canadensis ATCC 29591T Thermonema rossianu m AG3-1T (Y08958) Flavobacterium ferrugineu m ATCC 13524T Planctomyces limnophilus IFAM 1008T Geotoga subterrane a CC-1T (L10659)

0.05

FIGURE 73.  Neighbor-joining (NJ) tree based on 16S rRNA gene sequence (1337 nucleotides) showing the phy-

logenetic relationship between Dyadobacter and other related genera of the phylum Bacteroidetes. Bootstrap values (expressed as percentages of 1000 replicates) greater than 40% are given at nodes.

Taxonomic comments The genus Dyadobacter was established to accommodate the bacteria that are straight to curved, occur in pairs in young cultures and produce a non-diffusible, yellow, flexirubin-like pigment (Chelius and Triplett, 2000). The six currently known species (Dyadobacter fermentans, Dyadobacter crusticola, Dyadobacter hamtensis, Dyadobacter ginsengisoli, Dyadobacter beijingensis, and Dyadobacter koreensis) possess the above characteristics and, therefore, can be grouped within the genus. All six species have a 16S rRNA gene sequence similarity of 94.4–98.0%. The high-

est similarity of 98.0% was between Dyadobacter hamtensis and Dyadobacter koreensis, followed by Dyadobacter fermentans and Dyadobacter beijingensis (a similarity of 97.4%). Since their similarity was >97.0%, DNA–DNA hybridization was performed between Dyadobacter hamtensis vs Dyadobacter koreensis and Dyadobacter fermentans vs Dyadobacter beijingensis, and showed DNA–DNA similarities of 19.4 and 180 0.4–0.5 0.5–0.7 Bright orange Brick red − nd Saproxanthin Flexixanthin Aerobe Aerobe

F. ruber

2–60 0.4–0.5 White nd nd Facultative anaerobe

F. roseolus

10–60 0.5 Orange + Saproxanthin Aerobe

F. polymorphus

Length of threads (mm) Width of threads (mm) Color of cell mass Flexirubin reaction Carotenoid present Relation to oxygen

F. canadensis

Characteristic

F. flexilis

TABLE 95.  Characteristics differentiating the species of the genus Flexibactera,b

>200 0.6–1.1 Orange-peach nd Saproxanthin Aerobe

>50 nd Red nd + Aerobe

>50 nd Red nd + Aerobe

5 to >50 0.5 Orange nd Saproxanthin nd

+ − − − − nd − nd + nd 32 Marine 29

− + nd nd − + − − − − − + − − nd nd + − nd nd 40 40–45 Hot springs Hot springs 34–38 37

+ nd − +/− nd +/− nd nd + nd 30–45 Marine 35–40

Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive; +/−, most strains are positive; nd, not determined.

a

Data from Reichenbach (1989a, b) and Bergey’s Manual of Determinative Bacteriology, 9th edition.

b

white. Colonies often show a yellow-green-blue iridescence. ­Facultatively anaerobic. Oxidase- and catalase-positive. Casamino acids, gelatin, aspartate, glutamate, and NH4+ serve as sole nitrogen source but not NO3− or urea. Various sugars, glucose, and glycerol are utilized with acid production. Indole is not produced. H2S is produced. DNA, gelatin, and starch are hydrolyzed, but not agar, chitin, alginate, cellulose, or carboxymethyl cellulose. Pectin is weakly degraded. Nitrate is reduced. H2S is produced. Growth does not occur in the presence of 2% NaCl. The temperature range for growth is 10–40°C with optimum 18–30°C. The pH range for growth is 5–10 with an optimum of 6–8. DNA G+C content (mol%): 37 (Bd and Tm). Type strain: UASM 9D, ATCC 29591, CIP 104802, DSM 3403, JCM 21819, LMG 8368, NBRC 15130. Sequence accession no. (16S rRNA gene): AB078046. 2. Flexibacter elegans (ex Lewin 1969, non Soriano 1945) Reichenbach 1989b, 2067AL

acids serve as sole nitrogen source but not glutamate, NH4+, or NO3. Requires threonine. Growth on media containing peptone alone is very poor or absent but may be stimulated by the addition of a sugar such as glucose. In litmus milk, there is no acid production, coagulation, or reduction of litmus. Acid is produced from glucose. Gelatin is hydrolyzed, but not chitin, starch, or yeast cells. Indole and H2S are not produced. Nitrate is not reduced. Growth occurs on seawater media, but NaCl is not required. NaCl (2.4%) is tolerated. Optimum conditions for growth are around pH 7 and 30°C. The highest growth temperature is 40–45°C. The major carotenoid is saproxanthin. The flexirubin reaction is negative. DNA G+C content (mol%): 48 (Bd). Type strain: NZ-1, ATCC 23112, CIP 104801, DSM 3317, JCM 21159, LMG 10750, NBRC 15055. Sequence accession no. (16S rRNA gene): AB078048. 3. Flexibacter litoralis Lewin 1969, 199AL

e¢le.gans. L. masc. adj. elegans refined, fashionable, elegant.

li.tora¢lis. L. masc. adj. litoralis of or belonging to the seashore.

The characteristics are as described for the genus with the following additional features taken from the 1st edition of this Manual and Bergey’s Manual of Determinative Bacteriology, 9th edition. Very long, fine filaments with rounded ends, 0.4–0.5 mm wide and 50 mm long, often much longer, rarely shorter than 10–20 mm. On solid substrates the filaments tend to form loops and coils. Motile by gliding. On some media the colonies are spreading. The cell mass is bright orange. Aerobic. Oxidase-positive. Catalase-negative. Peptones and Casamino

The characteristics are as described for the genus with the following additional features taken from the 1st edition of this Manual and Bergey’s Manual of Determinative Bacteriology, 9th edition. Threads, 0.5–0.7 mm wide and up to 180 mm long, agile, gliding, and bending, apparently without cross-walls. The cell mass is brick red. Aerobic. Catalase-negative. Peptones serve as sole nitrogen source but not glutamate, or NO3. Requires many amino acids and thiamine. Sugars and organic acid is not utilized. Gelatin and starch are hydrolyzed, but not

Genus IX. Hymenobacter

agar, alginate, or carboxymethyl cellulose. H2S is not produced. Nitrate is not reduced. Growth does not occur below half-strength seawater. The highest growth temperature is 30–35°C. The major carotenoid is flexixanthin. DNA G+C content (mol%): 31 (Bd). Type strain: SIO-4, ATCC 23117, CIP 106402, DSM 6794, NBRC 15988. Sequence accession no. (16S rRNA gene): AB078056. 4. Flexibacter polymorphus Lewin 1974, 393AL po.ly.mor¢phus. N.L. masc. adj. polymorphus (from Gr. adj. polumorphos -on) of many shapes, variable in form. The characteristics are as described for the genus with the following additional features taken from the 1st edition of this Manual and Bergey’s Manual of Determinative Bacteriology, 9th edition. Long flexible filaments, 1.1 mm wide and up to several hundred mm long, with cross-walls 3.5–7 mm apart. The crosswalls are recognizable under the phase-contrast microscope. In media containing 0.1% NaHCO3, finer and shorter filaments 0.6 mm wide and 10–40 mm long appear, in addition to the long ones. At a pH above 8 the cells contain optically refractile granules, presumably some lipid material. Sometimes, inflated and branched filaments also occur. The filaments are very actively gliding with speeds up to 12 mm/s (23°C). The cell mass is orange. Aerobic. Catalase-negative. Peptones, Casamino acids, and glutamate serve as sole nitrogen source but not NH4+ or NO3. Requires cobalamine. Agar, alginate, cellulose, and starch are not hydrolyzed. H2S is not produced. Growth does not occur below half-strength seawater. The highest growth temperature is 32°C. The pH range for growth is 7–8.5. The major carotenoid is saproxanthin. DNA G+C content (mol%): 29 (Bd). Type strain: ATCC 27820, DSM 9678, LMG 13859, NBRC 16703. Sequence accession no. (16S rRNA gene): AB078059. 5. Flexibacter roseolus Lewin 1969, 199AL ro.se¢o.lus. L. adj. roseus rose-colored; L. masc. suff. -olus diminutive ending; N.L. masc. dim. adj. roseolus intended to mean with a rosy tinge. The characteristics are as described for the genus with the following additional features taken from the 1st edition of this Manual and Bergey’s Manual of Determinative Bacteriology, 9th edition. Very long threads, more than 50 mm long. The cell mass is red. Aerobic. Catalase-negative. Peptones and Casamino

397

acids serve as sole nitrogen source but not glutamate or NO3−. Gelatin is hydrolyzed but not starch. H2S is not produced. Seawater is tolerated. The highest growth temperature is 40°C. The major carotenoid is flexixanthin, demonstrated in a strain CR-141 (=ATCC 23087, NBRC 16030). DNA G+C content (mol%): 34–38 (Bd). Type strain: CR-155, ATCC 23088, CIP 106406, DSM 9546, LMG 13856, NBRC 16707. Sequence accession no. (16S rRNA gene): AB078063. 6. Flexibacter ruber Lewin 1969, 199AL ru¢ber. L. masc. adj. ruber red. The characteristics are as described for the genus with the following additional features taken from the 1st edition of this Manual and Bergey’s Manual of Determinative Bacteriology, 9th edition. Very long threads, more than 50 mm long. The cell mass is red. Aerobic. Catalase-negative. Peptones, Casamino acids, glutamate and NO3− serve as sole nitrogen source. Various sugars are metabolized with acid production. Gelatin is hydrolyzed. H2S is not produced. Nitrate is reduced. Growth does not occur on seawater media. The highest growth temperature is 40–45°C. The major carotenoid is flexixanthin. DNA G+C content (mol%): 37 (Bd). Type strain: GEY, ATCC 23103, DSM 9560, LMG 13857, NBRC 16677. Sequence accession no. (16S rRNA gene): AB078064. 7. Flexibacter tractuosus Leadbetter 1974, 106AL (“Microscilla tractuosa Lewin 1969, 199) trac.tu.o¢sus. L. masc. adj. tractuosus that draws to itself, drawn or clumped together. The characteristics are as described for the genus with the following additional features taken from the 1st edition of this Manual and Bergey’s Manual of Determinative Bacteriology, 9th edition. Threads, 0.5 mm wide and 5–50 mm long, or longer. The cell mass is orange. Peptones, Casamino acids, and glutamate serve as sole nitrogen source but not NO3−. Sugars and glycerol are usually utilized. Agar, alginate, and carboxymethyl cellulose are not degraded. Nitrate is reduced by a few strains. The highest growth temperature is 30–45°C. The major carotenoid is saproxanthin. DNA G+C content (mol%): 35–40 (Bd). Type strain: H-43, ATCC 23168, CIP 106410, DSM 4126, LMG 8378, NBRC 15989, VKM B-1430. Sequence accession no. (16S rRNA gene): AB078072.

Genus IX. Hymenobacter Hirsch, Ludwig, Hethke, Sittig, Hoffmann and Gallikowski 1999, 1VP emend. Buczolits, Denner, Kämpfer and Busse 2006, 2076VP (Effective publication: Hirsch, Ludwig, Hethke, Sittig, Hoffmann and Gallikowski 1998a, 374.) Sandra Buczolits and Hans-Jürgen Busse Hy.me.no.bac¢ter. Gr. masc. n. hymen pellicle, thin layer; N.L. masc. n. bacter the equivalent of Gr. neut. n. baktron a rod or staff; N.L. masc. n. Hymenobacter a rod growing in thin layers.

Rod-shaped, with polyphosphate granules near the cell poles. Cells aggregating with increasing formation of extracellular polymer and spreading in thin, red to pink layers on agar

s­ urfaces. Nonmotile. Gram-stain-negative. Colonies are flat with a small, raised center. Colony edges may show parallel arrangement of cells in the form of palisades. Aerobic. Heterotrophic,

398

Family I. Cytophagaceae

with a preference for oligotrophic media. Temperature range for growth, 4–37°C; optimum, 10–28°C. Some strains can grow at 42°C, but most strains do not. Cells do not grow anaerobically with or without light. Catalase- and oxidase-positive. Nonhemolytic. The carbon utilization spectrum is limited to some sugars, sugar alcohols, organic acids, and a few amino acids. Hydrolysis of gelatin, starch, xylan, Tween 80, and Tween 60 is common. DNA may also be hydrolyzed, but cellulose and pectin are not. Cells are highly sensitive towards the action of numerous antibiotics. meso-Diaminopimelic acid is present in the cell-wall murein. The principal menaquinone is MK-7. The fatty acid profile consists of predominantly branched fatty acids of the iso- and anteiso-type with C15:0 iso, C15:0 anteiso, C16:1 w7c/C15:0 iso 2-OH (summed feature), and C17:1 iso I/C17:1 anteiso B (summed feature), usually present in moderate to major amounts (except for Hymenobacter roseosalivarius, which lacks C15:0 anteiso). Unbranched fatty acids are usually present in moderate amounts, but their content is low in Hymenobacter ocellatus. All members of the genus contain a polar lipid profile consisting of phosphatidylethanolamine, an unknown aminophospholipid (APL3), two unknown polar lipids (L3, L5), and a mixture of several other unknown aminophospholipids, aminolipids, phospholipids, glycolipids, and polar lipids. sym-Homospermidine is the major polyamine. DNA G+C content (mol%): 55–65. Type species: Hymenobacter roseosalivarius Hirsch, Ludwig, Hethke, Sittig, Hoffmann and Gallikowski 1999, 1VP (Effective publication: Hirsch, Ludwig, Hethke, Sittig, Hoffmann and Gallikowski 1998a, 382.).

Further descriptive information The genus Hymenobacter was described with the single species Hymenobacter roseosalivarius that had been isolated from the Dry Valleys region in Antarctica (Hirsch et al., 1998a). In this study, affiliation of three strains - provisionally named “Taxeobacter ­chitinovorans”, “Taxeobacter gelupurpurascens”, and “Taxeobacter ocellatus” (Reichenbach, 1992b) — to this lineage was demonstrated by rRNA gene sequence similarity and phylogeny. So far, eight other species with validly published names have been effectively described as species of the genus: Hymenobacter actinosclerus isolated from irradiated pork (Collins et al., 2000); Hymenobacter aerophilus isolated during the examination of airborne bacteria in samples from the Museo Correr in Venice, Italy (Buczolits et al., 2002); Hymenobacter norwichensis isolated during the ­examination of airborne bacteria in samples from the ­Sainsbury Centre for Visual Arts in Norwich (UK; Buczolits et al., 2006); Hymenobacter chitinivorans (formerly designated “Taxeobacter chitinovorans”); Hymenobacter gelipurpurascens (formerly designated “Taxeobacter gelupurpurascens”); Hymenobacter ocellatus (formerly designated “Taxeobacter ocellatus”) isolated from dried soil which had been stored for several years (Baik et al., 2006; Reichenbach, 1992b); Hymenobacter rigui isolated from wetland freshwater in Woopo, South Korea (Baik et al., 2006); Hymenobacter xinjiangensis isolated from soil of the Xinjing desert, China following gamma-irradiation (Zhang et al., 2007). Sources of isolation suggest that at least some Hymenobacter species have developed strategies to survive under unfavorable conditions such as desiccation, radiation, and cold. Strains analyzed for their quinone systems and polyamine patterns contain menaquinone MK-7 and the predominant

compound sym-homospermidine, respectively. The contents of certain fatty acids such as C15:0 iso, C15:0 anteiso, C16:1 w5c, C16:1 w7c/C15:0 iso 2-OH (summed feature), and C17:1 iso I/ C17:1 anteiso B (summed feature), which were detected in the range 8–37, 0–26, 2–23, 5–30, and 8–27%, respectively, suggest a high potential for differentiation of Hymenobacter species and identification of newly isolated strains. Even polar lipid profiles exhibit a diversity that appears to be suitable for differentiation between Hymenobacter species (Baik et al., 2006). Physiological and biochemical traits useful for discrimination between Hymenobacter species are listed in Table 96. Nothing is known about pathogenic potential of Hymenobacter species.

Differentiation of the genus Hymenobacter from other genera Hymenobacter species do not exhibit extraordinary phenotypic traits which allow their differentiation from closely related genera such as Adhaeribacter, Pontibacter, and Effluviibacter, but differentiation may be accomplished by 16S rRNA gene sequence similarities below 88.0%. Certain quantitative differences in the fatty acid profiles might be useful for phenotypic differentiation of Hymenobacter species from related genera. The content of C15:0 iso 2-OH (16.5%) in the fatty acid profile of Adhaeribacter aquaticus (Rickard et al., 2005) is significantly higher than in Hymenobacter species (Table 97) and contents of C17:1 w6c and lack of “summed feature” (C15:0 iso 2-OH/C16:1 w7c) in the profiles of Adhaeribacter aquaticus and Effluviibacter roseus (Rickard et al., 2005; Suresh et al., 2006) might be useful for differentiation from Hymenobacter species. The high G+C content may distinguish Hymenobacter (>55 mol%) species from Pontibacter and Adaeribacter species (Nedashkovskaya et al., 2005c; Rickard et al., 2005; Zhou et al., 2007). However, the listed distinguishing traits cannot be considered to be highly reliable because each of the nearest related genera so far is represented by only one or two species, and hence the variability of the characteristics within these genera is unknown.

Taxonomic comments Sequence similarities in the 16S rRNA genes among established species of the genus Hymenobacter are above 90%. This value is rather low compared to many other genera where the threshold value for genus affiliation is approximately 95% similarity. This significantly lower threshold value for delineation of the genus suggests a higher mutation rate in the 16S rRNA coding genes or the need for dissection of the genus possibly on the basis of differences in the polar lipid and fatty acid profiles and other distinguishing traits as yet unidentified. However, if the threshold value of 90% for assignment to the genus Hymenobacter is considered to be acceptable, deposited 16S rRNA gene sequences indicate presence in institutional culture collections of several strains that might be described as novel species of the genus, as indicated under Other organisms. Phylogenetic relatedness shown in the maximum-likelihood tree after multiple alignments (Figure 75) are in good agreement with that suggested from 16S rRNA gene sequence similarities, demonstrating that Hymenobacter ocellatus represents the deepest branching within the genus. 16S rRNA gene based phylogenetic relatedness support placement of all unnamed strains

399

Genus IX. Hymenobacter

H. chitinivorans

H. gelipurpurascens

H. ocellatus

H. actinosclerus

H. aerophilus

H. rigui

H. xinjiangensis

Growth at/in: 1% NaCl 3% NaCl Starch Casein Tyrosine 4°C 37°C Assimilation of: N-Acetyl-d-glucosamine, gluconate p-Arbutin, d-ribose d-Cellobiose d-Fructose d-Galactose d-Xylose d-Glucose d-Mannose l-Rhamnose Maltitol Sucrose Salicin d-Maltose, d-trehalose d-Mannitol Acetate, propionate Glutarate, pyruvate cis-Aconitate, adipate, fumarate, dl-3-hydroxybutyrate, l-malate, itaconate, dl-lactate, mesaconate, oxoglutarate, suberate, l-alanine, l-proline, l-phenylalanine Citrate Hydrolysis of: pNP b-d-Glucuronide pNP Phosphate, l-proline, p-nitroanilide (pNA) pNP a-d-Glucopyranoside pNP b-Glucopyranoside bis-pNP Phosphate pNP Phenyl-phosphonate l-Alanine pNA l-Glutamate-g-3-carboxy-pNA

H. norwichensis

Characteristic

H. roseosalivarius

TABLE 96.  Differential characteristics among species of the genus Hymenobacter a

+b nr nr nr nr +b −

− − + + + + −

+ + + + + + −

+ + + + nr + −

+ + + + + − +

nr nr + nr nr −c +d

+ + + d + + −

+ − + nr nr + +

− nr − nr nr + +

− − d − − − − − − − − − − − + + −

− + + + + + + + − − − + + + − − −

− − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − −

+ + + − − − + + − − − − − − + + +

− − − + − − + + − − + − − − + − −

− − + + + − + − − nr + + + − nr nr nr

nr nr nr nr nr nr nr nr nr nr nr nr nr nr nr nr nr

−

−

−

−

−

+

−

−

nr

− nr + − + − + −

− nr + + + + + −

− + − − − − + −

− + + − − − + −

+ − + + + − + −

− + + − + + + +

− + + − + + + +

nr + − nr nr nr nr nr

nr + nr nr nr nr nr nr

a Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive; nr, not reported; PNP, para-nitrophenyl; pNA, p-­itroanilide.

Hirsch et al. (1998a).

b

Collins et al. (2000) reported no growth at 5°C.

c

Collins et al. (2000) reported growth at 42°C.

d

within the genus Hymenobacter, but strain Taxeobacter sp. SAFR033 appears to be a representative of a novel genus.

Maintenance procedures For long-term storage, Hymenobacter cultures may be lyophilized by common procedures that are used for many bacteria; alternatively, cultures can be stored at −80°C in potassium phosphate buffer/25% glycerol (v/v).

Enrichment and isolation procedures Temperatures between 20 and 28°C and, low nutrient media with neutral pH such as R2A* or PYE agar† appear to be most *R2A agar has the following composition (g/l): yeast extract, 0.5; casein hydrolysate, 0.5; glucose, 0.5; starch, 0.5; K2HPO4, 0.3; MgSO4, 0.024; Na-pyruvate, 0.3; agar, 15.0; pH 7.2 ± 0.2. † PYE agar has the following composition (g/l): yeast extract, 3.0; peptone from casein, 3.0; agar, 15.0; pH 7.2.

400

Family I. Cytophagaceae

H. chitinivorans

H. gelipurpurascens

H. rigui

H. ocellatus

H. aerophilus

H. actinosclerus

H. norwichensis

H. xinjiangenis

C13:0 iso C14:0 Unknown 13.565 C14:0 iso C15:1 iso G C15:1 anteiso A Summed feature: C15:1 iso I/C13:0 3-OH C15:0 iso C15:0 anteiso C15:0 iso 3-OH C15:0 iso 2-OH C15:1w6c C15:0 C16:1 iso H C16:0 iso 3-OH C16:1 w5c Summed feature: C16:1 w7c/C15:0 iso 2-OH C16:0 iso C16:0 anteiso C16:0 C16:0 3-OH Unknown 16.580 C17:0 iso C17:0 anteiso C17:0 iso 3-OH C17:0 2-OH C17:1 w6c C17:1 iso w9c Summed feature: C17:1 iso I/C17:1 anteiso B C18:1 w9c C18:1 w7c C18:0

H. roseosalivarius

TABLE 97.  Fatty acid profiles of Hymenobacter species a

− − − − − − − 8.3 − 2.7 − − − 2.7 1.0 23.3 29.8 2.1 − 1.1 1.2 − 1.7 − 5.8 − 1.1 − 18.5 − − −

0.6 − 0.5 0.5 0.6 − 2.1 31.1 3.7 4.1 − − − 2.0 − 8.9 13.9 1.4 − − 0.5 − 2.6 − 6.2 − − 0.7 20.8 − − −

− − 0.8 − 0.8 1.1 − 17.3 23.1 2.3 0.9 − − 3.7 − 11.3 17.6 2.7 − − − − 1.7 2.0 3.7 1.7 − − 9.4 − − −

− − − nr nr nr − 34.8 5.9 − − − nr − − 15.0 13.8 − 1.9 6.4 − − 5.0 − 3.1 − − nr 14.4 − − −

0.6 − − 1.5 − − 2.7 36.7 3.9 4.3 − − − 1.3 − 2.0 5.4 2.9 − 0.6 − − 2.8 − 6.8 − 1.2 0.6 26.9 − − −

− − − 0–0.4 − 0–0.5 0.8–1.4 10.8–15.0 18.6–22.3 1.3–1.6 0.5–0.9 0.8–1.2 nr 1.4–1.5 − 6.6–7.9 21.4–22.3 0.8 0–0.7 1.6–1.7 − − 2.7–4.5 1.3–2.3 2.6–3.5 0.8–1.3 0–0.7 0–0.5 17.7–18.8 − − −

− − − nr − − 2.3 22.3 25.8 1.6 0.8 0.8 nr 1.5 − 3.7 13.1 − − − − − 1.8 0.7 3.1 2.0 0.7 − 19.9 − − −

− 1.3 − 0.8 − − 0.8 27.3 10.6 2.2 − 0.5 0.5 1.4 − 13.6 23.6 1.2 − 2.2 0.6 − 1.6 − 3.0 − − − 8.3 − − −

− 1.3 − − 2.0 − 2.2 19.5 3.7 2.2 − − − 1.8 − 10.6 20.2 1.0 − 6.2 − − 1.8 tr 3.4 tr − − 8.5 1.1 4.8 6.5

Given as relative (%) amounts; tr, traces; nr, not reported.

a

appropriate for isolation of hymenobacters. The ability of several Hymenobacter species to survive under unfavorable ­conditions such as exposure to increased desiccation (Buczolits

et al., 2002; Hirsch et al., 1998a; Reichenbach, 1992b) or at high levels of radiation (Collins et al., 2000) may be useful for development of more specific isolation procedures.

List of species of the genus Hymenobacter 1. Hymenobacter roseosalivarius Hirsch, Ludwig, Hethke, Sittig, Hoffmann and Gallikowski 1999, 1VP (Effective publication: Hirsch, Ludwig, Hethke, Sittig, Hoffmann and Gallikowski 1998a, 382.) ro¢se.o.sa.li.va¢ri.us. L. adj. roseus rose colored; L. adj. salivarius salivary, slimy; N.L. masc. adj. roseosalivarius indicating a rose-colored bacterium surrounded by much polymer. The characteristics are as described for the genus and as listed in Tables 96 and 97, with the following additional features. Cells produce (often subpolarly) extracellular polymer and aggregate to form tight, thin, and spreading reddish layers on agar surfaces. Growth in liquid cultures is turbid. Does not grow at 37°C. l-Aspartate is used as a carbon

source, but not glutamate. The maximum salt tolerance is 0.5–2.0% NaCl. Pectin and cellulose are not hydrolyzed. The fatty acid profile is given in Table 97. Source: sandstone and soils of the Dry Valleys region of Antarctica. DNA G+C content (mol%): 56 (HPLC). Type strain: AA-718, CIP 106397, DSM 11622. Sequence accession no. (16S rRNA gene): Y18833. 2. Hymenobacter actinosclerus Collins, Hutson, Grant and ­Patterson 2000, 733VP ac.ti.no.scle¢rus. Gr. n. actis, actinos ray, beam; Gr. adj. scleros hard; N.L. masc. adj. actinosclerus hard against rays, pertaining to the organism’s radiation resistance.

Genus IX. Hymenobacter

401

Adhaeribacter aquaticus DSM 16391T (AJ626894) Flexibacter flexus IFO 15060T (AB078050) Taxeobacter sp. SAFR-033 (AY167829) 100

Pontibacter actiniarum KMM 6156T (AY989908) Effluviibacter roseus DSM 17521T (AM049256) Hymenobacter ocellatus DSM 11117T (Y18835) Cytophagales str. S23328 (D84607) 100

Hymenobacter sp. PB17 (AB251884)

70

Bacteroidete s bacterium P3 (DQ351728) 100

Hymenobacter actinosclerus CCUG 39621T (Y17356) Antarctic bacterium R-7572 (AJ440980) 71

Hymenobacter aerophilus DSM 13606T (AJ276901)

79

Hymenobacter chitinivorans DSM 11115T (Y18837) 80

Hymenobacter sp. zf-IRlt3 (DQ223662) 99

Hymenobacter norwichensis DSM 15439T (AJ549285)

81

Taxeobacter sp. SAFR-023 (AY167836)

Hymenobacter sp. N/S2 (AJ549284) Hymenobacter rigui KCTC 12533T (DQ089669) Hymenobacter gelipurpurascens DSM 11116T (Y18836) Hymenobacter sp. Tibet-IIU11 (DQ177475) 90

Hymenobacter xinjiangenisis CCTCC AB 206080T (DQ888329)

Taxeobacter sp. GIC34 (AY439245)

Hymenobacter sp. 21/4 (DQ365992) 100

0.1

Hymenobacter roseosalivarius DSM 11622T (Y18833)

Figure 75.  Maximum likelihood dendrogram showing phylogenetic relatedness among Hymenobacter species and

so far unnamed isolates considered to represent novel species of the genus. Numbers at the nodes indicate bootstrap values (500 replications). The scale bar represents 10% sequence divergence.

The characteristics are as described for the genus and as listed in Tables 96 and 97, with the following additional features. Cell dimensions are 0.5–0.6 × 2.0–3.6 mm. No variation in cell morphology is observed in old cultures. Colonies on yeast extract peptone agar are circular, entire, opaque, and not easily emulsified. A water-insoluble red pigment is produced. Fluorescent pigments are not produced on King’s A or B media. Chemo-organotrophic. Aerobic, having a respiratory type of metabolism, with an ability to grow under microaerobic conditions. Oxidase- and catalase-­positive. Acid and gas are not produced from d-glucose. Starch is hydrolyzed, but not esculin. Alkaline phosphatase, acid phosphatase, ester lipase C8, cystine arylamidase, leucine arylamidase, valine arylamidase, N-acetyl-b-glucosaminidase, and phosphoamidase activity are detectable with the API ZYM system. Lipase C14, chymotrypsin, trypsin, a-­galactosidase, b-galactosidase, b-glucuronidase, b-­glucosidase, a-­fucosidase,

a-­mannosidase, and urease activity are not detectable. Nitrate reduction, H2S production, and indole production are negative. Growth occurs at 42°C but not at 5°C. Optimal growth temperature, 25–30°C. Highly radiation-resistant with a D10 in sodium phosphate buffer of 3.45 kGy and on minced pork of 5.05 kGy. The fatty acid profile is given in Table 97. Source: pork chops irradiated with 1.75 kGy. DNA G+C content (mol%): 62 (Tm). Type strain: CCUG 39621, CIP 106628. Sequence accession no. (16S rRNA gene): Y17356. 3. Hymenobacter aerophilus Buczolits, Denner, Vybiral, Wieser, Kämpfer and Busse 2002, 454VP aer.o.phi¢lus. Gr. n. aer air; N.L. masc. adj. philus (from Gr. masc. adj. philos) friend, loving; N.L. masc. adj. aerophilus airloving, indicating its survival when suspended in air.

402

Family I. Cytophagaceae

The characteristics are as described for the genus and as listed in Tables 96 and 97, with the following additional features. Cells are 0.4–0.75 × 1.3–5.0 mm. Cells grow best on nutrient-reduced media such as CasMM* and R2A agar. Colonies on CasMM and R2A agar are translucent, red, circular, entire, low-convex, smooth, and slimy; the diameter is as much as 3.0 mm after 5 d at 28°C, and the colonies spread on CasMM agar. Colonies on standard bacteriological media such as PYES† agar and TSA are opaque, red, circular, entire, convex, and smooth; diameter is up to 2.0 mm after 5 d of incubation at 28°C. No growth occurs on MacConkey or Czapek–Dox agar. The optimum growth temperature is room temperature. The temperature range for growth is 4–28°C; no growth occurs at 37°C. Tolerance maximum for growth in the presence of NaCl is 2.0%. A water-insoluble red pigment is produced; the visible absorption spectrum of the acetone–extracted pigment shows a maximum at 482 nm and two slight inflexions at 453 and 505 nm. Cells assimilate acetate, propionate, d-fructose, d-glucose, d-mannose, and sucrose and display positive reactions in the following tests: catalase, l-alanine aminopeptidase, DNase, alkaline phosphatase, esterase (C4), esterase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, valine arylamidase, hydrolysis of Tween 80, p-nitrophenyl (pNP) a-d-glucopyranoside, pNP phenylphosphonate, l-proline p-nitroanilide (pNA), l-alanine pNA, l-glutamate g-3-carboxy-pNA, bis-pNP phosphate, and 2-deoxythymidine5¢-pNP phosphate. Depending on the sensitivity of the method applied, strains show either no catalase reaction or only a weakly positive one. Cells are susceptible to bacitracin, chloramphenicol, colistin sulfate, erythromycin, fusidic acid, gentamicin, kanamycin, nitrofurantoin, penicillin G, polymyxin B, tetracycline, and vancomycin. The fatty acid profile is given in Table 97. Source: air in the Museo Correr, Venice, Italy. DNA G+C content (mol%): 60–63 (HPLC). Type strain: I/26-Cor1, CCUG 49624, DSM 13606, LMG 19657. Sequence accession no. (16S rRNA gene): AJ276901. 4. Hymenobacter chitinivorans Buczolits, Denner, Kämpfer and Busse 2006, 2077VP (Taxeobacter chitinovorans Reichenbach 1992b, 182) chi.ti.ni.vo¢rans. N.L. neut. n. chitinum chitin; L. part. adj. vorans devouring; N.L. part. adj. chitinivorans devouring ­chitin. The characteristics are as described for the genus and as listed in 96 and 97, with the following additional features. Cells are approximately 0.8 × 4.0 mm. Colonies are brick red. Growth occurs on PYES agar, Czapek–Dox agar, and R2A but not on MacConkey agar. Oxidase- and catalase-positive. Nitrate reduction is weakly positive without production of N2. Negative for production of indole from tryptophan and for arginine dihydrolase and urease. Chitin is degraded. ­Positive

*CasMM agar (g/l): K2HPO4, 0.6; Na2HPO4·2H2O, 0.05; MgSO4·7H2O, 0.05; MgCl2·7H2O, 0.1; KNO3, 0.2; FeCl3·6H2O, 0.010; casein, 0.8; yeast extract, 0.4; agar, 15.0; pH 7.0. † PYES agar (g/l): yeast extract, 3.0; peptone from casein, 3.0; sodium succinate, 2.3; agar, 15.0; pH 7.2.

for alkaline phosphatase, esterase C4 (weakly), esterase lipase C8 (weakly), leucine arylamidase, and naphthol-AS-BIphosphohydrolase (weakly) with the API ZYM system. Negative for lipase C14, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase. Sensitive to bacitracin (10 IU), chloramphenicol (30 mg), colistin sulfate (10 mg), erythromycin (15 mg), fusidic acid (10 mg), gentamicin (10 mg), kanamycin (30 mg), penicillin G (10 IU), vancomycin (30 mg), polymyxin B sulfate (300 IU), and tetracycline (10 mg). The fatty acid profile is given in Table 97. Source: soil. DNA G+C content (mol%): ~61 (analytical ultracentrifuge). Type strain: strain Txc1, DSM 11115, LMG 21951. Sequence accession no. (16S rRNA gene): Y18837. 5. Hymenobacter gelipurpurascens Buczolits, Denner, Kämpfer and Busse 2006, 2077VP (Taxeobacter gelupurpurascens Reichenbach 1992b, 182) ge.lu.pur.pu.ras¢cens. L. masc. n. gelus -us (or L. neut. n. gelum -i) the ice cold; L. part. adj. purpurascens turning ­purple; N.L. part. adj. gelipurpurascens becoming purple in the cold. The characteristics are as described for the genus and as listed in Tables 96 and 97, with the following additional features. Size of cells is approximately 0.5 ´ 2.0 mm. ­Colonies are brick red, but at a growth temperature of 2–6°C, and colonies grown on a certain medium are deep blood-red (Reichenbach, 1992b). Growth occurs on PYES agar, ­Czapek–Dox agar, and R2A, but not on MacConkey agar. Oxidase- and catalase-positive. Nitrate reduction is weakly positive without production of N2. Negative for production of indole from tryptophan and for arginine dihydrolase and urease. Positive for alkaline phosphatase, esterase C4 (weakly), esterase lipase C8 (weakly), leucine arylamidase, valine arylamidase (weakly), and naphthol-AS-BI-­phosphohydrolase (weakly) with the API ZYM ­system. Negative for lipase C14, cystine arylamidase, trypsin, chymotrypsin, acid ­phosphatase, a-galactosidase, b-galactosidase, b-glucuronidase, a-­ glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase. Sensitive to bacitracin (10 IU), chloramphenicol (30 mg), colistin sulfate (10 mg), erythromycin (15 mg), fusidic acid (10 mg), gentamicin (10 mg), kanamycin (30 mg), penicillin G (10 IU), vancomycin (30 mg), polymyxin B sulfate (300 IU), and tetracycline (10 mg). The fatty acid profile is given in Table 97. Source: soil. DNA G+C content (mol%): ~57–58 (analytical ultracentrifuge). Type strain: Txg1, DSM 11116, LMG 21873. Sequence accession no. (16S rRNA gene): Y18836. 6. Hymenobacter norwichensis Buczolits, Denner, Kämpfer and Busse 2006, 2077VP nor.wi.chen¢sis. N.L. masc. adj. norwichensis of or belonging to Norwich, a city in England where the type strain was isolated. The characteristics are as described for the genus and as listed in Tables 96 and 97, with the following additional

Genus IX. Hymenobacter

f­ eatures. Size of cells is approximately 2–3 ´ 0.8 mm. ­Colonies are brick red. Growth occurs on PYES agar, and R2A but not on Czapek–Dox agar or MacConkey agar. Oxidase- and catalase-positive. Nitrate reduction is positive without production of N2. Negative for production of indole from tryptophan and for arginine dihydrolase and urease. Positive for alkaline phosphatase, esterase C4 (weakly), esterase lipase C8 (weakly), leucine arylamidase, valine arylamidase (weakly), acid phosphatase (weakly), naphthol-AS-BI-phosphohydrolase and b-glucosidase (weakly) with the API ZYM system. Negative for lipase C14, cystine arylamidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase. Sensitive to bacitracin (10 IU), chloramphenicol (30 mg), colistin sulfate (10 mg), erythromycin (15 mg), fusidic acid (10 mg), gentamicin (10 mg), kanamycin (30 mg), penicillin G (10 IU), vancomycin (30 mg), polymyxin B sulfate (300 IU), and tetracycline (10 mg). The fatty acid profile is given in Table 97. Source: air in the Sainsbury Centre for Visual Arts in Norwich (UK). DNA G+C content (mol%): not reported. Type strain: NS/50, DSM 15439, LMG 21876. Sequence accession no. (16S rRNA gene): AJ549285. 7. Hymenobacter ocellatus Buczolits, Denner, Kämpfer and Busse 2006, 2076VP (Taxeobacter ocellatus Reichenbach 1992b, 182) o.cel.la¢tus. L. masc. adj. ocellatus showing little eyes, referring to the bright granules at the cell poles. Cells are approximately 1 ´ 3–6 mm. Colonies are brick red. Growth occurs on PYES agar, Czapek–Dox agar, and R2A but not on MacConkey agar. Oxidase- and catalase-positive. Nitrate reduction is weakly positive without production of N2.Negative for production of indole from tryptophan and for arginine dihydrolase and urease. Positive for alkaline phosphatase, esterase lipase C8 (weakly), leucine arylamidase (weakly) and naphthol-AS-BI-phosphohydrolase (weakly) with the API ZYM system. Negative for esterase C4, lipase C14, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetylb-glucosaminidase, a-mannosidase, and a-fucosidase. Sensitive to bacitracin (10 IU), chloramphenicol (30 mg), colistin sulfate (10 mg), erythromycin (15 mg), fusidic acid (10 mg), gentamicin (10 mg), kanamycin (30 mg), penicillin G (10 IU), vancomycin (30 mg), polymyxin B sulfate (300 IU), and tetracycline (10 mg). The fatty acid profile is given in Table 97. Source: dung of an antelope. DNA G+C content (mol%): ~65 (analytical ultracentrifuge). Type strain: Myx 2105, Txo1, DSM 11117, LMG 21874. Sequence accession no. (16S rRNA gene): Y18835. 8. Hymenobacter rigui Baik, Seong, Moon, Park, Yi and Chun 2006, 2191VP ri¢gui. L. gen. n. rigui, of a well-watered place. Cells produce water-insoluble pinkish-red pigment. Cells grow best on R2A and TSA, and weakly on nutrient agar. Colonies on tryptic soy agar are translucent, low-convex,

403

circular, smooth, and slimy, with a diameter up to 3.0 mm after 5 d at 25–30°C (pH 7). Growth occurs in the presence of 0–2% (w/v) NaCl (optimum 0%), at pH 5–11 (optimum pH 6), and at 4–37°C (optimum 30°C). Oxidase-negative and catalase-positive. Nitrate is not reduced to nitrite. Esculin hydrolysis is weakly positive. Citrate is not utilized. Negative for fermentation of glucose. Gelatinase is produced but not arginine dihydrolase, urease, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, H2S, indole, or acetoin. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, valine arylamidase, and a-glucosidase, but not lipase (C14), trypsin, a-chymotrypsin, b-glucuronidase, a-galactosidase, b-galactosidase, b-glucosidase, a-mannosidase, or a-fucosidase. The following substrates are utilized as sole carbon and energy sources: galactose, esculin, starch, lactose, inulin, melezitose, raffinose, glycogen, and gentiobiose. The following substrates are not utilized: glycerol, erythritol, d-arabinose, l-arabinose, ribose, d-xylose, l-xylose, adonitol, b-methyl-d-xylopyranoside, sorbose, rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, a-methyl-d-mannopyranoside, a-methyl-d-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, melibiose, xylitol, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, and gluconate. The fatty acid profile is given in Table 97. Source: freshwater of Woopo wetland, Republic of Korea. DNA G+C content (mol%): 65 (Tm). Type strain: WPCB131, IMSNU 14116, KCTC 12533, NBRC 101118. Sequence accession no. (16S rRNA gene): DQ089669. 9. Hymenobacter xinjiangensis Zhang, Liu, Tang, Zhou, Shen, Fang and Yokota 2007, 1754VP xin.jiang.en¢sis. N.L. masc. adj. xinjiangensis pertaining to Xinjiang, an autonomous region in North-West China). Cells (~0.7 × 2–5 mm) are rod-shaped, Gram-stain-negative, aerobic, nonsporeforming bacteria. Motility is not observed. Cells grow best on nutrient-reduced media such as 0.1× TSA and PYES agar. Colonies on 0.1× TSA and PYES agar are translucent, pink, circular, entire, low-convex, and rough; diameter is up to 1.5 cm after 5 d at 28°C. The temperature range for growth is 4–37°C; optimum, 28°C. Oxidase- and catalase-positive. Esculin hydrolysis is positive. Negative for fermentation of glucose, nitrate reduction, H2S production, citrate utilization, indole production, and urease. Using the API ZYM system, the following enzyme activities are detectable: alkaline phosphatase, esterase C4, esterase lipase C8, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, N-acetyl-b-glucosaminidase, and a-mannosidase. The following are not detectable: lipase C14, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, aglucosidase, b-glucosidase, and a-fucosidase. The following are positive with the Biolog system: dextrin, d-cellobiose, i-erythritol, l-fucose, lactulose, maltose, d-mannitol, d-psicose, d-sorbitol, sucrose, d-trehalose, acetic acid, d-galacturonic acid, d-gluconic acid, d-glucosaminic, g-hydroxybutyric acid, propionic acid, succinic acid, glucuronamide, l-alanine, glycyl-l-glutamic acid, l-serine, dl-carnitine, thymidine, and

404

Family I. Cytophagaceae

a-d-glucose-1-phosphate. Negative in the Biolog system are: a-cyclodextrin, Tween 40, Tween 80, N-acetyl-d-galactosamine, adonitol, l-arabinose, d-arabitol, gentiobiose, a-d-glucose, a-d-lactose, b-methyl-d-glucoside, pyruvic acid methyl ester, succinic acid methyl ester, cis-aconitic acid, citric acid, formic acid, d-galactonic acid lactone, a-hydroxybutyric acid, b-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, a-ketobutyric acid, a-ketoglutaric acid, a-ketovaleric acid, dl-lactic acid, malonic acid, quinic acid, sebacic acid, bromosuccinic acid, succinamic acid, l-alaninamide, l-alanyl-glycine, l-asparagine, glycyl-l-aspartic acid, l-histidine, hydroxy-l-proline, l-leucine, l-ornithine, l-phenylalanine, l-proline, l-pyroglutamic acid, d-serine,

l-threonine, g-aminobutyric acid, urocanic acid, inosine, uridine, phenyethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, and glycerol. Sensitive to chloramphenicol, colistin sulfate, erythromycin, gentamicin, penicillin G, polymyxin B sulfate, tetracycline, and vancomycin. Tolerates high doses of gamma radiation with D10 of 4.8 kGy. The quinone system is menaquinone MK-7. Source: soil of the Xinjiang Desert, China following irradiation by gamma rays. DNA G+C content (mol%): 54 (Tm). Type strain: X2-1g, CCTCC AB 206080, IAM 15452, JCM 23206. Sequence accession no. (16S rRNA gene): DQ888329.

Other organisms If a threshold value of 16S rRNA similarity is considered to be 90% for assignment to the genus Hymenobacter, then deposited 16S rRNA gene sequences indicate the presence of several strains in institutional culture collections that might be described as novel species of the genus. These strains, and their % rRNA gene similarity to established species of the genus Hymenobacter, are listed below. 1. Antarctic bacterium R-7572 (accession no. AJ440980) isolated from a microbial mat, Fryxell Lake, McMurdo Dry Valleys, Antarctica (Van Trappen et al., 2002), has a 16S rRNA similarity value of 91.1–96.8%. 2. Taxeobacter sp. GIC34 (accession no. AY439245) isolated from glacial ice core, Greenland (Miteva et al., 2004), has a 16S rRNA similarity value of 90.3–98.1%. 3. Hymenobacter sp. 21/4 (accession no. DQ365992) isolated from soil in Victoria Land, Antarctica (Aislabie et al., 2006) has a 16S rRNA similarity value of 90.0–97.1%. 4. Hymenobacter sp. zf-IRlt3 (accession no. DQ223662) isolated from ice cores, East Rongbuk Glacier, Mt. Qomolangma, Himalaya (Zhang et al., 2006), has a 16S rRNA similarity value of 90.2–98.4%.

5. Taxeobacter sp. SAFR-023 (accession no. AY167836) isolated from spacecraft assembly facilities, has a 16S rRNA similarity value of 87.4–96.0%. 6. Hymenobacter sp. Tibet-IIU11 (accession no. DQ177475) isolated from permafrost Qinghai-Tibet Plateau, China has a 16S rRNA similarity value of 90.5–96.6%.* 7. Hymenobacter sp. PB17 (accession no. AB251884) isolated from soil in Daejeon, South Korea has a 16S rRNA similarity value of 90.2–92.4%.† 8. Cytophagales str. S23328 (accession no. D84607) isolated from paddy field soil near Sendai, Japan (Mitsui et al., 1997) has a 16S rRNA similarity value of 89.4–92.3%. 9. Bacteroidetes bacterium P3 (accession no. DQ351728) isolated from soil of La Gorce Mountains, Antarctica has a 16S rRNA similarity value of 90.3–91.3%. 10. Taxeobacter sp. SAFR-033 (accession no. AY167836) isolated from spacecraft assembly facilities, shares 86.7–90.0% 16S rRNA gene sequence similarity with established species of the genus Hymenobacter but slightly higher values with ­Effluviibacter roseus (90.7%), Pontibacter actiniarum (91.6%), and Adhaeribacter aquaticus (91.9%).

Genus X. Larkinella Vancanneyt, Nedashkovskaya, Snauwaert, Mortier, Vandemeulebroecke, Hoste, Dawyndt, Frolova, Janssens and Swings 2006, 239VP The Editorial Board Lar.ki.nel¢la. N.L. dim. fem. n. Larkinella named in honor of the American microbiologist John M. Larkin, who described the family Spirosomaceae in co-authorship with Renée Borrall.

Ring-like and horseshoe-shaped cells 0.5–0.9 mm wide and with an outer diameter of 1.5–3.0 mm. Coils or helices are not formed. Motile by gliding. Nonsporeforming. Gram-stain-negative. Strictly aerobic. Produce non-diffusible pale-pink pigments. Flexirubin-type pigments are absent. Chemo-organotrophic. Oxidase-, catalase-, and alkaline phosphatase-positive. Acid is not produced from a large variety of carbohydrates, including cellobiose, fructose, galactose, glucose, and sucrose. Dominant cellular fatty acids are C15:0 iso, C16:1 w5c, C17:0 iso 3-OH, and summed feature 3 (comprising C15:0 iso 2-OH, C16:1 w7c, and/or C16:1 w7t). The main isoprenoid quinone is MK-7. DNA G+C content (mol%): 53.

Type species: Larkinella insperata Vancanneyt, Nedashkovskaya, Snauwaert, Mortier, Vandemeulebroecke, Hoste, Dawyndt, Frolova, Janssens and Swings 2006, 239VP.

Enrichment and isolation procedures Larkinella insperata was isolated and purified from cooled water produced by a steam generator in a pharmaceutical company *Editorial note: Meanwhile, Hymenobacter sp. Tibet-IIU11 has been described as Hymenobacter psychrotolerans (Zhang et al., 2008; IJSEM 58, 1215–1220). † Editorial note: Meanwhile, Hymenobacter sp. PB17 has been described as Hymenobacter soli (Kim et al., 2008; IJSEM 58, 941–945).

Genus XI. Leadbetterella

in Belgium in 2004. Colonies on tryptic soy agar (BBL) at 28°C under aerobic conditions produce non-diffusible pale-pink pigments under aerobic conditions.

Differentiation of the genus Larkinella from other genera Characteristics differentiating the genus Larkinella from related or morphologically similar genera are listed in Table 93 in the chapter on the genus Flectobacillus.

405

Taxonomic comments Vancanneyt et al. (2006) reported that, by 16S rRNA gene sequence analysis, the nearest neighbor of Larkinella insperata was Spirosoma linguale, with the type strains of the two species sharing a sequence similarity of 88.8%. Similarity values with the type strains of the type species of the genera Arcicella, Dyadobacter, Flectobacillus, and Runella were lower: 83.6–86.3%.

List of species of the genus Larkinella 1. Larkinella insperata Vancanneyt, Nedashkovskaya, Snauwaert, Mortier, Vandemeulebroecke, Hoste, Dawyndt, Frolova, Janssens and Swings 2006, 239VP in.spe.ra¢ta. L. fem. adj. insperata unexpected, referring to the unexpected source from which the bacterium was isolated. The characteristics are as described for the genus, with the following additional features. Colonies are 1–2 mm in diameter, circular, and shiny with entire edges. Growth occurs at 10–40°C and with 0–2% NaCl. b-Galactosidase activity is present. Nitrate is not reduced. H2S, indole, and acetoin (Voges–Proskauer reaction) are not produced. Gelatin and Tween 40 are hydrolyzed. No hydrolysis occurs of agar, casein, starch, DNA, Tweens 20 and 80, urea, cellulose (CM-cellulose or filter paper), and chitin. No acid is produced from adonitol, l-arabinose, d-cellobiose, dulcitol, d-fructose, l-fucose, d-galactose, N-acetylglucosamine,

d-glucose, ­glycerol, ­inositol, d-lactose, d-maltose, mannitol, d-melibiose, l-raffinose, l-rhamnose, sorbitol, l-sorbose, d-sucrose, d-xylose, or l-xylose. l-Arabinose, d-glucose, d-­ lactose, d-mannose, and d-sucrose are utilized as sole carbon sources for growth, but not citrate, inositol, malonate, mannitol, and sorbitol. Susceptible to ampicillin, carbenicillin, and doxycycline; resistant to benzylpenicillin, chloramphenicol, erythromycin, gentamicin, kanamycin, lincomycin, neomycin, oleandomycin, polymyxin B, streptomycin, and tetracycline. Major fatty acid components (>1.0%) include C14:0, C15:0 iso, C15:0 anteiso, C15:0 iso 3-OH, C16:0, C16:1 w5c, C16:0 3-OH, C16:0 iso 3-OH, C17:0 iso 3-OH, and summed feature 3 (comprising C15:0 iso 2-OH, C16:1 w7c and/or C16:1 w7t). Source: water produced by a steam generator in a pharmaceutical company in Belgium. DNA G+C content (mol%): 53 (HPLC). Type strain: LMG 22510, NCIMB 14103. Sequence accession no. (16S rRNA gene): AM000022.

Genus XI. Leadbetterella Weon, Kim, Kwon, Park, Cha, Tindall, Stackebrandt, Trüper and Go 2005, 2299VP The Editorial Board Lead.bet.te.rel¢la. N.L. dim. fem. n. Leadbetterella named in honor of Edward R. Leadbetter, who studied bacteria belonging to the CFB group.

Rods 0.6–0.9 × 2–7 mm. Nonmotile. No gliding motility. Strictly aerobic. Gram-stain-negative. Oxidase- and catalase-positive. Colonies are orange. Flexirubin-type pigments are present. Growth occurs in the presence of 1% NaCl but not 3%. Several carbohydrates are used as sole carbon sources. Esculin, gelatin, starch, and tyrosine are degraded. Major fatty acids are C16:1 w7c/C15:0 iso 2-OH, C15:0 iso, C15:0 iso 2-OH/C16:1 w7c, C17:0 iso 3-OH, and C16:0. The respiratory quinone is MK-7. Isolated from cotton-waste composts. DNA G+C content (mol%): 33. Type species: Leadbetterella byssophila Weon, Kim, Kwon, Park, Cha, Tindall, Stackebrandt, Trüper and Go 2005, 2299VP.

Enrichment and isolation procedures Leadbetterella byssophila was isolated from cotton-waste composts by plating onto trypticase soy agar (TSA, pH 7.0; Difco) at 30°C.

Differentiation of the genus Leadbetterella from other genera In the family Cytophagaceae, Leadbetterella byssophila is the only species that has been isolated from cotton-waste composts. Its

failure to grow in the presence of 3% NaCl differentiates it from most marine genera. Leadbetterella byssophila differs from Belliella, Cytophaga, Cyclobacterium, Hongiella, and Persicobacter by its production of flexirubin-type pigments. Its lack of gliding motility distinguishes it from the members of the genus Cytophaga and Reichenbachiella. The cells of Leadbetterella are straight rods, unlike the curved or horseshoe-shaped cells of Cyclobacterium. The hydrolysis of gelatin and starch differentiates Leadbetterella byssophila from Dyadobacter, as well as the much lower mol% G+C of its DNA (33 vs 44–49). Leadbetterella byssophila does not hydrolyze agar, unlike Reichenbachiella and some species of Cytophaga; moreover, it exhibits a lower mol% G+C value than Reichenbachiella and Cytophaga (33 vs 44.5 and 39–46, respectively).

Taxonomic comments On the basis on 16S rRNA analyses, Weon et al. (2005) reported that the type strain of Leadbetterella byssophila could be positioned in the family “Flexibacteraceae”. Moreover, according to a clustal w alignment of the members of the phylum Bacteroidetes, of the named species the type strain showed highest sequence similarity (85.4%) to the type strain of Belliella baltica.

406

Family I. Cytophagaceae

List of species of the genus Leadbetterella 1. Leadbetterella byssophila Weon, Kim, Kwon, Park, Cha, ­Tindall, Stackebrandt, Trüper and Go 2005, 2299VP. bys.so¢phi.la. Gr. n. byssos cotton; N.L. fem. adj. phila (from Gr. fem. adj. philê) friend, loving; N.L. fem. adj. byssophila ­liking cotton. The characteristics are as described for the genus, with the following additional features. Colonies on TSA are initially light orange and convex with entire margins; with prolonged incubation, they become dark orange. Growth occurs at temperatures of 15–45°C and at pH 6.0–8.0. Growth occurs on 0.5% yeast extract medium. Positive for O/F test (glucose). Positive for indole production and b-galactosidase (API 20NE). Negative for nitrate reduction and arginine dihydrolase (API 20NE). Negative for hydrolysis of casein, cellulose, chitin, DNA, Tweens 40 and 80, and urea. Growth on carbohydrates (API 20NE) occurs with N-acetylglucosamine, arabinose, glucose, maltose, and mannose. The following enzyme activities are present (API ZYM system): alkaline phosphatase, leucine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, and a-fucosidase (API ZYM); weak activity is detected for a-galactosidase and b-galactosidase. The ­following

s­ ubstrates are oxidized or weakly oxidized (API 50CH system): N-acetylglucosamine, amygdalin, d-arabinose, arbutin, d-cellobiose, esculin, d-galactose, gentiobiose, d-glucose, d-lactose, d-maltose, d-mannose, d-melibiose, methyl-a-dglucopyranoside, methyl-a-d-mannopyranoside, l-rhamnose, salicin, starch, sucrose, and d-trehalose. The following compounds are assimilated or weakly assimilated (Biolog GN 20 system): acetic acid, N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, l-alaninamide, l-alanine, l-alanylglycine, l-arabinose, d-arabitol, l-asparagine, l-aspartic acid, d-cellobiose, a-cyclodextrin, dextrin, i-erythritol, d-fructose, d-galactose, d-galacturonic acid, gentiobiose, a-d-glucose, glucose-1phosphate, d-glucose 6-phosphate, l-glutamic acid, glycerol, a-dl-glycerol phosphate, glycogen, glycyl-l-aspartic acid, glycyl-l-glutamic acid, l-fucose, a-ketovaleric acid, dl-lactic acid, a-d-lactose, lactulose, maltose, d-mannose, d-melibiose, methyl-b-d-glucoside, methyl pyruvate, monomethylsuccinate, l-ornithine, d-raffinose, l-serine, sucrose, l-threonine, thymidine, d-trehalose, turanose, and uridine. Source: cotton-waste composts in the Republic of Korea. DNA G+C content (mol%): 33 (HPLC). Type strain: 4M15, DSM 17132, KACC 11308. Sequence accession no. (16S rRNA gene): AY854022.

Genus XII. Meniscus Irgens 1977, 42AL Roar L. Irgens Me.nis¢cus. N.L. masc. n. meniscus (from Gr. masc. n. mêniskos) crescent moon.

Curved or straight rods, 0.7–1.0 mm in diameter and 2.0–3.0 mm in length. Cultures may contain single cells, pairs, tightly coiled spirals, S shapes (two cells, one inverted), and doughnutshaped cells, where the ends are overlapping before division by binary fission. “Rings” have outer diameters of about 3.0 mm. Single polar flagellum. Stains Gram-negative. Nonmotile. Resting stages not known. Encapsulated. Gas vacuoles arranged at random within cells. Colonies chalky white. Chemo-organotrophic; strictly fermentative metabolism with no gas production. Catalase-and oxidase-negative. Aerotolerantly anaerobic; capable of growth under an air atmosphere when at least 1% CO2 is present. Vitamin B12, thiamin and CO2 are required for growth. Optimum temperature, 30°C. No growth at 10 or 40°C. Isolated from anaerobic digester sludge. DNA G+C content (mol%): 44.9 (Bd). Type species: Meniscus glaucopis Irgens 1977, 42AL.

Further descriptive information Cells of Meniscus appear as curved or straight rods. The curved rods often form ringlike shapes when the ends of a cell overlap prior to cell separation. Helical, filamentous forms may also be seen. Gas vacuoles (Figures 76 and 77) may be observed by phase-contrast microscopy, and the individual gas vesicles are resolved by observing whole cells or thin sections in the transmission electron microscope. Colonies are circular, convex in elevation, with an entire margin and smooth, glistening surface. The colonies may appear

translucent or opaque, chalky white. The larger the number of gas vacuoles within the cells of the colony, the whiter the colony. The consistency of the colonies is buttery when grown anaerobically and rubbery when grown aerobically. When grown to stationary phase in test tubes, the cells often rise to the surface where they form a white band. The cells apparently do not respire as aerobic and anaerobic cultures have the same cell yields. Fermentation end products when grown on maltose are acetic, propionic, and succinic acids (Irgens, 1977). Optimum growth occurs around 30°C at pH 7.0. Growth occurs at 15 and 35°C, but not at 10 or 40°C. Cobalamin (vitamin B12), thiamin, and CO2 are required for growth. Good growth in defined medium occurs with ammonium as the nitrogen source. The following characteristics are negative: deaminase (Casamino acids), urease, acetyl methyl carbinol, indole, H2S production and nitrate reduction. Cells ferment agar (weakly), dextrin, melezitose, raffinose, cellobiose, sucrose, lactose, maltose, melibiose, trehalose, fructose, galactose, glucose, rhamnose (weakly), CH3-a-d-glucoside, esculin, salicin, d-ribose, d-xylose, and arabinose. They do not ferment mannose, sorbose, glycerol, lactate, mannitol, sorbitol, adonitol, dulcitol, inositol, or amino acids. They do not hydrolyze starch, cellulose, DNA, gelatin, casein, pectin, inulin, gum arabic, tributyrin, chitin, xylan, or glycogen. Isolated from anaerobic digester sludge but probably also present in anaerobic hypolimnion of lakes.

Genus XII. Meniscus

407

sterilized by filtration, per 100 ml of medium. All solid media contain 1.0% CaCO3 and 1.5% agar. Stock media contain 0.3% agar. Maltose, at a concentration of 0.2–0.5%, is used as the carbon source for growth studies and stock cultures. Pour plates are prepared using anaerobic digester sludge as the inoculum. The plates are incubated in a GasPak anaerobic jar (BBL) at 25–30°C. After 10 d chalky white colonies indicative of the presence of gas vacuoles may be observed.

Maintenance procedures Stock cultures are maintained in test tubes rendered anaerobic by the pyrogallic acid technique, or they may be maintained on slants in cotton-stoppered test tubes in anaerobic jars. Lyophilized cultures may be stored indefinitely.

Procedures for testing special characters

FIGURE 76.  Meniscus glaucopis ATCC 29398. Phase-contrast. Note gas

vacuoles. Bar, 2.0 mm.

The presence of gas vacuoles may be demonstrated by the disappearance of the vacuoles upon the application of a sharp blow with a hammer to a wet mount of the cells. The cover slip is protected with a 3–4-mm-thick rubber pad. Phase-contrast microscopic examination of wet mounts made with dilute India ink is used to demonstrate capsules. The hydrolysis of glycogen, inulin, pectin, gum arabic, and dextrin is tested in BGM broth without maltose. Hydrolysis is considered positive if growth occurs and the pH drops.

Differentiation of the genus Meniscus from other genera Two genera of gas-vacuolated curved rods that may be confused with Meniscus are “Brachyarcus” and Ancyclobacter (formerly Microcyclus). “Brachyarcus” has never been isolated and differs from Meniscus in having cells arranged in groups (coenobia) consisting of two, four or more rings (Skuja, 1964). Ancyclobacter is differentiated from Meniscus as being a catalase-positive, obligate aerobe rather than an aerotolerant anaerobe and by having a mol% G+C of 66–69 (Van Ert and Staley, 1971).

Taxonomic comments

FIGURE 77.  Meniscus glaucopis strain R. Phase-contrast. Note gas vacu-

oles. Bar, 2.0 mm.

Enrichment and isolation procedures Members of this genus may be isolated on a complex medium (BGM) having the following composition (per liter): yeast extract, 1.0 g; KH2PO4, 0.5 g; NaCl, 0.4 g; NH4Cl, 0.4 g; CaCl2·2H2O, 0.01 g; sodium thioglycolate, 0.3 g; MgSO4·7H2O, 0.2 g; FeSO4·7H2O, 0.001 g; at least 1% CO2; trace elements solution (TES), 1.0 ml. The TES, modified from Pfennig’s formula (personal communication), contains (per liter): ZnSO4·7H2O, 0.10 g; MnCl·4H2O, 0.03 g; H3BO3, 0.3 g; CoCl2·6H2O, 0.2 g; CuCl2·2H2O, 0.01 g; NiCl2·6H2O, 0.02 g; Na2MoO4·2H2O, 0.03 g; pH 3.0–4.0. The pH of the medium is adjusted to 7.3 with 10% Na2CO3 before autoclaving, or the medium may be autoclaved with the pH unadjusted and then adjusted to about pH 7.0 after autoclaving by the addition of 2.0 ml of 5.0 % NaHCO3,

The assignment of Meniscus to the family Cytophagaceae is uncertain. Garrity et al. (2005) originally classified the genus within the “Flexibacteraceae” based upon phenotypic similarities to other members of this group. In preparation of this volume, the type genus of this family was replaced with Cytophaga because it has priority over Flexibacter (Ludwig et al., 2009), but most members of the group remained the same. In the absence of a sequence for its 16S rRNA gene, the phenotypic evidence was not sufficient to exclude Meniscus from the new family. However, the phenotypic evidence does not provide strong support for its assignment to this family. Most of the Cytophagaceae are catalase- and oxidase-positive aerobes, while Meniscus is catalase- and oxidase-negative and an aerotolerant anaerobe. However, within the Cytophagaceae, the genus Spirosoma contains a facultative anaerobic species, and a few other genera are either oxidase- or catalase-negative. Likewise, Arcicella, Larkinella and Runella form similar ring-like arrangements of cells, although all possess other morphological differences with Meniscus. In addition, gas vacuoles, which are abundant in Meniscus, have not been reported in other members of this family. Gas vesicles are also present in many morphologically and physiologically unrelated taxa and are not reliable taxonomic characteristics. At this time, it is also not possible to exclude a relationship

408

Family I. Cytophagaceae

of Meniscus to nonvacuolated vibrios within the Proteobacteria, such as Aquaspirillum and Vibrio, and possibly to other Gramstain-negative, aerotolerant anaerobes such as Zymomonas and Eikenella. While Meniscus was excluded from these genera on phenotypic grounds (Irgens, 1977), a familial relationship may

still exist. Meniscus glaucopis includes both a vibrioid strain and a straight rod strain. This is justified by the fact that all metabolic and physiological characteristics tested are identical for the two strains. These strains are merely morphovars of the same species.

List of species of the genus Meniscus 1. Meniscus glaucopis Irgens 1977, 42

AL

glau.co¢pis. N.L. masc. adj. glaucopis (from Gr. adj. glaukôpis) gleaming-eyed (an epithet of the warlike goddess Athena), perhaps a reference to the presence of refractile gas ­vacuoles.

The description of the species is the same as the genus. Isolated from anaerobic digester sludge. DNA G+C content (mol%): 44.9 (Bd). Type strain: ATCC 29398. Sequence accession no. (16S rRNA gene): not available.

Genus XIII. Microscilla Pringsheim 1951, 140 emend. Lewin 1969, 194AL Yasuyoshi Nakagawa Mic.ro.scil¢la. Gr. adj. micros small; L. n. oscillum swing; N.L. fem. n. Microscilla intended to mean small swinging organisms.

Long thin flexible threadlike rods usually 10–100 mm long or longer. Motile by gliding. Cell mass more or less intensely orange. Strictly aerobic and chemo-organotrophic. All grow on peptones as sole source of nitrogen. Chitin and cellulose are not attacked, but other polysaccharides including carboxymethyl cellulose may be decomposed. Marine organisms. No growth occurs in half-strength seawater. DNA G+C content (mol%): 37–44. Type species: Microscilla marina Pringsheim 1951, 140, emend. Lewin 1969, 201AL.

Further descriptive information The major quinone of Microscilla marina, “Microscilla furvescens”, and “Microscilla sericea” is MK-7 (Nakagawa, unpublished). The major polyamines of Microscilla marina are cadaverine, spermidine, and putrescine; that of “Microscilla furvescens” is homospermidine. “Microscilla sericea” contains mainly homospermidine in addition to agmatine (Hamana and Nakagawa, 2001).

Enrichment and isolation procedures No enrichment media have been designed for isolation of Microscilla strains. Standard procedures to isolate marine bacteria can be applied. Colonies of Microscilla are usually orange to yellow. Microscilla strains have been isolated from marine environments at widely separated sites (Lewin, 1969; Lewin and Lounsbery, 1969). The type strains of Microscilla marina and “Microscilla sericea” came from the marine aquarium in La Jolla, California. The type strain of “Microscilla furvescens” was isolated from marine sand in Samoa.

Maintenance procedures Cultures of Microscilla strains can be preserved by freezing at temperatures lower than −80°C. For freezing, cells are suspended in Marine broth 2216 (Difco) containing 10% glycerol or 7% DMSO. Microscilla strains are rather sensitive to drying; however, they can be preserved in a protective medium SM2 or SM3 (see the chapter on Flammeovirga for formulations) by the liquid drying method, or by freeze drying.

Differentiation of the genus Microscilla from other genera Phylogenetic analysis based on 16S rRNA sequences shows that Microscilla marina, the type species of the genus Microscilla, is phylogenetically independent in the phylum Bacteroidetes (see Figure 74 of the chapter Flexibacter). However, because few taxonomic characteristics have been investigated since the original descriptions, it is impossible to discriminate the genus Microscilla from other related genera only by phenotypic characteristics. It is hoped that additional strains will be isolated and useful differential chemotaxonomic, physiologic, and biochemical characteristics of the genus Microscilla will be investigated. All species possess MK-7 as the major quinone, which is useful for differentiating the genus from the family Flavobacteriaceae, which is characterized by MK-6 (Bernardet et al., 2002; Nakagawa and Yamasato, 1993).

Taxonomic comments The genus Microscilla was first described by Pringsheim (1951) for actively gliding organisms including the marine type species, Microscilla marina, and two freshwater species. From the beginning, it was realized that the genus Microscilla resembles the genus Flexibacter established by Soriano (1945). In addition, the original Pringsheim’s strains have been lost. Lewin (1969) reisolated morphologically similar organisms and redefined both genera by classifying marine organisms with longer filaments (20–100 mm) in the genus Microscilla. This definition was not followed in the Bergey’s Manual of Determinative Bacteriology, 8th edition (Leadbetter, 1974), and the genus Microscilla was included in the genus Flexibacter. Later, Reichenbach (1989a, b) tried to restrict the genus Microscilla to marine organisms with G+C content of the DNA of above 37 mol%. However, he also stated that too little was known about these bacteria, that these genera were still heterogeneous, and that future research including modern molecular taxonomy was a prerequisite. Those histories are described in detail in the 1st edition of this Manual. Only the type species, Microscilla marina, has been validly published to date. In addition, four non-validated species - “Microscilla aggregans” (synonym, Flexibacter aggregans), “­Microscilla

409

Genus XIII. Microscilla

furvescens”, “Microscilla sericea”, and “Microscilla tractuosa” (synonym, Flexibacter tractuosus) — were included in the genus, and three other species — “Microscilla arenaria”, Flexibacter litoralis, and Flexibacter polymorphus — were listed as Species Incertae Sedis in the 1st edition of the Manual. 16S rRNA sequencing analyses of the genera Microscilla and Flexibacter have shown a high degree of biological diversity of the genus (Nakagawa et al., 2002). The five strains in the four species Microscilla marina, “Microscilla furvescens”, “Microscilla sericea”, and “Microscilla arenaria” diverged into five distinct lineages, as shown in Figure 71 of the chapter on Flexibacter and Table 98 (see also Table 94 of the chapter on Flexibacter). Microscilla marina, the type species of the genus Microscilla, was a species secluded from the others, which means that other invalid Microscilla species should not be included in the genus. “Microscilla arenaria” NBRC 15982T, which was closely related with the genus Flammeovirga, was reclassified as Flammeovirga arenaria (Takahashi et al., 2006). “Microscilla furvescens” is an independent lineage located next to the cluster composed of Flexibacter tractuosus and “Microscilla sericea”. Two strains of “Microscilla sericea” were

­ hylogenetically different. The type strain of “Microscilla sericea”, p NBRC 15983T, clustered with Flexibacter tractuosus and may be classified in the same new taxon. Another strain of “Microscilla sericea”, NBRC 16561, occupied an independent position that seemed to require a new taxon. The marine Flexibacter species Flexibacter litoralis and Flexibacter polymorphus, in addition to Flexibacter aggregans (“Microscilla aggregans”) and Flexibacter tractuosus (“Microscilla tractuosa”), were not closely related to either the type species of the genus Microscilla or that of the genus Flexibacter. These marine Flexibacter species are discussed in the chapter on Flexibacter. In this chapter, Reichenbach’s definition of the genus is principally followed; however, based on 16S rRNA sequencing analysis, only the type species, Microscilla marina, can be justified as a member of the genus Microscilla. Other non-validated Microscilla species that should be reclassified in the future are listed under Other species.

Differentiation of species of the genus Microscilla Table 99 lists characteristics that distinguish the Microscilla ­species from one another.

List of species of the genus Microscilla at temperatures greater than 35°C. Peptones serve as sole nitrogen source, but not glutamate or NO3–. Glucose is not utilized. Gelatin is degraded, but not agar, alginate, carboxymethyl cellulose, or starch. Nitrate is not reduced. The major carotenoid is saproxanthin. Only one strain — the type strain — is available. DNA G+C content (mol%): 42 (Bd). Type strain: S10-8, ATCC 23134, DSM 4236, LMG 18923, NBRC 16560. Sequence accession no. (16S rRNA gene): AB078080.

1. Microscilla marina Pringsheim 1951, 140, emend. Lewin 1969, 201AL ma.ri¢na. L. fem. adj. marina of, or belonging to, the sea, marine. The characteristics are as described for the genus with the following additional features taken from the 1st edition of this Manual. Threads may become very long, more than 150 mm. The cell mass is orange. Growth occurs on media containing single- or double-strength seawater. Growth does not occur

Other species The species listed below should not be included in the genus Microscilla, because they are not closely related to the type species of the genus, based on 16S rRNA sequence analysis.

nitrogen sources. Various sugars, including glucose are utilized, but acetate is not. Agar, alginate, carboxymethyl cellulose, gelatin, and starch are degraded. Nitrate is not reduced. The major carotenoid is saproxanthin. Only one strain – the type strain – is available. DNA G+C content (mol%): 44 (Bd). Type strain: TV-2, ATCC 23129, LMG 13023, NBRC 15994, NCIMB 1419. Sequence accession no. (16S rRNA gene): AB078079.

1. “Microscilla furvescens” Lewin 1969, 201 fur.ves¢cens. L. adj. furvescens becoming black. The characteristics are as described for the genus with the following additional features taken from the 1st edition of this Manual. Long threads of 10–50 mm. The cell mass is orange. ­Peptones, Casamino acids, glutamate, and NO3– serve as sole

2. “Microscilla sericea” Lewin 1969, 201 se.ri¢cea. L. fem. adj. sericea silk-like, silky.

TABLE 98.  Phylogenetic groups of Microscilla strains and their present classification

Phylogenetic group 1 2 3 4 5 Takahashi et al. (2006).

a

Species and strain

Present classification

Microscilla marina NBRC 16560 “Microscilla arenaria” NBRC 15982T “Microscilla furvescens” NBRC 15994T “Microscilla sericea” NBRC 15983T “Microscilla sericea” NBRC 16561 T

Microscilla marina Flammeovirga arenariaa Excluded from the genus Microscilla Excluded from the genus Microscilla Excluded from the genus Microscilla

410

Family I. Cytophagaceae

TABLE 99.  Characteristics differentiating the species of the genus

Microscillaa,b Characteristic

M. marina “M. furvescens” “M. sericea”

Length of threads (mm) Salinity range (Sc) Glutamate NO3− Utilization of glucose Degradation of: Starch Alginate Carboxymethylcellulose DNA G+C content (mol%)

>150 1–2 − − −

10–50 nd + + +

30 to >100 0.5–2 − − +

− − − 42

+ + + 44

+ + − 38–39

Symbols: +, >85% positive; −, 0–15% positive; nd, not determined.

a

The characteristics are as described for the genus with the following additional features taken from the 1st edition of this Manual. Long threads of 30–100 mm. The cell mass is orange. Peptones serve as the sole nitrogen source, but not Casamino acids, glutamate, or NO3−. Various sugars, including glucose and glycerol are utilized. Alginate, gelatin, and starch are degraded, but agar and carboxymethyl cellulose are not. Nitrate is not reduced. The major carotenoid is saproxanthin. DNA G+C content (mol%): 38–39 (Bd). Type strain: SIO-7, ATCC 23182, LMG 13021, NBRC 15983, NCIMB 1403. Sequence accession no. (16S rRNA gene): AB078081.

Data from Reichenbach (1989a).

b

Expressed as strength of seawater (S); 0 = freshwater.

c

Genus XIV. Pontibacter Nedashkovskaya, Kim, Suzuki, Shevchenko, Lee, Lee, Park, Frolova, Oh, Bae, Park and Mikhailov 2005c, 2585VP Olga I. Nedashkovskaya and Seung Bum Kim Pon.ti.bac¢ter L. masc. n. pontus the sea; N.L. masc. n. bacter from Gr. n. baktron rod, N.L. masc. n. ­Pontibacter a marine bacterium.

Rods usually measuring 0.3–0.7 × 1.2–1.9 mm. Can move by gliding. Produce non-diffusible pink pigments. No flexirubin-type pigments are formed. Chemo-organotrophic. Strictly aerobic. Oxidase, catalase, alkaline phosphatase and b-galactosidasepositive. Esculin, gelatin, and DNA are hydrolyzed. Casein, Tween 80, chitin, and cellulose (CM-cellulose and filter paper) are not degraded, but agar, starch, and Tweens 20 and 40 may be decomposed. Carbohydrates are utilized. Can grow without seawater or sodium ions. Nitrate is not reduced to nitrite. Indole and H2S are not produced. The major respiratory quinone is MK-7. DNA G+C content (mol%): 48–52. Type species: Pontibacter actiniarum Nedashkovskaya, Kim, Suzuki, Shevchenko, Lee, Lee, Park, Frolova, Oh, Bae, Park and Mikhailov 2005c, 2585VP.

Further descriptive information Phylogenetic analysis of almost-complete 16S rRNA gene sequences of the genus Pontibacter revealed that its closest relative is a single species of the genus Adhaeribacter, Adhaeribacter aquaticus, with similarity of 89.2%. The level of 16S rRNA gene sequence similarity between strains Pontibacter actiniarum KMM 6156T and Adhaeribacter aquaticus MBRG 1.5T was 95%. The main cellular fatty acids are straight-chain unsaturated and branched-chain unsaturated fatty acids C15:1 iso, C17:0 iso 3-OH, summed feature 3 comprising C15:0 iso 2-OH and C16:1w7c or both, and summed feature 4 comprising C17:1 iso I and C17:1 anteiso B or both (Table 100). On rich nutrient media strains of the genus Pontibacter form regular, round, shiny, with entire edges, smooth, pink colonies with diameter of 1–3 mm after cultivation of 24 h. All strains grow at 6–36°C, at pH 7.0–10.0 and with 0–4% NaCl. According to Biolog GN2 tests, dextrin, glycogen, l-alanine, methyl b-d-glycoside, methyl pyruvate, a-ketobutyric acid, a-ketovaleric acid, dl-lactic acid, succinamic acid, alaninamide,

l-alanyl-glycine, l-asparagine, l-aspartic acid, l-glutamic acid, l-proline, and threonine are utilized, but Tween 80, adonitol, i-erythritol, uridine, urocanic acid, a-d-lactose, d-mannose, citric acid, formic acid, r-hydroxyphenylacetic acid, malonic acid, sebacic acid, glucuronamide, d-alanine, d-serine, phenylethylamine, 2-aminoethanol, dl-a-glycerol phosphate, and glucose-1-phosphate are not utilized. The strains of the genus Pontibacter are susceptible to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, carbenicillin, lincomycin, and tetracycline, and resistant to polymyxin B and streptomycin. The pontibacters were isolated from sea animals and from a desert soil sample collected in the temperate latitudes.

Enrichment and isolation procedures The strains of the genus Pontibacter were isolated from an unidentified actinian (sea anemone) and a desert soil sample using the dilution plating techniques on Marine agar (Difco) and on LB agar, respectively. All isolates have been grown on media containing 0.5% of a peptone and 0.1–0.2% yeast extract (Difco). The marine representative of the genus Pontibacter remains viable on Marine agar (Difco) or other rich media containing natural or artificial seawater for several weeks. They have survived storage in Marine broth or artificial seawater supplemented with 20% glycerol (v/v) at −80°C for at least 5 years. A soil isolate, cultivated on LB agar, can grow well under very dry environmental conditions.

Differentiation of the genus Pontibacter from other genera Strains of the genus Pontibacter differ from its closest phylogenetic neighbor, the freshwater bacterium Adhaeribacter aquaticus, by a higher DNA G+C content (48–52 mol% for Pontibacter strains compared with 40.0 mol% for Adhaeribacter aquaticus), and by the differences in fatty acid compositions (Nedashkovskaya et al., 2005c; Rickard et al., 2005) (Table 100).

411

Genus XIV. Pontibacter TABLE 100.  Cellular fatty acid content of Pontibacter actiniarum and its closest relative Adhaeribacter aquaticus a

Fatty acid

P. actiniarum KMM 6156T

A. aquaticus MBRG 1.5T

− 28.8 0.1 0.8 − 3.0 1.4 2.2 6.5 2.3 14.7 31.3 1.8

1.2 22.5 4.4 16.9 16.5 3.1 5.1 − 12.1 − − 11.2 −

C10:0 iso C15:0 iso C15:0 anteiso C16:1w5c C15:0 iso 2-OH C15:0 iso 3-OH C17:1 w6c C17:0 iso C17:0 iso 3-OH Summed feature 2 Summed feature 3 Summed feature 4 Summed feature 5

a Values are percentages and values of less than 1% are not shown. Summed feature 2 consisted of one or more of the following fatty acids which could not be separated by the Microbial Identification System: C15:1 iso I and C13:0 3-OH. Summed feature 3 contains one or more of the following fatty acids: C15:0 iso 2-OH, C16:1 w7c and C16:1 w7t. Summed feature 4 consisted of one or more of the following fatty acids: C17:1 iso I and C17:1 anteiso B. Summed feature 5 consisted of one or more of the following fatty acids: C18:0 anteiso and C18:2 w6,9c. Data from Nedashkovskaya et al. (2005c) and from Rickard et al. (2005).

TABLE 101.  Differentiating phenotypic properties of Pontibacter species a,b

Characteristic Hydrolysis of: Agar Starch Tweens 20 and 40 Acid production from N-acetylglucosamine d-fructose, l-fucose, d-glucose, inositol, d-maltose, l-raffinose, and d-sucrose Utilization of: Glycyl-l-aspartic acid, Tweens 20 and 40 a-Cyclodextrin, N-acetyl-d-galactosamine, l-arabinose, d-arabitol, cellobiose, d-galactose, gentiobiose, a-d-glucose, d-mannitol, d-melibiose, psicose, d-raffinose, l-rhamnose, d-sorbitol, d-trehalose, turanose, lactulose, xylitol, monomethyl succinate, acetic acid, d-galactonic acid, cis-aconitic acid, d-galacturonic acid, d-glucosaminic acid, a-, b- and g-hydroxybutyric acids, itaconic acid, a-ketoglutaric acid, propionic acid, quinic acid, d-saccharic acid, bromosuccinic acid, glycyl-l-glutamic acid, l-histidine, hydroxy-l-proline, l-leucine, l-ornithine, l-phenylalanine, l-pyroglutamic acid, l-serine, dl-carnitine, g-aminobutyric acid, inosine, thymidine, putrescine, 2,3-butanediol, and glucose 6-phosphate a-Galactosidase activity Susceptibility to: Kanamycin Gentamicin, neomycin

P. actiniarum

P. akesuensis

+ − + −

− + − +

+ −

− +

−

+

+ −

− +

Symbols: +, positive; −, negative.

a

Data are taken from Nedashkovskaya et al. (2005c) and Zhou et al. (2007).

b

Differentiation of the species of the genus Pontibacter Despite very different sources of isolation, the strains belonging to the two validly published species of the genus Pontibacter have

many common phenotypic features. However, they can clearly be differentiated from each other by the several phenotypic traits shown in Table 101.

List of species of the genus Pontibacter 1. Pontibacter actiniarum Nedashkovskaya, Kim, Suzuki, Shevchenko, Lee, Lee, Park, Frolova, Oh, Bae, Park and Mikhailov 2005c, 2586VP ac.ti.ni.a¢rum. N.L. gen. pl. n. actiniarum of sea anemones or related animals. Cells are 0.3–0.4 mm in width and 1.2–1.9 mm in length. On Marine agar, colonies are circular and 2–3 mm in diameter.

Growth occurs at 6–43°C (optimal temperature, 25–28°C) and with 0–10% NaCl. Agar is hydrolyzed (weakly). Acid is formed from arbutin (API 50 CH gallery, bioMérieux). No acid is formed from l-arabinose, d-cellobiose, d-galactose, d-lactose, d-melibiose, l-rhamnose, l-sorbose, dl-xylose, N-acetylglucosamine, glycerol, adonitol, dulcitol, inositol, and mannitol. Biolog GN2 tests show that the type strain does not

412

Family I. Cytophagaceae

utilize N-acetyl-d-glucosamine, d-fructose, l-fucose, myo-inositol, a-lactose, maltose, sucrose, d-gluconic acid, d-glucuronic acid, succinic acid, and glycerol. Acetoin (Voges–Proskauer reaction) production is negative. According to the API ZYM gallery (bioMérieux), the following enzymes are produced: acid phosphatase, esterase lipase (C8), leucine- and valinearylamidases, trypsin, naphthol-AS-BI-phosphohydrolase, a-glucosidase, and N-acetyl-b-glucosaminidase. The following enzymes are not produced: esterase (C4), lipase (C14), cystine arylamidase, a-chymotrypsin, b-glucosidase, b-glucuronidase, a-mannosidase, and a-fucosidase. Source: a single strain, KMM 6156, was isolated from unidentified actinians collected in the Rudnaya Bay, Sea of Japan, Pacific Ocean. DNA G+C content (mol%): 48.7 (Tm). Type strain: KMM 6156, KCTC 12367, LMG 23027. Sequence accession no. (16S rRNA gene): AY989908.

2. Pontibacter akesuensis Zhou, Wang, Liu, Zhang, Zhang, Lai and Li 2007, 324VP a.ke.su.en¢sis. N.L. masc. adj. akesuensis pertaining to Akesu, a city of XinJiang Province in the north-west of China from where the type strain was isolated. According to Zhou et al. (2007), cells are 0.7 mm × 1.5– 1.6 mm. On nutrient agar, colonies are round, 1–2 mm in diameter. Growth occurs at 4–36°C (optimum, 28–30°C), and with 0–4% NaCl. No acid is formed from d-cellobiose, d-galactose, d-lactose, d-melibiose, l-rhamnose, l-sorbose, and dl-xylose. Source: a single strain, AKS 1, was isolated from the surface layer of a desert soil from Akesu, XinJiang Province, China. DNA G+C content (mol%): 51.4 (Tm). Type strain: AKS 1, CCTCC AB 206086, KCTC 12758. Sequence accession no. (16S rRNA gene): DQ672723.

Genus XV. Runella Larkin and Williams 1978, 35AL The Editorial Board Ru.nel¢la. M.E. n. rune an ancient alphabet; L. fem. dim. ending -ella; N.L. fem. n. Runella that which resembles figures of the runic alphabet.

Rigid straight to curved rods, the degree of curvature varying among cells within a culture. In one species (Runella slithyformis), the cells range from nearly straight to crescent-shaped, but the ends of a cell may overlap, producing a ring-like structure with an outside diameter of 2.0–3.0 mm; in addition, filaments up to 14 mm long may also be produced, and on rare occasions, a coil of two to three turns may be produced. In other species, the cells are either long and filamentous or straight to slightly bent rods instead of crescent-shaped or ring-like as found with Runella slithyformis. Gram-stain-negative. Nonmotile. Resting stages are not known. Aerobic. Runella slithyformis has a strictly respiratory metabolism with oxygen as the terminal electron acceptor and produces acid oxidatively from a few carbohydrates, whereas Runella zeae can ferment sugars. Optimum temperature, 20–35°C. Colonies contain a pale pink or salmoncolored water-insoluble pigment. Catalase is either positive or weakly positive. The oxidase reaction differs among species. Chemo-organotrophic. In those species so far tested, the major quinone is MK-7. Isolated from freshwater (Runella slithyformis), activated sludge (Runella limosa and Runella defluvii), and the stems of Zea mays (Runella zeae). DNA G+C content (mol%): 40–49. Type species: Runella slithyformis Larkin and Williams 1978, 35AL.

Further descriptive information Cells of Runella slithyformis typically appear as rods whose degree of curvature varies from nearly straight to crescent shaped (Figure 3.2 in Larkin and Borrall, 1984b). An individual cell may be bent in more than one plane. Cells of Runella zeae are straight to slightly bent rods that form chains of irregular shapes. Colonies of Runella slithyformis on MS agar (see the chapter on Spirosoma for the recipe for this medium) produce a pale pink, water insoluble, nonfluorescent pigment. The colonies are circular and convex with an entire margin. Abundant growth occurs on MS agar and on nutrient agar. Scant growth occurs on chocolate agar, peptonized milk agar, and

yeast ­extract-acetate-tryptone agar. No growth occurs on blood agar, eosin-methylene blue agar, nutrient agar containing 5% sucrose, phenol red mannitol salt agar, phenylethyl alcohol agar, trypticase soy agar (with or without 5% sucrose or 3% glucose), MacConkey agar, bismuth sulfite agar, or SalmonellaShigella agar (Larkin and Williams, 1978). Colonies of Runella zeae are round, smooth, and salmon in color when grown on R2A medium* at 28°C. Runella is chemo-organotrophic. By the technique of Hugh and Leifson (1953), acid is produced aerobically only from glucose, maltose, sucrose, and inulin. Strains differ in their ability to produce an acid reaction from rhamnose, galactose, mannose, and raffinose. Sugar alcohols are not acidified. Starch and tributyrin are hydrolyzed; esculin, cellulose, agar, chitin, and casein are not hydrolyzed. None of 11 compounds tested in the medium of Gordon and Mihm (see the chapter on Spirosoma for the recipe for this medium) are utilized as sole carbon sources. Runella species are not known to be pathogenic. Only two strains of Runella slithyformis have been isolated and characterized. Both were isolated from eutrophic fresh waters. Similar organisms were seen in gelatinous deposits on wet planks from a mine (Kraepelin and Passern, 1980), in marine waters (Overbeck, 1974; Sieburth, 1978) and in fresh waters (Larkin, unpublished observations). Runella zeae was isolated from the stems of Zea mays (Chelius et al., 2002; Chelius and Triplett, 2000) and is not known to occur in other habitats. Only one strain has been isolated and characterized. Runella limosa was isolated from activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor. Only one strain has been isolated and characterized. *R2A medium of Reasoner and Geldreich (1985) contains (g/l): Yeast extract, 0.5; Proteose peptone no. 3 (Difco); Casamino acids, 0.5; glucose, 0.5; soluble starch, 0.5; K2HPO4, 0.3; MgSO4·7H2O, 0.05; sodium pyruvate, 0.3; and agar, 15.0. The pH is adjusted to 7.2 with crystalline K2HPO4 or KH2PO4. The medium is sterilized by autoclaving.

413

Genus XV. Runella

Runella defluvii was isolated from the activated sludge of a domestic wastewater treatment plant. Only one strain has been isolated and characterized.

Isolation procedures Runella slithyformis can be isolated by repeated streaking of water samples onto MS agar with incubation at room temperature for up to 2 weeks. The pale pink pigmentation of the colonies aids detection. Runella limosa was isolated by serial dilution of a sludge sample in 1% (w/v) saline solution, with subsequent spreading onto R2A agar (Difco) and incubation at 20°C for 5 d. Subcultivation was on R2A agar at 25°C for 3 d. For isolation of Runella defluvii, a sludge sample was serially diluted with 1% (w/v) saline solution, spread onto R2A agar (Difco) and incubated at 20°C for 5 d. Subcultivation was done on R2A agar at 30°C for 3 d. Runella zeae was isolated by the procedure used by Chelius and Triplett (2000) for Dyadobacter fermentans. Briefly, maize seeds are surface-sterilized with sodium hypochlorite, planted in a sterilized synthetic soil, and watered with a nitrogen-free nutrient solution. After 6 weeks, the plants are harvested and stems are surface-sterilized with sodium hypochlorite, washed with sterile water, and crushed. The fluid portion is plated onto R2A agar and incubated at 28°C.

Maintenance procedures Strains of Runella slithyformis are grown on MS agar or nutrient agar at room temperature for several days to allow abundant growth. They will then survive refrigeration (4°C) for at least 3 weeks. They may also be preserved indefinitely by lyophilization.

Procedures for testing special characters Utilization of carbohydrates.  For Runella slithyformis, the production of acid from carbohydrates by aerobic or anaerobic means is determined by the method of Hugh and Leifson (1953), in which MS agar is used but with the glucose replaced by 1% of the substrate to be tested and the agar concentration lowered to 0.3%. Incubation is continued for 8 weeks for cultures giving negative results. Utilization of single carbon sources.  For Runella slithyformis, the basal medium used is that of Gordon and Mihm, to which is added 0.2% of the substrate or the sodium salt of the substrate. If growth occurs through four successive subcultures, the results are considered positive even in the absence of a color change in the bromthymol blue indicator.

Differentiation of the genus Runella from other genera Table 102 in the chapter on the genus Flectobacillus provides the primary characteristics that can be used to differentiate

TABLE 102.  Characteristics differentiating the species of the genus Runellaa

Characteristic

R. slithyformisb

R. defluviic

R. limosac

R. zeaed

+ w + Oe

− + − nr

− + − nr

− + + F

+f −g +e w

− nr − −

+ nr nr −

+ − − −

−e w w − + − + w + −e

nr − − + w w − − + nr

nr − − − w + + − − nr

+ − w − + w + − + +

+ − − nr

− − + Phosphatidylethanolamine 40.1

− − + Phosphatidylglycerol 44.5

− + − nr

Rings formed Catalase Oxidase Oxidation/fermentation test with glucose and sucrose Acid production from: Glucose Ribose Growth at 4°C Starch hydrolysis Utilization of sole carbon sources: Acetate, starch, fumarate, d-lyxose, malate, tartrate d-Arabitol Dulcitol, inositol, sorbitol Glycerol Glycogen 5-Ketogluconate Maltose, trehalose Mannitol Methyl-b-xyloside Growth on peptone Habitat: Freshwater Stems of Zea mays Activated sludge Major phospholipid DNA G+C content (mol%)

49

Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive; w, weak reaction; nr, not reported.

a

Results for Runella slithyformis are taken from Larkin and Williams (1978), Larkin and Borrall (1984b), and Lu et al. (2007).

b

Results for Runella defluvii and Runella limosa are taken from Lu et al. (2007).

c

Results for Runella zeae are taken from Chelius et al. (2000, 2002).

d

Result from Chelius et al. (2000, 2002).

e

Larkin and Williams (1978) and Larkin and Borrall (1984b) reported a positive reaction; Chelius et al. (2000) reported a negative reaction.

f

Larkin and Williams (1978) and Larkin and Borrall (1984b) reported a negative reaction; Chelius et al. (2000) reported a positive reaction.

g

49

414

Family I. Cytophagaceae

Runella slithyformis and Runella zeae from the morphologically similar genera.

Taxonomic comments Analyses of the 16S rRNA gene sequences of these organisms support the classification of Runella as a distinct genus (Chelius and Triplett, 2000; Nikitin et al., 2004; Woese et al., 1990). Although Runella slithyformis and Runella zeae differ in morphology, fermentative ability, and other phenotypic characteristics, a 16S rRNA gene sequence similarity of 94% between the two strains suggests that they are sufficiently related to be included within a single genus (Chelius et al., 2002). The level of DNA–DNA hybridization value between Runella zeae and

Runella slithyformis is 19%, indicating separate species (Chelius et al., 2002). The addition of Runella limosa to the genus is supported by a 16S rRNA gene similarity of 94.8% to the type strain of Runella slithyformis (Ryu et al., 2006). Similarly, Runella defluvii exhibits a similarity value of 93.6% to Runella slithyformis, and a value of 97.1% to Runella limosa. The status of Runella defluvii as a species separate from Runella limosa is supported by a DNA–DNA hybridization value of 25% between the two species.

Differentiation of the species of the genus Runella Table 102 lists characteristics that differentiate the four species of Runella.

List of species of the genus Runella 1. Runella slithyformis Larkin and Williams 1978, 35AL slith.y.form¢is. slithy a nonsense word from Lewis Carroll¢s Jabberwocky for a fictional organism that is “slithy” (presumably a combination of slinky and lithe); L. adj. suff. -formis -is -e, -like, in the shape of; N.L. fem. adj. slithyformis slithy in form. The characteristics are as described for the genus and as listed in Table 102. The following additional features are taken from Larkin and Williams (1978) and Larkin and Borrall (1984b). Good growth occurs on MS agar. Moderate growth occurs on nutrient agar. Scant growth occurs on chocolate agar, peptonized milk agar, and yeast extract-acetate-tryptone agar. No growth occurs on blood agar, eosin-methylene blue agar, nutrient agar plus 5% sucrose, phenol red-mannitolsalt agar, phenylethyl alcohol agar, trypticase soy agar, trypticase soy agar plus 3% glucose, MacConkey agar, bismuth sulfite agar, and Salmonella-Shigella agar. A positive reaction is obtained in the following tests: b-galactosidase (ortho-nitrophenyl-b-galactoside [ONPG]); phosphatase; acid production from glucose, inulin, maltose, and sucrose; hydrolysis of tributyrin and of starch (weak). A negative reaction is obtained in the following tests: hydrolysis of gelatin, esculin, lecithin, urea, agar, cellulose, and chitin; lysine decarboxylase; ornithine decarboxylase; phenylalanine deaminase; hemolysin production; indole production; methyl red test, Voges–Proskauer test, nitrate reduction, H2S production from peptone; acid production from arabinose, a-methyl-d-glucoside, cellobiose, dextrin, dulcitol, erythritol, fructose, glycerol, lactose, mannitol, melibiose, ribose, salicin, sorbitol, sorbose, trehalose, and xylose; utilization of acetate, benzoate, citrate, formate, glycerol phosphate, malonate, methanol, methylamine, propionate, succinate, and tartrate as sole carbon sources. No change occurs in litmus milk. Acid production from galactose, mannose, raffinose, and rhamnose differs between strains. Source: freshwater. DNA G+C content (mol%): 49–50 (Tm; absorbance ratio). Type strain: ATCC 29530, LMG 11500. Sequence accession no. (16S rRNA gene): M62786.

2. Runella defluvii Lu, Lee, Ryu, Chung, Choe and Jeon 2007, 2602VP de.flu¢vi.i. L. gen. n. defluvii of sewage. The characteristics are as described for the genus and as listed in Table 102. The following additional features are taken from Lu et al. (2007). Rods 0.5–0.9 × 2.2–6.0 mm at 30°C on R2A agar. Colonies are slightly raised, circular and salmon pink on R2A agar. Temperature range 15–40°C, optimum, 30–35°C. pH range 6.0–9.5, optimum 7.5–8.0. Nitrate is not reduced to nitrite. Catalase-positive. Oxidasenegative. No anaerobic growth after 7 d at 30°C on R2A agar. Tyrosine, Tween 80, and esculin are hydrolyzed, but not casein, Tween 20, starch, gelatin, and urea. Acid is produced from raffinose, myo-inositol, lactose, l-arabinose, d-galactose, d-mannose, d-mannitol, and melibiose, but not from d-glucose, d-fructose, arbutin, or salicin. Indole, H2S and acetoin are not produced. Citrate is not utilized (API 20E system). Enzyme activities include alkaline phosphatase, trypsin, a-chymotrypsin, N-acetyl-b-glucosaminidase, and naphtholAS-BI-phosphohydrolase, but not tryptophan deaminase, esterase (C4), lipase (C14) and b-glucuronidase. Weak activities occur for esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, a-mannosidase, and a-fucosidase (API ZYM system). Glycerol, methyl b-xyloside, methyl a-d-mannoside, and esculin are used as sole carbon sources but not erythritol, d- or l-arabinose, d-xylose, adonitol, galactose, d-glucose, d-fructose, mannose, dulcitol, inositol, mannitol, sorbitol, N-acetylglucosamine, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, d-raffinose, d-turanose, d- or l-arabitol, gluconate, or 2-ketogluconate. Ribose, l-xylose, sorbose, rhamnose, methyl a-d-glucoside, amygdalin, arbutin, starch, glycogen, xylitol, b-gentiobiose, d-lyxose, d-tagatose, d- and l-fucose, and 5-ketogluconate are weakly utilized (API 50CH system). Cells contain a large amount of phosphatidylethanolamine and small amounts of phosphatidylcholine and an unknown phospholipid as polar lipids. The major quinone is menaquinone-7. The cellular fatty acids are C15:0 (29.0%), summed feature 3 (C16:1w7c and/ or C15:0 2-OH; 20.2%), C16:1w5c (10.8%), C17:0 3-OH (9.2%),

Genus XVI. Spirosoma

C15:0 3-OH (7.4%), C15:0 (6.5%), C15:1 G (3.4%), C16:0 3-OH (3.3%), C13:0 (2.2%), C15:1 w6c (1.8%), C16:0 (1.4%), C17:1w6c (0.9%), C14:0 (0.8%), C15:0 (0.6%), and unknown ECL 14.959 (2.7%). Source: activated sludge of a domestic wastewater treatment plant. DNA G+C content (mol%): 40.1 mol% (HPLC). Type strain: EMB13, DSM 17976, KCTC 12614. Sequence accession no. (16S rRNA gene): DQ372980. 3. Runella limosa Ryu, Nguyen, Park, Kim and Jeon 2006, 2759VP li.mo¢sa. L. fem. adj. limosa muddy, pertaining to sludge, the natural habitat of the species. The characteristics are as described for the genus and as listed in Table 102. The following additional features are taken from Ryu et al. (2006). Rods, 0.7–0.9 × 4.0–10.0 mm when grown at 25°C on R2A agar. Colonies are slightly raised, circular and salmon-pink on R2A agar. Optimum temperature, 25–30°C. Optimum pH, 7.5–8.0. Nitrate is not reduced to nitrite. Catalase-positive and oxidase-­negative. Tyrosine, Tween 80 and esculin are hydrolyzed but not casein, Tween 20, starch, gelatin, or urea. Acid is produced from d-glucose, d-raffinose, myo-inositol, d-lactose, d-mannitol, and melibiose, but not from sorbitol, sucrose, rhamnose, amygdalin, d-fructose, d-galactose, d-mannose, l-arabinose, arbutin, or salicin. Alkaline phosphatase, a-chymotrypsin, N-acetylb-glucosaminidase, and naphthol-AS-BI-phosphohydrolase activities are present, but not esterase (C4), esterase lipase (C8), lipase (C14), or cystine arylamidase. Weak activity occurs for leucine arylamidase, valine arylamidase, trypsin, b-galactosidase, a-glucosidase, b-glucosidase, acid phosphatase, a-galactosidase, b-glucuronidase, a-mannosidase, and a-fucosidase. A large amount of phosphatidylglycerol is present and small amounts of two unknown phospholipids (PL1, PL2). The major quinone is MK-7. The major fatty acids are C15:0, C16:1w5c, C17:0 3-OH, C15:0 3-OH, C16:0 3-OH, C16:0, and summed feature 3 (C16:1w7c and/or C15:0 2-OH).

415

Source: sludge performing enhanced biological phosphorus removal. DNA G+C content (mol%): 42.7 (HPLC). Type strain: EMB111, KCTC 12615, DSM 17973. Sequence accession no. (16S rRNA gene): DQ372985. 4. Runella zeae Chelius, Henn and Triplett 2002, 2062VP ze¢ae. L. n. zea a kind of grain, spelt, and also a botanical genus name (Zea); L. gen. n. zeae of Zea, named because the organism was isolated from maize, Zea mays). The characteristics are as described for the genus and as listed in Table 102. The following additional features are taken from Chelius and Triplett (2000) and Chelius et al. (2002). Straight to slightly bent rods that form chains of irregular shapes. Colonies are round, smooth, and salmon-colored when grown on R2A medium at 28°C. Temperature range for growth, 15–37°C. The following reactions are positive: growth on peptone water; growth on Ayer’s agar (weak); acid production from (and fermentation of) glucose and sucrose; growth on acetate, N-acetylglucosamine, amygdalin, d- and l-arabinose, arbutin, cellobiose, dulcitol (weak), erythritol, esculin, d-fructose, d- and l-fucose, fumarate, galactose, b-gentiobiose, d-glucose, glycogen, inositol (weak), inulin, 5-ketogluconate (weak), lactose, d-lyxose, malate, malonate, maltose, mannose, melezitose, melibiose, methyl-a-d-glucoside, methyl-ad-mannoside, b-methylxyloside, d-raffinose, rhamnose, salicin, sorbitol (weak), sorbose (weak), sucrose, d-tagatose, tartrate, trehalose, d-turanose, and xylitol as sole carbon sources. The following reactions are negative: nitrogenase; reduction of nitrate to nitrite; hydrolysis of agar, cellulose, and starch; growth in litmus milk; acid production from ribose; growth in the presence of 1.5% NaCl; growth on d- and l-arabitol, formate, glycerol, mannitol, and methanol as sole carbon sources. Source: the stems of Zea mays. DNA G+C content (mol%): 49 (renaturation rate). Type strain: NS12, ATCC BAA-293, LMG 21438. Sequence accession no. (16S rRNA gene): AF137381.

Genus XVI. Spirosoma Migula 1894, 237AL The Editorial Board Spi.ro.so¢ma. Gr. n. spira coil; Gr. neut. n. soma body; N.L. neut. n. Spirosoma coiled body.

Rigid straight to curved rods, the degree of curvature varying among individual cells within a culture. The cells measure 0.5–1.0 mm × 1.5–10.8 mm. Rings 1.5–3.0 mm in outer diameter are formed by overlapping of the ends of a cell in some species. Coils and helices may be present. Long sinuous filaments up to 50 mm long may be present. Gram-stain-negative. No swimming motility occurs. Gliding motility is present (Vancanneyt et al., 2006). Resting stages are not known. Obligate or facultative aerobes. Acids are produced aerobically from a variety of carbohydrates. Optimum temperature, 20–30°C. Colonies contain a pale to light yellow, water-insoluble carotenoid pigment. Catalase-positive and oxidase-negative or positive depending upon the species. Chemo-organotrophic. Isolated from soil and freshwater sources. DNA G+C content (mol%): 51–53 (Tm).

Type species: Spirosoma linguale (Eisenberg 1891) Migula 1894, 235AL.

Further descriptive information This chapter is taken largely from the previous treatment by Larkin and Borrall (1984d) in the 1st edition of Bergey’s Manual of Systematic Bacteriology. It has been updated where more recent information was available. Spirosoma linguale produces rings, coils, and helices and the morphology is illustrated in Figure 3.1 of Larkin and Borrall (1984d). Although a culture typically shows a ringlike morphology, it exhibits variations of shape and size under certain cultural conditions. Occasionally, cultures are composed mainly of relatively straight cells, especially after prolonged subculturing on MS agar. The curly form may be re-obtained by

416

Family I. Cytophagaceae

examining colonies from agar streaked for isolation ­(Larkin, unpublished observation). Maloy et al. (1978) showed that there is a relationship between morphology and the phosphate content of the medium. The rings, coils, and helices are produced when the phosphate level is below 20 mM at pH 7.2. At 20–60 mM phosphate, long nonseptate filaments develop and the ratio of diphosphatidyl glycerol to phosphatidyl glycerol is twofold higher in the filaments. Normal cells contain twice the amount of muramic acid in the peptidoglycan layer as do the filaments; furthermore, this layer in normal cells contains equal amounts of N-acetylglucosamine and O-acetylglucosamine, whereas that of filamentous cells has nearly twice as much N-acetylglucosamine as O-acetylglucosamine (Miller and Raj, 1978). The filamentous forms appear to be multinucleate (Redell et al., 1981). In contrast, the species Spirosoma rigui forms filaments but not rings, coils or helices (Baik et al., 2007b). Vancanneyt et al. (2006) reported that the type strain of Spirosoma linguale exhibits gliding motility. Spirosoma rigui also exhibits gliding motility. Colonies on MS agar* produce a yellow water-insoluble, nonfluorescent pigment, and flexirubin-type pigments are absent (Baik et al., 2007b; Vancanneyt et al., 2006). For Spirosoma rigui, the pigment extract had an absorption maximum of 451 nm and shoulder at 478 nm. Spirosoma linguale grows poorly or not at all on rich media such as chocolate agar or blood agar, or on enteric-selective media such as eosin methylene blue agar, MacConkey agar, or Salmonella-Shigella agar. Only one of the four available strains grows on Trypticase soy agar. Spirosoma rigui was isolated on PYGV medium (Staley, 1968) and R2A (Oxoid) agar. Best growth is obtained with R2A, MS, or PCA media. It also grows in tryptone-soy agar or TSA medium (Oxoid). Spirosoma is chemo-organotrophic. Spirosoma linguale is active in the acidification of carbohydrate media. Acidification occurs with all but one (sorbose) of the 20 carbohydrates tested using the medium of Hugh and Leifson (1953) and aerobic incubation, although up to 3 weeks is sometimes required. Acidification does not occur with any sugar alcohols. Cellulose and chitin are not hydrolyzed, but some strains hydrolyze esculin and (weakly) casein. All strains hydrolyze tributyrin, and (weakly) starch. Three of the available strains of Spirosoma linguale produce a soft curd in litmus milk, accompanied by the reduction and then reoxidation of the litmus. A fourth strain produces only an increased alkalinity in litmus milk. Although Spirosoma rigui uses glucose and esculin as sole carbon sources, it does not assimilate many other carbohydrates. Spirosoma linguale strains may be grown in the defined medium of Gordon and Mihm† (1957) with various sole carbon sources. Of 11 substrates that have been tested, only glycerol phosphate, succinate, tartrate, and malonate are utilized as sole carbon sources. Radiorespirometric studies by Kottel and Raj (1973) revealed that Spirosoma linguale ATCC 23276 catabolizes glucose almost entirely (96%) by the Embden–Meyerhof pathway

with a small amount (4%) being catabolized by the pentose phosphate pathway. The Entner–Doudoroff pathway is inducible, with 75% of radio-labeled gluconate being metabolized by that pathway. Gluconate recovery patterns of this strain showed evidence for the possibility of a 2–5-diketogluconate pathway or some other unorthodox pathway (Raj and Ordal, 1977). Radiorespirometric and enzymic data indicate that a functional tricarboxylic acid cycle occurs in this strain (Kottel and Raj, 1973). Spirosoma has the MK-7 menaquinone system (Baik et al., 2007b; Urakami and Komagata, 1986). The cellular fatty acids of Spirosoma include large amounts of straight-chain saturated C16:0, unsaturated C16:1, iso C15:0, and iso C17:0 (Urakami and Komagata (1986). Vancanneyt et al. (2006) reported the following fatty acid profile for the type strain of Spirosoma linguale (% in parentheses): C13:0 iso (3.8), C15:0 iso (7.6), C15:0 anteiso (trace), C15:0 iso 3-OH (4.9), C16:0 (7.2), C16:1 w5c (17.5), C16:0 3-OH (4.7), C17:0 iso 3-OH (6.5), and summed feature 3 (47.9). Summed feature 3 consisted of one or more fatty acids that could not be separated by the Microbial Identification System): C15:0 iso 2-OH, C16:1 w7c and/or C16:1 w7t. The composition of Spirosoma rigui was very similar: C15:0 iso (9.5), C15:0 anteiso (1.3), C15:0 iso 3-OH (2.6), C16:0 (8.8), C16:1 w5c (18.5), C16:0 3-OH (2.8), C17:0 iso 3-OH (3.5), and summed feature 3 (45.6). (Baik et al., 2007b). Analysis of polyamine profiles of Spirosoma indicate that spermidine is the major polyamine (Hamana and Nakagawa, 2001). Spirosoma is not known to be pathogenic. Four strains of Spirosoma linguale have been isolated and characterized: one from garden soil and three from freshwater. Spirosoma linguale has also been found in an activated sludge reactor established for the degradation of cutting fluids (Baker et al., 1983). Spirosoma rigui was isolated from fresh water at the Woopo wetland, Republic of Korea. Spirosoma-like organisms have been seen in gelatinous deposits on wet planks from a mine (Kraepelin and Passern, 1980). Although Spirosoma is considered noncellulolytic, cellulolytic “Spirosoma-like” bacteria have been isolated from the gut of the termite Zootermopsis angusticollis (Wenzel et al., 2002).

Isolation procedures Spirosoma may be isolated by repeated streaking of water or diluted soil samples onto MS agar or tryptone glucose extract agar (Difco) fortified with 0.1 % yeast extract (TGEY medium; Raj, 1970) with incubation at room temperature for up to 2 weeks. The yellow pigmentation of the colonies aids detection of Spirosoma.

Maintenance procedures Spirosoma linguale is grown on MS agar, TGEY or nutrient agar at room temperature until abundant growth occurs (usually 1–4 d). Cultures may then be stored at 4°C for at least 4 weeks. Preservation by lyophilization is effective for several years.

Differentiation of the genus Spirosoma from other genera *MS agar (g/l): peptone, 1.0; yeast extract, 1.0; glucose, 1.0; agar, 15.0. † Gordon and Mihm’s medium (g/l): MgSO4, 0.2; (NH4)2HPO4, 1.0; KH2PO4, 0.5; NaCl, 1.0; carbon source, 2.0; agar, 15.0; and bromthymol blue, 0.08.

Table 93 in the chapter on Flectobacillus provides the primary characteristics that can be used to differentiate this genus from several morphologically similar genera.

Genus XVI. Spirosoma

Taxonomic comments In the 8th edition of the Manual the genus Microcyclus consisted of three species which were placed together primarily because of their ability to form rings during growth. The DNA G+C values were quite different, being 39.5 mol% for “Microcyclus major”, 51 mol% for “Microcyclus flavus”, and 67 mol% for Microcyclus aquaticus (the type species). Claus (1967) and Claus et al. (1968) suggested that “Microcyclus flavus” and two additional isolates corresponded to the description of the forgotten genus Spirosoma. Moreover, Staley (1974) suggested that the grouping of the three species was unsatisfactory, as did Konopka et  al. (1976), who found only a 0–14% binding of the DNA from three Spirosoma strains to that of Microcyclus aquaticus. Larkin et  al. (1977) proposed the reintroduction of the genus Spirosoma and emended its description to include “Microcyclus flavus” and three other isolates. The formation of rings or coils is not a sufficiently restrictive character to delineate a single taxonomic group. Ring formation is a characteristic that occurs in several other aerobic, chemoorganotrophic bacteria, e.g., Flectobacillus, Runella, Cyclobacterium, Ancylobacter, Polaribacter, and Arcicella. However, analyses of the 16S rRNA gene sequences of these organisms support the classification of Spirosoma as a distinct

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genus ­(Nikitin et al., 2004; Woese et al., 1990). Moreover, as the description of Spirosoma rigui proves, rings and coils are not properties of all members of this genus. This observation serves to further illustrate the unreliability of this characteristic in taxonomic assignments. Vancanneyt et al. (2006) reported that, by 16S rRNA analyses, Spirosoma linguale was the nearest neighbor of Larkinella insperata, sharing a similarity of 88.8%. The literature concerning the utilization of sole carbon sources by Spirosoma linguale is conflicting. Raj (1970) reported an inability of the species to use malonate, succinate, or tartrate, in contrast to results obtained by Larkin et al. (1977). The discrepancy may be attributable to a difference in the media employed or to the method of detecting growth. Raj (1970) used Simmons citrate agar with various organic substrates substituted for citrate and with 0.2% yeast extract added; growth responses were indicated by a color change. Larkin et al. (1977) used the agar medium of Gordon and Mihm (1957) and estimated visible growth after four successive transfers even in the absence of a color change. The literature concerning gelatin hydrolysis also is conflicting. Larkin and Borrall (1984d) indicated that all strains hydrolyze gelatin, but Vancanneyt et al. (2006) reported that the type strain did not hydrolyze gelatin.

List of species of the genus Spirosoma 1. Spirosoma linguale (Eisenberg 1891) Migula 1894, 235AL (Vibrio lingualis Eisenberg 1891, 212; Microcyclus flavus Raj 1970, 62 lin.gua¢le. L. n. lingua the tongue; L. neut. suff. -ale suffix denoting pertaining to; N.L. neut. adj. linguale of the tongue. The characteristics are as described for the genus, with the following additional features. Morphological features are depicted in Figure 3.1 of Larkin and Borrall (1984d). Other features are as described for the genus, with the following additional characteristics. Colonies are circular and convex with an entire margin. Aerobic. Temperature range for growth of the type strain, 5–39°C (Vancanneyt et al., 2006). NaCl range for growth of the type strain, 0–1.25% (Vancanneyt et al., 2006). Starch is weakly hydrolyzed. Tweens 20, 40, and 80 are hydrolyzed by the type strain (Vancanneyt et al., 2006). Some strains hydrolyze esculin and (weakly) casein. The type strain produces b-galactosidase and alkaline phosphatase (Vancanneyt et al., 2006). The following tests are positive: acid production from arabinose, a-methyl-d-glucoside, cellobiose, dextrin, fructose, galactose, glucose, inulin, lactose, maltose, mannose, melibiose, raffinose, rhamnose, ribose, salicin, sucrose, trehalose, and xylose; utilization of glycerol phosphate, malonate, succinate, and tartrate as sole carbon sources. The type strain is reported to utilize mannose and sucrose (Vancanneyt et al., 2006). The following tests are negative: urease; lysine decarboxylase; phenylalanine deaminase; hemolysin production; indole formation;

methyl red test; Voges–Proskauer test; nitrate reduction; H2S production from peptone; acid production from sorbose, dulcitol, erythritol, glycerol, mannitol, and sorbitol; utilization of acetate, benzoate, citrate, formate, methanol, methylamine, and propionate. l-arabinose, d-glucose, and d-lactose are not utilized by the type strain (Vancanneyt et al., 2006). Found in soil and fresh water. DNA G+C content (mol%): 51–53 (Tm). Type strain: DSM 74, ATCC 33905, LMG 10896. Sequence accession no. (16S rRNA gene): AM000023. 2. Spirosoma rigui Baik, Kim, Park, Lee, Lee, Ka, Choi and Seong 2007b, 2872VP ri¢gu.i. L. gen. n. rigui of a well-watered place, referring to the site of isolation, the Woopo wetland, Korea The characteristics are as described for the genus, with the following additional features. Cells are rod shaped and do not form rings or coils. Colonies are circular, opaque, convex, smooth, wet and slimy. Facultatively anaerobic. Temperature range for growth, 4–37°C. NaCl range for growth, 0–1%. Uses aesculin and glucose as sole carbon and energy sources. Produces b-galactosidase but not alkaline phosphatase. Does not use the sugars cellobiose, fructose, galactose, lactose, maltose, mannose, melibiose, raffinose, rhamnose, starch, sucrose, and xylose. Does not produce H2S or acetoin. Isolated from fresh water. DNA G+C content (mol%): 53.3 (Tm). Type strain: WPCB118, KCTC 12531, NBRC 101117. Sequence accession no. (16S rRNA gene): EF507900.

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Family I. Cytophagaceae

Genus XVII. Sporocytophaga Stanier 1940, 629AL Edward R. Leadbetter Spo.ro.cy.toph¢a.ga. Gr. n. spora a seed, and in biology a spore; N.L. fem. n. Cytophaga genus name of a bacterium; N.L. fem. n. Sporocytophaga sporing Cytophaga.

Flexible rods with rounded ends, 0.3–0.5 × 5–8 mm, occurring singly. Sphaeroplasts and distorted cells occur in older cultures. A resting stage, the microcyst, is formed. Motile by gliding. Stains Gram-negative. Chemo-organotrophs. Strict aerobe. Metabolism is respiratory, with molecular oxygen used as terminal electron acceptor. Cellobiose, cellulose, glucose and, for some strains, mannose are the only known sources of carbon and energy. Agar and chitin are not known to be metabolized. Either ammonium or nitrate ions, or peptone, urea or yeast extract, can serve as sole nitrogen source. Amino acids, peptones, yeast extract or nutrient agar (Difco) cannot serve as sole carbon and energy sources. No organic growth factor requirements are known. Catalase-positive. Temperature optimum: ~30°C. DNA G+C content (mol%): 36 (Bd). Type species: Sporocytophaga myxococcoides (Krzemieniewska 1933) Stanier 1940, 629AL.

Further descriptive information Only one species of the genus has been extensively examined. Sporocytophaga myxococcoides was shown by Stanier (1942) to grow on glucose sterilized by filtration and by Kaars Sijpesteijn and Fåhraeus (1949) to grow on glucose autoclaved separately from other components of the medium, thus refuting the assertion that growth of the organism is obligately linked to cellulose utilization. Further studies indicate that the organism, when isolated from nature, is unable either to oxidize or to utilize glucose but putative mutants able to use glucose arise in the

population (Leadbetter, unpublished observations). These “mutants” are able to metabolize immediately either cellulose or glucose, irrespective of the substrate in which they are grown. These observations thus confirm and extend those of Kaars Sijpesteijn and Fåhraeus (1949). Recent studies of Sporocytophaga myxococcoides have demonstrated that the organism is able to form microcysts when either glucose or cellulose is the carbon and energy source (Gallin and Leadbetter, 1966; Leadbetter, 1963). During growth on glucose, the Embden–Meyerhof–Parnas pathway is used (Hanstveit and Goksøyr, 1974). Cells also produce a variety of cellulases, some of which have been partially ˇ g and Goksøyr, 1975; Vance et al., 1980). purified (Osmundsva Major cellular lipids are sulfonolipids (Godchaux and Leadbetter, 1983) which contain a variety of fatty acyl moieties (Godchaux and Leadbetter, 1984). The sulfonolipids predominate in the outer membrane, while phospholipids are present in the inner cell membrane. Phosphatidylethanolamine and lysophosphatidylethanolamine are the major phospholipids (Holt et al., 1979). Homospermidine is the most abundant polyamine, but small amounts of putrescine and spermidine are also present (Hamana and Nakagawa, 2001). The cell-wall peptidoglycan contains diaminopimelic acid (Verma and Martin, 1967). Moreover, cells produce a slime comprised of glucose, mannose, arabinose, xylose, galactose and glucuronic acid (Martin et al., 1968). The Bacteroides transposon Tn4351 can be introduced by conjugation from Escherichia coli into Sporocytophaga and could serve as the basis for a genetic system (McBride and Baker, 1996).

List of species of the genus Sporocytophaga 1. Sporocytophaga myxococcoides (Krzemieniewska 1933) Stanier 1940, 630AL (Cytophaga myxococcoides Krzemieniewska 1933, 400) myx.o.coc.coi¢des. N.L. masc. n. Myxococcus genus name of a bacterium; L. suff. -oides (from Gr. suff. eides from Gr. n. eidos that which is seen, form, shape, figure), resembling, similar; N.L. fem. adj. myxococcoides resembling Myxococcus. Single, often flexible rods with rounded ends, 0.3–0.5 × 5–8 mm. Sphaeroplasts and abnormally long forms may occur in old cultures. The resting stage, the microcyst, is spherical and about 1.5 mm in diameter. Both the growing (vegetative) rod and the microcyst have noticeably smaller dimensions when cultures are grown on cellulose rather than on glucose. Microcysts are notably more resistant to ultrasonic disruption than are vegetative cells.

Electron microscopic studies indicate that the vegetative cell has a fine structure typical of Gram-stain-negative bacteria, while the microcyst has a thick, fibrillar capsule exterior to a highly convoluted cell wall (Holt and Leadbetter, 1967). Growth on cellulose (filter paper) -salts agar (or silica gel) or glucose-salts agar is gummy, and liquid cultures become viscous as a result of extracellular slime production. Cell masses are yellow, reflecting the presence of carotenoid and flexirubin-type pigments. Filter paper on the agar or silica gel surface is eventually dissolved around colonies so that translucent areas result. Colonies on glucose-salts agar medium are raised. Other characteristics are the same as those of the genus.

Other species Other species of Sporocytophaga have been described, but they are incompletely characterized:

(c) “Sporocytophaga ellipsospora” (Imshenetski and Solntseva, 1936) Stanier 1942, 190.

(a) “Sporocytophaga cauliformis” Knorr and Gräf in Gräf 1962, 124.

(d) “Sporocytophaga ochracea” Ueda, Ishikawa, Itami and Asai 1952, 545.

(b) “Sporocytophaga congregata” subsp. maroonicum Akashi 1960, 899.

Genus XVII. Sporocytophaga

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Callies, E. and W. Mannheim. 1978. Classification of the FlavobacteriumCytophaga complex on the basis of respiratory quinones and fumarate respiration. Int. J. Syst. Bacteriol. 28: 14–19. Chaturvedi, P., G.S. Reddy and S. Shivaji. 2005. Dyadobacter hamtensis sp. nov., from Hamta glacier, located in the Himalayas, India. Int. J. Syst. Evol. Microbiol. 55: 2113–2117. Chelius, M.K. and E.W. Triplett. 2000. Dyadobacter fermentans gen. nov., sp. nov., a novel Gram-negative bacterium isolated from surface-sterilized Zea mays stems. Int. J. Syst. Evol. Microbiol. 50: 751–758. Chelius, M.K., J.A. Henn and E.W. Triplett. 2002. Runella zeae sp. nov., a novel Gram-negative bacterium from the stems of surface-sterilized Zea mays. Int. J. Syst. Evol. Microbiol. 52: 2061–2063. Christensen, P.J. 1977. The history, biology, and taxonomy of the Cytophaga group. Can. J. Microbiol. 23: 1599–1653. Christensen, P. 1980. Flexibacter canadensis sp. nov. Int. J. Syst. Bacteriol. 30: 429–432. Claus, D. 1967. Taxonomy of some highly pleomorphic bacteria Spisy Prirodoved Fak. Univ. J. E. Purkyne Brno 40: 254–257. Claus, D., J.E. Bergendahl and M. Mandel. 1968. DNA base composition of Microcyclus species and organisms of similar morphology. Arch. Mikrobiol. 63: 26–28. Collins, M.D., R.A. Hutson, I.R. Grant and M.F. Patterson. 2000. Phylogenetic characterization of a novel radiation-resistant bacterium from irradiated pork: description of Hymenobacter actinosclerus sp. nov. Int. J. Syst. Evol. Microbiol. 50: 731–734. Costerton, J.W., Z. Lewandowski, D.E. Caldwell, D.R. Korber and H.M. Lappin-Scott. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49: 711–745. DeLey, J., H. Cattoir and A. Reynaerts. 1970. The quantitative measurement of DNA hybridization from renaturation rates. Eur. J. Biochem. 12: 133–142. Dong, Z., X. Guo, X. Zhang, F. Qiu, L. Sun, H. Gong and F. Zhang. 2007. Dyadobacter beijingensis sp. nov., isolated from the rhizosphere of turf grasses in China. Int. J. Syst. Evol. Microbiol. 57: 862–865. Eisenberg, J. 1891. Bacteriologische Diagnostik. Hilfstabellen zum Gebrauche beim Praktischen Arbeiten. 3 Aufl, vols. VII-XXX. ­Leopold Voss, Hamburg. Euzéby, J.P. 1998. Taxonomic note: necessary correction of specific and subspecific epithets according to Rules 12c and 13b of the International Code of Nomenclature of Bacteria (1990 revision). Int. J. Syst. Bacteriol. 48: 1073–1075. Gallin, J.I. and E.R. Leadbetter. 1966. Morphogenesis of Sporocytophaga. Bacteriol. Proc. 75. Garcia-Pichel, F. 2002. Desert environments: biological soil crusts. Encyclopedia of Environmental Microbiology. John Wiley, New York. Garrity, G.M., J.A. Bell and T. Lilburn. 2005. The Revised Road Map to the Manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, The Proteobacteria, Part A, Introductory Essays (edited by Brenner, Krieg, Staley and Garrity). Springer, New York, pp. 159– 220. Godchaux, W., III and E.R. Leadbetter. 1983. Unusual sulfonolipids are characteristic of the Cytophaga-Flexibacter group. J. Bacteriol. 153: 1238–1246. Godchaux, W., III and E.R. Leadbetter. 1984. Sulfonolipids of gliding bacteria. Structure of the N-acylaminosulfonates. J. Biol. Chem. 259: 2982–2990. Gordon, R.E. and J.M. Mihm. 1957. A comparative study of some strains received as nocardiae. J. Bacteriol. 73: 15–27. Gosink, J.J., C.R. Woese and J.T. Staley. 1998. Polaribacter gen. nov., with three new species, P. irgensii sp. nov., P. franzmannii sp. nov., and P. filamentus sp. nov., gas vacuolate polar marine bacteria of the CytophagaFlavobacterium-Bacteroides group and reclassification of ‘Flectobacillus glomeratus’ as Polaribacter glomeratus comb. nov. Int. J. Syst. Bacteriol. 48: 223–235. Gräf, W. 1962. Über Wassermyxobakterien. (in German with English and French abstracts). Arch. Hyg. Bakteriol. 146: 114–125.

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Genus I. Cyclobacterium Xin, Y.H., Y.G. Zhou, H.L. Zhou and W.X. Chen. 2004. Ancylobacter rudongensis sp. nov., isolated from roots of Spartina anglica. Int. J. Syst. Evol. Microbiol. 54: 385–388. Yi, H. and J. Chun. 2004. Hongiella mannitolivorans gen. nov., sp. nov., Hongiella halophila sp. nov. and Hongiella ornithinivorans sp. nov., isolated from tidal flat sediment. Int. J. Syst. Evol. Microbiol. 54: 157–162. Yoon, J.H., S.J. Kang, C.H. Lee and T.K. Oh. 2005. Marinicola seohaensis gen. nov., sp. nov., isolated from sea water of the Yellow Sea, Korea. Int. J. Syst. Evol. Microbiol. 55: 859–863.

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Family II. Cyclobacteriaceae fam. nov. Olga I. Nedashkovskaya and Wolfgang Ludwig Cyc.lo.bac.te.ri.a¢ce.ae. N.L. neut. n. Cyclobacterium type genus of the family; suff. -aceae ending to denote a family; N.L. fem. pl. n. Cyclobacteriaceae the Cyclobacterium family. Regular and curved, ring-like or horseshoe-shaped rods that are 0.3–0.7 × 0.3–10 mm. Gram-stain-negative. Nonsporeforming. Nonflagellated and nonmotile in liquid media, but some of them can move by gliding on solid substrates. Aerobic, with respiratory type of metabolism. Optimal temperature is 16–37°C. Chemo-organotrophs. Carbohydrates are oxidized. One species ferments glucose. Colonies are pink, red, or orange in color. For most strains, an addition of seawater or NaCl to nutrient media sufficiently increases growth rates. Oxidase-, catalase-, and alkaline phosphatase-positive. Flexirubin-type pigments are absent. Produce no indole. One taxon

produces hydrogen sulfide. Nitrate may be reduced to nitrite. Most species cannot hydrolyze agar, casein, urea, or chitin. Menaquinone 7 is a major or single respiratory quinone. Predominant fatty acids are C15:0 iso, C17:1 iso w9c and C15:0 iso 2-OH and/or C16:1 w7c. Most species occur in seawater, marine sediments, seaweeds, or marine animals. Some taxa inhabitant algal mates of saline lakes, marine solar salterns, soil, or nonsaline groundwater. DNA G+C content (mol%): 35–49. Type genus: Cyclobacterium Raj and Maloy 1990, 345VP emend. Ying, Wang, Yang and Liu 2006, 2929.

Genus I. Cyclobacterium Raj and Maloy 1990, 345VP emend. Ying, Wang, Yang and Liu 2006, 2929 The Editorial Board Cy.clo.bac.ter¢i.um. Gr. n. cyclos a circle L. neut. n. bacterium a small rod; N.L. neut. n. Cyclobacterium a circle-shaped bacterium.

Cells are curved, ring-like or horseshoe-shaped. Nonmotile. Colonies on Marine agar (MA) are pink and shiny. Aerobic, having a strictly respiratory type of metabolism with O2 as the terminal electron acceptor. Neutrophilic and mesophilic. Chemo-organotrophic. Optimal growth temperature range is 25–30°C. Catalase- and oxidase-positive. The major cellular fatty acids are C15:0 iso, summed feature 3 (C15:0 iso 2-OH and/or C16:1 w 7c), C17:1 iso w 9c, C17:0 iso 3-OH, and C15:0 anteiso. Habitat: marine environments. DNA G+C content (mol%): 41–45. Type species: Cyclobacterium marinum corrig. (Raj 1976) Raj and Maloy 1990, 346VP [Flectobacillus marinus (Raj 1976) Borrall and Larkin 1978, 301AL; Microcyclus marinus Raj 1976, 540].

Further descriptive information The predominant cellular fatty acids of the type strains of Cyclobacterium amurskyense and Cyclobacterium marinum are straight-chain unsaturated, branched-chain unsaturated and saturated, namely C15:0 iso (22.2 and 23%, respectively), C15:0

anteiso (9.2 and 6.4%), C15:1 iso (8.4 and 9.7%), C17:1 iso w9c (4.3 and 6.3%), C17:0 iso 3-OH (10.7 and 12.7%), and summed feature 3 (24.3 and 23.4%), comprising C16:1 w7c and/or C15:0 iso 2-OH (Nedashkovskaya et al., 2005). The major cellular fatty acids of Cyclobacterium lianum are C15:0 iso (28.3%), summed feature 3 (C15:0 iso 2-OH and/or C16:1 w7c; 16.6%), C17:1 iso w9c (10.3%), C17:0 iso 3-OH (8.0%), and C15:0 anteiso (6.4%), similar to the profiles reported for Cyclobacterium marinum and Cyclobacterium amurskyense (Ying et al., 2006). The cellular polyamines of Cyclobacterium marinum contain homospermidine, whereas those of other ring-forming genera (Runella, Spirosoma, and Flectobacillus) contain spermidine (Hamana and Nakagawa, 2001). The oxidation of glucose in Cyclobacterium marinum occurs mainly via glycolysis, whereas gluconate is catabolized mainly via the Entner–Doudoroff pathway. These pathways act in conjunction with the tricarboxylic acid cycle and with some participation of the pentose phosphate pathway (Raj and Paveglio, 1983).

424

Family II. Cyclobacteriaceae

Enrichment and isolation procedures Cyclobacterium marinum can be isolated on MS agar* or on mZ medium.† Incubation is at room temperature (25°C) for several days to allow abundant growth. The cultures will then survive refrigeration at 4°C for at least 3 weeks. They may also be preserved indefinitely by lyophilization. Cyclobacterium amurskyense was isolated from a seawater sample collected in Amursky Bay, Gulf of Peter the Great. It can be cultured on Marine agar 2216 (Difco). Cyclobacterium lianum was isolated from sediment of the Xijiang oilfield in the South China Sea, near Fujian Province, China. For isolation, serially diluted sediment samples were spread onto low-organic marine agar 2216 plates (differs from regular Marine agar 2216 [Difco] only by decreasing the peptone concentration from 5 to 0.5 g/l and the yeast extract from 1 to 0.l g/l). A colony was selected after incubation at 30°C for 10 d and subcultured onto Marine agar 2216.

Differentiation of the genus Cyclobacterium from other genera The genera Runella, Flectobacillus, and Spirosoma have a ring-like or horseshoe-like morphology that resembles that of Cyclobacterium. However, Cyclobacterium has a marine habitat and can tolerate seawater (although not necessarily requiring NaCl), whereas the other three genera have a freshwater habitat and cannot grow in the presence of seawater or 3% NaCl. Moreover, the cellular polyamines of Cyclobacterium marinum contain homospermidine, whereas those of Runella, Spirosoma, and Flectobacillus contain spermidine (Hamana and Nakagawa, 2001).

Taxonomic comments Cyclobacterium marinum was initially classified in the Microcyclus by Raj (1976) as Microcyclus marinus. In many features, it resembled Flectobacillus major, the type species of the genus Flectobacillus Larkin, Williams and Taylor (1977), and it was assigned to that genus as Flectobacillus marinus by Borrall and Larkin (1978). H. D. Raj (1979) reported that Flectobacillus

major and ­Flectobacillus marinus were sufficiently distinct as to warrant separation of Flectobacillus marinus into a new genus; the name Cyclobacterium was suggested (H. D. Raj, cited by ­Larkin and Borrall, 1984a). However, Larkin and Borrall (1984a, b) believed that it would be best to retain the organism in the genus Flectobacillus because of the similarity in the mol% G+C content of the DNA and because the DNA–DNA hybridization value between the type strains of Flectobacillus marinus and Flectobacillus major was 71%, using the renaturation rate method of DeLey et al. (1970); consequently, Larkin and Borrall recommended that Flectobacillus marinus should not be used to create the new genus Cyclobacterium and that the latter name should be discarded. However, Raj and Maloy (1990) noted that analysis of the oligonucleotide sequence catalogues of the 16S rRNA of these organisms (Woese et al., 1990) indicated a relatively large evolutionary distance (ca. 20%) between Flectobacillus major and Flectobacillus marinus (Woese, 1987), supporting the idea that Flectobacillus marinus should be separated from Flectobacillus. major at the genus level. Consequently, Raj and Maloy (1990) reclassified Flectobacillus marinus as Cyclobacterium marinus (later corrected to marinum by Euzéby) (1998). Nedashkovskaya et al. (2005) reported that the level of 16S rRNA gene sequence similarity between the type strain of Cyclobacterium amurskyense and that of Cyclobacterium marinum was 96.6% (47 nucleotide differences). Ying et al. (2006) reported that the nearest neighbors of Cyclobacterium lianum, based on 16S rRNA gene sequence analysis, were Cyclobacterium marinum (93.8% sequence similarity) and Cyclobacterium amurskyense (92.8% similarity). Other related genera were Aquiflexum (89.7%), Belliella (89.7%), Hongiella (87.8–90.3%), Chimaereicella (88.4%), and Algoriphagus (88.3– 89.5%). Hongiella and Chimaereicella have since been reclassified into Algoriphagus by Nedashkovskaya et al. (2007b).

Differentiation of the species of the genus Cyclobacterium Table 103 lists reactions differentiating the three species of Cyclobacterium.

List of species of the genus Cyclobacterium 1. Cyclobacterium marinum corrig. (Raj 1976) Raj and Maloy 1990, 346VP [Flectobacillus marinus (Raj 1976) Borrall and Larkin 1978, 341AL; Microcyclus marinus Raj 1976, 540] ma.ri¢num. L. neut. adj. marinum of the sea, marine. The original spelling marinus was corrected by Euzéby (1998). The characteristics are as described for the genus, with the following additional information. In contrast to the original description, NaCl is not required for growth, and growth can occur at 42°C (Nedashkovskaya et al., 2005). The following reactions are positive (these results also apply to Cyclobacterium amurskyense below; Nedashkovskaya et al., 2005): b-galactosidase, alkaline and acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, naphthol-AS-BI-phos-

*MS agar for marine organisms contains (g/l): peptone, 1.0; yeast extract, 1.0; glucose, 1.0; NaCl, 30.0; agar, 15.0. † Modified Zobell 2216 (mZ) medium contains (g/l of seawater): peptone, 5.0; yeast extract, 1.0; ferrous sulfate, 0.2; agar, 20.0 (Raj, 1976).

phohydrolase, a- and b-galactosidase, a- and b-­glucosidase, N-acetyl-b-glucosaminidase, and a-mannosidase activities; growth at 0–10% NaCl and at 4–40°C; hydrolysis of esculin; acid formation from d-cellobiose, l-fucose, d-galactose, d-lactose, l-raffinose, d-melibiose, l-rhamnose, d-trehalose, d-maltose, and d-sucrose; susceptibility to ampicillin, ­carbenicillin, lincomycin and oleandomycin; and utilization of glucose, d-mannose, N-acetyl-d-glucosamine, d-fructose, methyl a-d-mannoside, methyl a-d-glucoside, amygdalin, arbutin, salicin, inulin, melezitose, gentiobiose, d-turanose, lyxose, tagatose, d-fucose, l-fucose, ribose, sorbose, d-xylose, and methyl b-d-xyloside. The following reactions are negative (these results also apply to Cyclobacterium amurskyense below; Nedashkovskaya et al., 2005): a-chymotrypsin, b-glucuronidase, a-fucosidase, arginine dihydrolase, lysine and ornithine decarboxylase acti­vities; gliding motility; Na+ requirement for growth; requirement for organic growth factors; nitrate reduction; flexirubin pigments; H2S, indole, and acetoin production; degradation of agar, casein, gelatin, DNA, starch, cellulose

Genus I. Cyclobacterium TABLE 103.  Characteristics differentiating the species of the genus Cyclobacterium a,b

Characteristic Lipase, trypsin Growth at 42°C Acid from: d-Glucose l-Arabinose, dl-xylose Starch N-Acetyl-d-glucosamine Utilization of: d-Gluconate l-Fucose, l-sorbose Mannitol Hydrolysis of: Tween 20 Tween 40 Susceptibility to: Benzylpenicillin, kanamycin Streptomycin, tetracycline DNA G+C content (mol%)

C. marinum C. amurskyense C. lianum − +

+ −

− +

− − − −

− + + +

+ + − nd

− + +

+ + −

+ − w

− +

− −

+ w

− + 41.9

+ − 41.3

+ − 45.2

Symbols: +, >85% positive; −, 0–15% positive; w, weak; nd, no data.

a

Data from Nedashkovskaya et al. (2005) and Ying et al. (2006).

b

(CM-­cellulose, filter paper), chitin, urea, and Tweens 20 and 80; acid ­production from d-glucose, l-sorbose, adonitol, dulcitol, glycerol, myo-inositol, mannitol, malate, fumarate, and citrate; utilization of glycerol, iso-erythritol, adonitol, dulcitol, myo-inositol, d-sorbitol, glycogen, xylitol, d-arabitol, l-arabitol, gentiobiose, 2-ketogluconate, 5-ketogluconate, caprate, adipate, malate, citrate, and phenylacetate; and susceptibility to gentamicin, neomycin, and polymyxin B. DNA G+C content (mol%): 41.9 (Tm) (Nedashkovskaya et al., 2005). Type strain: Raj, ATCC 25205, DSM 745, LMG 13164. Sequence accession no. (16S rRNA gene): AJ575266, AY533665. 2. Cyclobacterium amurskyense Nedashkovskaya, Kim, Lee, Park, Lee, Lysenko, Oh, Mikhailov and Bae 2005, 2392VP a.mur.sky.en¢se. N.L. neut. adj. amurskyense pertaining to Amursky Bay, from which the type strain was isolated. Cells have a width of 0.3–0.4 mm and an outer diameter of 0.9–1.2 mm. Colonies are pink, circular, low-convex, shiny with entire edges, and 1–3 mm in diameter on Marine agar 2216. Flexirubin pigments are absent. Grows in 0–10% NaCl. Growth occurs at 4–40°C. Esculin is hydrolyzed but not agar, casein, gelatin, starch, cellulose (CM-cellulose and filter paper), chitin, DNA, urea, or Tweens 20, 40, and 80. Nitrate is not reduced. H2S, indole and acetoin (Voges–Proskauer reaction) are not produced. Positive for b-galactosidase and alkaline phosphatase. Acid is produced from the following compounds: N-acetylglucosamine, l-arabinose, d-­cellobiose, l-fucose, d-galactose, d-lactose, d-maltose, d-melibiose, l-raffinose, l-rhamnose, d-sucrose, starch, d-trehalose, and dl-xylose. No acid is formed from adonitol, citrate, dulcitol, fumarate, d-glucose, d-glucuronic acid, glycerol, inositol, mannitol, malate, and l-sorbose. The following compounds are utilized (Biolog GN2 Microplate system): N-acetyl-dglucosamine, cellobiose, dextrin, d-fructose, d-galactose,

425

d-galacturonic acid, gentiobiose, a-d-glucose, glucose 1-phosphate, a-dl-glycerol phosphate, a-d-lactose, lactulose, maltose, d-mannose, d-melibiose, methyl b-d-glucoside, methylpyruvate, psicose, d-raffinose, l-rhamnose, sucrose, d-trehalose, and turanose. The following compounds are oxidized: acetic acid, N-acetyl-d-galactosamine, l-arabinose, glucuronamide, l-­glutamic acid, glycogen, l-fucose, glycerol, a-ketobutyric acid, a-ketoglutaric acid, dl-lactic acid, d-mannitol, monomethyl succinate, l-serine, and l-threonine. No oxidation occurs of cis-aconitic acid, adonitol, alaninamide, d-alanine, l-­alanine, l-alanyl glycine, g-aminobutyric acid, 2-amino­ ethanol, d-­arabitol, l-asparagine, l-aspartic acid, bromosuccinic acid, 2,3-butanediol, dl-carnitine, citric acid, a-cyclodextrin, iso-erythritol, formic acid, d-galacturonic acid, d-gluconic acid, d-glucosaminic acid, glucose 6-phosphate, glycyl-l-aspartic acid, glycyl-l-glutamic acid, l-histidine, a-hydroxybutyric acid, b-hydroxybutyric acid, g-hydroxybutyric acid, hydroxy-l-proline, p-hydroxyphenylacetic acid, inosine, myo-inositol, itaconic acid, a-ketovaleric acid, l-leucine, malonic acid, l-ornithine, l-phenylalanine, phenylethylamine, propionic acid, l-proline, putrescine, l-pyroglutamic acid, quinic acid, d-saccharic acid, sebacic acid, d-serine, d-sorbitol, succinamic acid, succinic acid, thymidine, Tweens 40 and 80, uridine, uronic acid, and xylitol. Susceptible to ampicillin, benzylpenicillin, carbenicillin, kanamycin, oleandomycin, and lincomycin. Resistant to neomycin, streptomycin, gentamicin, polymyxin B, and tetracycline. Source: seawater, collected in Amursky Bay, Gulf of Peter the Great, East Sea (also known as the Sea of Japan). DNA G+C content (mol%): 41.3 (Tm). Type strain: KMM 6143, KCTC 12363, LMG 23026. Sequence accession no. (16S rRNA gene): AY960985. 3. Cyclobacterium lianum Ying, Wang, Yang and Liu 2006, 2929VP li.a¢num. N.L. neut. adj. lianum pertaining to Li, named in honor of Ji-Lun Li, who devoted himself to microbiological research and education in China. The characteristics are as described for the genus, with the following additional features. Cells are 0.4–0.5 mm wide and the outer diameter of the rings is 1.5–1.8 mm. ­Colonies grown for 3 d on Marine agar are circular, 2–3 mm in dia­ meter, light rose in color, and shiny. Growth occurs at 15–42°C; optimum, 33°C. The pH range is 6.5–9.0; optimum, 7.5–8.0. Growth occurs in 0.1–12% NaCl; optimum, 1–4%. Arginine dihydrolase, urease, and lecithinase negative. Indole and H2S are not produced. Nitrate is not reduced. Esculin and Tween 20 are hydrolyzed, but Tweens 40 and 80 are only weakly hydrolyzed. No hydrolysis occurs of agar, casein, gelatin, starch, DNA, and carboxymethyl-cellulose. The following compounds are used as sole carbon sources: l-arabinose, cellobiose, galactose, gluconate, glucose, inulin, lactose, maltose, melezitose, d-melibiose, methyl a-d­glucoside, d-raffinose, l-rhamnose, ribose, sucrose, and trehalose. Weak utilization occurs of d-­fructose, l-glutamic acid, glycerol, lactate, malate, mannitol, d-­mannose, pyruvate, succinate, and d-xylose. The following are not used: adonitol, l-alanine, butyric acid, caprate, citrate, dulcitol,

426

Family II. Cyclobacteriaceae

formate, l-fucose, myo-inositol, l-lysine, malonate, and l-­sorbose. Acid is formed from l-arabinose, cellobiose, galactose, glucose, glycerol (weakly), inulin, ­lactose, maltose, melezitose, d-melibiose, methyl a-d-glucoside, d-raffinose, l-rhamnose, ribose (weakly), sucrose, trehalose, and d-xylose. The following show strong activity (API ZYM system): alkaline and acid phosphatases, leucine and valine arylamidases, naphthol-AS-BI-phosphohydrolase, b-galactosidase, a- and b-glucosidases and N-acetyl-b-glucosaminidase. Weak ­activity is exhibited for esterases C4 and C8, cystine arylamidase, a-galactosidase, and a-mannosidase. No activity is exhibited for trypsin, a-chymotrypsin, b-glucuronidase, a-fucosidase, or lipase (C14). The following compounds are oxidized (GN2 MicroPlate system): N-acetyl-d-glucosamine, l-­alaninamide, l-alanine, l-arabinose, 2,3-butanediol, glycerol, dl-­carnitine, d-cellobiose, dextrin, d-fructose, d-­galactose, d-­galacturonic acid, gentiobiose, a-d-glucose, ­glucose 1-phosphate, glucuronamide, dl-a-glycerol phosphate, dl-lactic acid, a-d-lactose, lactulose, maltose, d-­mannose, d-melibiose, methyl b-d-­glucoside, d-raffinose,

sucrose, d-trehalose, and turanose. Weak or variable oxidation occurs with glycogen, N-acetyl-d-galactosamine, l-alanyl glycine, g-aminobutyric acid, 2-aminoethanol, l-asparagine, l-aspartic acid, iso-erythritol, d-gluconic acid, glucose 6-phosphate, glutamic acid, a-ketovaleric acid, d-mannitol, monomethyl succinate, l-ornithine, l-proline, d-psicose, l-pyroglutamic acid, l-rhamnose, dl-serine, d-sorbitol, succinic acid, l-threonine, and uridine. Sensitivity is exhibited toward the following antibiotics (mg per disk): ampicillin (10), carbenicillin (100), vancomycin (30), ciprofloxacin (5), rifampicin (5), norfloxacin (10), chloramphenicol (30), benzyl penicillin (10), kanamycin (30), and erythromycin (15). Resistance is exhibited toward the following antibiotics (mg per disk): gentamicin (10), neomycin (30), polymyxin B (300), streptomycin (10), and tetracycline (30). Source: sediment from the Xijiang oilfield in the South China Sea. DNA G+C content (mol%): 45.2 (Tm). Type strain: HY9, CGMCC 1.6102, JCM 14011. Sequence accession no. (16S rRNA gene): DQ534063.

Genus II. Algoriphagus Bowman, Nichols and Gibson 2003, 1351VP, emend. Nedashkovskaya, Vancanneyt, Van Trappen, Vandemeulebroecke, Lysenko, Rohde, Falsen, Frolova, Mikhailov and Swings 2004, 1762VP, emend. Nedashkovskaya, Kim, Kwon, Shin, Luo, Kim and Mikhailov 2007b, 1993VP Olga I. Nedashkovskaya and Marc Vancanneyt Al.go.ri.pha¢gus. L. masc. n. algor cold; Gr. masc. n. phagos glutton; N.L. masc. n. Algoriphagus the cold eater

Rods usually measuring 0.3–0.7 × 0.3–10.0 mm. Gliding motility is not observed. Produce non-diffusible carotenoid pigments. No flexirubin type of pigments are formed. Chemo-organotrophs. Aerobic. Oxidase, catalase, alkaline phosphatase, and b-galactosidase-positive. Arginine dihydrolase and tryptophan deaminase are not produced. Esculin is hydrolyzed. Cellulose (CM-cellulose and filter paper) and urea are not degraded, but agar, casein, gelatin, starch, DNA, Tweens, and chitin may be decomposed. Carbohydrates are utilized. Can grow without seawater or sodium ions. Hydrogen sulfide and indole are not produced. The major respiratory quinone is MK-7. DNA G+C content (mol%): 35.0–49.0. Type species: Algoriphagus ratkowskyi Bowman, Nichols and Gibson 2003, 1351VP.

Further descriptive information The main cellular fatty acids are unsaturated and branchedchain unsaturated fatty acids C15:0 iso, C17:0 iso 3-OH, and summed feature 3 comprising C15:0 iso 2-OH, and C16:1 w7c or both (Table 104). On Marine agar (Difco) strains of the genus Algoriphagus form regular, circular, convex, shiny, smooth, with entire edges, and pink or orange colonies with diameter of 0.5–3 mm after cultivation for 48 h. All strains grow at 6–41°C, grow with 0–12% NaCl, and do not form acid from inositol or mannitol. Strains were isolated from sea animals and from seawater samples collected in ­temperate and tropic latitudes.

Enrichment and isolation procedures Three strains of the type species Algoriphagus ratkowskyi were isolated from the sea-ice algal assemblages on Marine agar 2216, and one strain was recovered from cyanobacterial mat collected in a meromictic lake in Antarctica (Bowman et al., 2003) using a seawater nutrient medium (SWN), containing of 0.05 g of yeast extract, 0.05 g of tryptone, 0.05 g of bacteriological peptone, 0.05g of soluble starch, and 0.02 g of sodium pyruvate dissolved in 1000 ml of natural seawater or artificial seawater (ASW), and supplemented with 0.1 ml of a sterile vitamin solution ­(Bowman et al., 2003). Six strains of another species, Algoriphagus antarcticus, were isolated from microbial mats and cultivated on Marine agar 2216 (Van Trappen et al., 2002, 2004). Strains of Algoriphagus chordae and Algoriphagus winogradskyi were isolated from the brown alga Chorda filum and the green alga Acrosiphonia sonderi, respectively, by the standard dilution-plating technique on Marine agar 2216 (Nedashkovskaya et al., 2004). Members of the two species, Algoriphagus ­mannitolivorans and Algoriphagus ornithinivorans, were isolated from tidal flat sediments samples using Marine agar 2216, and one strain of Algoriphagus halophilus was obtained on medium R2A (Difco) supplemented with artificial sea salts (Sigma) (Yi and Chun, 2004). The representatives of Algoriphagus aquimarinus, Algoriphagus locisalis, Algoriphagus marincola, and Algoriphagus yeomjeoni were isolated from samples of seawater and cultivated on Marine agar 2216 (Nedashkovskaya et al., 2004, 2005a, b; Yoon et al., 2004). Strain Algoriphagus vanfongensis KMM 6241T was isolated from 0.1 ml of tissue homogenates of the coral Palythoa sp. by direct plating on a medium containing [in g/l of a mixture of 30% (v/v)

427

Genus II. Algoriphagus

1.3

0.5 1.0

7.4

5.9

9.2 3.9

20.4 1.8

4.5 1.1 4.9 8.9

1.0 0.3 1.9 38.1

1.5 0.6 2.8 28.4

5.8 1.5

7.7 3.5

4.4 1.0 3.6 1.2 1.6

9.0 1.8 3.5 3.4 2.5

1.9 0.9

0.7 3.1

2.8 1.6 3.7 1.7 2.7 0.8 1.9 1.7

6.4

5.9

22.2 1.7

19.0 2.7

3.0 0.2

2.7 0.3

1.6 2.9

0.9 2.3 3.7 32.7 0.4 6.5 3.5 6.0 4.1 0.6 7.0 2.7

0.6 6.1 26.5 0.2 12.3 6.4 1.0 12.2 1.1 1.1 4.5 1.8

7.5

4.7 0.4 6.4

3.4 1.8 6.7

29.0 1.5

7.4 2.5

6.0 2.0

10. A. terrigena KCTC 12545T

6. A. locisalis KCTC 12310T

5. A. halophilus KCTC 12051T

4. A. chordae KMM 3957T

3. A. boritolerans T-22T 0.5 1.3 0.6

1.5 0.7 0.7 1.6 0.3 0.8 31.2 0.8 3.5 1.6

0.3

13. A. yeomjeoni KCTC 12309T

5.3 2.3 5.2 0.7 1.8

1.0

12. A. winogradskyi KMM 3956T

14.6 1.9 1.2 1.7 3.2

2.5 1.4

11. A. vanfongensis KMM 6241T

0.6 0.7 3.2 38.9 0.3 2.4 2.4

0.6 3.2 0.2 0.4 2.1 6.4 16.6 0.2 20.7 10.9 1.6 5.0

9. A. ratkowskyi ACAM 646T

1.6 1.7

8. A. ornithinivorans KCTC 12052T

1.6 0.4 0.9 2.6 0.3 3.4 32.4 1.4 3.2 3.8

7. A. mannitolivorans KCTC 12050T

C15:0 C16:0 C11:0 iso C11:0 anteiso C14:0 iso C15:0 anteiso C15:0 iso C15:1 iso G C16:0 iso C16:1 iso C17:1 anteiso w9c C17:1 iso w9c C15:1 w6c C16:1 w5c C17:1 w6c C15:0 iso 3-OH C16:0 2-OH C16:0 3-OH C16:0 iso 3-OH C17:0 2-OH C17:0 iso 3-OH C19:1 iso I Summed feature 3c Summed feature 4d

2. A. aquimarinus KMM 3958T

Fatty acid

1. A. alkaliphilus AC-74T

TABLE 104.  Fatty acid composition (%) of the Algoriphagus speciesa,b

0.7 0.3 2.2 2.6

1.2 1.0

0.6 0.8

2.1 1.4 3.6 30.5 1.1 3.4 1.7

1.1 1.6 1.0 2.4 35.3 1.0 1.0 2.5

1.5 2.1 5.8 0.5 2.9

8.5 1.0 2.8 0.7 3.2

4.2 0.5

1.9 2.8

0.6 0.6

0.6

0.8 1.9

2.3 0.8 3.3 1.0 3.0 1.3 1.1 3.3

9.2

6.9

6.4

7.8

22.3 0.9

24.5 2.5

10.7 1.3 32.6 4.9

24.6 1.6

33.7 1.3

1.6 21.5 6.8 0.3 0.6

0.7 5.1

1.2 0.3 1.6 36.6 2.9 3.9 2.1 4.0 1.3 3.6 0.8 2.0

1.6 1.2 28.6 4.8 1.9

Data are taken from Ahmed et al. (2007), Nedashkovskaya et al. (2004, 2007b), Schmidt et al. (2006), Tiago et al. (2006a), Yoon et al. (2005a, b, 2006).

a

Amount of the predominant fatty acids is shown in bold font. Values of less than 1% for all strains are not shown.

b

Summed feature 3 consisted of one or more of the following fatty acids which could not be separated by the Microbial Identification System: C15:0 iso 2-OH, C16:1 w7c, and C16:1 w7t.

c

Summed feature 4 consisted of one of the following fatty acids: C17:1 iso I and C17:1 anteiso B.

d

natural ­seawater and 70% (v/v) distilled water]: Bacto peptone (Difco), 5.0; sucrose, 5.0; glucose, 1.0; yeast extract (Difco), 2.5; KH2PO4, 0.1; MgSO4, 0.1 g; and Bacto agar (Difco), 15.0 (Nedashkovskaya et al., 2007a). Several members of the genus Algoriphagus have a terrestrial origin. Thus, strain Algoriphagus alkaliphilus AC-74T was recovered from sample of the alkaline artesian water and cultivated on a modified R2A medium without NaCl (Tiago et al., 2004, 2006a). Algoriphagus terrigena was isolated from soil sample using the dilution-plating on 10× diluted nutrient agar (Difco) with distilled water (Yoon et al., 2006). For isolation of a single strain of Algoriphagus boritolerans, soil samples (5 g) were incubated in 50 ml of phosphate-­buffered saline (PBS) solution at 30°C supplemented with 10 mM boron per day for several days. The supernatant was streaked on LuriaBertani (LB) agar plates containing different levels of boron up to 200 mM (Ahmed et al., 2007). Strain T-22T was isolated on LB agar medium containing high boron concentration and cultivated on modified R2A medium (designated R3A-V) (Tiago et al., 2004) or on marine agar 2216 at 30°C.

Maintenance procedures Almost all of the Algoriphagus strains remain viable on Marine agar (Difco) or other rich medium based on natural or artificial seawater for several weeks. They have survived storage at −80°C in Marine broth or artificial seawater supplemented with 20% glycerol (v/v) for at least 5 years. Strain Algoriphagus alkaliphilus AC-74T is cultivated on R3A-V medium at 30°C and maintained at −70°C in the same medium supplemented with 15% glycerol. Algoriphagus boritolerans is grown on R3A-V medium or on Marine agar 2216 at 30°C and maintained at −80°C in the same medium supplemented with 35% glycerol.

Differentiation of the genus Algoriphagus from other genera Bacteria belonging to the genus Algoriphagus have many similar phenotypic features with the representatives of their closest phylogenetic relatives, the genera Aquiflexum, Belliella, Cyclobacterium, Echinicola, and Rhodonellum (Table 105). All of them are

428

Family II. Cyclobacteriaceae

TABLE 105.  Phenotypic characteristics that differentiate the genus Algoriphagus from its close relatives in the family Cyclobacteriaceae a,b

Characteristics Cell morphology: Regular rods Ring-like/horseshoeshaped Cell size (mm) Gliding motility Oxidase activity Nitrate reduction Salinity range (%) Growth at: 25°C 40°C Hydrolysis of starch DNA G+C content (mol%)

Algoriphagus

Aquiflexum

Belliella

Cyclobacterium

Echinicola

Rhodonellum

+ −

+ −

+ −

− +

+ −

+ −

0.3–0.7 × 0.3–10.0 − + D 0–10

0.3–0.6 × 1.1–4.8 − + + 0–6

0.3–0.5 × 0.9–3.0 − + + 0–6

0.3–0.7 × 0.8–1.5 − + − 0–10

0.3–0.5 × 1.1–2.3 + + − 0–15

0.7–1.0 × 0.8–3.0 − − − 0–3

D D D 35–49

+ + + 38.4

+ − + 35.4

+ + − 41–45

+ + + 44–46

− − + 44.2

Symbols: +, >85% positive; −, 0–15% positive; D, different reactions occur in different taxa (species of a genus).

a

Data are taken from Ahmed et al. (2007): Bowman et al. (2003), Brettar et al. (2004a, b), Nedashkovskaya et al. (2004, 2005, 2006, 2007a, b), Schmidt et al. (2006), Tiago et al. (2006a), Van Trappen et al. (2004), Yi and Chun (2004), Ying et al. (2006), Yoon et al. (2004, 2005a, b, 2006). b

aerobic bacteria that form the pink-pigmented colonies on suitable solid nutrient media and grow without NaCl or seawater. However, Algoriphagus species may be easily differentiated from the genus Cyclobacterium by their inability to form ring-like or horseshoe-shaped cells on solid media (Raj and Maloy, 1990; Ying et al., 2006). Gliding motility and the absence of oxidase activity clearly distinguish representatives of the genus Algoriphagus from the genera Echinicola and Rhodonellum, respectively (Nedashkovskaya et al., 2006, 2007a; Schmidt et al., 2006). Notably, species of Algoriphagus are characterized by very diverse phenotypic features. These characteristics are suitable for the species differentiation (Table 104), but separation of members of the genus Algoriphagus from their close relatives, especially from members of the genera Aquiflexum and Belliella, is more difficult because their phenotypic properties are very variable. Therefore, to order discriminate Algoriphagus from its nearest neighbors, a polyphasic approach, including fatty acid methylester (FAME) (Table 106) and 16S rRNA-based phylogenetic analysis, should be used.

Taxonomic comments The genus Algoriphagus, consisting of a single species Algoriphagus ratkowskyi, was established for accommodation of marine, saccharolytic, and cold-adapted Cytophaga-like bacteria by Bowman and co-workers in 2003. Shortly thereafter, a new genus of marine bacteria, Hongiella, closely related to the genus Algoriphagus, was described by Yi and Chun (2004). One of them, Hongiella halophila, was moved to the genus Algoriphagus because of phylogenetic relatedness (96.8–97.5%) and phenotypic similarity with the Algoriphagus species (Nedashkovskaya et al., 2004), while Hongiella mannitolivorans and Hongiella ornithinivorans were more distantly related (93.7–94.0 and 94.3–94.6%, respectively). The descriptions of the genera Algoriphagus and Hongiella were also emended in that study (Nedashkovskaya et al., 2004). Later, the description of the genus Chimaereicella, comprising a single species Chimaereicella alkaliphila, isolated from alkaline artesian water, was reported by Tiago and colleagues (2006a). Despite a close phylogenetic relationship

between Chimaereicella alkaliphila and the Algoriphagus species (sequence similarity was 94.3–95.5%) and a resemblance in their fatty acid composition, the new bacterium was placed in a new genus based on a distinct isolation source. Later, another Chimaereicella species, Chimaereicella boritolerans, recovered from soil, was described (Ahmed et al., 2007). A level of 16S rRNA gene sequence similarity between Chimaereicella boritolerans and its closest relative, Chimaereicella alkaliphila, was 97.6%. The similarity values between Chimaereicella boritolerans and representative members of the genera Algoriphagus and Hongiella were 94.7–96.0 and 95.1–96.7%, respectively. DNA– DNA relatedness between a soil isolate and Chimaereicella alkaliphila AC-74T was 28.3%. This fact supported the affiliation of the new isolate with the genus Chimaereicella as a separate species, Chimaereicella boritolerans. In course of studying a novel marine bacterium, designated strain KMM 6241T, Nedashkovskaya et al. (2007b) carried out phylogenetic analysis based on sequencing of 16S rRNA gene. This analysis revealed an equidistant position of strains Chimaereicella alkaliphila AC-74T relative to members of the genera Algoriphagus and Hongiella, with sequence similarity of 94.7–97.0%. Similar phenotypic features, including the presence of oxidase activity and esculin hydrolysis and the absence of gliding motility and aerobic metabolism, may argue for moving the genus Chimaereicella to the genus Algoriphagus. In addition, the species of the genus Hongiella possess a close relatedness with validly published Algoriphagus species with sequence similarities ranging from 93.7 to 96.5%. These results taken together with similarity in fatty acid compositions and phenotypic properties could be considered strong confirmation of the proposal to transfer these species of the genus Hongiella to the genus Algoriphagus. Consequently, the phylogenetic evidence and the resemblance of phenotypic characteristics (Table 105) and fatty acid composition (Table 106) support the joining of the genera Algoriphagus, Chimaereicella, and Hongiella into the single genus Algoriphagus, thereby requiring an emended description of the genus Algoriphagus (Nedashkovskaya et al., 2007b). Phylogenetic analysis of the almost-complete 16S rRNA gene sequences reveal that species

429

Genus II. Algoriphagus TABLE 106.  Cellular fatty acid composition (%) of the genus Algoriphagus and related genera of the family Cyclobacteriaceae a

Fatty acid C11:0 iso C11:0 anteiso C13:1 AT C14:0 iso C15:1 iso G C15:0 iso C15:0 anteiso C15:0 C15:1 w6c C16:1 w5c C16:1 iso C16:0 iso 3-OH C16:0 iso C16:0 C15:0 iso 3-OH C15:0 3-OH C17:0 iso C17:0 cyclo C17:1 iso w9c C17:1 anteiso w9c C17:1 w8c C17:1 w6c C16:0 iso 3-OH C16:0 3-OH C17:0 iso 3-OH C17:0 2-OH C18:1 w7c C18:1 w5c C18:1 H C18:0 C19:1 iso Summed feature 3c Summed feature 4d Summed feature 5e

Algoriphagus

Aquiflexum

Echinicola

Belliella

Cyclobacterium

Rhodonellum

0–2.2 0–2.6 − 0–2.3 0–6.8 16.6–38.9 0.8–6.4 0.3–3.0 0–4.1 0–5.8 0.6–10.9 − 0.3–20.7 0–3.2 1.6–5.1 − 0–0.8 0–2.4 1.5–14.6 0–1.6 0.4–0.9 0.5–4.5 0–4.7 0–1.9 4.9–10.7 0–2.2 − 0.5–0.7 0–1.6 − 0–1.3 6.0–33.7 0–4.9 −

− 4.8b − − 9.4 22.6 18.3 − − 2.0 9.4 2.0 4.2 − 1.4 − − − 5.2 1.1 − 3.0 − − 1.4 − − − − − 1.5 6.1 2.5 −

− − 0–0.2 0.1–0.2 0–0.6 17.3–20.0 1.4–2.8 0.8–1.5 1.1–1.2 4.9–7.8 0.3–1.0 − 0.9–1.2 0.6–0.9 3.4–5.0 2.5–2.6 0.7–1.0 − 4.4–6.9 − 0.4 4.3–4.8 0.4–0.7 0.9–2.3 9.4–10.0 0.4 0.7–0.8 0.2 − − 0–0.7 30.7–34.5 0–5.0 −

− − 0–1.4 1.8–2.2 10.0–10.3 18.9–20.4 4.2–4.8 2.0–3.9 1.6–2.5 2.0–4.6 3.2–3.8 − 2.5–2.8 − 2.1–2.3 − 0–0.5 − 6.6–10.2 − 0.9–1.5 4.8–9.8 1.8–2.1 0–1.1 3.0–3.3 − − − − − 0.8–1.6 7.1–11.5 3.4–4.0 −

− − − 0–2.1 3.2–8.5 22.2–28.3 6.3–9.2 0–0.8 0.5–1.3 0–3.6 − − − 0.5–4.9 1.1–3.7 − − − 4.3–10.3 − − 1.3–1.4 0–1.0 1.3–1.7 8.0–10.7 1.5–2.9 0–3.0 1.2–1.4 − 0–1.3 − 16.2–25.1 2.5–4.4 −

− − − − 6.3 7.6 1.4 0.4 2.7 1.8 7.3 − 2.2 0.6 3.7 − 0.2 − 17.5 − 0.2 6.8 2.8 0.3 17.5 1.1 − − − − − 12.6 − 6.5

a Data are taken from Ahmed et al. (2007), Brettar et al. (2004a, b), Nedashkovskaya et al. (2004, 2005, 2006, 2007a, b), Schmidt et al. (2006), Ying et al. (2006), Yoon et al. (2005a, b, 2006).

Predominant fatty acids are shown in bold. Values of less than 1% for all strains are not shown.

b

Summed features consist of one or more fatty acids that could not be separated by the Microbial Identification System.

c–e

Summed feature 3 is C15:0 iso 2-OH and/or C16:1 w7c.

c

Summed feature 4 is C17:1 iso I and C17:1 anteiso B.

d

Summed feature 5 is C14:0 2-OH and/or C15:0 iso 2-OH.

e

of the genus Algoriphagus have a 16S rRNA gene sequence similarity of 93.5–99.6% and that the genus Algoriphagus forms a cluster with the genera Belliella, Rhodonellum, Aquiflexum, Echinicola, and Cyclobacterium with sequence similarities of 91.5–93.4, 91.1–93.0, 90.8–93.2, 88.8–92.4, and 88.7–91.7%, respectively.

Differentiation of species of the genus Algoriphagus The species of the genus Algoriphagus have many common phenotypic features. However, they can be differentiated from each other by several phenotypic traits as shown in Table 107.

List of species of the genus Algoriphagus 1. Algoriphagus alkaliphilus (Tiago, Mendes, Pires, Morais and Veríssimo 2006a) Nedashkovskaya, Kim, Kwon, Shin, Luo, Kim and Mikhailov 2007b, 1993VP (Chimaereicella alkaliphila Tiago, Mendes, Pires, Morais and Veríssimo 2006b, 925VL; effective publication: Tiago, Mendes, Pires, Morais and Veríssimo 2006a, 107.) al.ka.li.phi¢la. N.L. n. alkali (from Arabic article al the; Arabic n. qaliy ashes of saltwort, soda), alkali; Gr. adj. philos loving; N.L. masc. adj. alkaliphilus loving alkaline environments.

Cells are 0.5 × 2.1–3.3 mm. Colonies are small and red. Optimal growth occurs at about 30°C, at pH 8.0 and without NaCl. Hippurate is hydrolyzed, but elastin is not. Xylanase is not produced. d-Arabitol, l-arabitol, ribitol, a-methylmannoside, and 2-ketogluconate are utilized but erythritol, galactitol, b-methyl-xyloside, amygdalin, ribose, l-sorbose, glycogen, and inulin are not. Acid is formed from a-methylglucoside, arbutin, salicin, b-gentiobiose, d-turanose, and 5-ketogluconate. Susceptible to ceftazidin and cephalothin.

a

3. A. aquimarinus

4. A. boritolerans

5. A. chordae

6. A. halophilus

7. A. locisalis

8. A. mannitolivorans

9. A. marincola

10. A. ornithinivorans

11. A. ratkowskyi

12. A. terrigena

13. A. vanfongiensis

14. A. winogradskyi

15. A. yeomjeoni

Nitrate reduction NaCl requirement for growth Salinity range for growth (%) Temperature range (°C) Hydrolysis of: Agar Casein Gelatin Starch DNA Tween 20 Tween 40 Tween 80 Acid production from: N-Acetylglucosamine l-Arabinose d-Cellobiose d-Fructose d-Galactose d-Glucose d-Lactose d-Maltose d-Mannose d-Melibiose d-Raffinose l-Rhamnose d-Sucrose d-Trehalose dl-Xylose Utilization of: l-Arabinose d-Galactose d-Glucose d-Lactose d-Maltose d-Mannose d-Xylose d-Mannitol Sorbitol Inositol Glycerol N-Acetylglucosamine Susceptibility to: Ampicillin Benzylpenicillin Carbenicillin Gentamicin Kanamycin Lincomycin Oleandomycin Neomycin Polymyxin B Streptomycin Tetracycline DNA G+C content (mol%)

2. A. antarcticus

Characteristic

1. A. alkaliphilus

TABLE 107.  Phenotypic characteristics of the Algoriphagus speciesa,b

+ −

− −

− −

− −

− +

− −

− +

+ −

d +

− −

d d

+ +

− −

+ −

− +

0–3

0–5

0–10

0–3

1–10

0–8

1–9

0–7

1–9

0–10

0–6

1–7

0–8

0–6

1–9

11–39

5–25

4–34

17–37

4–32

10–41

4–35

10–42

10–45

10–40

−2–25

10–36

12–35

4–39

4–35

− − + + + nd nd nd

− − − − − nd nd nd

+ + + − + + + +

− nd − + nd nd nd nd

+ − − − − + + −

− − + + − + + +

− − − − nd + + +

− − + + + − − −

− + − + + + + +

− − + + + − + +

d + d d d + + −

− + − − nd + + +

− − + − − − − −

+ − + + − + + −

− + d − − + + +

− + + + + + + + + + − − + + −

− − − − − − − − − − − − − − −

+ − + nd − + + − + + − + + nd +

+ − + − + + + + + − + + − − −

− − + nd + + + + + + + + + nd +

+ − − nd − + − + nd − − − + nd +

nd + + + + + + + + + + + + d +

− − + − − − − − − − − − + nd −

− + + d + + + + + + + − + + +

− − − − − + − − − − − − − − −

+ + + + + + − + + + + + − + +

nd + + + + + + + + + + − + + +

+ − + − − + − + + − − + − − +

+ − + nd + + + + + + + + + nd +

− − + + + + + + d (−) + d (+) + + + +

+ + + + + + − − + − − −

− nd − nd − − nd − nd nd nd −

+ nd + + + + + + − − − +

− + + + + + − + − − nd −

+ + + + + + + − − − − +

+ + + + + + + − − − − +

+ + + + + + + − nd − − nd

+ − + + + + + + − − − +

+ + + + + + + − − − − −

− − + + + + + − − − − +

+ + + + + + + + + + − +

+ + + + + + + − nd − nd nd

+ nd + + + + + − − − − +

+ + + + + + + − − − − +

− + + + + + + − nd − − −

nd nd nd nd + nd nd nd nd nd nd 43.5

nd nd nd nd nd nd nd nd nd nd nd 39–41

− − + − − + + − − − + 41

nd + nd + − nd nd nd nd + + 42.5

− − − − − + − − − − − 37–40

− − + − − + + − − − + 37

− − − − − + + − − − − 42

+ + + − − + + − − + + 42

+ + − − − − + − + + + 43

+ + + − + + + − + − + 38

− − + − − + + − − − + 35

− − − − − − − − − − − 49

− − + + − + + + − + + 43.8

− − v − − v + − − − d 39–42

− − + − − + + − − − − 41

Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive; v, variable reaction; nd, not determined.

Data from Ahmed et al. (2007), Bowman et al. (2003), Nedashkovskaya et al. (2004, 2007b), Tiago et al. (2006a), Van Trappen et al. (2004), Yi and Chun (2004), Yoon et al. (2004, 2005a, b, 2006). b

Genus II. Algoriphagus

Source: artesian water collected at Cabeço de Vide, ­Southern Portugal. DNA G+C content (mol%): 43.5 (HPLC). Type strain: AC-74, CIP 108470, LMG 22694. Sequence accession no. (16S rRNA gene): AJ717393. 2. Algoriphagus antarcticus Van Trappen, Vandecandelaere, Mergaert and Swings 2004, 1972VP ant.arc¢ti.cus. L. masc. adj. antarcticus southern, of the Antarctic, the environment from where the strains were isolated. Cells are up to 0.5 mm in width and 2–3 mm in length. On Marine agar, colonies are 0.5–3 mm in diameter, opaque, and orange-red after 6 d incubation. Optimal growth occurs at 20°C and with 2% NaCl. Pectin and tyrosine are not hydrolyzed. No acid is produced from carbohydrates. Gluconate, caprate, adipate, and phenylacetate are not utilized. Source: microbial mats from lakes Reid, Fryxell, and Ace, Antarctica. DNA G+C content (mol%): 39.9–41.0 (HPLC). Type strain: R-10710, LMG 21980, DSM 15986. Sequence accession no. (16S rRNA gene): AJ577141. 3. Algoriphagus aquimarinus Nedashkovskaya, Vancanneyt, Van Trappen, Vandemeulebroecke, Lysenko, Rohde, ­Falsen, Frolova, Mikhailov and Swings 2004, 1762VP a.qui.ma.ri¢nus. L. fem. n. aqua water, L. masc. adj. marinus marine, of the sea; N.L. masc. adj. aquimarinus, of seawater. Cells range from 0.5–0.7 mm in width and 1–10 mm in length. On Marine agar, colonies are 2–3 mm in diameter and pale-pink. Optimal temperature for growth is 23–25°C. No acid is produced from l-sorbose, adonitol, or glycerol. Source: seawater from Amursky Bay, Gulf of Peter the Great, Sea of Japan. DNA G+C content (mol%): 41.0 (Tm). Type strain: KMM 3958, LMG 21971, CCUG 47101. Sequence accession no. (16S rRNA gene): AJ575264. 4. Algoriphagus boritolerans (Ahmed, Yokota and Fujiwara 2007) Nedashkovskaya, Kim, Kwon, Shin, Luo, Kim and Mikhailov 2007b, 1993VP (Chimaereicella boritolerans Ahmed, Yokota and Fujiwara 2007, 991VP) bo¢ri.to.le.rans. N.L. n. borum boron; L. part. adj. tolerans tolerating; N.L. part. adj. boritolerans boron-tolerating). Cells are 0.3–0.4 × 1.2–3.4 mm, occurring singly and occasionally in pairs. Colonies are red and small in diameter after several days of incubation at 30°C. Growth is observed at pH 6.5–10.0. Optimal growth occurs at 28–30°C and pH 8.0–9.0. Tolerant up to 300 mM boron but grows optimally without boron supply. Acid is produced from d-turanose, gentiobiose, inulin, potassium 5-ketogluconate, methyl a-dmannopyranoside, glycogen, and d-lyxose but not from salicin, glycerol, erythritol, d-ribose, l-sorbose, d-tagatose, d- and l-fucose, d- and l-arabitol, potassium gluconate, potassium 2-ketogluconate, methyl b-d-xylopyranoside, d-adonitol, dulcitol, d-sorbitol, amygdalyn, d-melezitose, or xylitol. Amygdalin, arbutin, and melezitose are utilized, but erythritol, d-ribose, l-xylose, methyl b-d-xylopyranoside, l-sorbose, d-melibiose, d-trehalose, d-raffinose, potassium

431

gluconate, l-fucose or d-tagatose are not. a-Chymotrypsin, leucine arylamidase, valine arylamidase, naphthol-AS-BIphosphohydrolase, esterase (C4), esterase lipase (C8), trypsin, acid phosphatase, and a-glucosidase are produced. Weakly susceptible to rifampin and cefoperazon but resistant to oxacillin, sulfamethizol, and metronidazole. Susceptible to amoxycycline and ofloxacin; resistant to cephalothin and chloramphenicol. Source: naturally boron-contaminated soil of the Hisarcik area in the Kutahya province of Turkey. DNA G+C content (mol%): 42.5 (HPLC). Type strain: T-22, ATCC BAA-1189, DSM 17298, NBRC 101277. Sequence accession no. (16S rRNA gene): AB197852. 5. Algoriphagus chordae Nedashkovskaya, Vancanneyt, Van Trappen, Vandemeulebroecke, Lysenko, Rohde, Falsen, Frolova, Mikhailov and Swings 2004, 1763VP chor¢dae. N.L. gen. n. chordae of Chorda, the generic name of the brown alga Chorda filum, from which the type strain was isolated. Cells are 0.5–0.7 mm in width and 1–10 mm in length. On Marine agar, colonies are 2–3 mm in diameter, bright pink, and sunken into the agar. Optimal temperature for growth is 23–25°C. No acid is produced from l-sorbose, adonitol, or glycerol. Source: brown alga Chorda filum, Troitsa Bay, Gulf of Peter the Great, Sea of Japan. DNA G+C content (mol%): 37–40 (Tm). Type strain: KMM 3957, LMG 21970, CCUG 47095. Sequence accession no. (16S rRNA gene): AJ575265. 6. Algoriphagus halophilus (Yi and Chun 2004) Nedashkovskaya, Vancanneyt, Van Trappen, Vandemeulebroecke, ­Lysenko, Rohde, Falsen, Frolova, Mikhailov and Swings 2004, 1763VP (Hongiella halophila Yi and Chun 2004, 160VP) ha.lo.phi¢lus. Gr. n. hals halos salt; Gr. adj. philos loving; N.L. masc.adj. halophilus, salt-loving. Cells are 0.3–0.5 mm in width and 1.0–1.8 mm in length. On Marine agar, colonies are flat, translucent, and pink-orange. Optimal growth occurs at 35°C, pH 7.0, and with 1–2% NaCl or 1–2% artificial sea salts. Alginic acids and egg yolk are not decomposed. No acid is produced from l-sorbose, adonitol or glycerol. d-Cellobiose, d-fructose, and d-salicin are utilized but acetamide, acetate, benzoate, citrate, d-ribose, ethanol, glycine, inulin, 2-propanol, l-arginine, l-ascorbate, l-asparagine, l-lysine, l-ornithine, polyethylene glycol, salicylate, succinate, tartrate, or thiamin is not. Leucine arylamidase, acid phosphatase, a-chymotrypsin, naphthol-AS-BI-phosphohydrolase, b-glucuronidase and a- and b-glucosidases activities are present, but esterase (C4), esterase lipase (C8), lipase (C14), cystine arylamidase, valine arylamidase, trypsin, a-mannosidase, and a-fucosidase activities are absent. Maximum absorption of pigment occurs at 475 nm. Source: sediment sample of getbol, the Korean tidal flat, Sea of Japan. DNA G+C content (mol%): 37 (HPLC). Type strain: JC 2051, KCTC 12051, DSM 15292, IMSNU 14013. Sequence accession no. (16S rRNA gene): AY264839.

432

Family II. Cyclobacteriaceae

7. Algoriphagus locisalis Yoon, Kang and Oh 2005b, 1638VP lo.ci.sa¢lis. L. n. locus place, locality; L. gen. n. salis, of salt; N.L. gen. n. locisalis, of a place of salt. Cells are rods 0.4–0.7 mm in width and 1.5–3.0 mm in length. On Marine agar, colonies are 1–2 mm in diameter and orange after 3 d incubation at 30°C. Optimal growth occurs at 30°C, pH 7.0–8.0, and with 2% (w/v) NaCl. Hypoxanthine, xanthine, and tyrosine are not decomposed. No acid is produced from d-melezitose or d-sorbitol. Salicin is utilized but succinate, formate, and l-glutamate are not. Utilization of d-trehalose is strain-dependent (positive for the type strain). Susceptible to chloramphenicol. Source: seawater from a marine solar saltern of the Yellow Sea in Korea. DNA G+C content (mol%): 42 (HPLC). Type strain: MSS-170, KCTC 12310, JCM 12597. Sequence accession no. (16S rRNA gene): AY835922. 8. Algoriphagus mannitolivorans (Yi and Chun 2004) Nedashkovskaya, Kim, Kwon, Shin, Luo, Kim and Mikhailov 2007b, 1993VP (Hongiella mannitolivorans Yi and Chun 2004, 160VP) man.ni.to.li.vo¢rans. N.L. n. mannitolum mannitol; L. v. vorare to devour; N.L. part. adj. mannitolivorans mannitoldevouring, utilizing mannitol.

­succinate are not. Leucine arylamidase, valine ­arylamidase, cystine arylamidase, esterase (C4), esterase lipase (C8), acid phosphatase, trypsin, a-chymotrypsin, naphthol-ASBI-phosphohydrolase, a-glucosidase, and b-glucosidase are produced, but lipase (C14), a-galactosidase, a-mannosidase, a-fucosidase, or b-glucuronidase is not. Susceptible to chloramphenicol, doxycycline, and erythromycin. Source: seawater from the East Sea of Korea. DNA G+C content (mol%): 43 (HPLC). Type strain: SW-2, DSM 16067, JCM 12319, KCTC 12180. Sequence accession no. (16S rRNA gene): AY533663. Reference strain: SW-26. Sequence accession no. (16S rRNA gene): AY533664. 10. Algoriphagus ornithinivorans (Yi and Chun 2004) Nedashkovskaya, Kim, Kwon, Shin, Luo, Kim and Mikhailov 2007b, 1993VP (Hongiella ornithinivorans Yi and Chun 2004, 160VP) or¢ni.thi.ni.vo¢rans. N.L. n. ornithinum ornithine; L. v. vorare to devour; N.L. part. adj. ornithinivorans ornithine-devouring, utilizing ornithine. Cells are 0.3–0.4 × 0.8–2.6 mm. Colonies are pink-orange on Marine agar. Optimal growth is observed at 35–40°C, pH 7.0, and with 1% NaCl or 1.0–2.5% artificial sea salts. Maximum absorption of pigment occurs at 480 nm. Hydrolysis of alginic acids and egg yolk is not detected. Leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BY-phosphohydrolase, trypsin, a-chymotrypsin, a-galactosidase, a-glucosidase, and b-glucosidase are produced, but esterase (C4), esterase lipase (C8), lipase (C14), cystine arylamidase, b-glucuronidase, a-mannosidase, or a-fucosidase are not. Acid is not produced from d-adonitol, glycerol or d-sorbitol. d-Fructose, d-trehalose, d-salicin, and l-ornithine are utilized, but acetamide, acetate, benzoate, citrate, d-ribose, d-sorbitol, ethanol, glycine, inulin, 2-propanol, l-arginine, l-ascorbate, l-asparagine, l-lysine, polyethylene glycol, salicylate, succinate, tartrate, and thiamine are not. Susceptible to chloramphenicol, doxycycline, and erythromycin. Source: sediment of getbol, of the Korean tidal flat. DNA G+C content (mol%): 38 (HPLC). Type strain: JC 2052, DSM 15282, IMSNU 14014, KCTC 12052. Sequence accession no. (16S rRNA gene): AY264840.

Cells are 0.4–0.5 × 1.1–1.7 mm. Colonies are pink-orange on Marine agar. Optimal growth is observed at 35–40°C, pH 7.0 and with 1% NaCl or 0.5–1.5% artificial sea salts. Alginic acids and egg yolk are not decomposed. Acid is not formed from d-adonitol, glycerol or d-sorbitol. d-Fructose, acetamide, acetate, benzoate, citrate, d-ribose, d-sorbitol, ethanol, glycine, inulin, 2-propanol, l-arginine, l-ascorbate, l-asparagine, l-lysine, l-ornithine, polyethylene glycol, salicylate, tartrate, and thiamine are not utilized. Leucine arylamidase, valine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and a-galactosidase are produced, but esterase lipase (C8), lipase (C14), cystine arylamidase, b-glucuronidase, a- and b-glucosidases, a-mannosidase, and a-fucosidase are not. Susceptible to chloramphenicol, doxycycline, and erythromycin. Maximum absorption of pigment occurs at 480 nm. Source: sediment of getbol, of the Korean tidal flat. DNA G+C content (mol%): 42 (HPLC). Type strain: JC 2050, DSM 15301, IMNSNU 14012, KCTC 12050. Sequence accession no. (16S rRNA gene): AY264838.

11. Algoriphagus ratkowskyi Bowman, Mancuso, Nichols and Gibson 2003, 1352VP

9. Algoriphagus marincola (Yoon, Yeo and Oh 2004) ­Nedashkovskaya, Kim, Kwon, Shin, Luo, Kim and Mikhailov 2007b, 1993VP (Hongiella marincola Yoon, Yeo and Oh 2004, 1848VP)

rat.kow¢sky.i. N.L. gen. masc. n. ratkowskyi of Ratkowsky, named in honor of David A. Ratkowsky, who made significant contributions to growth modelling of bacteria, including psychrophilic bacteria.

ma.rin¢co.la. L. n. mare -is the sea; L. n. incola inhabitant; N.L. n. marincola inhabitant of the sea.

Cells are 0.3–0.4 × 0.3–0.9 mm. On Marine agar, colonies are 1–3 mm in diameter and salmon-pink. Optimal growth occurs at 16–19°C and with 0–6% NaCl. Tributyrin is not hydrolyzed. a-Fucosidase and glutamyl glycine arylamidase activities are present; some strains produce b-glucuronidase. Acid is not formed from sugar alcohols. Salicin, b-glycerol phosphate, d-gluconate, propionate, isobutyrate, succinate, pimelate, azelate, l-proline, 2-aminobutyrate, and l-serine are utilized. Utilization of l-ornithine, glycogen, n-butyrate, glutarate, aconitate, and hydroxyl-l-proline

Cells are 0.4–0.6 × 2.0–3.0 mm. Colonies are low convex, reddish-orange, and 1–2 mm in diameter after 72 h incubation at 37°C on Marine agar. Optimal growth occurs at 37°C, pH 6.5–7.5, and with 2–3% NaCl. Tyrosine is hydrolyzed weakly. Hypoxanthine, xanthine and birchwood xylan are not degraded. Acid is produced from d-melezitose but not from d-ribose, glycerol or adonitol. d-Trehalose is ­utilized but d-fructose, acetate, benzoate, citrate, formate, and

Genus III. Aquiflexum

is strain-­dependent. The following substrates are not utilized as sole carbon sources: l-fucose, 2-ketogluconate, adonitol, d-arabitol, dulcitol, iso-erythritol, methanol, itaconate, n-­valerate, suberate, 3-dl-hydroxybutyrate, oxaloacetate, dl-lactate, dl-tartrate, methylamine, isovalerate, heptanoate, caproate, nonanoate, adipate, 2-oxoglutarate, l-alanine, l-aspartate, l-asparagine, l-phenylalanine, l-glutamate, l-histidine, l-threonine, l-tyrosine, l-leucine, putrescine, and urate. Source: cold marine and marine-derived habitats, including sea ice and algal mats of saline lakes. DNA G+C content (mol%): 35–36 (Tm). Type strain: IC025, ACAM 646, LMG 21435, CIP 107452. Sequence accession no. (16S rRNA gene): U85891. 12. Algoriphagus terrigena Yoon, Lee, Kang and Oh 2006, 779VP ter.ri.ge¢na. L. masc. or fem. n. terrigena child of the earth, referring to the isolation of the type strain from soil. Cells are 0.4–0.6 × 0.8–2.5 mm. On Marine agar, colonies are 1–2 mm in diameter and light orange after incubation for 7 d at 25°C. Optimal growth occurs at 25°C, pH 6.5–7.5, and with 2% (w/v) NaCl. Hypoxanthine, xanthine, and tyrosine are not decomposed. a-Mannosidase is present but b-glucuronidase and a-fucosidase are absent. Acid is formed from d-melezitose and d-ribose but not from d-sorbitol. Salicin is utilized as a sole carbon and energy source but not succinate or l-glutamate. Susceptible to chloramphenicol and novobiocin but resistant to cephalothin. Source: soil of island Dokdo, Korea. DNA G+C content (mol%): 49.0 (HPLC). Type strain: DS-44, KCTC 12545, CIP 108837. Sequence accession no. (16S rRNA gene): DQ178979. 13. Algoriphagus vanfongensis Nedashkovskaya, Kim, Kwon, Shin, Luo, Kim and Mikhailov 2007b, 1990VP van.fong.en¢sis. N.L. masc. adj. vanfongensis pertaining to the Vanfong Bay, from which the type strain was isolated. Cells are 0.4–0.5 × 1.0–2.5 mm. Colonies are light-pink on Marine agar. Optimal growth is observed with 1–4% NaCl. Acid is not produced from l-sorbose, glycerol, adonitol or dulcitol. Gluconate, caprate, adipate, malate,

433

citrate, or ­phenylacetate is not utilized. Susceptible to ­chloramphenicol, doxycycline, and erythromycin. Source: coral Palithoa sp. collected in Vanfong Bay, South China Sea, Vietnam. DNA G+C content (mol%): 43.8 (Tm). Type strain: KMM 6241, DSM 17529, KCTC 12716. Sequence accession no. (16S rRNA gene): EF392675. 14. Algoriphagus winogradskyi Nedashkovskaya, Vancanneyt, Van Trappen, Vandemeulebroecke, Lysenko, Rohde, Falsen, Frolova, Mikhailov and Swings 2004, 1763VP wi.no.grad¢sky.i. N.L. gen. masc. n. winogradskyi, of Winogradsky, named to honor Sergey N. Winogradsky, for his contributions to the study of Cytophaga-like bacteria. Cells are 0.5–0.7 × 1–10 mm. On Marine agar, colonies are 2–4 mm in diameter, bright pink, and sunken into the agar. Optimal temperature for growth is 25–28°C. No acid is produced from l-sorbose, adonitol, or glycerol. Source: green alga Acrosiphonia sonderi, Troitsa Bay, Gulf of Peter The Great, Sea of Japan. DNA G+C content (mol%): 39–42 (Tm). Type strain: KMM 3956, LMG 21969, JCM 13505, CCUG 47094. Sequence accession no. (16S rRNA gene): AJ575263. 15. Algoriphagus yeomjeoni Yoon, Kang, Jung, Lee and Oh 2005a, 869VP yeom.jeo¢ni. N.L. gen. n. yeomjeoni, of a yeomjeon, the Korean name for a marine solar saltern. Cells are 0.4–0.7 × 1.5–2.5 mm. On Marine agar, colonies are 0.8–1.0 mm in diameter and vivid orange after 3 d incubation at 30°C. Optimal growth occurs at 25–30°C, pH 7.0–8.0, and with 2% (w/v) NaCl. Hypoxanthine, xanthine, and tyrosine are not decomposed. No acid is produced from d-melezitose, d-ribose, or d-sorbitol. Salicin is utilized but succinate and l-glutamate are not. Susceptible to c­ hloramphenicol. Source: seawater in a marine solar saltern of the Yellow Sea in Korea. DNA G+C content (mol%): 41 (HPLC). Type strain: strain MSS-160, KCTC 12309, JCM 12598. Sequence accession no. (16S rRNA gene): AY699794. Reference strain: MSS-161.

Genus III. Aquiflexum Brettar, Christen and Höfle 2004b, 2339VP Ingrid Brettar, Richard Christen and Manfred G. Höfle A.qui.fle¢xum. L. fem. n. aqua water; L. part. adj. flexus -a -um bent, winding; N.L. neut. n. Aquiflexum to indicate the bacterium’s aquatic origin and its long flexible rods.

Rods, occurring singly or in chains of up to five cells. Nonmotile (no flagella, no gliding activity), Gram-stain-negative. Aerobic. Heterotrophic. Oxidase- and catalase-positive. Cells contain a high percentage of branched-chain fatty acids (>80%), and of C15:0 anteiso. Cells contain carotenoids but no flexirubin. NaCl is not needed for growth, but growth is improved by its presence. Nitrate is reduced to nitrite. Gelatin is hydrolyzed. DNA G+C content (mol%): 38.4 (HPLC). Type species: Aquiflexum balticum Brettar, Christen and Höfle 2004b, 2339VP.

Further descriptive information The genus Aquiflexum is so far represented by a single strain and a single species, Aquiflexum balticum BA160T.

Enrichment and isolation procedures Strain BA160T was isolated during a cruise onboard the research vessel (RV) Aranda in September 1998 from surface water (5 m, 15°C, 6% salinity, pH 8.2) from a site in the Central Baltic Sea at the entrance of the Gulf of Finland (station LL12, 59.2900° N, 22.5398° E). The strain was isolated by spreading 0.1 ml

434

Family II. Cyclobacteriaceae

of ­seawater on 1/5 diluted Marine agar (Difco 2216, Marine broth or agar diluted by a factor of 5; final concentration of agar, 1.8%) and subsequently purified and cultured on this medium.

Maintenance procedures The strain can be kept alive for months at 4°C on agar plates (dilute or regular Marine agar), mixed with glycerol and stored at −70°C, or freeze-dried. For growing the strain from old or preserved biomass, incubation at 30–35°C is recommended. At room temperature, cultivation is often not successful or shows long lag-periods.

Differentiation of the genus Aquiflexum from other genera According to 16S rRNA gene sequence analysis, Aquiflexum is most closely related to the genera Belliella, Algoriphagus, Hongiella, and Cyclobacterium (see phylogenetic tree in the genus description of Belliella). The nearest relative is Belliella baltica (Brettar et al., 2004a), having a 16S rRNA gene sequence similarity of 92.4%. By phenotypic traits, Aquiflexum can be distinguished from Belliella by its temperature range and optimum, hydrolysis of gelatin, acid production from 13 substrates, assimilation of three substrates, and utilization of eight substrates. Compared to Belliella baltica, BA160T showed a higher versatility in using organic substrates,

except for amino acids. Compared to Belliella baltica, Aquiflexum has a higher fraction of branched-chain fatty acids (BA160T: 87%, Belliella baltica: 70%), lower number of detectable fatty acid compounds, and considerably different composition. The major difference is the higher abundance of anteiso branched fatty acids (Aquiflexum balticum, 22%; Belliella baltica, 8%). The most abundant was C15:0 anteiso (Aquiflexum balticum, 19%; Belliella baltica, 4.5%). Additionally, C17:1 anteiso w9c was detectable for Aquiflexum balticum, but not for Belliella baltica. A table of comparison is given in the chapter on Belliella.

Taxonomic comments The genus Aquiflexum belongs to the family Cyclobacteriaceae of the class Cytophagia. The phylogenetic tree in the chapter on Belliella reflects the phylogenetic relationships within the family Cyclobacteriaceae.

Acknowledgements We are grateful to H. Kuosa and the crew of the Finnish RV Aranda for their assistance with sampling and seawater analysis of the Baltic Sea, to J. Bötel for excellent technical assistance, and to the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) staff for analysis of fatty acids and ­physiological tests.

List of species of the genus Aquiflexum 1. Aquiflexum balticum Brettar, Christen and Höfle 2004b, 2339VP bal¢ti.cum. N. L. neut. adj. balticum of or belonging to the Baltic sea, referring to the source of the type strain. The characteristics are as given for the genus with the following additional characteristics. Cells are 0.3–0.6 mm × 1.1–4.8 mm. The dominant fatty acids are of C15:0 iso, C15:0 anteiso, C15:1 iso G, and C16:1 iso H. Colonies are circular, smooth, convex, and entire; they are red and transparent when young but become opaque with ongoing incubation (>1 week, 30°C, on ½× Marine agar). The temperature range is 4–40°C; optimum, around 30°C. Growth occurs in 0–6% NaCl; optimum, around 1.5%. The pH range is 7–9; optimum, at neutral pH. Esculin, starch, and gelatin are hydrolyzed. No degradation of cellulose or tyrosine occurs. Indole is not produced. Growth does not occur on media with 0.5% yeast, casein, DNA, chitin, or pectin. Acid is produced (using the API 50CHE system) from galactose, d-glucose, d-fructose, d-mannose, rhamnose, a-methyl-d-mannoside, a-methyl-dglucoside, N-acetylglucosamine, amygdalin, arbutin, esculin salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, d-raffinose, starch, glycogen, ­xylitol,

b-gentiobiose, d-turanose, l-fucose, and 5-ketogluconate. Enzymic activities (i.e., positive results using the API 20NE and API ZYM systems) include a- and b-glucosidase; b-galactosidase; acid and alkaline phosphatase; leucine-, valine-, and cystine-arylamidase; trypsin; chymotrypsin; naphthol-phosphohydrolase; and N-acetyl-b-glucosaminase. The following substrates are assimilated (using the API 20NE system): glucose, arabinose, mannose, N-acetylglucosamine, maltose, and gluconate. The following substrates are utilized (using the Biolog GN2 system): l-arabinose, cellobiose, l-fructose, d-galactose, gentiobiose, a-d-glucose, a-d-lactose, lactulose, maltose, d-mannose, b-methyl-d-glucoside, d-psicose, d-sorbitol, sucrose, d-trehalose, turanose, mono-methyl-succinate, acetic acid, a-ketoglutaric acid, lactic acid, and propionic acid. All unmentioned tests using the API 20CHE, API ZYM, API 20NE, and Biolog GN2 test systems were negative. No utilization of amino acids was detected, but aminopeptidase was produced. Source: marine or estuarine. DNA G+C content (mol%): 38.4 (HPLC). Type strain: BA160, CIP 108445, DSM 16537, LMG 22565. Sequence accession no. (16S rRNA gene): AJ744861.

Genus IV. Belliella Brettar, Christen and Höfle 2004a, 69VP Ingrid Brettar, Richard Christen and Manfred G. Höfle Bel.li.el¢la. N.L. fem. dim. n. Belliella named in honor the aquatic microbiologist Russell Bell of the University of Uppsala.

Rods occurring as single cells or chains of up to five cells. Nonmotile (no flagella, no gliding activity). Gram-stain-negative. Aerobic. Oxidase- and catalase-positive. Chemoheterotrophic. The dominant fatty acids are C15:0 iso, C15:1 iso G, C17:1 iso w9c,

and C17:1 w6c. Cells contain carotenoids but no flexirubin. NaCl is not required for growth and does not influence growth up to 3%. Nitrate is reduced to nitrite. Gelatin is not hydrolyzed. DNA G+C content (mol%): 35.3–35.5.

435

Genus IV. Belliella

Type species: Belliella baltica Brettar, Christen and Höfle 2004a, 69VP.

Maintenance procedures The strain can be kept alive for months at 4°C on agar plates (dilute or regular Marine agar), mixed with glycerol and stored at −70°C, or freeze-dried. Recovery from old biomass or from preserved biomass is recommended on Marine agar (halfstrength) at 20–25°C.

Further descriptive information The genus Belliella is so far represented by a single species comprising two strains, Belliella baltica BA1 and BA134T, both isolated from the central Baltic Sea.

Differentiation of the genus Belliella from other genera According to 16S rRNA gene sequence analysis, Belliella is most closely related to the genera Aquiflexum, Algoriphagus, Hongiella, and Cyclobacterium (see Figure 78). The nearest relative is Aquiflexum balticum (Brettar et al., 2004b), having a 16S rRNA gene sequence similarity of 92.4%. Belliella can be distinguished from Aquiflexum on the basis of its temperature range and optimum, hydrolysis of gelatin, acid production from 13 substrates, assimilation of three substrates, and utilization of eight substrates (Table 108). Compared to Aquiflexum balticum, Belliella is less able to use organic substrates. The cellular fatty acids show significant differences between Aquiflexum and Belliella. The major

Enrichment and isolation procedures The strains of Belliella baltica were isolated during a cruise onboard the research vessel (RV) Aranda in September 1998 from surface water (5 m, 15°C, 7% salinity, pH 8.4) of two stations in the Central Baltic Sea (Gotland Deep BY-15 [57.1920°N, 20.0302°E] and TEILI1 [59.2607°N, 21.3002°E]). Strains were isolated by spreading 0.1 ml of seawater on 1/5 diluted Marine agar (Difco 2216; Marine broth or agar diluted by a factor of 5; final concentration of agar, 1.8%), and subsequently purified and cultured on this medium.

Belliella baltica BA134T {AJ564643} Aquiflexum balticum BA160T {AJ744861} 0.02 +* 75% +* 91% +* 93% + 68%

+* 99%

Algoriphagus winogradskii LMG 21969T {AJ575263} Algoriphagus locisalis MSS 170T {AY835922} Algoriphagus yeomjeoni MSS 160T {AY699794} Algoriphagus chordae LMG 21970T {AJ575265} Algoriphagus antarcticus LMG 21980T {AJ577141}

+* 65% +* 99%

Algoriphagus ratkowskyi LMG 21435T {AJ608641} Algoriphagus aquimarinus KMM 3958T {AJ575264} Algoriphagus terrigena DS-44T {DQ178979}

+* 100%

Algoriphagus halophilus JC2051T {AY264839}

+* 98%

Hongiella mannitolivorans JC2050T {AY264838} 90%

Hongiella ornithinivorans JC2052T {AY264840} Hongiella marincola SW2T {AY533663}

+* 100%

Cyclobacterium amurskyense KMM 6143T {AY960985} Cyclobacterium marinum LMG 13164T {AJ575266} Flexibacter flexilis ATCC 23079T{M62794} Flexibacter flexilis IFO 15060T {AB078050}

FIGURE 78.  Phylogenetic position of Belliella baltica and Aquiflexum balticum within the family Cyclobacteriaceae. According to the phylogenetic

analysis of almost complete 16S rRNA gene sequences, Aquiflexum balticum BA160T and Belliella baltica BA134T belong to a well defined clade (all three phylogenetic methods – see below) that also includes genera such as Algoriphagus, Hongiella, and Cyclobacterium. Aquiflexum balticum and Belliella baltica fail to cluster robustly with any of these genera or with each other, strongly supporting their position as independent genera. A domain (positions 85–1416 of the Aquiflexum balticum sequence, corresponding to a domain sequenced for every species) was chosen for phylogenetic analysis. Parsimony and maximum-likelihood (G option) were performed using programs from the phylip package. For neighbor-joining, distances were calculated with dnadist (from phylip) and the Kimura 2 parameters correction. A tree was generated using the BioNJ algorithm (Gascuel, 1997). Bootstrap analysis (1000 replications) was performed using the NJ algorithm as described previously. The topology of the tree is based on NJ analysis, with the results of the other analyses reported on the figure: *, for a branch also found by maximum-likelihood; +, for a branch also found by parsimony; %, indicates % of bootstrap.

436

Family II. Cyclobacteriaceae TABLE 108.  Differential features of Belliella baltica and Aquiflexum balticum a,b

Characteristic

Belliella baltica

Aquiflexum balticum

Color DNA G+C content (mol%) Optimum NaCl concentration (%) Growth at 40°C Temperature optimum (°C) Growth at pH 10 Gelatin hydrolysis Acid production from: c l-Arabinose d-Xylose d-Mannose Rhamnose a-Methyl-d-mannoside a-Methyl-d-glucoside N-Acetylglucosamine Amygdalin Melezitose Xylitol d-Turanose l-Fucose 5-Ketogluconate Assimilation of: d Mannose N-Acetylglucosamine Gluconate Utilization of: e d-Mannose b-Methyl-d-glucoside d-Psicose d-Sorbitol Monomethylsuccinate a-Ketobutyric acid a-Ketovaleric acid l-Glutamic acid Cellular fatty acids: f C15:0 C15:0 anteiso C15:1 w6c C17:1 w8c C17:1 anteiso w9c

Pink/orange 35.4 0–3.4 − 20–30 W −

Red 38.4 1–2 + 30–35 − +

+ + − − − − − − − − − − −

− − w + w w w w w w w w w

− − −

+ + +

− − − − − + + +

w w w w w − − −

2.94 4.53 2.07 1.19 −

− 18.53 − − 1.10

a

Symbols: +, positive; −, negative; w, weak reaction. Data from Brettar et al. (2004a, b).

b

Using the API50CHE system.

c

Using the API 20NE system.

d

Using the BIOLOG GN2 system.

e

Values represent percent of the total fatty acids.

f

differences are the higher abundance of C15:0 anteiso for Aquiflexum balticum, and the occurrence or absence or absence of four fatty acids (Table 108).

Taxonomic comments

Acknowledgements We are grateful to H. Kuosa and the crew of the Finnish RV Aranda for their assistance with sampling and seawater analysis of the Baltic Sea.

The genus Belliella belongs to the family Cyclobacteriaceae of the class Cytophagia. The phylogenetic affiliation within the family is reflected by the phylogenetic tree (Figure 78).

List of species of the genus Belliella 1. Belliella baltica Brettar, Christen and Höfle 2004a, 69VP bal¢ti.ca. N. L. fem. adj. baltica of or belonging to the Baltic Sea (the source of the type strain).

The characteristics are as given for the genus with the following additional information. The cells are 0.3–0.5 × 0.9–3.0 mm. Colonies are circular, smooth, convex, and entire; they are pink and transparent when young but become orange

Genus V. Echinicola

and opaque with ongoing incubation (>2 weeks, 20°C, on ½× Marine agar). The temperature range is 4–37°C; optimum, around 25°C. NaCl is not needed for growth; growth occurs a t salinities up to 6% NaCl; optimum, 0–3%. The pH range is 7–10; optimum, around pH 7. Growth occurs on 0.5% yeast extract. Esculin, starch, and DNA are hydrolyzed. Indole is not produced. Tyrosine, cellulose, and chitin are not degraded. Growth does not occur on media with casein and pectin. Acid is produced (using the API 50CHE system) from l-arabinose, d-xylose, galactose, d-glucose, d-fructose, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, d-raffinose, and starch. The following enzymic activities occur (using the API 20NE and API ZYM systems): a- and b-glucosidase; b-galactosidase; acid and alkaline

437

­ hosphatase; lipase (C8); leucine-, valine-, and cystine-arylamp idase; trypsin, chymotrypsin, and naphthol-phosphohydrolase. The following substrates are assimilated (using the API 20NE system): glucose, arabinose, and maltose. The following substrates are utilized (using the Biolog GN2 system): d-galactose, gentobiose, a-d-­glucose, a-d-lactose, lactulose, maltose, d-trehalose, acetic acid, a-ketobutyric/glutaric/ valeric-acid, and l-glutamic acid. All other tests using the API 20CHE, API ZYM, API 20NE, and Biolog GN2 test systems were negative. Source: marine or estuarine. DNA G+C content (mol%): 35.3–35.5 (HPLC). Type strain: BA134, CIP 108006, DSM 15883, LMG 21964. Sequence accession no. (16S rRNA gene): AJ564643.

Genus V. Echinicola Nedashkovskaya, Kim, Vancanneyt, Lysenko, Shin, Park, Lee, Jung, Kalinovskaya, Mikhailov, Bae and Swings 2006, 955VP Olga I. Nedashkovskaya and Seung Bum Kim E.chi.ni.co¢la. L. masc. n. echinus -i a sea urchin; L. suff. -cola derived from L. masc. or fem. n. incola a dweller; N.L. fem. n. Echinicola a sea-urchin dweller.

Rods usually measuring 0.3–0.5 × 1.1–2.3 mm. Motile by gliding. Produce non-diffusible carotenoid pigments. No flexirubintype pigments are formed. Chemo-organotrophs. Aerobic. Can ferment d-glucose. Oxidase-, catalase-, alkaline phosphatase-, and b-galactosidase-positive. Starch is hydrolyzed. Casein, urea, chitin, and cellulose (CM-cellulose and filter paper) are not decomposed, but agar, gelatin, and Tweens may be decomposed. Carbohydrates are utilized. Can grow without seawater or sodium ions. Nitrate is not reduced to nitrite. Indole is not produced. The major respiratory quinone is MK-7. DNA G+C content (mol%): 44–s. Type species: Echinicola pacifica Nedashkovskaya, Kim, Vancanneyt, Lysenko, Shin, Park, Lee, Jung, Kalinovskaya, Mikhailov, Bae and Swings 2006, 955VP.

Enrichment and isolation procedures

Further descriptive information

Echinicola strains remain viable for several weeks on Marine agar or other rich media based on natural or artificial seawater. They have survived storage −80°C for at least 5 years in Marine broth or artificial seawater supplemented with 20% glycerol (v/v).

The main cellular fatty acids are straight-chain unsaturated and branched-chain unsaturated fatty acids C15:0 iso, C16:1 w5c, C15:0 iso 3-OH, C17:1 iso w9c, C17:1 w6c, C17:0 iso 3-OH, summed feature 3 comprising C15:0 iso 2-OH and C16:1 w7c or both (Table 109). On Marine agar (Difco), strains of the genus Echinicola form regular, circular, convex, shiny, smooth, and pink colonies with entire edges and a diameter of 2–3 mm after cultivation for 48 h. All strains grow at 6–41°C and with 0–12% NaCl. The Echinicola strains do not form acid from melibiose, raffinose, sorbose, glycerol, adonitol, dulcitol, inositol, or mannitol. They utilize arabinose, glucose, lactose, mannose, and sucrose, but not myoinositol, mannitol, or sorbitol. The strains are susceptible to lincomycin and resistant to ampicillin, benzylpenicillin, gentamicin, kanamycin, neomycin, polymyxin B, streptomycin, and tetracycline. Strains of the genus Echinicola were isolated from sea animals and from seawater samples collected in the temperate and tropic latitudes.

Strains of Echinicola pacifica were isolated from a sea urchin, Strongylocentrorus intermedius, using the dilution plating ­technique on Marine agar 2216 (Nedashkovskaya et al., 2006). A single strain Echinicola vietnamensis, KMM 6221T, was isolated from seawater by direct plating on a medium containing (in g/l of a 1:1 mixture of natural seawater and distilled water): Bacto peptone (Difco), 5.0; casein hydrolysate (Merck), 2.0; Bacto yeast extract (Difco), 2.0; glucose, 1.0; KH2PO4, 0.2; MgSO4, 0.05; Bacto agar (Difco), 15.0. All isolates have been grown on media containing 0.5% of a peptone and 0.1–0.2% yeast extract (Difco) (Nedashkovskaya et al., 2007a).

Maintenance procedures

Differentiation of the genus Echinicola from other genera The genus Echinicola differ from its closest phylogenetic relatives, the genera Algoriphagus, Belliella, and Cyclobacterium by its gliding motility and by its ability to grow in the presence of 15% NaCl (Table 110). The absence of nitrate reductase and a higher mol% G+C content of its DNA (44–46 vs 35.4) separate members of the genus Echinicola from their nearest neighbor, Belliella baltica. Differences in fatty acid composition may also be helpful for discrimination of the Echinicola strains from their closest relatives (Table 109).

Taxonomic comments Phylogenetic analysis of almost-complete 16S rRNA gene sequences of the genus Echinicola indicates that its closest

438

Family II. Cyclobacteriaceae TABLE 109.  Cellular fatty acid compositions (%) of the genus Echinicola and related genera

of the phylum Bacteroidetesa,b Fatty acid

Echinicola

Algoriphagus

Belliella

Cyclobacterium

C11:0 iso C11:0 anteiso C13:1 AT C14:0 iso C15:1 iso C15:0 iso C15:0 anteiso C15:0 C15:1 w6c C16:1w5c C16:1 iso C16:0 iso C16:0 C15:0 iso 3-OH C15:0 3-OH C17:0 iso C17:0 Cyclo C17:1 iso w9c C17:1 anteiso w9c C17:1 w8c C17:1 w6c C16:0 iso 3-OH C16:0 3-OH C17:0 iso 3-OH C17:0 2-OH C18:1 w7c C18:1 w5c C18:1 H C18:0 C19:1 iso Summed feature 3 Summed feature 4

−c − 0–0.2 0.1–0.2 0–0.6 17.3–20.0 1.4–2.8 0.8–1.5 1.1–1.2 4.9–7.8 0.3–1.0 0.9–1.2 0.6–0.9 3.4–5.0 2.5–2.6 0.7–1.0 − 4.4–6.9 − 0.4 4.3–4.8 0.4–0.7 0.9–2.3 9.4–10.0 0.4 0.7–0.8 0.2 − − 0–0.7 30.7–34.5 0–5.0

0–2.2 0–2.6 − 0–2.3 0–6.8 16.6–38.9 0.8–6.4 0.3–3.0 0–4.1 0–5.8 0.6–10.9 0.3–20.7 0–3.2 1.6–5.1 − 0–0.8 0–2.4 1.5–14.6 0–1.6 0.4–0.9 0.5–4.5 0–4.7 0–1.9 4.9–10.7 0–2.2 − 0.5–0.7 0–1.6 − 0–1.3 6.0–33.7 0–4.9

− − 0–1.4 1.8–2.2 10.0–10.3 18.9–20.4 4.2–4.8 2.0–3.9 1.6–2.5 2.0–4.6 3.2–3.8 2.5–2.8 − 2.1–2.3 − 0–0.5 − 6.6–10.2 − 0.9–1.5 4.8–9.8 1.8–2.1 0–1.1 3.0–3.3 − − − − − 0.8–1.6 7.1–11.5 3.4–4.0

− − − 0–2.1 3.2–8.5 22.2–28.3 6.3–9.2 0–0.8 0.5–1.3 0–3.6 − − 0.5–4.9 1.1–3.7 − − − 4.3–10.3 − − 1.3–1.4 0–1.0 1.3–1.7 8.0–10.7 1.5–2.9 0–3.0 1.2–1.4 − 0–1.3 − 16.2–25.1 2.5–4.4

a Values of less than 1% for all strains are not shown. The percentages of predominant fatty acids are shown in bold. Summed features consist of one or more fatty acids that could not be separated by the Microbial Identification System. Summed feature 3 is C15:0 iso 2-OH and/or C16:1 w7c, summed feature 4 is C17:1 iso I and C17:1 anteiso B. b Data are taken from Ahmed et al. (2007), Brettar et al. (2004a), Nedashkovskaya et al. (2004, 2005, 2006, 2007a, b), and Ying et al. (2006).

TABLE 110.  Phenotypic characteristics that differentiate the genus Echinicola from its close relatives in the family Cyclobacteriaceae a,b

Characteristic Cell morphology: Regular rods Curved, ring-like, or horseshoe-shaped Cell size (mm) Gliding motility Nitrate reduction Salinity range (%) Growth at 40°C Hydrolysis of starch DNA G+C content (mol%) a

Echinicola

Algoriphagus

Belliella

Cyclobacterium

+ − 0.3–0.5 × 1.1–2.3 + − 0–15 + + 44–46

+ − 0.3–0.7 × 0.3–10.0 − D 0–10 D D 35–49

+ − 0.3–0.5 × 0.9–3.0 − + 0–6 − + 35.4

− + 0.3–0.7 × 0.8–1.5 − − 0–10 + − 41–45

Symbols: +, >85% positive; −, 0–15% positive; D, different reactions occur in different taxa (species of a genus).

Data are taken from Ahmed et al. (2007), Bowman et al. (2003), Brettar et al. (2004a), Nedashkovskaya et al. (2004, 2005, 2006, 2007a, b), Raj and Maloy (1990), Tiago et al. (2006a), Van Trappen et al. (2004), Yi and Chun (2004), Ying et al. (2006), and Yoon et al. (2004, 2005a, b, 2006).

b

439

Genus V. Echinicola TABLE 111.  Differential phenotypic characteristics of species of the genus Echinicolaa,b

Characteristic

E. vietnamensis KMM 6221T

E. pacifica (n = 3)

− − + + − −

+ + − − + +

+

−

45.9

44–45

Fermentation of d-glucose Production of H2S Growth with 15% NaCl Growth at 44°C Hydrolysis of agar, gelatin, and Tween 40 Acid production from l-arabinose, d-cellobiose, d-glucose, d-lactose, d-maltose, d-mannose, l-rhamnose, and dl-xylose Susceptibility to carbenicillin, chloramphenicol, doxycycline, erythromycin, and oleandomycin DNA G+C content (mol%) a

Symbols: +, >85% positive; −, 0–15% positive; n, a number of the strains studied. Data are from Nedashkovskaya et al. (2006, 2007a).

b

­relatives are the genera Belliella, Algoriphagus, and Cyclobacterium, with sequence similarity values of 91.7–92.1, 88.8–92.4, and 89.5–91.5%, respectively. A level of 16S rRNA gene sequence similarity between the two species of the genus, Echinicola pacifica and Echinicola vietnamensis, is 94.7–95.0%. The 16S rRNA gene sequence similarity between the type strain Echinicola pacifica KMM 6172T and reference strains KMM 6166 and KMM

6173 ranges from 99.4 to 99.9%. The DNA–DNA hybridization values between these strains vary from 93 to 98%.

Differentiation of the species of the genus Echinicola The strains of the two species of the genus Echinicola have many similar phenotypic features. However, they can be differentiated from each other by the several phenotypic traits shown in Table 111.

List of species of the genus Echinicola 1. Echinicola pacifica Nedashkovskaya, Kim, Vancanneyt, Lysenko, Shin, Park, Lee, Jung, Kalinovskaya, Mikhailov, Bae and Swings 2006, 955VP pa.ci¢fi.ca. L. fem. adj. pacifica pacific, and by extension referring to the Pacific Ocean, from which the type strain was isolated. Cells are 0.3–0.4 mm in width and 1.2–1.9 mm in length. On Marine agar, colonies are sunken into the agar. Growth occurs at 6–41°C and with 0–12% NaCl. The optimal temperature for growth 25–28°C. Decomposes gelatin (weakly) and esculin. Hydrolysis of Tweens 20 and 80 is strain-dependent. DNA is not hydrolyzed. Produces acid from N-acetylglucosamine. Can oxidize d-galactose and d-sucrose. Does not form acid from l-fucose. According to the API 20E gallery (bioMérieux), strain KMM 6172T utilizes citrate, forms acid from amygdalin, and is negative for arginine dihydrolase, lysine decarboxylase, and ornithine decarboxylase. Results of Biolog GN2 (Biolog) testing show that strain KMM 6172T utilizes a-cyclodextrin, dextrin, glycogen, a-d-glucose, d-fructose, l-fucose, d-galactose, gentiobiose, a-lactose, a-d-lactose, lactulose, d-melibiose, methyl b-d-glucoside, psicose, d-raffinose, d-trehalose, turanose, d-galacturonic acid, d-glucuronic acid, a-ketobutyric acid, alaninamide, l-alanine, l-alanylglycine, l-asparagine, l-aspartic acid, l-glutamic acid, hydroxy-l-proline and l-threonine. The following compounds are not utilized: Tween 80, N-acetyl-dgalactosamine, adonitol, l-arabitol, i-erythritol, myo-inositol, d-mannitol, d-sorbitol, xylitol, methyl pyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, citric acid, formic acid, d-galactonic acid, d-gluconic acid, d-glucosaminic acid,

a-, b-, and g-hydroxybutyric acids, p-hydroxyphenylacetic acid, itaconic acid, a-ketoglutaric acid, a-ketovaleric acid, dl-lactic acid, malonic acid, propionic acid, quinic acid, d-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, d-alanine, glycyl-laspartic acid, glycyl-l-glutamic acid, l-histidine, l-leucine, l-ornithine, l-phenylalanine, l-proline, l-pyroglytamic acid, d-serine, l-serine, dl-carnitine, g-aminobutyric acid, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, dl-a-glycerol phosphate, glucose 1-phosphate, and glucose-6-phosphate. H2S is ­produced. Indole and acetoin (Voges–Proskauer reaction) are not produced. According to API ZYM gallery (bioMérieux), the following enzyme activities are present: a-galactosidase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, naphthol-AS-BI-phosphohydrolase, a- and b-glucosidases, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase. Lipase (C14) and b-glucuronidase are not present. The predominant fatty acids are C15:0 iso (17.3–18.0%), C16:1 w5c (6.7–7.8%), C17:1 iso w9c (6.3–6.9%), C17:1 w6c (4.3–4.8%), C15:0 iso 3-OH (3.4–5.0%), C17:0 iso 3-OH (9.4–10.0%) and summed feature 3 (30.7–30.8%), comprising C16:1 w7c and/ or C15:0 iso 2-OH (Table 109). Source: sea urchin Strongylocentrotus intermedius collected in Troitsa Bay, Gulf of Peter the Great, the East Sea (also known as the Japan Sea). DNA G+C content (mol%): 44–45 (Tm). Type strain: KMM 6172, KCTC 12368, LMG 23350. Sequence accession no. (16S rRNA gene): DQ185611.

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Family II. Cyclobacteriaceae

2. Echinicola vietnamensis Nedashkovskaya, Kim, Hoste, Shin, Beleneva, Vancanneyt and Mikhailov 2007a, 763VP vi.et.nam.en¢sis. N. L. fem. adj. vietnamensis of or belonging to Vietnam, the country of origin of the type strain. Cells are 0.4–0.5 × 1.1–2.3 mm. Colonies are light pink on Marine agar. Growth occurs at 6–44°C and with 0–15% NaCl. The optimal temperature for growth is 30–32°C. Tweens 20 and 80 are not hydrolyzed. Acid is not produced from d-fructose, d-galactose, d-sucrose, or N-acetylglucosamine. The fatty acids accounting for more than 1% of the total

are C15:0 anteiso (1.4%), C15:0 iso (20.0%), C15:1 w6c (1.2%), C15:0 (1.5%), C16:1 w5c (4.9%), C17:1 iso w9c (4.4%), C17:0 iso (1.0%), C17:1 w6c (4.5%), C15:0 iso 3-OH (3.7%), C16:0 3-OH (2.3%), C17:0 iso 3-OH (10.0%), summed feature 3 (34.5%), comprising C16:1 w7c and/or C15:0 iso 2-OH and summed feature 4 (5%), comprising C17:1 iso I and/or C17:1 anteiso B. Source: seawater collected in a mussel farm located in a lagoon of Nha Trang Bay, South China Sea, Vietnam. DNA G+C content (mol%): 45.9 (Tm). Type strain: KMM 6221, DSM 17526, LMG 23754. Sequence accession no. (16S rRNA gene): AM406795.

Genus XV. Rhodonellum Schmidt, Priemé and Stougaard 2006, 2891VP The Editorial Board Rho.do.nell.um. Gr. neut. n. rhodon a rose; L. neut. dim. ending -ellum; N.L. dim. neut. n. Rhodonellum a small rose, referring to the red color of the colonies.

Rods 0.7–1.0 × 0.8–3.0 mm. Gram-stain-negative. Oxidase-negative. Catalase-positive. Aerobic. Chemoheterotrophic. Colonies are pink to red due to carotenoids. Temperature range, 0–22°C; optimum, ca. 5°C. pH range, 7.5–10.7 when grown at 5–10°C. NaCl range for growth, 0–3%; optimum, 0.6%. Predominant fatty acids are C17:1 iso w9c, C17:0 iso 3-OH (12.5–18.5%), and summed feature 3. Cells contain red pigment in the form of carotenoids. Optimal growth occurs above pH 9. NaCl is not required for growth, but growth is enhanced by the presence of up to 0.6% NaCl. DNA G+C content (mol%): 44.2. Type species: Rhodonellum psychrophilum Schmidt, Priemé and Stougaard 2006, 2891VP.

Further descriptive information Predominant fatty acids of Rhodonellum psychrophilum are C15:1 iso G (6.3%), C15:0 iso (7.6%), C16:1 iso H (7.3%), C17:1 iso w9c (17.5%), C17:1 w6c (6.8%), C17:0 iso 3-OH (17.5%), and summed feature 3, comprising C16:1 w7c and/or C15:0 iso 2-OH (12.6%), and summed feature 4, comprising C14:0 2-OH and/or C15:0 iso 2-OH (6.5%), which could not be distinguished by the method used (Schmidt et al., 2006).

Enrichment and isolation procedures Rhodonellum psychrophilum was isolated from submarine ikaite tufa columns collected from the Ikka Fjord, southwest Greenland (Schmidt et al., 2006). The columns were conserved in 15% glycerol and kept at −20°C. Isolation was achieved on agar plates containing 0.1× R2A medium (Difco).

Differentiation of the genus Rhodonellum from other genera Rhodonellum psychrophilum can be differentiated from Belliellia baltica by its pH range for growth (7.5–10.7 vs 6–10), negative oxidase reaction, temperature range of 0–22°C vs 4–37°C, an optimum temperature of 5°C vs 25–30°C, and a higher mol% G+C of the DNA (43.1 vs 35).

Taxonomic comments Schmidt et al. (2006) reported that, based on 16S rRNA gene sequence analysis, the type strain of Rhodonellum psychrophilum, together with five related isolates from ikaite columns, formed a separate cluster with 86–93% gene sequence similarity to their closest relative, Belliella baltica.

List of species of the genus Rhodonellum 1. Rhodonellum psychrophilum Schmidt, Priemé and Stougaard 2006, 2891VP psy.chro¢phi.lum. Gr. adj. psychros cold; N.L. neut. adj. philum (from Gr. neut. adj. philon), friend, loving; N.L. neut. adj. psychrophilum cold-loving. The characteristics are as described for the genus, with the following additional features. Colonies are smooth, circular, and red due to the presence of carotenoids when grown under low light intensity. Colonies are white to light red when grown at a light intensity of 20–40 mE/m2/s. Growth occurs from pH 7.5 to above pH 10.7, with an optimum at pH 9.2–10.0. At optimal growth temperature, the range of

tolerated pH is largest, whereas at, below, and above the optimal growth temperature, a narrower pH range is tolerated. NaCl is not required for growth, but up to 3% (w/v) NaCl is tolerated. Optimal growth occurs around 0.6% (w/v) NaCl. Strains can use a wide spectrum of carbon sources such as galactose, glycerol, lactose, maltose, mannose, sorbitol, and starch. Source: the permanently alkaline and cold ikaite columns in the Ikka Fjord in southwest Greenland. DNA G+C content (mol%): 44.2 (HPLC). Type strain: GCM71, DSM 17998, LMG 23454. Sequence accession no. (16S rRNA gene): DQ112660.

Genus XV. Rhodonellum

References Ahmed, I., A. Yokota and T. Fujiwara. 2007. Chimaereicella boritolerans sp. nov., a boron-tolerant and alkaliphilic bacterium of the family Flavobacteriaceae isolated from soil. Int. J. Syst. Evol. Microbiol. 57: 986–992. Borrall, R. and J.M. Larkin. 1978. Flectobacillus marinus (Raj) comb. nov., a marine bacterium previously assigned to Microcyclus. Int. J. Syst. Bacteriol. 28: 341–343. Bowman, J.P., C. Mancuso, C.M. Nichols and J.A.E. Gibson. 2003. Algoriphagus ratkowskyi gen. nov., sp. nov., Brumimicrobium glaciale gen. nov., sp. nov., Cryomorpha ignava gen. nov., sp. nov. and Crocinitomix catalasitica gen. nov., sp. nov., novel flavobacteria isolated from various polar habitats. Int. J. Syst. Evol. Microbiol. 53: 1343–1355. Brettar, I., R. Christen and M.G. Höfle. 2004a. Belliella baltica gen. nov., sp. nov., a novel marine bacterium of the Cytophaga-FlavobacteriumBacteroides group isolated from surface water of the central Baltic Sea. Int. J. Syst. Evol. Microbiol. 54: 65–70. Brettar, I., R. Christen and M.G. Höfle. 2004b. Aquiflexum balticum gen. nov., sp. nov., a novel marine bacterium of the Cytophaga-Flavobacterium-Bacteroides group isolated from surface water of the central Baltic Sea. Int. J. Syst. Evol. Microbiol. 54: 2335–2341. DeLey, J., H. Cattoir and A. Reynaerts. 1970. The quantitative measurement of DNA hybridization from renaturation rates. Eur. J. Biochem. 12: 133–142. Euzéby, J.P. 1998. Taxonomic note: necessary correction of specific and subspecific epithets according to Rules 12c and 13b of the International Code of Nomenclature of Bacteria (1990 Revision). Int. J. Syst. Bacteriol. 48: 1073–1075. Gascuel, O. 1997. BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol. Biol. Evol. 14: 685–695. Hamana, K. and Y. Nakagawa. 2001. Polyamine distribution profiles in the eighteen genera phylogenetically located within the Flavobacterium-Flexibacter-Cytophaga complex. Microbios 106: 7–17. Larkin, J.M. and R. Borrall. 1984a. Family I. Spirosomaceace. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 125–132. Larkin, J.M. and R. Borrall. 1984b. Deoxyribonucleic acid base composition and homology of Microcyclus, Spirosoma, and similar organisms. Int. J. Syst. Bacteriol. 34: 211–215. Larkin, J.M., P.M. Williams and R. Taylor. 1977. Taxonomy of genus Microcyclus Ørskov 1928, reintroduction and emendation of genus Spirosoma Migula 1894, and proposal of a new genus, Flectobacillus. Int. J. Syst. Bacteriol. 27: 147–156. Nedashkovskaya, O.I., M. Vancanneyt, S. Van Trappen, K. Vandemeulebroecke, A.M. Lysenko, M. Rohde, E. Falsen, G.M. Frolova, V.V. Mikhailov and J. Swings. 2004. Description of Algoriphagus aquimarinus sp. nov., Algoriphagus chordae sp. nov. and Algoriphagus winogradskyi sp. nov., from sea water and algae, transfer of Hongiella halophila Yi and Chun 2004 to the genus Algoriphagus as Algoriphagus halophilus comb. nov. and emended descriptions of the genera Algoriphagus Bowman et al. 2003 and Hongiella Yi and Chun 2004. Int. J. Syst. Evol. Microbiol. 54: 1757–1764. Nedashkovskaya, O.I., S.B. Kim, M.S. Lee, M.S. Park, K.H. Lee, A.M. Lysenko, H.W. Oh, V.V. Mikhailov and K.S. Bae. 2005. Cyclobacterium amurskyense sp. nov., a novel marine bacterium isolated from sea water. Int. J. Syst. Evol. Microbiol. 55: 2391–2394. Nedashkovskaya, O.I., S.B. Kim, M. Vancanneyt, A.M. Lysenko, D.S. Shin, M.S. Park, K.H. Lee, W.J. Jung, N.I. Kalinovskaya, V.V. Mikhailov, K.S. Bae and J. Swings. 2006. Echinicola pacifica gen. nov., sp. nov., a novel flexibacterium isolated from the sea urchin Strongylocentrotus intermedius. Int. J. Syst. Evol. Microbiol. 56: 953–958. Nedashkovskaya, O.I., S.B. Kim, B. Hoste, D.S. Shin, I.A. Beleneva, M. Vancanneyt and V.V. Mikhailov. 2007a. Echinicola vietnamensis sp. nov.,

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a member of the phylum Bacteroidetes isolated from seawater. Int. J. Syst. Evol. Microbiol. 57: 761–763. Nedashkovskaya, O.I., S.B. Kim, K.K. Kwon, D.S. Shin, X. Luo, S.J. Kim and V.V. Mikhailov. 2007b. Proposal of Algoriphagus vanfongensis sp. nov., transfer of members of the genera Hongiella Yi and Chun 2004 emend. Nedashkovskaya et al., 2004 and Chimaereicella Tiago et al. 2006 to the genus Algoriphagus, and emended description of the genus Algoriphagus Bowman et al. 2003 emend. Nedashkovskaya et al. 2004. Int. J. Syst. Evol. Microbiol. 57: 1988–1994. Raj, H.D. 1976. A new species: Microcyclus marinus. Int. J. Syst. Bacteriol. 26: 528–544. Raj, H.D. 1979. Adansonian analysis of Microcyclus and related bacteria. Abstract 1–31. Proceedings of the Annu. Meet. Am. Soc. Microbiol.. Raj, H.D. and K.A. Paveglio. 1983. Contributing carbohydrate catabolic pathways in Cyclobacterium marinus. J. Bacteriol. 153: 335–339. Raj, H.D. and S.R. Maloy. 1990. Proposal of Cyclobacterium marinus gen. nov., comb. nov. for a marine bacterium previously assigned to the genus Flectobacillus. Int. J. Syst. Bacteriol. 40: 337–347. Schmidt, M., A. Priemé and P. Stougaard. 2006. Rhodonellum psychrophilum gen. nov., sp. nov., a novel psychrophilic and alkaliphilic bacterium of the phylum Bacteroidetes isolated from Greenland. Int. J. Syst. Evol. Microbiol. 56: 2887–2892. Tiago, I., A.P. Chung and A. Veríssimo. 2004. Bacterial diversity in a nonsaline alkaline environment: heterotrophic aerobic populations. Appl. Environ. Microbiol. 70: 7378–7387. Tiago, I., V. Mendes, C. Pires, P.V. Morais and A. Veríssimo. 2006a. Chimaereicella alkaliphila gen. nov., sp. nov., a Gram-negative alkaliphilic bacterium isolated from a nonsaline alkaline groundwater. Syst. Appl. Microbiol. 29: 100–108. Tiago, I., V. Mendes, C. Pires, P.V. Morais and A. Veríssimo. 2006b. In List of new names and new combinations previously effectively, but not validly, published. List no. 109. Int. J. Syst. Evol. Microbiol. 56: 925–927. Van Trappen, S., J. Mergaert, S. Van Eygen, P. Dawyndt, M.C. Cnockaert and J. Swings. 2002. Diversity of 746 heterotrophic bacteria isolated from microbial mats from ten Antarctic lakes. Syst. Appl. Microbiol. 25: 603–610. Van Trappen, S., I. Vandecandelaere, J. Mergaert and J. Swings. 2004. Algoriphagus antarcticus sp. nov., a novel psychrophile from microbial mats in Antarctic lakes. Int. J. Syst. Evol. Microbiol. 54: 1969– 1973. Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev. 51: 221–271. Woese, C.R., S. Maloy, L. Mandelco and H.D. Raj. 1990. Phylogenetic placement of the Spirosomaceae. Syst. Appl. Microbiol. 13: 19–23. Yi, H. and J. Chun. 2004. Hongiella mannitolivorans gen. nov., sp. nov., Hongiella halophila sp. nov. and Hongiella ornithinivorans sp. nov., isolated from tidal flat sediment. Int. J. Syst. Evol. Microbiol. 54: 157– 162. Ying, J.Y., B.J. Wang, S.S. Yang and S.J. Liu. 2006. Cyclobacterium lianum sp. nov., a marine bacterium isolated from sediment of an oilfield in the South China Sea, and emended description of the genus Cyclobacterium. Int. J. Syst. Evol. Microbiol. 56: 2927–2930. Yoon, J.H., S.H. Yeo and T.K. Oh. 2004. Hongiella marincola sp. nov., isolated from sea water of the East Sea in Korea. Int. J. Syst. Evol. Microbiol. 54: 1845–1848. Yoon, J.H., S.J. Kang, S.Y. Jung, C.H. Lee and T.K. Oh. 2005a. Algoriphagus yeomjeoni sp. nov., isolated from a marine solar saltern in the Yellow Sea, Korea. Int. J. Syst. Evol. Microbiol. 55: 865–870. Yoon, J.H., S.J. Kang and T.K. Oh. 2005b. Algoriphagus locisalis sp. nov., isolated from a marine solar saltern. Int. J. Syst. Evol. Microbiol. 55: 1635–1639. Yoon, J.H., M.H. Lee, S.J. Kang and T.K. Oh. 2006. Algoriphagus terrigena sp. nov., isolated from soil. Int. J. Syst. Evol. Microbiol. 56: 777–780.

442

Family III. Flammeovirgaceae

Family III. Flammeovirgaceae fam. nov. Olga I. Nedashkovskaya and Wolfgang Ludwig Flam.me.o.vir.ga¢ce.a.e. N.L. fem. n. Flammeovirga type genus of the family; suff. -aceae ending to denote a family; N.L. fem. pl. n. Flammeovirgaceae the Flammeovirga family. Cells are straight, flexible or curved rods, that are 0.2–1.0×1.5– 100 mm or longer. Gram-stain-negative. Chemo-organotrophs. Nonsporeforming. Nonflagellated and nonmotile in liquid media, but most of them move by gliding on solid substrates. Cells of one species can form sheathed filaments. Colonies are pink, red, orange or apricot in color. Colonies of the majority of strains are characterized by a spreading growth and some of them produce gelase fields and form deep craters in agar plates. Strains of the majority species are strictly aerobic, but strains of one genus are characterized as facultatively anaerobic organisms. Optimal grown temperature is 20–33°C. Carbohydrates are oxidized. Two species ferment glucose. Most strains require seawater or NaCl for growth. All strains are alkaline phosphatasepositive and arginine dihydrolase-, lysine decarboxylase-, and ornithine decarboxylase-negative. The most strains produce oxidase and catalase. Only one species produces flexirubin type pigments. The majority of the strains cannot produce acetoin, hydrogen sulfide or indole. Nitrate may be reduced to nitrite. Most species hydrolyze esculin, gelatin, and DNA. Agar, casein, starch, carboxymethylcellulose and Tweens may be decomposed, but crystalline cellulose is not. Menaquinone 7 is a major or single respiratory quinone. Predominant fatty acids are C15:0 iso, C15:1 iso, C17:0 iso 3-OH and C15:0 iso 2-OH and/or C16:1 w 7c. The main polyamines for the majority species are spermidine or homospermidine. The most strains produced carotenoid pigment saproxanthin. All species occur in different marine environments including sea water, marine sediments, seaweeds, or marine animals, one of them was isolated from marine aquarium outflow. DNA G+C content (mol%): 31–45. Type genus: Flammeovirga Nakagawa, Hamana, Sakane and Yamasato 1997, 221VP emend. Takahashi, Suzuki and Nakagawa 2006, 2097VP.

Taxonomic comments Family Flammeovirgaceae comprises the recognized genera Flammeovirga, Fabibacter, Flexithrix, Marinoscillum, Perexilibacter, Persicobacter, Rapidithrix, Reichenbachiella, and Roseivirga, and generically misclassified species [Flexibacter] litoralis, [Flexibacter] polymorphus, [Flexibacter] roseolus, “Microscilla sericea”, and “Microscilla tractuosa”, which are phylogenetically distant from the type species of their genera. In the last two decades the

strains currently affiliated with the family Flammeovirgaceae have been subjected to intensive taxonomic investigation by using a polyphasic approach and, especially, a phylogenetic analysis based on comparison of 16S rRNA gene sequences (Nakagawa et  al., 2002). The species described as [Cytophaga] aprica and [Cytophaga] diffluens by Reichenbach (1989a) in the previous edition of Bergey’s Manual of Systematic Bacteriology, were placed in the novel genera Flammeovirga and Persicobacter, respectively (Nakagawa et al., 1997). Later, Takahashi et al. (2006) emended the description of the genus Flammeovirga to include misclassified species “Microscilla arenaria” and the novel species Flammeovirga yaeyamensis; Flammeovirga kamogawensis was subsequently described by Hosoya and Yokota (2007a). A species with an uncertain taxonomic position, “[Flexibacter] aggregans” (Lewin, 1969) Leadbetter 1974, was reclassified as a later heterotypic synonym of Flexithrix dorotheae Lewin 1970 because of the very close phylogenetic relationship between them (Hosoya and Yokota, 2007b). In order to accommodate the single and type strain of species “Microscilla furvescens” and a representative of a novel species, the genus Marinoscillum was created (Seo et al., 2009). Recently a precise taxonomic position of the misclassified species “Microscilla sericea” and “Microscilla tractuosa” was determined, and it was proposed to reclassify them in the novel genus “Marivirga” as two distinct species (Nedashkovskaya et al., 2009). In addition, the single and type strain of Fabibacter halotolerans forms a coherent phylogenetic cluster with species of the other member of the family, the genus Roseivirga. They share many phenotypic features in common and, perhaps, will be joined to the single genus. From a view of the heterogeneity of the family Flammeovirgaceae, it should be noted that the representatives of all recognized and newly proposed genera such as Flexithrix, Perexilibacter, Persicobacter, Rapidithrix, Roseivirga, and generically misclassified species [Flexibacter] litoralis, [Flexibacter] polymorphus and [Flexibacter] roseolus can be considered as members of novel families in future. Differential characteristics of the genera of Flammeovirgaceae are given in Table 112. Please note that Marinoscillum (Seo et al., 2009), “Marivirga” (Nedashkovskaya et  al., 2009), Perexilibacter (Yoon et al., 2007), and Rapidithrix (Srisukchayakul et al., 2007) were proposed after the valid publication deadline for this volume and are not described in greater detail than given here.

Genus I. Flammeovirga Nakagawa, Hamana, Sakane and Yamasato 1997, 221VP emend. Takahashi, Suzuki and Nakagawa 2006, 2097VP Yasuyoshi Nakagawa Flam.me.o.vir¢ga. L. adj. flammeus fire-colored; L. fem. n. virga rod; N.L. fem. n. Flammeovirga fire-colored rod.

Rods 0.4–0.9 mm wide and 1.7–96 mm long or longer. Motile by gliding. Nonsporeforming. Gram-stain-negative. Aerobic, having a strictly respiratory type of metabolism with oxygen as the

terminal electron acceptor. Chemo-organotrophic. Colonies spread and produce large gelase fields and deep craters in agar plates. Cell mass is orange to reddish orange. Saproxanthin is

Genus I. Flammeovirga

present as the major carotenoid pigment. Flexirubin-type pigments are absent. Oxidase and catalase activities differ among species. Marine organisms. Seawater is required for growth; NaCl alone can substitute. The optimum pH for growth is 7. Nitrate is reduced. Agar, alginic acid, esculin, and starch are degraded. The major respiratory quinone is MK-7. Predominant cellular fatty acids are C15:0 iso, C20:4 w6,9,12,15c, and C16:0 3-OH. DNA G+C content (mol%): 31–36. Type species: Flammeovirga aprica (Reichenbach 1989b) ­Nakagawa, Hamana, Sakane and Yamasato 1997, 221VP emend. Takahashi, Suzuki and Nakagawa 2006, 2099VP [Cytophaga aprica (ex Lewin 1969) Reichenbach 1989c, 495VP; Cytophaga diffluens var. aprica Lewin 1969, 197].

Further descriptive information All species are negative for acid production from API 50CH system substrates including adonitol, d-arabinose, l-arabinose, d-arabitol, l-arabitol, dulcitol, erythritol, d-fucose, gluconate, glycerol, inositol, inulin, 2-ketogluconate, d-lyxose, methyl b-d-xyloside, d-tagatose, d-turanose, and l-xylose. All species are negative for the utilization of Biolog GN2 Microplate system substrates including N-acetyl-d-galactosamine, cis-aconitic acid, adonitol, d-alanine, g-aminobutyric acid, 2-aminoethanol, l-arabinose, d-arabitol, bromosuccinic acid, 2,3-butanediol, dl-carnitine, citric acid, i-erythritol, formic acid, d-fructose, d-galactonic acid lactone, d-galacturonic acid, d-gluconic acid, d-glucosaminic acid, glucuronamide, d-glucuronic acid, l-glutamic acid, glycerol, dl-a-glycerol phosphate, a-hydroxybutyric acid, b-hydroxybutyric acid, g-hydroxybutyric acid, hydroxyl-proline, p-hydroxyphenylacetic acid, inosine, myo-inositol, itaconic acid, a-ketoglutaric acid, a-ketovaleric acid, l-leucine, malonic acid, d-mannitol, methyl-b-d-glucoside, methyl pyruvate, l-phenylalanine, phenylethylamine, d-psicose, putrescine, l-pyroglutamic acid, quinic acid, d-raffinose, d-saccharic acid, sebacic acid, d-serine, d-sorbitol, succinamic acid, succinic acid, sucrose, Tween 40, Tween 80, turanose, and xylitol.

Enrichment and isolation procedures No enrichment media have been designed for isolation of Flammeovirga strains. Standard procedures to isolate marine bacteria can be applied. Colonies of Flammeovirga are usually orange to reddish orange and spread rapidly.

443

Flammeovirga strains have been isolated from marine e­ nvironments at widely separated sites (Lewin, 1969; Nakagawa, 2004; Takahashi et  al., 2006). The type strain of Flammeovirga aprica came from Kailua, Hawaii. The type strain of Flammeovirga arenaria was isolated from marine sand in Mexico. Strains of Flammeovirga yaeyamensis have been isolated from seaweeds, coastal sands, and dead leaves along the seashores of Iriomote and Ishigaki Islands (24° 20¢ N 123° 45¢ E and 24° 20¢ N 124° 9¢ E, respectively).

Maintenance procedures Cultures of Flammeovirga strains can be preserved by freezing at lower than −80°C. For freezing, cells are suspended in Marine broth 2216 (Difco) containing 10% glycerol or 7% DMSO. Flammeovirga strains are rather sensitive to drying; however, they can be preserved by the liquid drying method using a protective medium such as SM2* or SM3† (Sakane et al., 1996), or by freeze drying.

Differentiation of the genus Flammeovirga from other genera Characteristics differentiating the genus Flammeovirga from other mesophilic genera in the family Flammeovirgaceae are listed in Table 113. Presence of C15:0 iso, C16:0 3-OH, and C20:4 w6,9,12,15c as major fatty acids and saproxanthin (the major carotenoid) are useful characters to discriminate the genus Flammeovirga from other genera.

Taxonomic comments The genus Flammeovirga was created by Nakagawa et al. (1997) to accommodate the misclassified species Cytophaga aprica. Until 2006, the genus contained a single species, Flammeovirga aprica. However, Nakagawa et al. (2002) reported that “Microscilla arenaria” Lewin 1969 was related to the genus Flammeovirga, and then Takahashi et al. (2006) reclassified it as Flammeovirga arenaria, with the proposal of a third species, Flammeovirga yaeyamensis isolated from the Yaeyama Islands, Japan. These three species are clearly differentiated from each other by DNA–DNA hybridizations and phenotypic characteristics (Table 114).

Differentiation of the species of the genus Flammeovirga Table 114 lists characteristics that distinguish the Flammeovirga species from one another.

List of species of the genus Flammeovirga‡ 1. Flammeovirga aprica (Reichenbach 1989b) Nakagawa, Hamana, Sakane and Yamasato 1997, 221VP emend. Takahashi, Suzuki and Nakagawa 2006, 2099VP [Cytophaga aprica (ex Lewin 1969) Reichenbach 1989c, 495VP; Cytophaga diffluens var. aprica Lewin 1969, 197] a¢pri.ca. L. fem. adj. aprica sunlit, sun-loving. The characteristics are as described for the genus and as listed in Table 113, with the following additional features. Cells are 0.5–0.9  mm wide and 1.7–96  mm long or longer. The cell mass is orange to reddish orange. Growth occurs at 15–30°C; optimum, 25°C. The pH range for growth is 6–8; optimum, 7. Growth occurs in the presence of 1–5% NaCl; optimum, 3%. Oxidase- and catalase-positive. Urease-negative. Agar, ­alginic acid, carboxymethylcellulose, DNA, esculin and

starch are degraded, but not cellulose, chitin, gelatin, inulin, Tween 80, or tyrosine. Casein is weakly degraded. H2S is produced. Indole-negative. The oxidation/fermentation test for cata­bolism of glucose is fermentative. Utilization (with the Biolog GN2 microplate system) is positive for cellobiose, a-cyclodextrin, dextrin, l-fucose, d-galactose, gentiobiose, * SM2 consists of: monosodium glutamate monohydrate, 50 g; adonitol, 15 g; d(−)sorbitol, 10 g; artificial seawater, 750 ml; and distilled water, 250 ml; pH 7.0. †  SM3 consists of: monosodium glutamate monohydrate, 50 g; adonitol, 15 g; d(−)sorbitol, 10 g; l(−)-proline, 2 g; 2% methyl cellulose [4000 centipoise (cps)] solution, 100 ml; artificial seawater, 900 ml; pH 7.0. ‡  Flammeovirga kakegawaensis was published after completion of the manuscript. Refer to Hosoya S. and A. Yokota. 2007. Flammeovirga kamogawensis sp. nov., isolated from coastal seawater in Japan. Int. J. Syst. Evol. Microbiol. 57: 1327–1330.

Table 112.  Phenotypic characteristics differentiating members of the family Flammeovirgaceae a,b

Characteristic Source of isolation

Metabolism Cell shape Cell size (mm) Sheathed filaments formation Colony pigmentation Gliding motility Tryptophan deaminase Oxidase/catalase b-Galactosidase Carotenoid pigments Flexirubin type pigments production Nitrate reduction Indole/acetoin production Hydrogen sulfide production Temperature range for growth (°C) (optimum) pH range for growth (optimum) Salinity range for growth (% NaCl) (optimum) Mg2+ and Ca2+ requirement Hydrolysis of: Esculin Agar Alginic acid Casein Crystalline cellulose/ carboxymethylcellulose Chitin Gelatin Starch DNA Tween 20 Tween 40 Tween 80 Tyrosine Urea Acid from carbohydrates Carbohydrates utilization Citrate utilization DNA G+C content (mol%) Main fatty acids

Flammeovirga

Fabibacter

Flexithrix

Marinoscillum

Rocky sand, Hawaii, USA; brown sand, Norse Beach Puerto Penasco Sonora, Mexico; seaweed, sand, dead leaves, Yaeyama Islands, Japan sea water, Kamogawa, Japan, Pacific Ocean Fermentative/aerobic Flexible rods

Sponge Tedania ignis, Bahamas

Marine silt, Kerala, India; green-brown sand, Canoe Beach, Tema, Ghana

Sand, Samoa; unidentified sponge, Micronesia, Pacific Ocean

Aerobic Curved rods

Aerobic Flexible rods

Aerobic Flexible rods

0.4–1.0×1.7–96 or longer

0.5×1.5

2–100

−

−

d

0.2–0.5×10–100 or longer −

Orange-reddish orange + − d/d d Saproxanthin −

Pink + − +/+ + nd −

Yellow + − +/+ + Zeaxanthin −

Orange or apricot + + +/+ + Saproxanthin −

+ −/− d

− −/+ −

− −/− −

− d/d −

10–35 (25–30)

12–36 (28–30)

17–40

15–45 (33–33.5)

6.0–10.0 (7.0)

5.0–10.0

6.5–8.0

5.0–9.5 (7.5)

1–5 (3)

0–12

2–5

0.5–12

−

−

−

+

+ + + d −/d

+ − nd − −

+ − − − −/+

+c − + d −/+c

− d + d d d d − − + + − 31–36 C15:0 iso, C20:4 w6,9,12,15c, C16:0 iso 3-OH

− − + + + + + nd − + +

− + − + + + + d − + + − 35.6 C16:1 w5c, C15:0 iso, summed feature 3, C17:0 iso 3-OH, C16:0

− d − d nd − − nd − + + − 41–44 C15:0 iso, C16:1 w5c, C17:0 iso 3-OH, summed feature 3

nd

nd

Spermidine, agmatine

Homospermidinec

Polar lipids

Main polyamines

nd

42.5 C15:0 iso, C15:1 iso, C15:0 iso 3-OH, C16:0 iso 3-OH, C17:0 iso 3-OH, summed feature 3 nd

−

nd

Symbols: +, >90% positive; d, different strains give different reactions (11–89% positive); −, 0–0% positive; w, weak reaction; nd, not determined.

a

Data from: Hamana and Nakagawa (2001), Hamana et al. (2008), Hosoya and Yokota (2007a, b), Lau et al. (2006), Nakagawa et al. (1997), Nedashkovskaya et al. (2003),

b

Data for species Marinoscillum furvescens only.

c

Table 112.  (Continued)

“Microscilla” sericea

“Microscilla” tractuosa

Perexilibacter

Persicobacter

Rapidithrix

Reichenbachiella

Roseivirga

Marine aquarium outflow, La Jolla, California

Beach sand, South China Sea

Sediments, Palau, Pacific Ocean

Black sandy mud, India

Seashell materials, Andaman Sea

Sea water, Sea of Japan

Green alga Ulva fenestrata, sea urchin Strongylocentrotus intermedius, Sea of Japan; sea water, Yellow Sea; sponge Tedania ignis, Bahamas

Aerobic Flexible rods

Aerobic Flexible rods

Fermentative Flexible rods

Aerobic Flexible rods

Aerobic Flexible rods

Aerobic Straight rods

0.4–0.5×10–100 or longer −

0.4–0.5×10–50

0.5×4–30

0.7×20–100

0.5–0.7×5–15

0.2–0.5×2.0–4.0

−

Aerobic Straight rods 0.3–0.5×10– 20 −

−

−

−

−

Orange + − +/+ + Saproxanthin −

Orange + − +/+ + Saproxanthin −

Orange + − +/+ − + nd

Orange + − +/− + Saproxanthin −

Olive-gray + nd +/− − nd nd

Orange + − +/+ + nd +

Pink-orange d − +/+ d nd −

− −/− −

− −/− d

− −/− −

+ −/− −

nd nd nd

− −/nd −

d −/d −

10–38

10–40

4–45 (30–37)

15–40 (25–30)

nd (25–30)

4–35 (25–28)

4–44 (20–30)

nd

nd

5–10 (7)

nd

5–10

nd

5.0–10.0 (7–8)

0.5–12 (4–6)

0.5–10 (4–7)

0–3.5

1–6

nd

1–6

0–16 (2–3)

−

−

+

−

+

−

−

+ − + + −/−

+ − − − −/−

+ − nd nd nd

+ + + − −/+

+ − nd − nd

+ + + − −/−

d − − − −/−

− + − + + + + nd − + + − 36.1 C15:1 iso, C15:0 iso, C17:0 iso 3-OH, C15:0 iso 3-OH and summed feature 3 nd

− + − + + + + nd − + + + 36–37 C15:1 iso, C15:0 iso, C17:0 iso 3-OH and C15:0

nd + − − nd nd nd nd nd + + − 43 C15:0 iso, C16:17c, C16:15c

− + − d d d d − − d + d 40–44 C15:1 iso, C15:0 iso, C17:0 iso 3-OH, C16:0 iso 3-OH, C15:0 iso 3-OH

nd

nd + + nd + nd + + nd + + + 40–43 C16:15c, C15:0 iso, C17:0 iso 3-OH, C15:0 iso 3-OH nd

− + + + + − −

nd

− + + + nd nd nd nd − + + + 40–42 C15:0 iso, C17:0 iso 3-OH, C15:0, C16:0 iso, C16:0 iso 3-OH, C20:4 w6, 9,12,15c, nd

Homospermidine, agmatine

nd

Spermidine

Spermidine

Spermidine

− + + − 44.5 Summed feature 3, C16:15c, C15 :0 nd

Spermidine

Phosphatidylethanolamine, diphosphatidylglycerol, unidentified phospholipids, ninhydrin-positive lipid Spermidine

Nedashkovskaya et al. (2005a-c, 2008, 2009), Reichenbach (1989a), Seo et al. (2009), Srisukchayakul et al. (2007), Takahashi et al. (2006), and Yoon et al. (2007).

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Family III. Flammeovirgaceae Table 113.  Characteristics that differentiate the mesophilic genera belonging to Flammeovirgaceae a

Characteristic Color of cell mass Aerobic Facultatively anaerobic Oxidase activity Catalase activity Urease activity Nitrate reduction H2S production DNA G+C content (mol%) Major fatty acids (%):d C15:0 iso C16:0 3-OH C16:1 w5c C17:0 iso 3-OH C20:4 w6,9,12,15c Major carotenoids: Saproxanthin Zeaxanthin

Flammeovirga b

Flexithrix c

Persicobacter c

Orange to reddish orange + − v v nd + + 31–36

Golden yellow to yellow + − + + w or − − − 35–37

Orange to pink + or − + − + or w − + − 42–44

30–54 8–12 2–9 0–3 8–25

22–28 0–1 39–44 0–2 0

47–56 3–7 1–5 6–17 2–7

+ −

− +

+ −

Symbols: +, >85% positive; −, 0–15% positive; w, weak reaction; nd, not determined. Data from Takahashi et al. (2006). c Unpublished data. d Percentage of total fatty acids. a

b

F. yaeyamensis

Growth at 10°C Growth at 35°C Optimum temperature (°C) Growth at pH 10 Hydrolysis of: Carboxymethylcellulose, DNA Casein Gelatin, inulin, Tween 80 Oxidase Catalase O-F test (glucose): Fermentation Oxidation Assimilation of: N-acetyl-d-glucosamine, d-melibiose l-Alanyl glycine, l-ornithine, l-threonine Glucose 6-phosphate Glucose 1-phosphate Monomethyl succinate l-Rhamnose, urocanic acid Acid production from: Arbutin, melibiose Mannitol, melezitose, sorbitol Methyl a-d-glucoside Raffinose, xylitol Rhamnose Ribose d-Xylose DNA G+C content (mol%)

F. arenaria

Characteristic

F. aprica

Table 114.  Characteristics differentiating the species of the genus Flammeovirga  a,b

− − 25 −

+ − 25 −

− + 30 +

+ w − + +

− w − − −

+ − + + or w d

+ −

− +

+ −

− − w + w −

+ w + + − −

+ + or w − − − +

− − − − − w + 34.2

+ w w w − w − 31.8

+ − w + or w + − + 33.4–35.7

a Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive; w, weak reaction. b

Data from Takahashi et al. (2006).

Genus II. Fabibacter

a-d-glucose, glucose 1-phosphate, glucose 6-phosphate, glycogen, glycyl-l-glutamic acid, dl-lactic acid, a-d-lactose, lactulose, maltose, d-mannose, monomethyl succinate, and d-trehalose, but negative for N-acetyl-d-glucosamine, l-alanyl glycine, d-melibiose, l-ornithine, l-rhamnose, l-threonine, and urocanic acid. Acid production (API 50CH system) is positive for N-acetylglucosamine, amygdalin, cellobiose, esculin, galactose, gentiobiose, glycogen, lactose, maltose, salicin, starch, and d-xylose, and weakly positive for glucose, l-fucose, mannose, and ribose, but negative for arbutin, mannitol, melibiose, melezitose, methyl a-d-glucoside, raffinose, rhamnose, sorbitol, and xylitol. The major cellular fatty acids are C15:0 iso, C20:4w6, 9, 12, 15c, C16:0 3-OH, and C14:0. DNA G+C content (mol%): 34.2 (HPLC). Type strain: JL-4, ATCC 23126, CIP 104807, IFO (now NBRC) 15941, JCM 21138, NCIMB 13348, NRRL B-14729. Sequence accession no. (16S rRNA gene): AB247553. 2. Flammeovirga arenaria (ex Lewin 1969) Takahashi, Suzuki and Nakagawa 2006, 2099VP (Microscilla arenaria Lewin 1969, 197) a.re.na¢ria. L. fem. adj. arenaria of or pertaining to sand, (referring to the source of the organism). The characteristics are as described for the genus and as listed in Table 113, with the following additional features. Cells are 0.5–0.9 mm wide and 2.0–40 mm long or longer. The cell mass is orange. Temperature range for growth, 10–30°C; optimum, 25°C. The pH range for growth, 6–8; optimum, 7. Growth occurs at 1–5% NaCl; optimum, 3%. Oxidase, catalase, and urease activities are negative. Agar, alginic acid, esculin, and starch are hydrolyzed, but not carboxymethylcellulose, cellulose, chitin, DNA, inulin, gelatin, Tween 80, or tyrosine. Casein is weakly degraded. H2S is produced. Indolenegative. The O-F test of glucose catabolism is oxidative. Utilization (with the Biolog GN2 microplate system) is positive for N-acetyl-d-glucosamine, cellobiose, a-cyclodextrin, dextrin, l-fucose, d-galactose, gentiobiose, a-d-glucose, glucose-1-phosphate, glucose 6-phosphate, l-glutamic acid, glycogen, glycyl-l-aspartic acid, glycyl-l-glutamic acid, a-dlactose, lactulose, maltose, d-mannose, and d-melibiose, and weakly positive for alaninamide, l-alanine, l-alanylglycine, l-aspartic acid, dl-lactic acid, l-ornithine, and l-threonine, but negative for monomethyl succinate, l-rhamnose, and urocanic acid. Acid production (API 50CH system) is positive for N-acetylglucosamine, amygdalin, arbutin, cellobiose, esculin, l-fucose, galactose, gentiobiose, glucose, glycogen,

447

lactose, maltose, mannose, melibiose, starch, and trehalose, and weakly positive for fructose, 5-ketogluconate, mannitol, methyl a-d-glucoside, methyl-a-d-mannoside, melezitose, raffinose, ribose, salicin, sorbitol, sucrose, and xylitol, but negative for rhamnose and d-xylose. The major cellular fatty acids are C15:0 iso, C14:0, C16:0 3-OH, C20:4 w6, 9, 12, 15c, C16:1 w5c, and C15:0 iso 3-OH. DNA G+C content (mol%): 31.8 (HPLC). Type strain: HJ-1, CIP 109101, JCM 21777, LMG 18922, NBRC 15982, NCIMB 1413. Sequence accession no. (16S rRNA gene): AB078078. 3. Flammeovirga yaeyamensis Takahashi, Suzuki and Nakagawa 2006, 2099VP ya.e.ya.men¢sis. N.L. fem. adj. yaeyamensis of or belonging to the Yaeyama Islands, from where the organisms were isolated. The characteristics are as described for the genus and as listed in Table 113, with the following additional features. Cells are 0.4–0.9 mm wide and 1.7–90 mm long or longer. The cell mass is orange. Temperature range for growth, 15–35°C; optimum, 30°C. The pH range for growth is 6–10; optimum, 7. Growth occurs at 1–5% NaCl; optimum, 3%. Oxidase ­activity is positive. Catalase activity differs among strains. Urease-negative. Agar, alginic acid, carboxymethylcellulose, DNA, esculin, inulin, gelatin, starch and Tween 80 are degraded, but not casein, cellulose, chitin, or tyrosine. H2S is produced. Indolenegative. The O-F test of glucose catabolism is fermentative. Utilization (with the Biolog GN2 microplate system) is positive for N-acetyl-d-glucosamine, cellobiose, d-galactose, gentiobiose, glycyl-l-glutamic acid, d-melibiose, l-rhamnose, and urocanic acid, and weakly ­positive for l-alanylglycine, a-d-glucose, maltose, l-ornithine, and l-threonine, but negative for glucose 1-phosphate, ­glucose 6-phosphate, and monomethyl succinate. Acid production (API 50CH system) is ­positive for N-acetylglucosamine, amygdalin, arbutin, cellobiose, esculin, galactose, gentiobiose, glucose, glycogen, ­lactose, maltose, mannose, melibiose, rhamnose, salicin, starch, and d-xylose, and positive or weakly positive for l-fucose, methyl a-d-glucoside, raffinose, and xylitol, but negative for mannitol, melezitose, ribose, and sorbitol. The major cellular fatty acids are C15:0 iso, C20:4 w6,9,12,15c, C16:0 3-OH, C15:0 iso 3-OH, C16:0, and C14:0. DNA G+C content (mol%): 33.4–35.7 (HPLC). Type strain: IR25-3, CIP 109099, NBRC 100898. Sequence accession no. (16S rRNA gene): AB247554.

Genus II. Fabibacter Lau, Tsoi, Li, Plakhotnikova, Dobretsov, Wu, Wong, Pawlik and Qian 2006, 1062VP The Editorial Board Fa.bi.bac¢ter. L. fem. n. faba bean; N.L. masc. n. bacter rod; N.L. masc. n. Fabibacter bean(-like) rod.

Curved rods 0.5 × 1.5 mm. Gram-stain-negative. Strictly aerobic. Gliding motility is present. Chemo-organotrophic. Colonies on marine agar are pink. The major respiratory quinone is MK-7. Flexirubin-type pigments are not produced. Acid is produced from carbohydrates. Oxidase- and catalase-positive. Tween 80

is hydrolyzed but not gelatin. NaCl is not required for growth. Isolated from the marine sponge Tedania ignis. DNA G+C content (mol%): 42.5. Type species: Fabibacter halotolerans Lau, Tsoi, Li, Plakhotnikova, Dobretsov, Wu, Wong, Pawlik and Qian 2006, 1062VP.

448

Family III. Flammeovirgaceae

Enrichment and isolation procedures Fabibacter halotolerans was isolated on marine agar* from the marine sponge Tedania ignis in the Bahamas. The strains appeared as pinkpigmented after 48 h of cultivation at 30°C on marine agar.

Differentiation of the genus Fabibacter from other genera Unlike Roseivirga echinicomitans and Roseivirga ehrenbergii, Fabibacter halotolerans exhibits a different cell shape (curved rod vs straight rod), gliding motility, no requirement for NaCl for growth, a higher range of tolerance to NaCl (0–12% vs 1–8%), an ability to hydrolyze Tween 80 but not gelatin, the production of acid from carbohydrates, a narrower temperature range for growth (12–36°C vs 4.0–39°C), and a slightly higher mol% G+C value for its DNA (42.5 vs 40.2–41.3). Moreover, the fatty acid profile of Fabibacter halotolerans differs from those described for the members of Roseivirga mainly by having larger quantities of C15:0 iso 3-OH, C16:0 iso 3-OH, and summed feature 3 (SF3) and by the additional presence of C14:0 iso 3-OH, C15:0 2-OH, and C15:0 3-OH (Lau et al., 2006).

Lau et  al. (2006) indicated that Fabibacter was also closely related to the Marincola seohaensis; however, Marinicola seohaensis has been formally reclassified to the genus Roseivirga as Roseivirga seohaensis (Lau et al., 2006). Fabibacter halotolerans differs from Roseivirga seohaensis by having a different cell shape (curved rod vs straight rod), orange colonies, lack of flexirubin pigments, a slightly higher mol% G+C value for its DNA (42.5 vs 40.3), no requirement for NaCl for growth, a higher range of tolerance to NaCl (0–12% vs 2–8%), the production of acid from carbohydrates, and by exhibiting activities for arginine dihydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, and a-mannosidase.

Taxonomic comments Lau et  al. (2006) found that the type strain of Fabibacter was most closely related to the members of the genera Marinicola (see above) and Roseivirga, with 93.1–93.3% 16S rRNA gene sequence similarity.

List of species of the genus Fabibacter 1. Fabibacter halotolerans Lau, Tsoi, Li, Plakhotnikova, Dobretsov, Wu, Wong, Pawlik and Qian 2006, 1062VP ha.lo.to¢le.rans. Gr. masc. n. hals, halos salt; L. part. adj. tolerans tolerating; N.L. part. adj. halotolerans salt-tolerating. The description is as given for the genus, with the following additional features. All the characteristics described below are based on cultures grown on marine agar at 30°C for 48  h. Colonies are pink, circular, 2.0–4.0 mm in diameter, convex with a smooth surface and an entire margin. No diffusible pigment is formed. Growth occurs between 12 and 36°C (optimum, 28–30°C) and between pH 5.0 and 10.0. Sodium ions are not required for growth. The organisms ­tolerate up to 12% NaCl. Predominant fatty acids (>5%) are C15:0 iso, C15:1, C15:0 iso 3-OH, C16:0 iso 3-OH, C17:0 iso 3-OH, and SF 3 (comprising C15:0 iso 2-OH and/or 16:1 w7c). These fatty acids represent 80.7% of the total. Produces acetoin, but not indole or H2S. Nitrate is not reduced. DNA and Tweens 20, 40, and 80 are hydrolyzed, but not agar, casein, carboxymethylcellulose, chitin, or gelatin. Starch is weakly hydrolyzed. The following enzyme activities are present: N-Acetyl-b-glucosaminidase, acid phosphatase, alkaline phosphatase, arginine dihydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, a-chymotrypsin, cystine arylamidase, leucine arylamidase, valine arylamidase, esterase (C4), esterase lipase (C8), lipase (C14), a-mannosidase, trypsin, and naphthol-AS-BI-phosphohydrolase. No

activities are exhibited for a-fucosidase, b-glucuronidase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, or urease. Growth occurs on the following sole carbon sources (API 20E, 20NE and 50CH systems): d-cellobiose, d-lactose, d-maltose, and starch. Acid is produced from the following sole carbon sources in the API 20E and 50CH systems: amygdalin, arbutin, d-cellobiose, esculin ferric citrate, d-galactose, d-glucose, gentiobiose, maltose, methyl a-d-glucopyranoside, d-raffinose, salicin, sucrose, starch, and d-trehalose. The following carbon sources are utilized (MicroLog 3 system): l-alaninamide, l-alanine, l-alanyl­ glycine, l-aspartic acid, d-cellobiose, dextrin, d-galacturonic acid, gentiobiose, a-d-glucose, d-glucose 6-phosphate, l-glutamic acid, glycogen, glycyl-l-aspartic acid, glycyl-l-glutamic acid, a-ketobutyric acid, a-ketoglutaric acid, a-ketovaleric acid, dl-lactic acid, a-d-lactose, lactulose, maltose, d-melibiose, methyl-b-d-glucoside, l-ornithine, l-proline, l-pyroglutamic acid, d-raffinose, succinamic acid, sucrose, d-trehalose, turanose, and l-threonine. In disc-diffusion tests, susceptibility is shown toward to ampicillin (1  mg), chloramphenicol (1 mg), penicillin (1 mg), streptomycin (0.1 mg), and tetracycline (5 mg), but not to kanamycin (tested up to 100 mg). Source: marine sponge Tedania ignis in the Bahamas. DNA G+C content (mol%): 42.5±0.3 (HPLC). Type strain: UST030701-097, JCM 13334, NRRL B-41220. Sequence accession no. (16S rRNA gene): DQ080995.

Genus III. Flexithrix Lewin 1970, 513VP emend. Hosoya and Yokota 2007b, 1087VP emend. Yasuyoshi Nakagawa Flex¢i.thrix. L. part. adj. flexus flexible; Gr. fem. n. thrix hair; N.L. fem. n. Flexithrix flexible hair (flexible rod).

Rods 0.4–0.9  mm wide and 1.5–70  mm long or longer. Motile by gliding. Nonsporeforming. Sheath is present or not. Gramstain-negative. Aerobic. Chemo-organotrophic. The cell mass

* Marine agar medium (Lau et al., 2006) composed of (g/l of filter-sterilized seawater): peptone (Oxoid), 5.0; and yeast extract (Oxoid), 3.0.

is golden yellow to yellow. Zeaxanthin is present as the major carotenoid pigment. Flexirubin-type pigments are absent. Oxidase- and catalase-positive. Urease activity is negative or weakly positive. Marine organisms. Seawater is required for growth; NaCl alone can substitute. The optimum pH for growth is 7. Nitrate is not reduced. H2S and indole are not produced. ­Esculin, gelatin, starch, and Tween 80 are degraded. The major

Genus III. Flexithrix

respiratory quinone is MK-7. Predominant cellular fatty acids are C15:0 iso and C16:1 w5c. DNA G+C content (mol%): 35–37. Type species: Flexithrix dorotheae Lewin 1970, 511AL emend. Hosoya and Yokota 2007b, 1087VP emend.

Further descriptive information All strains are negative for utilization of Biolog GN2 Microplate system substrates including cis-aconitic acid, adonitol, g-amino butyric acid, 2-amino ethanol, d-arabitol, bromosuccinic acid, 2,3-butanediol, dl-carnitine, citric acid, i-erythritol, formic acid, d-galactonic acid lactone, d-glucosaminic acid, glucose6-phosphate, glycerol, dl-a-glycerol phosphate, l-histidine, a-hydroxybutyric acid, b-hydroxybutyric acid, g-hydroxybutyric acid, p-hydroxy phenylacetic acid, hydroxy l-proline, inosine, m-inositol, itaconic acid, a-ketobutyric acid, a-ketoglutaric acid, a-ketovaleric acid, l-leucine, malonic acid, methylpyruvate, l-phenylalanine, phenyl ethylamine, propionic acid, putrescine, l-pyroglutamic acid, quinic acid, d-saccharic acid, sebacic acid, d-serine, d-sorbitol, succinamic acid, thymidine, Tween 40, Tween 80, uridine, urocanic acid, and xylitol. All strains are negative for acid production from API 50CH system substrates including adonitol, d-arabitol, l-arabitol, dulcitol, erythritol, d-fucose, gluconate, glycerol, glycogen, inositol, 2-keto-gluconate, 5-keto-glucotate, mannitol, b-methyl-d-xyloside, sorbitol, sorbose, d-tagatose, xylitol, and l-xylose. All strains are negative for API ZYM system substrates including b-glucosidase and b-glucuronidase. All strains are sensitive to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, nalidixic acid, nitrofurantoin, novobiocin, and oleandomycin, but resistant to bacitracin, carbenicillin, colistin sulfate, gentamicin, kanamycin, neomycin, polymyxin B, streptomycin, and tetracycline.

Enrichment and isolation procedures No special enrichment media have been designed for isolation of Flexithrix strains. Standard procedures to isolate marine bacteria can be applied. Colonies of Flexithrix are usually yellow and spread on agar media. Flexithrix strains have been isolated from marine environments at widely separated sites (Lewin, 1969, 1970; Lewin and Lounsbery, 1969). The type strain of Flexithrix dorotheae came from Ernakulum, India.

Maintenance procedures Cultures of Flexithrix strains can be preserved by freezing at lower than −80°C. For freezing, cells are suspended in Marine

449

broth 2216 (Difco) containing 10% glycerol or 7% DMSO. ­Flexithrix strains are rather sensitive to drying; however, they can be preserved by the liquid drying method using a protective medium such as SM2 or SM3 (see the chapter on Flammeovirga for the formulations), or by freeze drying.

Differentiation of the genus Flexithrix from other genera Characteristics differentiating the genus Flexithrix form other mesophilic genera in the family Flammeovirgaceae are listed in Table 113 in the chapter on Flammeovirga. The presence of C15:0 iso and C16:1 w5c as major fatty acids and zeaxanthin as a major carotenoid are useful characters to discriminate the genus Flammeovirga from other genera.

Taxonomic comments The genus Flexithrix was created by Lewin (1970) to accommodate a sheathed gliding bacteria strain QQ-3 (NBRC 15987T) isolated from brown silt from the coast of Kerala, India. Until the 1st edition of Bergey’s Manual of Systematic Bacteriology, the genus contained a single strain belonging to a single species Flexithrix dorotheae; however, Reichenbach (1989d) mentioned that the genus Flexithrix closely resembles Flexibacter aggregans (“Microscilla aggregans”) when it lacks sheathed filaments. Nakagawa et  al. (2002) reported that three strains belonging to Flexibacter aggregans (NBRC 15973, NBRC 15976T, and NBRC 15990) were closely related to the genus Flexithrix by 16S rRNA gene sequence analysis. Hosoya and Yokota (2007b) proposed reclassification of Flexibacter aggregans as Flexithrix dorotheae and emended the description of the genus Flexithrix and the species Flexithrix dorotheae. However, the emended descriptions did not cover characteristics of all Flexithrix strains, because they were based only on type strains (NBRC 15976T and NBRC 15987T). We have investigated all four strains belonging to the genus Flexithrix, phenotypically, chemotaxonomically, and genotypically (unpublished). The type strain of Flexithrix dorotheae exhibits more than 70% DNA–DNA relatedness with the three strains of Flexibacter aggregans, and they also share many not previously known phenotypic and chemotaxonomic characteristics. Those characteristics are described in this chapter. In addition, we found that presence of zeaxanthin (the major carotenoid) can be used to differentiate the genus Flexithrix from other mesophilic genera of Flammeovirgaceae. Therefore, we propose the emendation of the genus Flexithrix and the species Flexithrix dorotheae as indicated in this chapter.

List of species of the genus Flexithrix 1. Flexithrix dorotheae Lewin 1970, 511 emend. Hosoya and Yokota 2007b, 1087VP emend. (Microscilla aggregans Lewin 1969, 197; Flexibacter aggregans (Lewin 1969) Leadbetter 1974, 106AL) do.ro.the¢ae. N.L. gen. fem. n. dorotheae of Dorothy; named after a deceased technical assistant, Dorothy White. The characteristics are as described for the genus, with the following additional features. Cells are 0.4–0.9 mm wide and 1.5–70 mm long or longer. The cell mass is golden yellow to yellow. Growth occurs at 10–40°C; optimum, 25–30°C. The pH range for growth is 6–11; optimum, 7. Growth occurs in the presence of 1–5% NaCl; optimum, 3%. Some strains can grow AL

in the presence of 7% NaCl. Oxidase- and catalase-­positive. ­Esculin, gelatin, starch, and Tween 80 are degraded, but not agar, cellulose, chitin, inulin, and yeast cells. ­Alginate is strongly or weakly degraded. Sole nitrogen sources include ammonium sulfate, Casamino acids, ­peptone, sodium ­glutamate, and sodium nitrate. Utilization (with the Biolog GN2 microplate system) is positive for N-acetyl-d-galactosamine, alaninamide, l-alanylglycine, dextrin, d-fructose, l-fucose, d-galactose, dllactic acid, d-melibiose, b-methyl d-glucoside, d-raffinose, l-rhamnose, d-trehalose, and turanose, and ­positive or weakly positive for acetic acid, N-acetyl-d-glucosamine, lalanine, l-arabinose, l-asparagine, l-aspartic acid, gentiobiose,

450

Family III. Flammeovirgaceae

a-d-glucose, glucuronamide, d-glucuronic acid, l-glutamic acid, glycyl-l-aspartic acid, glycyl-l-glutamic acid, a-d-lactose, lactulose, maltose, l-ornithine, d-psicose, l-serine, sucrose, and l-threonine. Acid production (API 50CH system) is positive for cellobiose, esculin, fructose, galactose, lactose, maltose, melibiose, salicin, sucrose, trehalose, and d-turanose, and positive or weakly positive for N-acetylglucosamine, amygdalin, l-arabinose, arbutin, glucose, inulin, d-lyxose, mannose, melezitose, a-methyl-d-glucoside, a-methyl-d-mannoside,

raffinose, and d-xylose. Enzyme production (API ZYM system) is positive for N-acetyl-b-glucosaminidase, acid phosphatase, alkaline phosphatase, b-galactosidase, leucine allyl amidase, and valine allyl amidase. The major cellular fatty acids are C15:0 iso, C16:1 w5c, and C16:0. DNA G+C content (mol%): 35.9–36.1 (HPLC). Type strain: QQ-3, ATCC 23163, DSM 6795, NBRC 15987, NBRC 102100, NCIMB 1390. Sequence accession no. (16S rRNA gene): AB078077.

Genus IV. Persicobacter Nakagawa, Hamana, Sakane and Yamasato 1997, 221VP Yasuyoshi Nakagawa Per.si.co.bac¢ter. Gr. neut. n. persikon peach; N.L. masc. n. bacter rod; N.L. masc. n. Persicobacter peach rod, because the organism is a peach-colored rod.

Slender rods 0.4–0.6 mm wide and 0.9–30 mm long or longer. Motile by gliding. Nonsporeforming. Gram-stain-negative. Facultatively anaerobic. Chemo-organotrophic. Colonies spread and produce large gelase fields and deep craters in agar plates. The cell mass is pink to orange. Saproxanthin is present as the major carotenoid pigment. Flexirubin-type pigments are absent. Oxidase- and urease-negative. Strongly or weakly catalase-­positive. Marine organisms. Seawater is required for growth; NaCl alone can substitute. The optimum pH for growth is 7. Nitrate is reduced. H2S and indole are not produced. Agar, alginic acid, esculin, and gelatin are degraded. Starch is weakly degraded. The major respiratory quinone is MK-7. Predominant cellular fatty acids are C15:0 iso and C17:0 iso 3-OH. DNA G+C content (mol%): 42–44. Type species: Persicobacter diffluens (Reichenbach 1989b) Nakagawa, Hamana, Sakane, Yamasato 1997, 222VP [Cytophaga diffluens (ex Stanier 1940) Reichenbach 1989c, 495VP; Cytophaga diffluens Stanier 1940, 623 emend. mut. char. Lewin 1969, 197].

Further descriptive information All species are negative for acid production from API 50CH system substrates including adonitol, l-arabinose, d-arabitol, l-arabitol, dulcitol, erythritol, d-fucose, l-fucose, gluconate, glycerol, inositol, inulin, 2-ketogluconate, 5-ketoglucomate, mannitol, melezitose, b-methyl-d-xyloside, raffinose, ribose, sorbitol, d-tagatose, d-turanose, and l-xylose. All species are negative for the utilization of Biolog GN2 Microplate system substrates including N-acetyl-d-galactosamine, cis-aconitic acid, adonitol, d-alanine, g-amino butyric acid, 2-amino ethanol, l-arabinose, d-arabitol, bromosuccinic acid, 2,3-butanediol, dl-carnitine, citric acid, i-erythritol, formic acid, d-galactonic acid lactone, d-gluconic acid, glucuronamide, glycerol, dl-a-glycerol phosphate, l-histidine, a-hydroxybutyric acid, b-hydroxybutyric acid, g-hydroxybutyric acid, p-hydroxy phenylacetic acid, hydroxy-lproline, inosine, m-inositol, itaconic acid, a-ketoglutaric acid, malonic acid, d-mannitol, mono-methyl succinate, l-phenylalanine, phenyl ethylamine, propionic acid, d-psicose, putrescine, l-pyroglutamic acid, quinic acid, d-saccharic acid, sebacic acid, d-serine, d-sorbitol, succinamic acid, succinic acid, Tween 80, uridine, and xylitol. All species are sensitive to chloramphenicol, lincomycin, nitrofurantoin, novobiocin, oleandomycin,

and tetracycline, but resistant to colistin sulfate, gentamicin, kanamycin, polymxin B, and streptomycin.

Enrichment and isolation procedures No enrichment media have been designed for isolation of Persicobacter strains. Standard procedures to isolate marine bacteria can be applied. Colonies of Persicobacter are usually orange to pink, degrade agar, and spread rapidly. Persicobacter strains have been isolated from marine environments at widely separated sites (Lewin, 1969; Lewin and Lounsbery, 1969; Nakagawa, 2004). The type strain of Persicobacter diffluens came from Bombay, India. The type strain of “Persicobacter psychrovividus” was isolated from a clam, Ruditapes philippinarum, collected off a seacoast in Chiba, Japan.

Maintenance procedures Cultures of Persicobacter strains can be preserved by freezing at lower than −80°C. For freezing, cells are suspended in Marine broth 2216 (Difco) containing 10% glycerol or 7% DMSO. Persicobacter strains are rather sensitive to drying; however, they can be preserved by a liquid drying method using a protective medium SM2 or SM3 (see the chapter on Flammeovirga for formulations), or by freeze drying.

Differentiation of the genus Persicobacter from other genera Characteristics differentiating the genus Persicobacter from other mesophilic genera in the family Flammeovirgaceae are listed in Table 115 in the chapter on Flammeovirga. The presence of C15:0 iso and C17:0 iso 3-OH as major fatty acids and saproxanthin as the major carotenoid are useful characters to discriminate the genus Persicobacter from other genera.

Taxonomic comments The genus Persicobacter was created by Nakagawa et al. (1997) to accommodate the misclassified species Cytophaga diffluens. Until now, the genus contained a single species, Persicobacter diffluens. However, we isolated a new Persicobacter strain Asr22-19 from the gut of the clam (Ruditapes philippinarum) (Muramatsu et al., in press). This strain could grow both aerobically and anaerobically. Strain Asr22-19 with two other strains, NBRC 101035

Genus IV. Persicobacter Table 115.  Characteristics differentiating the species of the genus

Persicobacter  a,b Characteristic Growth at 5–10°C Optimum temperature (°C) Optimum NaCl concentration (%) Hydrolysis of: Carboxymethyl cellulose, yeast cells Chitin Assimilation of: N-Acetyl-d-glucosamine, l-glutamic acid l-Aspartic acid, l-ornithine l-Fucose, glucose 1-phosphate DNA G+C content (mol%)

P. diffluens “P. psychrovividus” − 30–35 3

+ 25 5

+ w

− +

−

+

− w 42.6–43.8

+/w − 42.0–42.7

451

and NBRC 101041, constituted a single independent species in the genus Persicobacter by DNA–DNA hybridization. In addition, those three strains could be phenotypically differentiated from Persicobacter diffluens (Table 115). The genus Persicobacter was originally described as oxidase-positive, catalase-negative, and aerobic. However, we found that both species produce catalase but not oxidase. Thus, we conclude that the three strains [Asr22-19 (NBRC 101262), NBRC 101035, and NBRC 101041] should be assigned to a new species of the genus Persicobacter for which the name Persicobacter psychrovividus sp. nov. is proposed.

Differentiation of the species of the genus Persicobacter Table 115 lists characteristics that distinguish the Persicobacter species from one another.

Symbols: +, >85% positive; −, 0–15% positive; w, weak reaction.

a

Data from Takahashi et al. (unpublished).

b

List of species of the genus Persicobacter 1. Persicobacter diffluens (Reichenbach 1989b) Nakagawa, Hamana, Sakane and Yamasato 1997, 222VP [Cytophaga diffluens (ex Stanier 1940) Reichenbach 1989c, 495VP; Cytophaga diffluens Stanier 1940, 623 emend. mut. char. Lewin 1969, 197] dif¢flu.ens. L. part. adj. diffluens flowing away. The characteristics are as described for the genus and as listed in Table 115, with the following additional features. Cells are 0.4–0.5  mm wide and 0.9–30  mm long or longer. The cell mass is pink to orange. Growth occurs at 15–40°C; optimum, 30–35°C. The pH range for growth is 6–11; optimum, 7. Growth occurs in the presence of 1–5% NaCl; optimum, 3%. Some strains can grow at 45°C, pH 3, or 7% NaCl. Oxidase-negative. Catalase-positive. Agar, alginate, carboxymethyl cellulose, DNA, esculin, gelatin, and yeast cells are degraded, but not cellulose and tyrosine. Starch and chitin are weakly degraded. Utilization (with the Biolog GN2

microplate system) is positive for l-alanine, l-asparagine, c­ ellobiose, a-cyclodextrin, dextrin, d-galactose, a-d-glucose, glycogen, glycyl-l-aspartic acid, glycyl-l-glutamic acid, dl-­lactic acid, a-d-lactose, maltose, d-mannose, l-threonine, and d-­trehalose, positive or weakly positive for alaninamide, d-­fructose, l-fucose, gentiobiose, glucose 1-phosphate, lactulose, and l-rhamnose, but negative for N-acetyl-d-­glucosamine, l-aspartic acid, and l-glutamic acid. Acid production (API 50CH system) is positive for N-acetyl ­glucosamine, amygdalin, arbutin, cellobiose, esculin, galactose, gentiobiose, ­glucose, glycogen, lactose, maltose, ­mannose, salicin, starch, and trehalose. The major cellular fatty acids are C15:0 iso, C17:0 iso 3-OH, C15:0 anteiso, and C16:0. DNA G+C content (mol%): 42.6–43.8 (HPLC). Type strain: strain B-1 Lewin, ATCC 49458, DSM 3658, JCM 8513, LMG 13036, NBRC 15940, NCIMB 1402. Sequence accession no. (16S rRNA gene): AB260929.

Other organisms 1. “Persicobacter psychrovividus” Muramatsu, Takahashi, Kaneyasu, Iino, Suzuki and Nakagawa (in press) psy.chro¢viv.idus. Gr. adj. psychros cold; L. adj. vividus full of life, vigorous, active; N.L. masc. adj. psychrovividus active at low temperatures. Cells are 0.4–0.6 mm wide and 0.9–6 mm long or longer. The cell mass is orange. Growth occurs at 5–45°C; optimum, 25°C. Some strains can grow at 45°C. The pH range for growth is 3–11; optimum 7. Growth occurs in the presence of 1–7% NaCl; optimum 5%. Oxidase-negative. Catalase-positive or weakly catalase-positive. Agar, alginate, esculin, gelatin, chitin, and Tween 80 are degraded, but not cellulose, carboxymethyl cellulose, yeast cells, or tyrosine. Starch is degraded weakly. Utilization (with the Biolog GN2 microplate system) is positive for N-acetyl-d-glucosamine, l-asparagine, a-cyclodextrin,

dextrin, d-galactose, ­gentiobiose, a-d-glucose, l-glutamic acid, glycogen, glycyl-l-aspartic acid, glycyl-l-glutamic acid, dl-lactic acid, a-d-lactose, maltose, d-mannose, l-threonine, and d-trehalose, and positive or weakly positive for l-alanine, l-alanylglycine, l-aspartic acid, cellobiose, a-ketobutyric acid, and lactulose, but negative for l-fucose and glucose 1-phosphate. Acid production (API 50CH system) is positive for N-acetyl glucosamine, amygdalin, arbutin, cellobiose, esculin, galactose, gentiobiose, glucose, glycogen, lactose, maltose, mannose, rhamnose, salicin, starch, trehalose, and d-xylose. The major cellular fatty acids are C15:0 iso, C17:0 iso 3-OH, C15:0 iso 3-OH, C16:0 3-OH, and C16:0. DNA G+C content (mol%): 42.0–42.7 (HPLC). Type strain: Asr 22-19, CIP 109100, NBRC 101262. Sequence accession no. (16S rRNA gene): AB260934.

452

Family III. Flammeovirgaceae

Genus V. Reichenbachiella Nedashkovskaya, Kim, Suzuki, Shevchenko, Lee, Lee, Park, Frolova, Oh, Bae, Park and Mikhailov 2005c, 2587VP (Reichenbachia Nedashkovskaya, Suzuki, Vysotskii and Mikhailov 2003, 82) Olga I. Nedashkovskaya and Makoto Suzuki Rei.chen.bach.i.el¢la. N.L. fem. dim. n. Reichenbachiella named after Hans Reichenbach, a German ­microbiologist who has made a great contribution to the taxonomy of bacteria belonging to the phylum Bacteroidetes.

Thin rods 0.5–0.7 × 5–15 mm. Motile by gliding. Produce nondiffusible, orange, flexirubin pigments. Chemo-organotrophs. Strictly aerobic. Oxidase-, catalase-, and alkaline phosphatasepositive. Agar, alginate, gelatin, casein, starch, urea, DNA, and Tween 20 are hydrolyzed, but casein, chitin, cellulose (CMcellulose and filter paper), and Tweens 40, 60, and 80 are not hydrolyzed. The major respiratory quinone is MK-7. Marine, isolated from coastal habitats in temperate latitudes. Growth does not occur without seawater or sodium ions. DNA G+C content (mol%): 44–45. Type species: Reichenbachiella agariperforans (Nedashkovskaya, Suzuki, Vysotskii and Mikhailov 2003) Nedashkovskaya, Kim, Suzuki, Shevchenko, Lee, Lee, Park, Frolova, Oh, Bae, Park and Mikhailov 2005c, 2587VP (Reichenbachia agariperforans Nedashkovskaya, Suzuki, Vysotskii and Mikhailov 2003, 83).

Further descriptive information Affiliation of the genus Reichenbachiella with known families of the phylum Bacteroidetes is not yet clear. It has no near neighbors among members of the phylum, and its closest relatives are the type strains of [Flexibacter] tractuosus and Persicobacter diffluens, with similarities of 16S rRNA genes of 88.1 and 86.5%, respectively. DNA–DNA hybridization values between Reichenbachiella and other representatives of the phylum Bacteroidetes are less than 86.2%. The main cellular fatty acids are straight-chain unsaturated and branched-chain saturated fatty C15:0 iso, C16:1 w5c and summed feature 3 comprising C15:0 iso 2-OH and/or C16:1 w7c. On Marine agar 2216 (Difco), Reichenbachiella agariperforans forms regular, round, smooth, bright orange colonies that are sunken into the agar. They have entire edges and a diameter of 3–5 mm after 48 h at 28°C. Reichenbachiella agariperforans grows on media containing 0.5% of a peptone and 0.1–0.2% yeast extract (Difco), prepared with natural or artificial seawater or supplemented with 2–3% NaCl. Good growth is observed on Marine agar 2216. Growth occurs at 4–35°C (optimum, 25–28°C). Growth occurs with 1–6% NaCl. The pH range for growth is 5.5–10.0 (optimum, 7.5–8.5).

The single available strain of Reichenbachiella agariperforans is susceptible to carbenicillin, oleandomycin, lincomycin, and tetracycline and resistant to ampicillin, benzylpenicillin, streptomycin, gentamicin, neomycin, and polymyxin B.

Enrichment and isolation procedures The organisms were isolated from seawater by direct plating on Marine agar. Natural or artificial seawater is suitable for their cultivation. Strain KMM 3525T has been grown on media containing 0.5% of a peptone and 0.1–0.2% yeast extract (Difco). The organisms may remain viable on Marine agar or other rich medium based on natural or artificial seawater for 1 week. They have survived storage at −80°C for at least 5 years.

Differentiation of the genus Reichenbachiella from other genera Reichenbachiella differs from its closest relative [Flexibacter] tractuosus ATCC 23168T by its production of flexirubin type pigments and agarase, NaCl requirement for growth, and the lower G+C content of its DNA. The production of catalase and flexirubin type pigments clearly distinguishes Reichenbachiella from its nearest neighbor, Persicobacter diffluens.

Taxonomic comments Previously, the genus Reichenbachia and the species Reichenbachia agariperforans were described to accommodate strain KMM 3525T (Nedashkovskaya et al., 2003). However, the name Reichenbachia is illegitimate according to the International Code of Bacteriological Nomenclature because it is a later homonym of a plant genus, and also a later homonym of an insect genus. Therefore, replacement of the names Reichenbachia and Reichenbachia agariperforans with Reichenbachiella and Reichenbachiella agariperforans, respectively, was proposed by Nedashkovskaya et al. (2005c). Nedashkovskaya et al. (2003) reported that strain KMM 3525T did not utilize carbohydrates but could produce acid from several of them (Nedashkovskaya et al., 2005c).

List of species of the genus Reichenbachiella 1. Reichenbachiella agariperforans (Nedashkovskaya et  al., 2003) Nedashkovskaya, Kim, Suzuki, Shevchenko, Lee, Lee, Park, Frolova, Oh, Bae, Park and Mikhailov 2005c, 2587VP (Reichenbachia agariperforans Nedashkovskaya, Suzuki, Vysotskii and Mikhailov 2003, 83). a.ga.ri.per.fo¢rans. N.L. n. agarum, agar (algal polysaccharide); L. part. adj. perforans perforating (making holes); N.L. part. adj. agariperforans, making holes in agar, i.e., bacterium making deep hollows in agar. Cells are 0.5–0.7 × 5–15 mm. Colonies are 3–5 mm in dia­ meter, circular, sunken into agar, shiny, with entire edges and

­ range-pigmented on solid media containing high ­nutrient o levels. Growth occurs at 4–35°C, with an optimum of 25–28°C and with 1–6% NaCl. Agar, starch, alginate, gelatin, DNA, urea, and Tween 20 are hydrolyzed, but cellulose (CM-­ cellulose and filter paper), chitin, casein, and Tweens 40, 60, and 80 are not. Acid is formed from l-arabinose, d-cellobiose, l-fucose, d-glucose, arbutin, esculin, and N-acetylglucosamine, but not from d-galactose, d-lactose, d-maltose, d-melibiose, l-rhamnose, d-sucrose, dl-xylose, adonitol, ­dulcitol, inositol, or mannitol. Lactose, mannose, and mannitol are utilized, but citrate, fumarate, and malate are not. Leucine- and

Genus VI. Roseivirga

v­ aline-­arylamidases, trypsin, naphthol-AS-BI-phosphohydrolase, a- and b-galactosidases, a- and b-glucosidases, N-acetylb-glucosaminidase, and alkaline and acid phosphatases are present, but not esterase (C4), esterase lipase (C8), lipase (C14), crystine arylamidase, a-chymotrypsin, b-glucuronidase, a-mannosidase, and a-fucosidase. Flexirubin pigments are produced. Nitrate is not reduced. H2S and indole production are negative. The predominant fatty acids are C15:0 iso (28.6%),

453

C16:1 w5c (21.9%), and summed feature 3 (20.7%; comprising C15:0 iso 2-OH and/or C16:1 w7). The single available strain was isolated from seawater sample collected in the Amursky Bay, the Gulf of Peter the Great, the Sea of Japan. Source: coastal marine environments. DNA G+C content (mol%): 44.5 (Tm). Type strain: JCM 11238, KMM 3525, NBRC 16625. Sequence accession no. (16S rRNA gene): AB058919.

Genus VI. Roseivirga Nedashkovskaya, Kim, Lee, Lysenko, Shevchenko, Frolova, Mikhailov, Lee and Bae 2005a, 232VP, emend. Nedashkovskaya, Kim, Lysenko, Park, Mikhailov, Bae and Park 2005b, 1800VP Olga I. Nedashkovskaya and Seung Bum Kim Ro.se.i.vir¢ga. L. adj. roseus pink-colored; L. fem. n. virga rod; N.L. fem. n. Roseivirga a pink-colored and rod-shaped marine bacterium.

Thin rods usually measuring 0.2–0.5 × 2.0–4.0  mm. Gliding motility can be observed. Produce nondiffusible pink-orange pigments. Flexirubin type of pigments can be formed. Chemoorganotrophs. Strictly aerobic. Can require seawater or sodium ions for growth. Oxidase-, catalase-, and alkaline phosphatasepositive. Arginine dihydrolase, lysine, and ornithine decarboxylases, and tryptophan deaminase are absent. Agar, casein, starch, urea, cellulose (CM-cellulose and filter paper), and chitin are not attacked, but gelatin, DNA, and Tweens may be decomposed. H2S and indole are not produced. The major respiratory ­quinone is MK-7. Marine, from coastal habitats. DNA G+C content (mol%): 40–45. Type species: Roseivirga ehrenbergii Nedashkovskaya, Kim, Lee, Lysenko, Shevchenko, Frolova, Mikhailov, Lee and Bae 2005a, 233VP.

Further descriptive information Phylogenetic analysis based on 16S rRNA gene sequencing indicates that the genus Roseivirga forms a distinct lineage within the phylum Bacteroidetes and the class Cytophagia (Figure 6). The genus Fabibacter is the closest relative of the Roseivirga species, showing sequence similarities of 93.9–95%. The range of 16S rRNA gene sequence similarities between the Roseivirga species is 96–99.8%. Dominant cellular fatty acids are straight-chain unsaturated and branched-chain unsaturated fatty acids C15:1 iso, C15:0 anteiso, C15:0 iso, C15:0 iso 3-OH, and C17:0 iso 3-OH. On Marine agar 2216 (Difco), colonies are circular, glistening, convex, smooth, with entire edges, pink or pink-orangepigmented, and 1–3 mm in diameter after 72 h at 25–30°C. All isolated strains have been grown on media containing 0.5% of a peptone and 0.1–0.2% yeast extract (Difco), prepared with natural or artificial seawater or supplemented by 2–3% NaCl. Growth occurs at 4–40°C, with 1–8% NaCl and at pH 5.5–10.0. Optimal growth is observed at 21–30°C, with 2–3% NaCl and at pH 7.0–8.0. According to API ZYM testing, all studied strains of the genus Roseivirga produce esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, a-chymotrypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, but not b-glucuronidase, and a-fucosidase. Strains of the genus Roseivirga are susceptible to carbenicillin, lincomycin, and oleandomycin, and resistant to ­benzylpenicillin,

gentamicin, kanamycin, neomycin, polymyxin B, streptomycin, and tetracycline. Sensitivity to ampicillin is variable. The strains of the genus Roseivirga inhabit coastal marine environments. They were isolated from seaweeds, seawater, sponges, and echinoderms in the temperate latitudes.

Enrichment and isolation procedures The roseivirgas were isolated by direct or standard dilution plating technique on marine agar (Difco). Natural or artificial seawater is suitable for cultivation of the representatives of some species. They can grow on Casamino acids, peptone, and tryptone as the sole carbon and nitrogen sources (Yoon et al., 2005). All isolated strains have been grown on media containing 0.5% peptone and 0.1–0.2% yeast extract (Difco). Strains remain viable on Marine agar (Difco) or other rich medium based on natural or artificial seawater for one or several weeks. They survive storage at −80°C for at least 5 years.

Differentiation of the genus Roseivirga from other genera The genus Roseivirga and its closest relative, Fabibacter halotolerans, have many common traits (Lau et al., 2006; Nedashkovskaya et al., 2005a). However, Roseivirga strains can be differentiated from Fabibacter halotolerans by their inability to hydrolyze starch and by their distinctive fatty acid composition (Table 116).

Taxonomic comments Since a description of the genus Roseivirga was published (Nedashkovskaya et al., 2005a), Yoon et al. (2005) have described a novel genus, Marinicola. We have found high levels of 16S rRNA gene similarity between Marinicola seohaensis SW-152T and Roseivirga ehrenbergii KMM 6017T and Roseivirga echinicomitans (99.8 and 99.1%, respectively) (Nedashkovskaya et al., 2005b). Consequently, we proposed placement of the genus Marinicola in the genus Roseivirga. This conclusion was supported by the results of genomic, chemotaxonomic, and phenotypic analyses, which revealed many common features between members of the genera Marinicola and Roseivirga. These data have been incorporated in the emended description of the genus Roseivirga (Nedashkovskaya et al., 2005b), and Marinicola seohaensis has been formally reclassified to the genus Roseivirga as Roseivirga seohaensis (Lau et al., 2006). Notably, DNA–DNA hybridization experiments were not conducted for genomic comparison

454

Family III. Flammeovirgaceae

1.7

2.9 1.9 0.8 13.1 20.2 0.8 0.3 0.4 2.4 20.2 4.1 0.5 1.8 2.0 4.2 1.4 2.0 1.1 12.1 1.0 1.0

5.2

0.7 –

2.4 33.5 1.1

12.5 18.6

1.6 4.7 1.1 2.5 18.3

R. seohaensis LMG 1343T

F. halotolerans UST 030701-097T

3.2 0.4 0.1 4.5 24.4 0.9 0.2 0.2 1.8 34.2 3.0 0.6 1.1 0.9 4.1 1.6 0.9 – 7.7

R. spongicola UST 030701-084T

C13:0 iso C14:0 iso C14:0 iso 3-OH C15:0 anteiso C15:0 iso C15:0 C15:0 2-OH C15:0 3-OH C15:1 anteiso C15:1 iso C15:0 iso 3-OH C16:0 C16:0 iso C16:1 iso C16:0 iso 3-OH C16:0 3-OH C17:0 2-OH C17:1 iso w9c C17:0 iso 3-OH C17:0 iso Summed feature 3c

R. echinicomitans KMM 6058T

Fatty acid

R. ehrenbergii KMM 6017T

Table 116.  Fatty acid content of Roseivirga species and Fabibacter halotolerans a,b

1.9

20.5 5.6 1.2 7.2 1.8

11.2 4.8

– 12.5 4.9

1.9 1.3 0.8 14.2 12.5

2.0 – 1.2 – 10.1 10.8 18.3 – 5.5

1.2 1.2 12.7 1.2 1.3 – 9.3 0.5 13.7

Values represent the percentage of the total fatty acids. Fatty acids amounting to less than 1% in all taxa are not given.

a

Data are taken from Lau et al. (2006), Nedashkovskaya et al. (2005a, b), and Yoon et al. (2005).

b

Summed features consist of one or more fatty acids that could not be separated by the Microbial Identification System. Summed feature 3: C15:0 iso 2-OH and/or 16:1w7.

c

of the type strains of Roseivirga seohaensis SW-152T and its nearest phylogenetic neighbors, Roseivirga ehrenbergii KMM 6017T and Roseivirga echinicomitans KMM 6058T. Therefore, an additional study on the determination of a level of DNA–DNA reassociation between the above-mentioned Roseivirga strains is needed to clarify the species status of strain SW-152T.

Differentiation of the species of the genus Roseivirga Although species of the genus Roseivirga are very close phylogenetically to each other and have similar fatty acid compositions (Table 116) and phenotypic characteristics, they can be differentiated by a set of phenotypic traits (Table 117).

List of species of the genus Roseivirga 1. Roseivirga echinicomitans Nedashkovskaya, Kim, Lysenko, Park, Mikhailov, Bae and Park 2005b, 1799VP e.chi.ni.co¢mi.tans. L. n. echinus -i sea urchin; L. pres. part. comitans (from L. v. comito) accompanying; N.L. part. adj. echinicomitans accompanying a sea urchin. Cells are 0.3–0.5 mm wide and 2.1–3.2 mm long. On Marine agar, colonies are 2–3 mm in diameter, circular, shiny with entire edges, and pink-pigmented. The optimal temperature for growth is 21–23°C. Acid is not formed from l-arabinose, d-cellobiose, l-fucose, d-galactose, d-glucose, d-lactose, d-maltose, d-melibiose, l-raffinose, l-rhamnose, l-sorbose, d-sucrose, dl-xylose, N-acetylglucosamine, citrate, adonitol, dulcitol, glycerol, inositol, or mannitol. l-Arabinose, d-­lactose, d-mannose, mannitol, inositol, malonate, gluconate, caprate, malate, and phenylacetate are not utilized.

­ roduces (API ZYM kit) cystine arylamidase and trypsin. UtiP lizes (Microlog GN2 [Biolog] system) i-erythritol, d-galactose, d-sorbitol, l-leucine, l-ornithine, l-phenylalanine, l-proline, l-pyroglutamic acid, and 2,3-butanediol but not a-cyclodextrin, dextrin, glycogen, Tween 80, N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, adonitol, d-arabitol, cellobiose, d-fructose, l-fucose, gentiobiose, a-d-glucose, m-inositol, a-lactose, a-d-lactose lactulose, d-mannitol, d-melibiose, methyl b-d-glucoside, psicose, d-raffinose, l-rhamnose, sucrose, d-trehalose, turanose, xylitol, methylpyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, citric acid, formic acid, d-galactonic acid, d-gluconic acid, d-glucosaminic acid, d-glucuronic acid, a-hydroxybutyric acid, b-hydroxybutyric acid, g-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, a-ketobutyric acid, a-ketoglutaric acid, dl-lactic acid, malonic acid, propionic acid, quinic

455

Genus VI. Roseivirga

R. saehaensis

R. spongicola

Gliding motility Flexirubin type pigments production Nitrate reduction Acetoin production Temperature range for growth (°C) Salinity range for growth (%) Hydrolysis of: Esculin, gelatin DNA Tween 20 Tween 40 Tween 80 Acid from amygdalin Utilization of: Citrate d-Galacturonic acid, glycerol, dl-a-glycerol phosphate, inosine, thymidine l-Alanine l-Glutamic acid a-Ketovaleric acid Assimilation of: Glucose, N-acetylglucosamine, maltose, adipate Enzyme activity: a-Galactosidase b-Galactosidase a-Glucosidase b-Glucosidase N-Acetyl-b-glucosaminidase Lipase (C14) a-Mannosidase Susceptibility to: Ampicillin Benzylpenicillin, streptomycin Tetracycline DNA G+C content (mol%)

R. erhenbergii

Characteristic

R. echinicomitans

Table 117.  Phenotypic characteristics differentiating Roseivirga species a,b

− − + − 4–31 1–8

− − − − 4–39 1–8

+ + − 4–40 1–8

+ − − + 12–44 0–16

+ − − + − +

+ + + − − −

− − + + + nd

+ + + + + −

+ +

− −

nd nd

+ −

− − −

+ + −

nd − nd

− − +

+

−

−

−

+ + + + + − −

− + + − − + +

− − − − + + −

− − + + + + −

+ + + 41.3

+ − − 40.2

− − + 40.3

− − − 43–45

Symbols: see standard definitions; nd, not detected.

a

Data are taken from Lau et al. (2006), Nedashkovskaya et al. (2005a, b), and Yoon et al. (2005).

b

acid, d-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, ­alaninamide, d-alanine, l-alanyl glycine, l-asparagine, l-aspartic acid, glycyl l-aspartic acid, glycyl l-glutamic acid, l-histidine, hydroxy-l-proline, d-serine, l-serine, l-threonine, dl-carnitine, g-aminobutyric acid, urocanic acid, uridine, phenylethylamine, putrescine, 2-aminoethanol, glucose 1-phosphate, and glucose 6-phosphate. Only one strain has been isolated. Source: the sea urchin Strongylocentrotus intermedius in the Troitsa Bay, Sea of Japan. DNA G+C content (mol%): 41.3 (Tm). Type strain: KCTC 12370, KMM 6058, LMG 22587. Sequence accession no. (16S rRNA gene): AY753206.

2. Roseivirga ehrenbergii Nedashkovskaya, Kim, Lee, Lysenko, Shevchenko, Frolova, Mikhailov, Lee and Bae 2005a, 233VP eh.ren.ber¢gi.i. N.L. gen. masc. n. ehrenbergii of Ehrenberg, named after the German biologist Christian Gottfried Ehrenberg (1795–1876) for his contribution to the development of microbiology. Cells are 0.3–0.5 mm wide and 2.1–3.2 mm long. On Marine agar, colonies are 2–4 mm in diameter, circular, shiny with entire edges and pink-pigmented. Optimal growth occurs at 23–25°C. No acid is formed from l-arabinose, d-cellobiose, l-fucose, d-galactose, d-glucose, d-lactose, d-maltose, d-melibiose, l-raffinose, d-sucrose, l-rhamnose, dl-xylose,

456

Family III. Flammeovirgaceae

adonitol, dulcitol, glycerol, inositol, mannitol, or sorbitol. The following substrates are not utilized: d-lactose, d-mannose, d-sucrose, mannitol, inositol, sorbitol, malonate, ­gluconate, caprate, malate, or phenylacetate. Utilizes (Microlog GN2 [Biolog] system) glycogen, l-arabinose, methyl pyruvate, a-ketoglutaric acid, glucuronamide, alaninamide, l-alanylglycine, l-asparagine, l-aspartic acid, glycyl-l-aspartic acid, l-histidine, l-ornithine, l-proline, l-serine, l-threonine, and putrescine. API ZYM galleries indicate the presence of ­cystine arylamidase and trypsin. Source: the green alga Ulva fenestrata collected in the ­Pallada Bay of the Gulf Peter the Great of the Sea of Japan. DNA G+C content (mol%): 40.2 (Tm). Type strain: JCM 13514, KCTC 12282, KMM 6017, LMG 22567. Sequence accession no. (16S rRNA gene): AY608410. 3. Roseivirga seohaensis Lau, Tsoi, Li, Plakhotnikova, Dobretsov, Wu, Wong, Pawlik and Qian 2006, 1064VP (Marinicola seohaensis Yoon, Kang, Lee and Oh 2005, 862VP) seo.ha.en¢sis. N.L. fem. adj. seohaensis of Seohae, the Korean name for the Yellow Sea in Korea, from where the organism was isolated. Cells are 0.2–0.3 mm wide and 2.0–4.0 mm long and move by gliding. On marine agar, colonies are 1–2 mm in diameter, circular, shiny with entire edges, and pink-orange in color. Optimal growth is observed at 30°C and with 2–3% NaCl. Tween 60 is hydrolyzed but not hypoxanthine, xanthine, or l-tyrosine. No acid is formed from l-arabinose, d-cellobiose, d-fructose, l-fucose, d-galactose, d-glucose, d-lactose, d-maltose, d-mannose, d-melibiose, d-melezitose, d-raffinose, l-rhamnose, d-ribose, d-sucrose, d-trehalose, d-xylose, N-acetylglucosamine, adonitol, d-sorbitol, myo-­inositol, or d-mannitol. The following substrates are not utilized: l-arabinose, d-lactose, d-mannose, d-sucrose, d-ribose, inositol,

References Hamana, K. and Y. Nakagawa. 2001. Polyamine distribution profiles in the eighteen genera phylogenetically located within the Flavobacterium-Flexibacter-Cytophaga complex. Microbios 106: 7–17. Hamana, K., T. Itoh, Y. Benno and H. Hayashi. 2008. Polyamine distribution profiles of new members of the phylum Bacteroidetes. J. Gen. Appl. Microbiol. 54: 229–236. Hosoya, S. and A. Yokota. 2007a. Flammeovirga kamogawensis sp. nov., isolated from coastal seawater in Japan. Int. J. Syst. Evol. Microbiol. 57: 1327–1330. Hosoya, S. and A. Yokota. 2007b. Reclassification of Flexibacter aggregans (Lewin 1969) Leadbetter 1974 as a later heterotypic synonym of Flexithrix dorotheae Lewin 1970. Int. J. Syst. Evol. Microbiol. 57: 1086–1088. Lau, S.C., M.M. Tsoi, X. Li, I. Plakhotnikova, S. Dobretsov, M. Wu, P.K. Wong, J.R. Pawlik and P.Y. Qian. 2006. Description of Fabibacter halotolerans gen. nov., sp. nov. and Roseivirga spongicola sp. nov., and reclassification of [Marinicola] seohaensis as Roseivirga seohaensis comb. nov. Int. J. Syst. Evol. Microbiol. 56: 1059–1065. Leadbetter, E.R. 1974. Genus II Flexibacter. In Bergey’s Manual of Determinative Bacteriology, 8th edn (edited by Buchanan and Gibbons). Williams & Wilkins, Baltimore, pp. 105–107.

mannitol, sorbitol, dl-aspartate, l-leucine, or l-proline. ­Cystine arylamidase and trypsin are not produced. Susceptible to chloramphenicol, doxycycline, and erythromycin. Polar ­lipids are phosphatidylethanolamine, diphosphatidylglycerol, an unidentified glycolipid, an unidentified phospholipid, and a ninhydrin-positive lipid. Source: seawater of the Yellow Sea, Korea. DNA G+C content (mol%): 40.3 (HPLC). Type strain: SW-152, KCTC 12312, JCM 12600. Sequence accession no. (16S rRNA gene): AY739663. 4. Roseivirga spongicola Lau, Tsoi, Li, Plakhotnikova, Dobretsov, Wu, Wong, Pawlik and Qian 2006, 1063VP spon.gi¢co.la. L. n. spongos -i sponge; L. masc./fem. suff. -cola (from L. n. incola) inhabitant; N.L. n. (nominative in apposition) spongicola inhabitant of sponges. Cells are 0.5 mm in width and 2.0 mm in length and move by gliding. On Marine agar, colonies are pink, 2–4  mm in diameter, and circular with entire edges. Growth occurs at pH 5.0–10.0. Optimal growth is observed at 20–30°C. Tween 60 is hydrolyzed but not hypoxanthine, xanthine, or l-tyrosine. No acid is formed from l-arabinose, d-­cellobiose, d-fructose, l-fucose, d-galactose, d-glucose, d-lactose, d-maltose, d-mannose, d-melibiose, d-melezitose, d-raffinose, l-rhamnose, d-ribose, d-sucrose, d-trehalose, d-xylose, N-acetylglucosamine, adonitol, d-sorbitol, myo-inositol, or d-mannitol. The following substrates are not utilized: l-arabinose, d-lactose, d-mannose, d-sucrose, d-ribose, inositol, mannitol, sorbitol, dl-aspartate, l-leucine, or l-proline. Cystine arylamidase and trypsin are not produced. Susceptible to chloramphenicol, doxycycline, and erythromycin. Source: the marine sponge Tedania ignis in the Bahamas. DNA G+C content (mol%): 43.7 (HPLC). Type strain: UST030701-084, JCM 13337, NRRL B-41219. Sequence accession no. (16S rRNA gene): DQ080996.

Lewin, R.A. 1969. A classification of Flexibacteria. J. Gen. Microbiol. 58: 189–206. Lewin, R.A. 1970. Flexithrix dorotheae gen. et sp. nov. (Flexibacterales); and suggestions for reclassifying sheathed bacteria. Can. J. Microbiol. 16: 511–515. Lewin, R.A. and D.M. Lounsbery. 1969. Isolation, cultivation and ­characterization of Flexibacteria. J. Gen. Microbiol. 58: 145–170. Nakagawa, Y. 2004. Taxonomic studies of Cytophaga-like bacteria (in Japanese). Microbiol. Cult. Coll. 20: 41–51. Nakagawa, Y., K. Hamana, T. Sakane and K. Yamasato. 1997. Reclassification of Cytophaga aprica (Lewin 1969) Reichenbach 1989 in Flammeovirga gen. nov. as Flammeovirga aprica comb. nov. and of Cytophaga diffluens (ex Stanier 1940; emend. Lewin 1969) Reichenbach 1989 in Persicobacter gen. nov. as Persicobacter diffluens comb. nov. Int. J. Syst. Bacteriol. 47: 220–223. Nakagawa, Y., T. Sakane, M. Suzuki and K. Hatano. 2002. Phylogenetic structure of the genera Flexibacter, Flexithrix, and Microscilla deduced from 16S rRNA sequence analysis. J. Gen. Appl. Microbiol. 48: 155–165. Nedashkovskaya, O.I., M. Suzuki, M.V. Vysotskii and V.V. Mikhailov. 2003. Reichenbachia agariperforans gen. nov., sp. nov., a novel marine

Family I. Rhodothermaceae bacterium in the phylum Cytophaga-Flavobacterium-Bacteroides. Int. J. Syst. Evol. Microbiol. 53: 81–85. Nedashkovskaya, O.I., S.B. Kim, D.H. Lee, A.M. Lysenko, L.S. Shevchenko, G.M. Frolova, V.V. Mikhailov, K.H. Lee and K.S. Bae. 2005a. Roseivirga ehrenbergii gen. nov., sp. nov., a novel marine bacterium of the phylum ‘Bacteroidetes’, isolated from the green alga Ulva fenestrata. Int. J. Syst. Evol. Microbiol. 55: 231–234. Nedashkovskaya, O.I., S.B. Kim, A.M. Lysenko, M.S. Park, V.V. Mikhailov, K.S. Bae and H.Y. Park. 2005b. Roseivirga echinicomitans sp. nov., a novel marine bacterium isolated from the sea urchin Strongylocentrotus intermedius, and emended description of the genus Roseivirga. Int. J. Syst. Evol. Microbiol. 55: 1797–1800. Nedashkovskaya, O.I., S.B. Kim, M. Suzuki, L.S. Shevchenko, M.S. Lee, K.H. Lee, M.S. Park, G.M. Frolova, H.W. Oh, K.S. Bae, H.Y. Park and V.V. Mikhailov. 2005c. Pontibacter actiniarum gen. nov., sp. nov., a novel member of the phylum ‘Bacteroidetes’, and proposal of Reichenbachiella gen. nov. as a replacement for the illegitimate prokaryotic generic name Reichenbachia Nedashkovskaya et  al. 2003. Int. J. Syst. Evol. Microbiol. 55: 2583–2588. Nedashkovskaya, O.I., Kim S.B., Lysenko A.M., Kalinovskaya N.I. and Mikhailov V.V. 2008. Reclassification of Roseivirga seohaensis (Yoon et al. 2005) Lau et al. 2006 as a later synonym of Roseivirga ehrenbergii Nedashkovskaya et al. 2005 and emendation of the species description. Int. J. Syst. Evol. Microbiol. 58: 1194–1197. Nedashkovskaya, O.I., M. Vancanneyt, Kim S.B. and K.S. Bae. 2009. Reclassification of ‘Microscilla tractuosa’ (ex Lewin 1969) Reichenbach 1989 and ‘Microscilla sericea’ (ex Lewin 1969) Reichenbach 1989 in the genus Marivirga gen. nov. as Marivirga tractuosa nom. rev., comb. nov. and Marivirga sericea nom. rev., comb. nov. Int. J. Syst. Evol. Microbiol. DOI: ijs.0.016121-0. Reichenbach, H. 1989a. Order Cytophagales Leadbetter 1974, 99AL. In Bergey’s Manual of Systematic Bacteriology, 8th edn, vol. 3 (edited by Staley, Bryant, Pfennig and Holt). Williams & Wilkins, Baltimore, pp. 2011–2073.

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Reichenbach, H. 1989b. Genus I. Cytophaga. In Bergey’s Manual of Systematic Bacteriology, vol. 3 (edited by Staley, Bryant, Pfennig and Holt). Williams & Wilkins, Baltimore, pp. 2015–2050. Reichenbach, H. 1989c. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 31. Int. J. Syst. Bacteriol. 39: 495–497. Reichenbach, H. 1989d. Genus III Flexithrix. In Bergey’s Manual of Systematic Bacteriology, vol. 3 (edited by Staley, Bryant, Pfennig and Holt). Williams & Wilkins, Baltimore, pp. 2058–2060. Sakane, T., T. Nishii, T. Itoh and K. Mikata. 1996. Protocols for longterm preservation of microorganisms by L-drying (in Japanese). Microbiol. Cult. Coll. 12: 91–97. Seo, H.S., K.K. Kwon, S.H. Yang, H.S. Lee, S.S. Bae, J.H. Lee and S.J. Kim. 2009. Marinoscillum gen. nov., a member of the family ‘Flexibacteraceae’, with Marinoscillum pacificum sp. nov. from a marine sponge and Marinoscillum furvescens nom. rev., comb. nov. Int. J. Syst. Evol. Microbiol. 59: 1204–1208. Srisukchayakul, P., C. Suwanachart, Y. Sangnoi, A. Kanjana-Opas, S. Hosoya, A. Yokota and V. Arunpairojana. 2007. Rapidithrix thailandica gen. nov., sp. nov., a marine gliding bacterium isolated from samples collected from the Andaman sea, along the southern coastline of Thailand. Int. J. Syst. Evol. Microbiol. 57: 2275–2279. Stanier, R.Y. 1940. Studies on the cytophagas. J. Bacteriol. 40: 619–635. Takahashi, M., K. Suzuki and Y. Nakagawa. 2006. Emendation of the genus Flammeovirga and Flammeovirga aprica with the proposal of Flammeovirga arenaria nom. rev., comb. nov. and Flammeovirga yaeyamensis sp. nov. Int. J. Syst. Evol. Microbiol. 56: 2095–2100. Yoon, J.H., S.J. Kang, C.H. Lee and T.K. Oh. 2005. Marinicola seohaensis gen. nov., sp. nov., isolated from sea water of the Yellow Sea, Korea. Int. J. Syst. Evol. Microbiol. 55: 859–863. Yoon, J., S. Ishikawa, H. Kasai and A. Yokota. 2007. Perexilibacter aurantiacus gen. nov., sp. nov., a novel member of the family ‘Flammeovirgaceae’ isolated from sediment. Int. J. Syst. Evol. Microbiol. 57: 964–968.

Order II. Incertae sedis The genera Rhodothermus and Salinibacter were previously assigned to the “Crenotrichaceae” by Garrity et  al. (2005), but subsequent ­analyses transferred Crenothrix to the Proteobacteria. Phylogenetic analyses of the 16S rRNA suggests that these genera represent a very deep group and are only distantly related to any of the previously described orders within the Bacteroidetes. In view of their ambiguous status, they have been assigned to their own order incertae sedis.

Reference Garrity, G.M., J.A. Bell and T. Lilburn. 2005. The Revised Road Map to the Manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, The Proteobacteria, Part A, Introductory Essays (edited by Brenner, Krieg, Staley and Garrity). Springer, New York, pp. 159–220.

Family I. Rhodothermaceae fam. nov. Wolfgang Ludwig, Jean Euzéby and William B. Whitman Rho.do.ther.ma.ce¢a.e. N.L. masc. n. Rhodothermus type genus of the family; suff. -aceae ending to denote a family; N.L. fem. pl. n. Rhodothermaceae the Rhodothermus family. Straight or curved rods that stain Gram-negative. Nonmotile or motile with flagella. Chemoheterotrophic aerobes that preferentially utilize sugars or amino acids. Catalase-positive. Most strains form red or orange colonies, due to a carotenoid ­pigment. Moderately (0.6 to >6% NaCl) or extremely halophilic (>15% NaCl). May be thermophilic, growing at

54–77°C, or mesophilic Common habitats include hot springs and salterns. DNA G+C content (mol%): 64–68. Type genus: Rhodothermus Alfredsson, Kristjansson, Hjörleifsdottir and Stetter 1995, 418VP (Effective publication: Alfredsson, Kristjansson, Hjörleifsdottir and Stetter 1988, 304.).

458

Family I. Rhodothermaceae

Genus I. Rhodothermus Alfredsson, Kristjansson, Hjörleifsdottir and Stetter 1995, 418VP (Effective publication: ­Alfredsson, Kristjansson, Hjörleifsdottir and Stetter 1988, 304.) The Editorial Board Rho.do.ther¢mus. Gr. n. rhodon rose; Gr. masc. adj. thermos hot; N.L. masc. n. Rhodothermus the red ­thermophile.

Straight rods about 0.5 × 2.0–2.5 mm, with curved ends. Occur singly, never in chains or filaments. Nonmotile. Gram-stain­negative. A slime capsule is formed on carbohydrate-rich medium. Most strains form red colonies, due to a carotenoid pigment. Colonies are low convex, 3–4 mm in diameter with an entire edge. Aerobic. Thermophilic, growing at 54–77°C. Neutrophilic. Heterotrophic. Growth is strictly salt-dependent, occurring in the range of 0.6 to >6% NaCl. Catalase-positive. The oxidase reaction varies among strains. No dissimilatory nitrate reduction occurs. Growth occurs on most common sugars. The major cellular fatty acids are C15 iso, C15 anteiso, C17 iso and C17 anteiso. The major quinone is menaquinone 7. The habitat is submarine freshwater alkaline hot springs, marine hot springs, geothermal sites, and borehole effluents. DNA G+C content (mol%): 64–66 (Tm). Type species: Rhodothermus marinus Alfredsson, Kristjansson, Hjörleifsdottir and Stetter 1995, 418VP (Effective publication: Alfredsson, Kristjansson, Hjörleifsdottir and Stetter 1988, 304; Rhodothermus obamensis Sako, Takai, Ishida, Uchida and Katayama 1996, 1103.).

Further descriptive information Although Rhodothermus marinus is considered to be nonmotile, the presence of a polar flagellum has been reported (Nunes et al., 1992a). Red colonies are formed by most strains of Rhodothermus marinus, but two distinct subgroups of colorless isolates that were correlated with their geographic origin were found by Petursdottir et al. (2000). Rhodothermus marinus accumulates osmolytes in response to increasing salinity of its growth medium. Mannosylglycerate, which occurs in some hyperthermophilic organisms and protects enzymes against inactivation by temperature and freezedrying (Ramos et  al., 1997), accumulates in Rhodothermus marinus in response to growth at supraoptimal temperature and salinity. The amide form, mannosylglyceramide, which has been found only in Rhodothermus marinus (Santos and da Costa, 2002), accumulates exclusively in response to salt stress (Nunes et al., 1995; Silva et al., 1999). Martins et al. (1999) elucidated the biosynthetic pathways of mannosylglycerate in Rhodothermus marinus and characterized the enzyme mannosylglycerate ­synthase, which catalyzes the final step in the synthesis of mannosylglycerate. Rhodothermus marinus is the only organism known to have two distinct pathways for the synthesis of mannosylglycerate: a two-step pathway and a single-step pathway. The level of mannosylglycerate synthase involved in the single-step pathway was selectively enhanced by heat stress, whereas mannosylglyceramide was overproduced in response to osmotic stress. The two alternative pathways for the synthesis of mannosylglycerate are regulated differently at the level of expression to play specific roles in the adaption of Rhodothermus marinus to two different types of stress (Borges et al., 2004).

Rhodothermus marinus is noted for its production of thermostable enzymes; a detailed review has been provided by Bjorns­ dottir et  al. (2006). Some examples of the most heat-tolerant enzymes and their optimum temperature for activity include cellulase (100°C; Halldorsdottir et al., 1998); b-glucanase (85°C; Spilliaert et al., 1994); laminarinase (85°C; Krah et al., 1998); mannanase (85°C; Politz et  al., 2000), mannosylglycerate ­synthase (85–90°C; Martins et  al., 1999), and xylanase (80°C; Nordberg Karlsson et al. (1997, 1998). The original isolation of Rhodothermus marinus was from s­ubmarine alkaline freshwater hot springs in Iceland by Alfredsson et al. (1988). Additional strains have since been isolated from various marine hot springs, geothermal sites, and borehole effluents. Nunes et  al. (1992a) isolated ten strains from hot springs on a beach on the island of Sao Miguel, Azores. Moreira et  al. (1996) isolated Rhodothermus marinus from hot springs in Naples, Italy. Petursdottir et al. (2000) isolated 81 strains from four different geothermal sites in Iceland. These locations were coastal springs at Reykjanes in Isafjardardjup in northwest Iceland, an effluent from the geothermal power plant at the Blue Lagoon in southwest Iceland, the effluent from the salt factory at Reykjanes in southwest Iceland, and coastal springs and effluent from a borehole in Oxarfjordur in northeast Iceland. Petursdottir et  al. (2000) found that even though Rhodothermus marinus strains were isolated at different geographic ­locations, they exhibited a close genetic relatedness, based on high DNA–DNA reassociation values (>68%) and almost identical 16S rRNA gene sequences. However, other measures of genetic diversity, viz., ribotyping and pulsed-field gel electrophoresis (Moreira et al., 1996) and electrophoretic analysis of allelic variation in 13 genes encoding enzymes (Pétursdóttir et al., 2000) have indicated a relatively high degree of genetic variance. Petursdottir et al. (2000) concluded that this variance is most likely the result of genetic changes occurring independently in the different geographic locations studied. Tindall (Tindall, 1991) reported that the major cellular fatty acids of Rhodothermus marinus were iso-even, i.e., C14 iso, C16 iso, and C18 iso, in the type strain and another strain. Contrasting results were reported by Nunes et al. (1992b), who found the predominant acids to be iso-odd, i.e., C15 iso and C17 iso) and anteiso-odd (C15 and C17) in the type strain and in the type strain and four other strains. Chung et al. (1993) found that the culture medium affected the fatty acid composition of five strains of Rhodothermus marinus. They cultured the organisms on four different media: basal salts medium 162 (Degryse et al., 1978) supplemented with yeast extract plus tryptone (medium A); yeast extract plus sodium glutamate (medium B); yeast extract alone (medium C) and glutamate alone (medium D). In media A and C, branched-chain C15 iso and C17 iso fatty acids were the major fatty acids, and C16 iso and normal-chain fatty acids were minor components. In medium B, the relative proportion of C16 iso increased to high levels. Glutamate had a ­profound effect on

Genus I. Rhodothermus

the fatty acid composition: in medium D, with glutamate as sole source of carbon and energy, normal-chain C16 constitued the major component, and normal-chain fatty acids reached about 50% of the total fatty acids. Moreira et al. (1996) reported that the fatty acid composition of 21 strains of Rhodothermus marinus isolated from three different locations, including a nonpigmented variant, were all highly similar. The medium used was agar-solidified basal salts medium 162 supplemented with NaCl, tryptone, and yeast extract. The major fatty acids were iso- and anteiso-branched fatty acids. Under the conditions used, C17:0 iso was the major fatty acid, but C15:0 anteiso was also relatively high. Pereira et  al. (2004) summarized the types of respiratory complexes from thermophilic aerobic prokaryotes. The components so far identified in the respiratory chain of Rhodothermus include NADH:quinone oxidoreductase type 1 (NDH-1); succinate:quinone oxidoreductasae (type B); quinol:cytochrome c oxidoreductase bc; a caa3 oxygen reductase (type A2) and a cbb3 oxygen reductase (type C); menaquinone 7; and HiPIP (the electron carrier between the bc complex and the caa3 oxygen reductase). The genome size of Rhodothermus marinus is approximately 3.3–3.6 Mbp (Moreira et al., 1996). A gene transfer system for Rhodothermus marinus has been established by Bjornsdottir et al. (2005). A thorough review of the physiology and molecular biology of Rhodothermus has been given by Bjornsdottir et al. (2006).

Enrichment and isolation procedures Alfredsson et  al. (1988) isolated Rhodothermus marinus from ­samples collected directly from the openings of alkaline submarine hot springs in Iceland at a depth of 2–3 m. It was not possible to exclude seawater completely when sampling. The samples, which consisted of fine gravel and water, were kept refrigerated until processed further. Water samples were ­filtered directly, but sand and gravel samples were washed with sterile seawater, which was then filtered through membrane ­filters having a pore size of 0.45 mm. The filters were put on plates containing nutrient agar medium 162 of Degryse et al. (1978) containing 3% (w/v) NaCl and incubated at 72°C for 4 d. Redpigmented colonies were selected and purified by streaking

459

onto the same medium. The bacteria were routinely cultured on nutrient agar medium 162 containing 1% NaCl. Sako et  al. (1996) obtained strains previously classified as Rhodothermus obamensis from samples of effluent water and sediment from hydrothermal vents and sedimentary materials adjacent to vents. The samples were inoculated into a series of media, including Jx medium.* All of the Jx medium tubes containing sediment were turbid after 1 d of incubation at 80°C. Cultures were then streaked onto Jx medium plates and incubated at 70°C. Well-defined colonies were streaked onto another plate, and this plate was incubated at 70°C. This procedure was repeated at least five times and red colonies were selected.

Differentiation of the genus Rhodothermus from other genera The combination of a high optimum temperature and a salt requirement distinguish Rhodothermus from other genera in the Bacteroidetes. The other aerobic thermophilic genus in this phylum is Thermonema, some strains of which require a low concentration of Na+. Rhodothermus can also be differentiated from Thermonema by its lack of filament formation and its ability to use most common sugars and starch.

Taxonomic comments Sako et al. (1996) isolated a thermophilic bacterium from a shallow marine hydrothermal vent environment in Tachibana Bay, Japan, that belonged to the genus Rhodothermus on the basis of rRNA gene sequencing. This organism was named Rhodothermus obamensis. However, Silva et  al. (2000) reported that the type strain of Rhodothermus obamensis, JCM 9785, has a DNA–DNA reassociation value of 78% with the type strain of Rhodothermus marinus, DSM 4252. On the basis of the DNA–DNA reassociation value, 16S rRNA gene sequence comparison, and fatty acid profiles, Silva et al. concluded that Rhodothermus obamensis and Rhodothermus marinus represent the same species and that the name Rhodothermus obamensis should be regarded as a later synonym of Rhodothermus marinus. Antón et al. (2002) reported that the Rhodothermus marinus was the closest cultivated relative of Salinibacter ruber, with a 16S rRNA gene sequence similarity of about 89%.

List of species of the genus Rhodothermus 1. Rhodothermus marinus Alfredsson, Kristjansson, ­Hjörleifsdottir and Stetter 1995, 418VP (Effective publication: Alfredsson, Kristjansson, Hjörleifsdottir and Stetter 1988, 304; Rhodothermus obamensis Sako, Takai, Ishida, Uchida and Katayama 1996, 1103VP.) ma.ri¢nus, L. masc. adj. marinus of the sea, marine. The characteristics are as given for the genus, with the following additional features. Optimum temperature is 65–80°C. Optimum pH is 7; optimum NaCl concentration is 2–3%. Growth occurs in the presence of 5–6% NaCl. The major cellular fatty acid components are anteiso- and C17 and anteisoand C15 acids, and the major quinone is menaquinone 7, with

smaller amounts of menaquinones 6 and 5 (Sako et al., 1996). The natural habitat is submarine alkaline hot springs. Source: the type strain was isolated from a marine hot spring at Reykjanes, NW Iceland. DNA G+C content (mol%): 65–67 (Tm). Type strain: ATCC 43812, DSM 4252. Sequence accession no. (16S rRNA gene): AF17493.

*Jx medium contains (per liter): Jamarin S synthetic seawater powder (Jamarin Laboratory, Osaka, Japan ), 35 g; Jamarin S synthetic seawater solution, 5 ml; yeast extract (Difco), 1 g; and Trypticase peptone (BBL), 1 g; pH adjusted to 6.8–7.2 with H2SO4.

460

Family I. Rhodothermaceae

Genus II. Salinibacter Antón, Oren, Benlloch, Rodríguez-Valera, Amann and Rosselló-Mora 2002, 490VP Josefa Antón, Rudolf Amann and Ramon Rosselló-Mora Sa.li.ni.bac¢ter. L. fem. pl. n. salinae salterns, salt-works; N.L. masc. n. bacter masc. equivalent of the Gr. neut. n. baktron a rod; N.L. masc. n. Salinibacter a rod from salt-works.

Rod-shaped, often slightly curved. Gram-stain-negative. Motile by means of flagella. Obligately aerobic. Catalase- and oxidasepositive. Nitrate is not reduced. Extremely halophilic, requiring at least 15% NaCl (w/v) to grow; optimum, 20–30%. Chemoheterotrophic. Grows best at low substrate concentrations; high levels of organic compounds may be inhibitory. Amino acids are the preferred substrates for growth. Salinibacter strains form a deep branch of the phylum Bacteroidetes of the domain ­Bacteria. Colonies are bright red/orange-pigmented. Among the most salt-tolerant and salt-requiring strains within the bacterial domain. DNA G+C content (mol%): 66.3–67.7 (HPLC). Type species: Salinibacter ruber Antón, Oren, Benlloch, Rodríguez-Valera, Amann and Rosselló-Mora 2002, 490VP.

Further descriptive information The typical cells are slightly curved rods, but larger, round structures at the ends of the cells can also be found in some cultures (Figure 79). Maintenance of cell shape does not depend on the presence of high salt concentrations. Salt concentrations of at least 15% (w/v) are required and all strains grow at NaCl concentrations up to saturation. Salinibacter apparently uses KCl to provide osmotic balance and lacks high concentrations of organic solutes (Oren et  al., 2002). In this aspect, its physiology resembles that of the halophilic Archaea more than other aerobic bacteria. The potassium content relative to cell protein in Salinibacter is in the same order as for haloarchaea (in mmol/mg: 11.4–15.2 in Salinibacter, 13.2

Figure 79.  Micrograph of a Salinibacter ruber culture grown in 23%

MGM (modified growth media with 23% total salts), showing the round structures that occasionally appear at the end of the cells. Picture courtesy of Dr. Mike Dyall-Smith, Max Planck Institute for Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18a, Martinsried D82152, Germany. Scale bar: 5 mm.

in Haloarcula marismortui, and 12.0 in Halobacterium salinarum). The only other Bacteria known to use KCl rather than organic solutes to provide osmotic balance are the anaerobic fermentative members of the order Halanaerobiales. The optimal pH range for growth is 6.5–8.0 with an optimum temperature of 35–45°C. Under optimal growth conditions, the generation time is 14–18 h. Although the original description of Salinibacter ruber stated that simple sugars and organic acids (acetate, succinate) did not support growth as sole carbon and energy sources, Oren and Mana (2003) showed that the addition of glucose, maltose, and starch to a medium with yeast extract had a pronounced stimulatory effect on growth. However, sugars are not the preferred growth substrates and their consumption starts after the exhaustion of other substrates (Oren and Mana, 2003). Glycerol is probably a readily available carbon and energy source in hypersaline water bodies, for it is produced in high quantities as an osmotic solute by unicellular green algae of the genus Dunaliella (Oren, 2005). However, the use of radioactive substrates did not indicate an ability of Salinibacter to metabolize glycerol during a period of 24–48 h (Rosselló-Mora et al., 2003). On the other hand, Sher et al. (2004) showed that growth in pure culture is stimulated by glycerol, although glycerol alone was not sufficient to support growth. After 190 h of incubation, 25% of the radioactive label was incorporated into the cells, with part of the glycerol transformed into CO2. As with haloarchaea, Salinibacter proteins showed a high content of acidic amino acids, a low amount of basic amino acids, a low content of hydrophobic amino acids, and a high content of serine (Oren and Mana, 2002). In addition, biochemical studies of enzymes such as NAD-dependent isocitrate dehydrogenase, NADP-dependent isocitrate dehydrogenase, NAD-dependent malate dehydrogenase, NAD-dependent glutamate dehydrogenase, and two distinct glutamate dehydrogenases showed that Salinibacter enzymes are adapted to function in the presence of high salt concentrations (Bonete et al., 2003; Oren and Mana, 2002). On the other hand, Madern and Zaccai (2004) found that malate dehydrogenase behaves like a non-halophilic protein, since it is completely stable in the absence of salts, its amino acid ­composition does not display the strong acidic character specific of halophilic proteins, and its activity is reduced by high salt concentration. As pointed out by Oren (2005), the general trend of salt dependence of Salinibacter proteins is clear, although there are significant differences for individual enzymes. Salinibacter is sensitive to penicillin G, ampicillin, chloramphenicol, streptomycin, novobiocin, rifampin and ciprofloxacin. No growth inhibition was found with kanamycin, bacitracin, tetracycline and colistin. The cells are resistant to anisomycin and aphidicolin, two potent growth inhibitors of halophilic Archaea of the order Halobacteriales. Salinibacter harbors pigment concentrations in the same order of magnitude as halophilic Archaea (Oren and Rodríguez-Valera, 2001). The principal pigment of Salinibacter is salinixanthin, a C40-carotenoid acyl glycoside [(all-E, 2¢S)-2¢-hydroxy-1¢-[6-

Genus II. Salinibacter

O-(13-methyltetradecanoyl)-b-d-glycopyranosyloxy]-3¢,4¢didehydro-1¢,2¢-dihydro-b,y-caroten-4-one], that accounts for more than 96% of the total pigments (Lutnæs et al., 2002). The remarkable structural conformity between salinixanthin and the major carotenoid acyl glucoside from Rhodothermus marinus is consistent with the 16S rRNA phylogeny (Lutnæs et al., 2004). Bright-red pigmentation is common in microorganisms inhabiting salt lakes and saltern ponds; for instance, the Halobacteriaceae possess bacterioruberins, and Dunaliella, synthesizes b-carotene. Thus, colony color alone is not a reliable trait to allow classification of extremely halophilic prokaryotes as members of the domains Archaea or Bacteria. Salinibacter has a membrane lipid composition with glycerophospholipids containing ester-linked fatty acyl chains that are typical of Bacteria. According to Peña et  al. (2005) the major lipids in Salinibacter ruber strains are diphosphatidylglycerol and the unknown polar lipid L7. Phosphatidylethanolamine, as well as three unknown glycolipids, several polar lipids, and one phospholipid were usually found in moderate to minor amounts. Contrary to previous observations (Oren et al., 2004), no phosphatidylglycerol was detected nor could phosphatidylcholine (PC) be unambiguously identified in any of the 17 Salinibacter ruber strains analyzed. In addition, a spot staining with Dragendoff reagent (specific for quaternary nitrogen found in PC but rarely in other lipids) with similar but not identical chromatographic behavior was found also by Peña et  al. (2005). Salinibacter ruber M31 contains a novel sulfonolipid with the structure 2-carboxy-2-amino-3-O-(13¢-methyltetradecanoyl)4-hydroxy-18-methylnonadec-5-ene-1-sulfonic acid. This lipid accounts for 10% of total cellular lipids and appears to be a structural variant of sulfonolipids found in the cell walls of gliding Cytophaga and diatoms (Corcelli et al., 2004). A peak at m/z 660, corresponding to this sulfonolipid, has been proposed as the lipid signature of Salinibacter. The genome of the type strain of the type species, Salinibacter ruber M31, has been completely sequenced by Mongodin et al. (2005). It harbors one 3,551,823 bp chromosome (62.29 mol% G+C, slightly lower, but in good agreement with earlier HPLC measurements) and one plasmid of 35,505 bp (57.9 mol% G+C) containing 2934 and 33 ORFs, respectively. The calculated isoelectric point (5.2) for the proteome of Salinibacter is nearer to those of haloarchaea than its closest sequenced relatives, Bacteroides fragilis and Chlorobium tepidum. M31, as do all other 16 strains characterized so far (Peña et al., 2005), has a single rRNA operon. The 16S rRNA gene sequence is almost identical for all the analyzed Salinibacter ruber strains, while the similarity for their 16S–23S gene spacer regions is above 97% (Peña et al., 2005). Embden-Meyerhoff glycolytic pathway genes have been found in the chromosome, contrary to previous growth studies that suggested that Salinibacter uses the classic Entner-Doudoroff pathway for catabolism of glucose (Oren and Mana, 2003). Genes related to the transport and metabolism of glycerol and glycine betaine, as well as some genes related to adaptions to microoxic environments have been annotated. One so-called “hypersalinity island” encoding proteins of crucial importance to a hyperhalophilic lifestyle (such as K+ uptake/efflux systems and cationic amino acid transporters) was also found. The M31 genome harbors genes encoding four retinal proteins: halorhodopsin (Antón et  al., 2005), two sensory rhodospins and ­xanthorhodopsin (Balashov

461

et  al., 2005). Genome analysis suggested that the convergence on an aerobic hyperhalophilic lifestyle between haloarchaea and Salinibacter has arisen through convergence at the physiological level (different genes producing similar overall phenotype) and the molecular level (independent mutations yielding similar sequences or structures). Mongodin et  al. (2005) hypothesize that several genes and gene clusters might have suffered lateral transfer from (or may have been laterally transferred to) haloarchaea, although the total number of apparent lateral gene transfers between Salinibacter and haloarchaea appears to be modest. Without a doubt, one of the most striking features of Salinibacter is the presence of xanthorhodopsin (XR), a proton pump with a light-harvesting carotenoid antenna XR contains two chromophores, retinal and salinixanthin, in a molar ratio of about 1:1. Light energy absorbed by the carotenoid is transferred to the retinal, extending the wavelength range of the collection of light for uphill transmembrane proton transport. Thus XR shares characteristics with the archaeal (e.g., bacteriorhodopsin and archaeorhodpsin), bacterial (proteorhodopsin), and eukaryal (letospheria rhodopsin) retinal-based light-driven proton pumps, as well as with the chlorophyll-based light-harvesting complexes and reaction centers. As pointed out by Balashov et al. (2005), “the XR complex represents the simplest electrogenic pump with an accessory antenna pigment, and it might be an early evolutionary development in using energy transfer for energy capture.” Strictly speaking, heterotrophy is not the only source of energy for Salinibacter ruber M31. In 2000, the high abundance and growth of a group of uncultured Bacteria was reported for samples from crystallizer ponds (salinity 30–37%) in a saltern in Alicante, Spain. This group, that was then named as “Candidatus Salinibacter” gen. nov. accounted for 5–25% in crystallizers from different locations and was affiliated with the phylum Bacteroidetes (Antón et al., 2000). This study followed the first evidence that Bacteria could be present in high numbers in crystallizer ponds that was reported in 1999 with the use of fluorescence in situ hybridization (FISH) with Archaea- and Bacteria-specific probes (Antón et al., 1999). This kind of approach allows for direct quantification of prokaryotes in the environment and lacks the pitfalls associated with a PCR based approach. FISH with Bacteriaspecific probes showed that this group accounted for 11–18% in crystallizer samples from a Spanish solar saltern. The finding of abundant Bacteria with high cellular rRNA content in such a hypersaline environment was unexpected in light of previous reports that suggested that almost all the active biomass was of archaeal origin. In a pioneering study of the molecular microbial ecology of the salterns using a PCR-based approach, Martínez-Murcia et al. (1995) reported that the bacterial population present in crystallizers was very different (based on similarities of 16S rDNA-RFLP) from the populations inhabiting lower salinity ponds. Although these authors stated that crystallizers probably represented an extremely specialized niche for Bacteria, they also pointed out that only a very small proportion of the crystallizer biomass could correspond to Bacteria, and that the bacterial DNA detected by PCR could come from allochthonous microbiota carried over from lower salinity ponds. In 1995, Benlloch et al. analyzed 16S rRNA gene clone libraries from a crystallizer pond and obtained five bacterial clones that were partially sequenced (around 200 bp); all were related (82.6– 83.6% similarities) to the a-proteobacterium R ­ hodopseudomonas

462

Family I. Rhodothermaceae

marina. These sequences (accession nos: X84322, X84323 and X84324) could indeed correspond to Salinibacter since they have from 89 to 96% similarity with Salinibacter ruber in the analyzed 16S rRNA gene sequence fragment. However, the authors pointed out that “considering the salt concentration in the pond (30.8%) no known Bacteria could be physiologically active”. Indeed, the idea that only Archaea could thrive in hypersaline environments was very strong. We must point out, however, that direct proof of activity of Salinibacter species in the highest salinity (37%) ponds has not been obtained so far. In fact, Gasol et  al. (2004) found that above 32% salinity, all the prokaryotic activity was carried out by haloarchaea in the very same saltern ponds where Salinibacter ruber represented up to 18% of the DAPI (4¢,6-diamidino-2-phenylindole) counts (total counts). This observation is based on the assumption that Salinibacter species are not inhibited by taurocholate, which is a potent haloarchaeal inhibitor. The occurrence of Salinibacter in the environment has been studied by several methods: FISH (Antón et al., 2000; RossellóMora et  al., 2003), pigment analysis (Oren and RodríguezValera, 2001), total DNA melting profiles and reassociation techniques (Øvrėas et al., 2003), Denaturing gradient gel electrophoresis (DGGE) (Benlloch et al., 2002), and 16S rRNA gene clone library analysis, among others. These techniques show that there is a considerable degree of microdiversity among the environmental sequences related to Salinibacter ruber. Using the above-mentioned culture-independent approaches, Salinibacter has been found in crystallizer pond salterns from locations in Santa Pola (mainland Spain), Balearic (Mallorca and Ibiza) and Canary Islands, which accounted for 5–27% of the total prokaryotic community. In Santa Pola salterns, Salinibacter was not detected in ponds having up to 22.4% salinity, whereas it was found in increasingly high numbers (3.5–12%) in three ponds of 25, 31.6 and 37% salinity. The Salinibacter lipid signature peak at m/z 660 was evident in the ESI-MS profile in the lipid extract from a crystallizer pond in the Margherita di Savoia salterns (Italy) (Corcelli et al., 2004), indicating the presence of Salinibacter in this environment. In some instances, e.g., Andean and Eilat (Israel) salterns, Salinibacter was not detected by culture-independent analyses, although it could be readily isolated (Elevi-Bardavid et  al., 2007; Maturrano et  al., 2006). Although crystallizers are the most frequently reported habitat for Salinibacter, sequences with a similarity of 92–97% to those of Salinibacter were very abundant in 16S rRNA gene libraries constructed with DNA extracted from the different layers of an endoevaporite (crystallized gypsum-halite matrix in nearsaturated salt water) from salt-works in Guerrero Negro, Mexico (Spear et al., 2003). Partial 16S rRNA gene sequences with similarities of less than 92% to Salinibacter have been retrieved from biofilms colonizing ancient limestone Mayan monuments in Uxmal (Mexico) (Ortega-Morales et  al., 2004). Salinibacter 16S rRNA gene sequences were also found in clone libraries obtained from a hypersaline endoevaporite microbial mat from Eilat salterns (Sørensen et  al., 2005). These sequences were most abundant in the green layer of the mat. Finally, 16S rRNA gene sequences that fall within the radiation of the genus Salinibacter, but represent distinct novel lineages have been recovered from evaporite crusts in brine pools at the Badwater site in Death Valley National Park, California (Elevi-Bardavid et al., 2007). Isolates having 93–94% 16S rRNA gene similarity with Salinibacter ruber have been obtained from these samples

(­ Hollen et al., 2003). These authors claimed that they have isolated a new species of Salinibacter.

Enrichment and isolation procedures The strains used for the genus description and for intraspecific comparative analyzes, were all isolated by plating dilutions of crystallizer samples on agar plates. They were recognized as members of the genus by 16S rRNA gene analysis or by polar lipids thin layer chromatography (TLC). The fact that Salinibacter is insensitive to the haloarchaeal inhibitors anisomycin and bacitracin has been used to design selective enrichment and isolation protocols (Elevi-Bardavid et al., 2007). Strains of Salinibacter can be preserved by lyophilization.

Differentiation of the genus Salinibacter from closely related genera No extreme halophiles are known so far among taxa closely related to Salinibacter, thus the differentiation from these taxa can be made on the basis of salt needed for optimum growth. However, Salinibacter shares its habitats with haloarchaea (i.e., the ­family Halobacteriaceae). Interestingly, there is a surprising similarity between these two groups: both are aerobic heterotrophs, maintain high intracellular K+ concentrations, require high salt concentrations for growth with optima in the range of 12–25% total salts, are red pigmented, and have a similar mol% G+C content of their DNA. For these reasons, Salinibacter can be easily mistaken as haloarchaea based only on phenotypic characteristics. Therefore, we recommend phylogenetic identification as the easiest way to identify Salinibacter strains. Lipid and pigment analyzes (Elevi-Bardavid et al., 2007) can also be used for strain identification.

Taxonomic comments The closest 16S rRNA sequence similarity (86.4%) relative to Salinibacter is Rhodothermus, a genus of slightly halophilic (optimum 0.5–2% NaCl), thermophilic (optimum 65–70°C) bacteria isolated from marine hot springs (Alfredsson et al., 1988). The phylogenetic branch comprising the two genera Salinibacter and Rhodothermus bifurcates close to the divergence node between the two main phyla Bacteroidetes and Chlorobi. This observation made upon 16S rRNA gene sequence analysis has been corroborated by the use of independent phylogenetic approaches based on concatenating two sets of 22 and 74, respectively, protein sequences (Soria-Carrasco et al., 2007). From the single protein phylogenies it can be observed that in nearly 30% of the cases the affiliation was with Chlorobi. This may be an indication of an early divergence from Bacteroidetes, and a future classification as a single phylum cannot be d ­ iscarded (Figure 80). Note added in proof. A novel halophilic member of the Bacteroidetes, Salisaeta longa gen. nov., sp.nov., was published after acceptance of the present chapter. It is able to grow at concentrations of 5–20% NaCl, with an optimum at 10% plus 5% MgCl·6H2O and has been described as halophilic (not extremely halophilic, as Salinibacter ruber). The 16S rRNA genes of Salinibacter ruber and Salisaeta longa have 88.3% similarity and thus, according to this parameter, this new species is now the closest relative of ­Salinibacter ruber, instead of Rhodothermus marinus. They can be differentiated based on their morphology, 16S rRNA gene sequences and salt optimum needed for growth, among other characteristics. However, a 16S rRNA primer originally designed as specific for Salinibacter ruber (Antón et al., 2002) is no longer specific.

Genus II. Salinibacter

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Figure 80.  Tree reconstruction based on 16S rRNA gene sequences showing the affiliation of Salinibacter ruber strain M31 with its closest relative genus Rhodothermus, and to the hitherto uncultured Salinibacter species EHB-2 (Antón et al., 2000). The tree also shows the branch position relative to the rest of the members of the phyla Bacteroidetes and Chlorobi. The reconstruction was based on a dataset of more than 50,000 primary aligned 16S rRNA gene structures as implemented in the arb software package, and corresponding to the released database available at http://www.arb-home.de (Ludwig et al., 2004). The phylogenetic analyzes were performed by using the sequences corresponding to all type strains of both phyla, and by using the neighbor-joining, maximum-parsimony, and maximum-likelihood algorithms. All treeing approaches rendered an identical topology. The bar indicates 10% of estimated sequence divergence.

Acknowledgements We thank Dr Hans-Jürgen Busse, Institut fur Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität Wien, Vienna, Austria, for his help with the lipid data.

Further reading Antón, J., A. Peña, M. Valens, F. Santos, F.O. Glöckner, M. Bauer, J. ­Dopazo, J. Herrero, R. Rosselló-Mora and R. Amann. 2005. Salinibacter ruber: genomics and biogeography. In Adaptation to life in high salt concentrations in Archaea, Bacteria and Eukarya (edited by Gunde-Cimerman, Plemenitasand Oren). Kluwer Academic Publishers, ­Dordrecht, The Netherlands, pp. 257–266. Mongodin, E.F., K.E. Nelson, S. Daugherty, R.T. DeBoy, J. Wister, H. Khouri, J. Weidman, D.A. Walsh, R.T. Papke, G. Sanchez Perez, A.K. Sharma, C.L. Nesbø, D. MacLeod,

E. Bapteste, W.F. Doolittle, R.L. Charlebois, B. Legault and F. Rodríguez-Valera. 2005. The genome of Salinibacter ruber. Convergence and gene exchange among hyperhalophilic bacteria and archaea. Proc. Natl. Acad. Sci. U. S. A. 102: 18147–18152. Oren, A. 2005. The genera Rhodothermus, Thermonema, Hymenobacter and Salinibacter. In The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, 3rd edn (edited by Dworkin, Falkow, Rosenberg, Schleifer and Stackebrandt). Springer, New York. Peña, A., M. Valens, F. Santos, S. Buczolits, J. Antón, P. Kämpfer, H.-J. Busse, R. Amann and R. Rosselló-Mora. 2005. Intraspecific comparative analysis of the species Salinibacter ruber. Extremophiles 9: 151–161.

List of species of the genus Salinibacter 1. Salinibacter ruber Antón, Oren, Benlloch, Rodríguez-Valera, Amann and Rosselló-Mora 2002, 490VP ru¢ber. L. masc. adj. ruber red. The description is as given for the genus, with the following additional features.

References Alfredsson, G.A., J.K. Kristjansson, S. Hjörleifsdottir and K.O. Stetter. 1988. Rhodothermus marinus, gen. nov., sp. nov., a thermophilic, halophilic bacterium from submarine hot springs in Iceland. J. Gen. Microbiol. 134: 299–306. Alfredsson, G.A., J.K. Kristjansson, S. Hjörleifsdottir and K.O. Stetter. 1995. In Validation of new names and new combinations previously

Source: crystallizer pond salterns. DNA G+C content (mol%): 66.5 (HPLC). Type strain: M31, DSM 13855, CECT 5946. Sequence accession no. (16S rRNA gene): AF323500.

effectively published outside the IJSB. List no. 60. Int. J. Syst. Bacteriol. 45: 418–419. Antón, J., E. Llobet-Brossa, F. Rodríguez-Valera and R. Amann. 1999. Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds. Environ. Microbiol. 1: 517–523. Antón, J., R. Rosselló-Mora, F. Rodríguez-Valera and R. Amann. 2000. Extremely halophilic Bacteria in crystallizer ponds from solar salterns. Appl. Environ. Microbiol. 66: 3052–3057.

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Family I. Rhodothermaceae

Antón, J., A. Oren, S. Benlloch, F. Rodríguez-Valera, R. Amann and R. Rosselló-Mora. 2002. Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds. Int. J. Syst. Evol. Microbiol. 52: 485–491. Antón, J., A. Peña, M. Valens, F. Santos, F.O. Glöckner, M. Bauer, J. Dopazo, J. Herrero, R. Rosselló-Mora and R. Amann. 2005. Salinibacter ruber: genomics and biogeography. In Adaptation to life in high salt concentrations in Archaea, Bacteria and Eukarya (edited by GundeCimerman, Plemenitas and Oren). Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 257–266. Balashov, S.P., E.S. Imasheva, V.A. Boichenko, J. Antón, J.M. Wang and J.K. Lanyi. 2005. Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna. Science 309: 2061–2064. Benlloch, S., A.J. Martínez-Murcia and F. Rodríguez-Valera. 1995. Sequencing of bacterial and archaeal 16S rRNA genes directly ­amplified from a hypersaline environment. Syst. Appl. Microbiol. 18: 574–581. Benlloch, S., A. López-López, E.O. Casamayor, L. Øvreas, V. Goddard, F.L. Daae, G. Smerdon, R. Massana, I. Joint, F. Thingstad, C. PedrósAlió and F. Rodríguez-Valera. 2002. Prokaryotic diversity throughout the salinity gradiente of a coastal solar saltern. Environ. Microbiol. 4: 349–360. Bjornsdottir, S.H., S.H. Thorbjarnardottir and G. Eggertsson. 2005. Establishment of a gene transfer system for Rhodothermus marinus. Appl. Microbiol. Biotechnol. 66: 675–682. Bjornsdottir, S.H., T. Blondal, G.O. Hreggvidsson, G. Eggertsson, S. Petursdottir, S. Hjörleifsdottir, S.H. Thorbjarnardottir and J.K. Kristjansson. 2006. Rhodothermus marinus: physiology and molecular biology. Extremophiles 10: 1–16. Bonete, M.J., F. Perez-Pomares, S. Diaz, J. Ferrer and A. Oren. 2003. Occurrence of two different glutamate dehydrogenase activities in the halophilic bacterium Salinibacter ruber. FEMS Microbiol. Lett. 226: 181–186. Borges, N., J.D. Marugg, N. Empadinhas, M.S. da Costa and H. Santos. 2004. Specialized roles of the two pathways for the synthesis of mannosylglycerate in osmoadaptation and thermoadaptation of Rhodothermus marinus. J. Biol. Chem. 279: 9892–9898. Chung, A.P., O.C. Nunes, B.J. Tindall and M.S. da Costa. 1993. The effect of the growth medium composition on the fatty acids of ­Rhodothermus marinus and ‘Thermus thermosphilus’ HB-8. FEMS ­Microbiol. Lett. 112: 13–18. Corcelli, A., V.M. Lattanzio, G. Mascolo, F. Babudri, A. Oren and M. Kates. 2004. Novel sulfonolipid in the extremely halophilic bacterium Salinibacter ruber. Appl. Environ. Microbiol. 70: 6678–6685. Degryse, W., N. Glansdorff and A. Piérard. 1978. A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus. Arch. Microbiol. 117: 189–196. Elevi-Bardavid, R., D. Ionescu, A. Oren, F.A. Rainey, B.J. Hollen, D.R. Bagaley, A.M. Small and C.M. McKay. 2007. Selective enrichment, isolation and molecular detection of Salinibacter and related extremely halophilic Bacteria from hypersaline environments. Hydrobiologia 576: 207. Gasol, J.M., E.O. Casamayor, I. Joint, K. Garde, K. Gustavson, S. Benlooch, B. Díez, M. Schauer, R. Massana and C. Pedrós-Alió. 2004. Control of heterotrophic prokaryotic abundance and growth rate in hypersaline planktonic environments. Aquat. Microb. Ecol. 34: 193–206. Halldorsdottir, S., E.T. Thorolfsdottir, R. Spilliaert, M. Johansson, S.H. Thorbjarnardottir, A. Palsdottir, G.O. Hreggvidsson, J.K. Kristjansson, O. Holst and G. Eggertsson. 1998. Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12. Appl. Microbiol. Biotechnol. 49: 277–284. Hollen, B.J., D.R. Bagaley, A.M. Small, A. Oren, C.P. McKay and F.A. Rainey. 2003. Investigation of the microbial community of the salt surface layer at Badwater, Death Valley National Park. Proceedings of the American Society for Microbiology Annual Meeting, Washington, DC. Karlsson, E.N., L. Dahlberg, N. Torto, L. Gorton and O. Holst. 1998. Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xynl from Rhodothermus marinus. J. Biotechnol. 60: 23–35.

Krah, M., R. Misselwitz, O. Politz, K.K. Thomsen, H. Welfle and R. Borriss. 1998. The laminarinase from thermophilic eubacterium Rhodothermus marinus - conformation, stability, and identification of active site carboxylic residues by site-directed mutagenesis. Eur. J. Biochem. 257: 101–111. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Forster, I. Brettske, S. Gerber, A.W. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. Konig, T. Liss, R. Lussmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N. Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode and K.H. Schleifer. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32: 1363–1371. Lutnæs, B.F., A. Oren and S. Liaaen-Jensen. 2002. New C40-carotenoid acyl glycoside as principal carotenoid in Salinibacter ruber, an extremely halophilic eubacterium. J. Nat. Prod. 65: 1340–1343. Lutnæs, B.F., A. Strand, S.K. Petursdottir and S. Liaaen-Jensen. 2004. Carotenoids of thermophilic bacteria - Rhodothermus marinus from submarine Icelandic hot springs. Biochem. Syst. Ecol. 32: 455–468. Madern, D. and G. Zaccai. 2004. Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein. Biochimie 86: 295–303. Martínez-Murcia, A.J., S.G. Acinas and F. Rodríguez-Valera. 1995. Evaluation of prokaryotic diversity by restrictase digestion of 16S rDNA directly amplified from hypersaline environments. FEMS. Microb. Ecol. 17: 247–256. Martins, L.O., N. Empadinhas, J.D. Marugg, C. Miguel, C. Ferreira, M.S. da Costa and H. Santos. 1999. Biosynthesis of mannosylglycerate in the thermophilic bacterium Rhodothermus marinus. Biochemical and genetic characterization of a mannosylglycerate synthase. J. Biol. Chem. 274: 35407–35414. Maturrano, L., F. Santos, R. Rosselló-Mora and J. Antón. 2006. Microbial diversity in Maras salterns, a hypersaline environment in the Peruvian Andes. Appl. Environ. Microbiol. 72: 3887–3895. Mongodin, E.F., K.E. Nelson, S. Daugherty, R.T. DeBoy, J. Wister, H. Khouri, J. Weidman, D.A. Walsh, R.T. Papke, G. Sanchez Perez, A.K. Sharma, C.L. Nesbø, D. MacLeod, E. Bapteste, W.F. Doolittle, R.L. Charlebois, B. Legault and F. Rodríguez-Valera. 2005. The genome of Salinibacter ruber. Convergence and gene exchange among hyperhalophilic bacteria and archaea. Proc. Natl. Acad. Sci. U. S. A. 102: 18147–18152. Moreira, L., M.F. Nobre, I. Sa-correia and M.S. da Costa. 1996. Genomic typing and fatty acid composition of Rhodothermus marinus. Syst. Appl. Microbiol. 19: 83–90. Nordberg Karlsson, E., E. Bartonek-Roxa and O. Holst. 1997. Cloning and sequence of a thermostable multidomain xylanase from the bacterium Rhodothermus marinus. Biochim. Biophys. Acta. 1353: 118–124. Nunes, O.C., M.M. Donato and M.S. da Costa. 1992a. Isolation and characterization of Rhodothermus strains from S. Miguel Azores. Syst. Appl. Microbiol. 15: 92–97. Nunes, O.C., M.M. Donato, C.M. Manaia and M.S. da Costa. 1992b. The polar lipid and fatty acid composition of Rhodothermus strains. Syst. Appl. Microbiol. 15: 59–62. Nunes, O.C., C.M. Manaia, M.S. Da Costa and H. Santos. 1995. Compatible Solutes in the thermophilic bacteria Rhodothermus marinus and “Thermus thermophilus”. Appl. Environ. Microbiol. 61: 2351–2357. Oren, A. 2005. The genera Rhodothermus, Thermonema, Hymenobacter and Salinibacter. In The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, 3rd edn (edited by Dworkin, Falkow, Rosenberg, Schleifer and Stackebrandt). Springer, New York. Oren, A. and F. Rodríguez-Valera. 2001. The contribution of halophilic Bacteria to the red coloration of saltern crystallizer ponds. FEMS Microbiol. Ecol. 36: 123–130. Oren, A. and L. Mana. 2002. Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber, an extremely halophilic bacterium. Extremophiles 6: 217–223. Oren, A. and L. Mana. 2003. Sugar metabolism in the extremely halophilic bacterium Salinibacter ruber. FEMS Microbiol. Lett. 223: 83–87. Oren, A., M. Heldal, S. Norland and E.A. Galinski. 2002. Intracellular ion and organic solute concentrations of the extremely halophilic bacterium Salinibacter ruber. Extremophiles 6: 491–498.

Genus I. Thermonema Oren, A., F. Rodríguez-Valera, J. Antón, S. Benlloch, R. RossellóMora, R. Amann, J. Coleman and N.J. Russell. 2004. Red, extremely ­halophilic, but not archaeal: the physiology and ecology of Salinibacter ruber, a bacterium isolated from saltern crystallizer ponds. In Halophilic Microorganisms (edited by Ventosa). Springer, New York, pp. 63–76. Ortega-Morales, B.O., J.A. Narváez-Zapata, A. Schmalenberger, A. SosaLópez and C.C. Tebbe. 2004. Biofilms fouling ancient limestone Mayan monuments in Uxmal, Mexico: a cultivation-independent analysis. Biofilms 1: 79–90. Øvreas, L., F.L. Daae, V. Torsvik and F. Rodríguez-Valera. 2003. Characterization of microbial diversity in hypersaline environments by melting profiles and reassociation kinetics in combination with terminal restriction fragment length polymorphism (T-RFLP). Microb. Ecol. 46: 291–301. Peña, A., M. Valens, F. Santos, S. Buczolits, J. Antón, P. Kämpfer, H.-J. Busse, R. Amann and R. Rosselló-Mora. 2005. Intraspecific comparative analysis of the species Salinibacter ruber. Extremophiles 9: 151–161. Pereira, M.M., T.M. Bandeiras, A.S. Fernandes, R.S. Lemos, A.M. Melo and M. Teixeira. 2004. Respiratory chains from aerobic thermophilic prokaryotes. J. Bioenerg. Biomembr. 36: 93–105. Pétursdóttir, S.K., G.O. Hreggvidsson, M.S. da Costa and J.K. Kristjansson. 2000. Genetic diversity analysis of Rhodothermus reflects geographical origin of the isolates. Extremophiles 4: 267–274. Politz, O., M. Krah, K.K. Thomsen and R. Borriss. 2000. A highly thermostable endo-(1,4)-beta-mannanase from the marine bacterium Rhodothermus marinus. Appl. Microbiol. Biotechnol. 53: 715–721. Ramos, A., N. Raven, R.J. Sharp, S. Bartolucci, M. Rossi, R. Cannio, J. Lebbink, J. Van Der Oost, W.M. De Vos and H. Santos. 1997. Stabilization of enzymes against thermal stress and freeze-drying by mannosylglycerate. Appl. Environ. Microbiol. 63: 4020–4025. Rosselló-Mora, R., N. Lee, J. Antón and M. Wagner. 2003. Substrate uptake in extremely halophilic microbial communities revealed by microautoradiography and fluorescence in situ hybridization. Extremophiles 7: 409–413.

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Sako, Y., K. Takai, Y. Ishida, A. Uchida and Y. Katayama. 1996. Rhodothermus obamensis sp. nov., a modern lineage of extremely thermophilic marine bacteria. Int. J. Syst. Bacteriol. 46: 1099–1104. Santos, H. and M.S. da Costa. 2002. Compatible solutes of organisms that live in hot saline environments. Environ. Microbiol. 4: 501–509. Sher, J., R. Elevi, L. Mana and A. Oren. 2004. Glycerol metabolism in the extremely halophilic bacterium Salinibacter ruber. FEMS Microbiol. Lett. 232: 211–215. Silva, Z., N. Borges, L.O. Martins, R. Wait, M.S. da Costa and H. Santos. 1999. Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis. Extremophiles 3: 163–172. Silva, Z., C. Horta, M.S. da Costa, A.P. Chung and F.A. Rainey. 2000. Polyphasic evidence for the reclassification of Rhodothermus obamensis Sako et al. 1996 as a member of the species Rhodothermus marinus Alfredsson et al. 1988. Int. J. Syst. Evol. Microbiol. 50: 1457–1461. Sørensen, K.B., D.E. Canfield, A.P. Teske and A. Oren. 2005. Community composition of a hypersaline endoevaporitic microbial mat. Appl. Environ. Microbiol. 71: 7352–7365. Soria-Carrasco, V., M. Valens-Vadell, A. Peña, J. Antón, R. Amann, J. Castresana and R. Rosselló-Mora. 2007. Phylogenetic position of Salinibacter ruber based on concatenated protein alignments. Syst. Appl. Microbiol. 30: 171–179. Spear, J.R., R.E. Ley, A.B. Berger and N.R. Pace. 2003. Complexity in natural microbial ecosystems: the Guerrero Negro experience. Biol. Bull. 204: 168–173. Spilliaert, R., G.O. Hreggvidsson, J.K. Kristjansson, G. Eggertsson and A. Palsdottir. 1994. Cloning and sequencing of a Rhodothermus marinus gene, bglA, coding for a thermostable b-glucanase and its expression in Escherichia coli. Eur. J. Biochem. 224: 923–930. Tindall, B.J. 1991. Lipid composition of Rhodothermus marinus. FEMS Microbiol. Lett. 80: 65–68. Vaisman, N. and A. Oren. 2009. Salisaeta longa gen. nov., sp. nov., a red, halophilic member of the Bacteroidetes. Int. J. Syst. Evol. Microbiol. 59: 2571–2574.

Order III. Incertae sedis Thermonema was previously assigned to the “Flammeovirgaceae” by Garrity et al. (2005), but subsequent analyses of the 16S rRNA suggests that it represents a very deep group and is only dis-

tantly related to any of the previously described orders within the Bacteroidetes. In view of its ambiguous status, it has been assigned to its own order incertae sedis.

Genus I. Thermonema Hudson, Schofield, Morgan and Daniel 1989, 487VP The Editorial Board Ther.mo.ne¢ma. Gr. adj. thermos hot; Gr. neut. n. nema a thread; N.L. neut. n. Thermonema a thermophilic thread.

Apparently unicellular filaments ~0.7 mm in diameter and ~ 60 to several hundred mm long. Motile by gliding. Gram-stain-negative. Aerobic. Optimum temperature, ~60°C; poor or no growth occurs at 70°C. Colonies are orange (Schofield et al., 1987) or yellow (Tenreiro et al., 1997), depending on the medium. Cells possess acetone-extractable pigments with an absorbance peak at 450 nm. Flexirubin-type pigments are not produced. Oxidase- and catalase-positive. Aminopeptidase-positive. Casein, gelatin, and hippurate are hydrolyzed but not starch, xylan, or cellulose. Growth occurs on vitamin-free Casamino acids and on a mixture of the 20 natural amino acids, but not on single amino acids. Growth does not occur on pentoses, hexoses, N-acetylglucosamine, disaccharides, polyols, or organic acids. Na+ may not be required; but if required, only low concentrations are needed. Menaquinone 7 is the major respiratory quinone. Sphingolipids are present. Source: hot springs. DNA G+C content (mol%): 47–51.

Type species: Thermonema lapsum Hudson, Schofield, Morgan and Daniel 1989, 487VP.

Further descriptive information The initial isolate of Thermonema lapsum was obtained by ­Schofield et  al. (1987) from hot-spring water samples collected from Kuirau Park, Rotorua, New Zealand. Marteinsson et  al. (2001) isolated seven marine strains belonging to the genus Thermonema from the concentrated fluid issuing from giant geothermal cones on the seafloor at a depth of 65 m in Eyjafjordur, Northern Iceland. The isolates were obtained only from the outer zone of the chimney, where cold seawater mixes into the vent fluid with increasing salinity and decreasing temperature. Mountain et al. (2003) obtained Thermonema isolates from ­sinters in New Zealand hot springs. Tenreiro et al. (1997) isolated Thermonema rossianum strains from saline hot springs along the Bay of Naples, Italy.

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Family I. Rhodothermaceae

Schofield et  al. (1987) indicated that colonies are orange on Castenholz medium D (CMD; Ramaley and Hixson, 1970). ­Tenreiro et  al. (1997) reported colonies as bright yellow on medium 162 (Degryse et al., 1978) agar containing 1.0% NaCl. Patel et al. (1994) determined that the major normal fatty acid components of the phospholipids and lipopolysaccharide of Thermonema lapsum were (in decreasing order of abundance) C15:0 iso, C15:0 anteiso, and C15:0. No monounsaturated fatty acids occurred. Tenreiro et al. (1997) reported that the fatty acid composition of Thermonema rossianum strains was dominated by C15:0 iso and C17:0 iso 3-OH. C15:0 anteiso, C15:0 iso 2-OH, and C15:0 iso 3-OH also occurred in moderate relative concentrations, but the concentrations of other fatty acids were minor or ­negligible. The total relative proportion of hydroxy fatty acids was very high, about 40% of the total fatty acids. Homospermidine and homospermine have been detected in Thermonema lapsum as the major polyamines (Hamana et al., 1992).

Enrichment and isolation procedures Schofield et al. (1987) spread hot-spring water onto Castenholz medium D solidified with 3% agar and incubated the cultures for 24 h at 70°C. Single colonies were selected, and subsequent transfers grew well at 60°C but poorly at 70°C. Spreading occurred on media containing 1.5% agar but not 3% agar. In static broth cultures, the organisms grew as a pellicle that later formed clumps. Gliding motility was observed in hanging drop preparations at room temperature. Mountain et  al. (2003) inoculated media with samples (0.1–1.0 ml) of water collected from sinters in New Zealand hot springs, and incubated the cultures at temperatures in the range 30–60°C. Microbial growth, as indicated by turbidity in

liquid media (composition not specified), or by colonies on agar plates, was subcultured on solid media. Pure cultures were stored in glycerol at –80°C. Tenreiro et al. (1997) isolated Thermonema rossianum strains from saline hot springs along the Bay of Naples, Italy. Water samples were filtered through membrane filters (0.45 mm pore diameter) and the filters were placed on the surfaces of medium 162 agar containing 1.0% NaCl. The plates were wrapped in plastic bags and incubated at 60°C for up to 7 d. Cultures were purified by subculturing and were maintained at –80°C in medium 162 containing 1.0% NaCl and 15.0% glycerol.

Differentiation of the genus Thermonema from other genera The other aerobic thermophilic genus in the phylum Bacteroidetes is Rhodothermus marinus, which is slightly halophilic, requiring about 0.25% NaCl for growth. It can be differentiated from Thermonema by its lack of filament formation and its ability to use most common sugars and starch.

Taxonomic comments Based on 1490 nucleotides constituting 97% of the 16S rRNA gene of Thermonema lapsum, Patel et al. (1994) concluded that Thermonema lapsum was the deepest member of what is now the phylum Bacteroidetes. Tenreiro et  al. (1997) found Thermonema rossianum strains to be related to other members of the phylum Bacteroidetes by 79–97.5% similarity in 16S rRNA gene sequences. The highest relatedness was exhibited toward Thermonema lapsum (97.2–97.5% sequence similarity). DNA–DNA reassociation values among Thermonema rossianum strains were high (>91%), but they were lower (37–41%) between these strains and Thermonema lapsum.

List of species of the genus Thermonema 1. Thermonema lapsum Hudson, Schofield, Morgan and Daniel 1989, 487VP lap¢sum. L. neut. part. adj. lapsum gliding, from L. v. labor to glide. The characteristics are as given for the genus, with the following additional features. Optimum growth in medium 162 occurs without added NaCl (thus differentiating this species from Thermonema rossianum); addition of NaCl causes a decrease in growth, and no growth occurs at 4% NaCl (Tenreiro et al., 1997). Negative for a- and b-galactosidasenegative. Positive for DNase. Proteolytic. Casein, gelatin, and hippurate are hydrolyzed but not cellulose, starch, and xylan. The following basal medium supplements do not support growth: acetate, l-alanine, casein, l-cystine, galactose, gelatin, gluconate, glucose, inositol, lactose, l-malate, l-proline, propan-1-ol, pyruvate, rhamnose, ribose, skim milk, sorbitol, succinate, sucrose, and yeast extract (all at concentrations of 1 g/l) plus the glutamate amino acid family (glutamate, proline, and arginine) (all at concentrations of 3.3 g/l). The following basal medium supplements do support growth: Casamino acids, amino acid mixture 1 (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, and arginine), and amino acid mixture 2

(the same as amino acid mixture 1 but lacking methionine, phenylalanine, tyrosine, and leucine). Source: New Zealand hot springs. DNA G+C content (mol%): 47 (Tm). Type strain: 23/9, ATCC 43542, DSM 5718. Sequence accession no. (16S rRNA gene): L11703. 2. Thermonema rossianum Tenreiro, Nobre, Rainey, Miguel and da Costa 1997, 125VP ros.si.a¢num. N.L. neut. adj. rossianum pertaining to Rossi, in honor of Mosé Rossi, the noted Neapolitan biochemist. The characteristics are as described for the genus, with the following additional features. Colonies on medium 162 are yellow and 2 mm in diameter after 48 h. Optimum temperature, approximately 60°C; no growth occurs at 30 and 70°C in medium 162. Optimum pH, 7.0–7.5; no growth occurs at pH 5.0 or 10.0. The optimum NaCl concentration for growth is 1.0–3.0%; no growth occurs in medium 162 without added NaCl (thus differentiating this species from Thermonema lapsum) or with >6.0% NaCl. The major fatty acids are C15:0 iso and C17:0 iso 3-OH. Nitrate is not reduced to nitrite. Casein, elastin, and gelatin are degraded; starch, xylan, and cellulose are not degraded. Casamino acids and complex amino acid mixtures are utilized for growth. Growth

Genus I. Toxothrix

DNA G+C content (mol%): 50.9 (HPLC). Type strain: NR-27, DSM 10300. Sequence accession no. (16S rRNA gene): Y08956.

does not occur on single amino acids, sugars, organic acids, and polyols. Source: thermal water tap at the Stufe di Nerone.

References Degryse, W., N. Glansdorff and A. Piérard. 1978. A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus. Arch. Microbiol. 117: 189–196. Garrity, G.M., J.A. Bell and T. Lilburn. 2005. The Revised Road Map to the Manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, Part A, Introductory Essays (edited by Brenner, Krieg, Staley and Garrity). Springer, New York, pp. 159–206. Hamana, K., H. Hamana, M. Niitsu, K. Samejima and S. Matsuzaki. 1992. Distribution of unusual long and branched polyamines in thermophilic eubacteria belonging to Rhodothermus, Thermus and ­Thermonema. J. Gen. Appl. Microbiol. 38: 575–584. Hudson, J.A., K.M. Schofield, H.W. Morgan and R.M. Daniel. 1989. Thermonema lapsum gen. nov., sp. nov., a thermophilic gliding bacterium. Int. J. Syst. Bacteriol. 39: 485–487. Marteinsson, V.T., J.K. Kristjánsson, H. Kristmannsdóttir, M. Dahlkvist, K. Saemundsson, M. Hannington, S.K. Petursdóttir, A. Geptner and P. Stoffers. 2001. Discovery and description of giant submarine smectite cones on the seafloor in Eyjafjordur, northern Iceland, and

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a novel thermal microbial habitat. Appl. Environ. Microbiol. 67: 827–833. Mountain, B.W., L.G. Benning and J.A. Boerema. 2003. Experimental studies on New Zealand hot spring sinters; rates of growth and textural development. Can. J. Earth Sci. 40: 1643–1667. Patel, B.K., D.S. Saul, R.A. Reeves, L.C. Williams, J.E. Cavanagh, P.D. Nichols and P.L. Bergquist. 1994. Phylogeny and lipid composition of Thermonema lapsum, a thermophilic gliding bacterium. FEMS Microbiol. Lett. 115: 313–317. Ramaley, R.F. and J. Hixson. 1970. Isolation of a nonpigmented, ­thermophilic bacterium similar to Thermus aquaticus. J. Bacteriol. 103: 527–528. Schofield, K.M., J.A. Hudson, H.W. Morgan and R.M. Daniel. 1987. A thermophilic gliding bacterium from New Zealand hot springs. FEMS Microbiol. Lett. 40: 169–172. Tenreiro, S., M.F. Nobre, F.A. Rainey, C. Miguel and M.S. da Costa. 1997. Thermonema rossianum sp. nov., a new thermophilic and slightly halophilic species from saline hot springs in Naples, Italy. Int. J. Syst. Bacteriol. 47: 122–126.

Order IV. Incertae sedis Toxothrix was previously assigned to the “Crenotrichaceae” by Garrity et al. (2005), but subsequent analyses transferred Crenothrix to the Proteobacteria. Phylogenetic analyses of the 16S rRNA suggests that Toxothrix represents a very deep group and is only

­ istantly related to any of the previously described orders d within the Bacteroidetes. In view of its ambiguous status, it has been assigned to its own order incertae sedis.

Genus I. Toxothrix Molisch 1925, 144AL* Peter Hirsch To.xo¢thrix. Gr. n. toxon a bow; Gr. fem. n. thrix a thread; N.L. fem. n. Toxothrix bent thread.

Cells cylindrical, colorless, 0.5–0.75×3–6  mm, in filaments (trichomes) up to 400  mm long. A dense body (polyphosphate?) is often located at either end of the cell (Figure 81). Gram reaction not recorded. Filaments often U-shaped ­(Figure 81a and b) and rotating while slowly moving forward with the rounded part in the lead; a mucoid substance, excreted from several sites on the trailing ends, is deposited as a double track (“railroad track”) of twisted strings each 0.2 mm wide (Figure 81). Fan-shaped structures may be deposited laterally along the tracks, as the arms of the U move from side to side, and between the tracks, as a result of the middle section being lifted and then touched down again (Figure 81; Krul et  al., 1970). Oxidized iron may be deposited on the mucoid threads, rendering them yellowish brown and brittle and giving them a diameter of 2.5 mm. Pure cultures have not been obtained, but chemoorganothrophic and psychrophilic cultures have been maintained for long periods at 5 and 10°C. ­Filaments are extremely fragile during laboratory ­examination, and explosive disintegration of filaments has been observed *Editorial note: this chapter is reproduced from Vol. 1 of the 1st edition of Bergey’s Manual of Systematic Bacteriology.

after short periods under the microscope. Grow attached to surfaces and develop best at reduced oxygen tensions ­(Hässelbarth and Lüdemann, 1967) and slightly below neutrality (pH 5.1–7.7). Originally found in water reservoir near the Biological Station at the Dnjepr River in the U.S.S.R. Widely distributed in cold iron springs, brooks, forest ponds, and lakes containing ferrous iron and with reduced oxygen tension (Hirsch, 1981). DNA G+C content (mol%): not known. Type species: Toxothrix trichogenes (Cholodny 1924) Beger in Beger and Bringmann 1953, 332AL.

Further descriptive information The normal trichome does not appear to have cross-walls when viewed with the phase microscope (Figure 81). Cholodny (1924) thought the organisms had a thin, tubular sheath that split repeatedly longitudinally, thus giving rise to the “twisted thread rope.” However, Krul et al. (1970) followed the formation of the double tracks and fan-shaped structures on living, undisturbed and actually growing specimens. Toxothrix trichogenes has been reported to be cultivated by Teichmann (1935). Hirsch (1981) kept natural samples

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Family I. Rhodothermaceae

Figure 81.  Toxothrix trichogenes observed in a small iron spring catch basin. (a and b) Laboratory wet mounts of living trichomes during the first minute. Phase-contrast micrographs. (c and d) Excreted polymer coated with iron oxides, through the peculiar type of motion, form fan-shaped structures (c) or double tracks (d). (Reproduced with permission from J.M. Krul et al., 1970. Antonie van Leeuwenhoek Journal of Microbiology 36: 409–420.)

c­ ontaining Toxothrix in the laboratory for several months; Toxothrix cells survived if the samples contained sediment and organic detritus and were kept cold (5°C) and dark. The appearance of Toxothrix throughout the year (except for May and June) has been reported (Hirsch, 1981). Usually, it is found where Gallionella ferruginea grows and in waters with Fe2+ (1–2.7  mg/l). But Toxothrix cells, contrary to Gallionella cells, prefer habitats with a slightly higher concentration of organic compounds. An iron spring catch basin with cold, Fe-containing water and decaying leaves appears to be the optimal Toxothrix habitat.

Differentiation of the genus Toxothrix from other genera In the absence of Fe deposition, the Toxothrix filaments closely resemble Herpetosiphon, Haliscomenobacter, or “Achroonema” ­filaments. But Herpetosiphon filaments are extremely long

(300–1200 mm) and vary in their cell diameter. Also, transparent sections at the filament tips of Herpetosiphon (called necridia) are not present in Toxothrix. Strains of Haliscomenobacter are not known to glide or to show true branches, and their optimum pH is 7.0–8.0; also, they do not deposit iron oxides. “Achroonema” filaments do not glide in a U-shaped way but remain straight and fairly rigid.

Taxonomic comments Balashova (1968) has pointed out that in some respects Toxothrix resembles Gallionella. The great differences in cell shape do not seem to support this view. Beger and Bringmann (1954) described “Toxothrix gelatinosa” on the basis of smaller filaments (diameter with slime threads: 1.5–1.7 mm) and the fan-shaped arrangements of individual filaments in a gelatinous matrix. However, the individual cell size (0.5×3  mm) falls within the range given for Toxothrix trichogenes.

List of species of the genus Toxothrix 1. Toxothrix trichogenes (Cholodny 1924) Beger in Beger and Bringmann 1953, 332AL (Leptothrix trichogenes Cholodny 1924, 296; Toxothrix ferruginea Molisch 1925, 144; Chlamydothrix trichogenes (Cholodny 1924) Naumann 1929, 513; Sphaerotilus trichogenes (Cholodny 1924) Pringsheim 1949, 234)

tri.cho¢ge. nes. Gr. n. thrix trichos hair; N.L. suff. -genes (from Gr. v. gennaô to produce) producing; N.L. adj. trichogenes hair-producing. Description is the same as for the genus. DNA G+C content (mol%): unknown. Type strain: no culture isolated.

Genus I. Toxothrix

References Balashova, V.V. 1968. [On taxonomy of Gallionella genus]. Mikrobiologiya 37: 715–723. Beger, H. and G. Bringmann. 1953. Die Scheidenstruktur des Abwasserbakteriums Sphaerotilus und des Eisenbakteriums Leptothrix im elektronenmikroskopischen Bilde und ihre Bedeutung für die Systematik dieser Gattungen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. II 107: 318–334. Cholodny, N. 1924. Über neue Eisenbakterienarten aus der Gattung Leptothrix Kütz. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. II 61: 292–298. Garrity, G.M., J.A. Bell and T. Lilburn. 2005. The Revised Road Map to the Manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, Part A, Introductory Essays (edited by Brenner, Krieg, Staley and Garrity). Springer, New York, pp. 159–206. Hässelbarth, U. and D. Lüdemann. 1967. Die biologische Verockerung von Brunnen durch Massenentwicklung von Eisen- und Manganbakte-

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rien. Bohrtechnik-Brunnenbau-Rohr-leitungsbau 10/11 (Ber. DVGW Fachausschuss “Wasserfassung und Wasseranreicherung”). Hirsch, P. 1981. The genus Toxothrix. In The Prokaryotes: A Handbook on Habitats, Isolation, and Identification of Bacteria (edited by Starr, Stolp, Trüper, Balows and Schlegel). Springer, New York, pp. 409–411. Krul, J.M., P. Hirsch and J.T. Staley. 1970. Toxothrix trichogenes (Chol.) Beger et Bringmann: the organism and its biology. Antonie van Leeuwenhoek 36: 409–420. Molisch, H. 1925. Botanische Beobachtungen in Japan. VIII. Eisenorganismen Japan. Sci. Rep. Tohoku Imp. Univ. Ser. IV Biol. 1: 135–168. Naumann, E. 1929. Die eisenspeichernden Bakterien. Kritische Übersicht der bisher bekannten Formen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. II 78: 512–515. Pringsheim, E.G. 1949. Iron bacteria. Bacteriol. Rev. 24: 200–245. Teichmann, P. 1935. Vergleichende Untersuchungen über die Kultur und Morphologie einiger Eisenorganismen. PhD thesis, Prague.

Phylum XV. Spirochaetes Garrity and Holt 2001 Bruce J. Paster Spi.ro.chae¢tes. N.L. fem. pl. n. Spirochaetales type order of the phylum; dropping the ending to denote a phylum; N.L. fem. pl. n. Spirochaetes the phylum of Spirochaetales.

Spirochetes are Gram-stain-negative, helical or spiral-shaped, motile cells that can flex, rotate and translate through liquid and semisolid environments. Most spirochetes possess a cellular ultrastructure unique to bacteria in that they have internal organelles of motility, namely periplasmic flagella. Consequently, the spirochetes are one of the few bacterial phyla whose phenotypic characteristics, e.g., cell morphology, reflect its phylogenetic relationships as based on 16S rRNA gene sequence comparisons (Garrity and Holt, 2001), forming a distinct line of evolutionary descent among the bacteria (Garrity et al., 2005). Spirochetes are chemo-organotrophic and, depending upon the species or phylogenetic group, may grow under anaerobic, microaerophilic, facultatively anaerobic or aerobic conditions. Some are free-living and others are host-associated, e.g.,

a­ rthropods, mollusks, and mammals, including humans. Some species are known to be pathogenic. Type order: Spirochaetales Buchanan 1918, 163AL.

References Buchanan, R.E. 1918. Studies in the nomenclature and classification of the bacteria: X. Subgroups and genera of the Myxobacteriales and Spirochaetales. J. Bacteriol. 3: 541–545. Garrity, G.M. and J.G. Holt. 2001. The Road Map to the Manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 1, The Archaea and the Deeply Branching and Phototrophic Bacteria (edited by Boone, Castenholz and Garrity). Springer, New York, pp. 119–166. Garrity, G.M., J.A. Bell and T. Lilburn. 2005. The Revised Road Map to the Manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, The Proteobacteria, Part A, Introductory Essays (edited by Brenner, Krieg, Staley and Garrity). Springer, New York, pp. 159–206.

Class I. Spirochaetia class. nov. Bruce J. Paster Spi.ro.chae¢ti.a. N.L. fem. n. Spirochaeta type genus of the type order Spirochaetales; suff. -ia ending proposed by Gibbons and Murray and by Stackebrandt et al. to denote a class; N.L. neut. pl. n. Spirochaetia the class of Spirochaetales. Members of the class Spirochaetia are as described for the phylum. The class contains spirochetes in one order, Spirochaetales, which is presently comprised of four families, namely Spirochaetaceae, Brachyspiraceae, Brevinemataceae, and Leptospiraceae. Type order: Spirochaetales Buchanan 1918, 163AL.

Reference Buchanan, R.E. 1918. Studies in the nomenclature and classification of the bacteria: X. Subgroups and genera of the Myxobacteriales and Spirochaetales. J. Bacteriol. 3: 541–545.

Order I. Spirochaetales Buchanan 1917, 163AL Bruce J. Paster Spi.ro.chae.ta¢les. N.L. fem. n. Spirochaeta type genus of the order; -ales suffix to denote an order; N.L. fem. pl. n. Spirochaetales the Spirochaeta order. Based on 16S rRNA gene sequence comparisons, spirochetes form a coherent phylogenetic phylum (Paster et  al., 1991). The order Spirochaetales contains the families Spirochaetaceae, Brachyspiraceae, Brevinemataceae, and Leptospiraceae, as shown in Figure 82. Helically shaped, motile bacteria, 0.1–3 mm in diameter and 4–250  mm in length. Cells have internal organelles of motility called periplasmic flagella (which have been previously called axial fibrils, axial filaments, flagella, endoflagella, and periplasmic fibrils) (Canale-Parola, 1978; Paster and Canale-Parola, 1980). Periplasmic flagella are inserted subterminally at each end of the

protoplasmic cylinder and extend along most of the length of the cell overlapping in the central region, but the other end of the flagella are inserted (Figure 82). This results in a n:2n:n flagellar arrangement where n ranges from 1 to 100s depending upon the species. However, the periplasmic flagella do not overlap in cells of members of the family Leptospiraceae. The protoplasmic cylinder and flagella are encased by an “outer sheath” which has some features analogous the outer membrane of traditional Gram-stainnegative bacteria (Figure 82). Under certain growth conditions, periplasmic flagella of some species protrudes outside of the cell (Charon et al., 1992) (Figure 82). 471

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Phylum XV. Spirochaetes 5%

Figure 82.  Schematic representation of a spirochete. The dotted line

indicates the outer sheath encasing the ­helical protoplasmic cell and the periplasmic flagella which are inserted at each end of the cell.

Chemoheterotrophic. Carbohydrates, amino acids, long-chain fatty acids, or long-chain fatty alcohols serve as carbon and energy sources. Anaerobic, facultatively anaerobic, microaerophilic, and aerobic. Stains Gram-negative. Free-living or in association with animal, insect, and human hosts. Some species are pathogenic. DNA G+C content (mol%): 25–66. Type genus: Spirochaeta Ehrenberg 1835, 313AL.

Further descriptive information Spirochetes have three main types of movements in liquids, namely locomotion, rotations about their longitudinal axis, and flexing (Canale-Parola, 1978). Cells can translocate in highly viscous environments, such as methyl cellulose or in media containing 1% agar. Cells also have been reported to creep or crawl on solid surfaces. Some “free-living pleomorphic spirochetes”, referred to as FLiPS (Ritalahti and Löffler, 2004), have been recently described. One species from the termite hindgut has been named Spirochaeta coccoides (Dröge et al., 2006). These species do not have the characteristic ultrastructural and behavioral features of spirochetes; namely helical, motile cells with protoplasmic cylinder and periplasmic flagella enclosed in an outer sheath. However based on 16S rRNA gene sequence analysis, these species fall within the family Spirochaetaceae (see chapter on Spirochaeta, below). “Spironema culicis” was isolated recently from the mosquito and is the only species of the genus (Cechová et al., 2004). Although it has not yet been formally named, it falls phylogenetically within the family Spirochaetaceae (Figure 83) and likely warrants separate genus designation from other genera of the family. Sequences of species of the genera Treponema and Spirochaeta in the family Spirochaetaceae, Brevinema in the family Brevinemataceae, and Leptospira in the family Leptospiraceae are unusual among bacteria in that they possess a 20–30 base 5¢ extension of 16S rRNA molecule (Paster et al., 1991). The function of this 5¢ extension is unknown, but the region is highly variable and was proposed to form helices of 2–12 bases. The 5¢ extension has not been reported in other spirochetal species.

Key to the families of the order Spirochaetales 1. Cell diameter 0.1–3 mm. Ends of cells are usually not hooked. Periplasmic flagella overlap in the central region of the cell.

References Buchanan, R.E. 1917. Studies on the Nomenclature and Classification of the Bacteria: III. The Families of the Eubacteriales. J. Bacteriol. 2: 347– 350. Canale-Parola, E. 1978. Motility and chemotaxis of spirochetes. Annu. Rev. Microbiol. 32: 69–99.

Spirochaeta alkalica Spirochaeta halophila Spirochaeta litoralis "Spironema culicis" ; AF166259 Borrelia burgdorferi Borrelia hermsii Cristispira pectinis Treponema azotonutricum Treponema denticola Treponema pallidum Brevinema andersonii Brachyspira hyodysenteria e Brachyspira aalborgi Leptospira interrogans Leptonema illini Leptospira parva

Spirochaetacea e

Brevinematacea e Brachyspiraceae Leptospiraceae

Figure 83.  Phylogeny of the order Spirochaetales. The order is comprised of four families, namely Spirochaetaceae, Brachyspiraceae, Brevinemataceae, and Leptospiraceae. Bar = 5% difference in 16S rRNA gene sequences.

The diamino acid in the peptidoglycan is l-ornithine. Anaerobic or facultatively anaerobic. Use carbohydrates and/or amino acids as carbon and energy sources. Free-living and host associated.   → Family I. Spirochaetaceae 2. Cell diameter 0.2–0.4 mm. Cell ends may be blunt or pointed, and are not hooked. Periplasmic flagella overlap in the central region of the cell. The diamino acid in the ­peptidoglycan is l-ornithine. Obligately anaerobic, aerotolerant. Use monosaccharides, disaccharides, the trisaccharide trehalose, and amino sugars as carbon and energy sources. Does not use polysaccharides. Host-associated.   → Family II. Brachyspiraceae 3. Cell diameter 0.2–0.3 mm. Cells are short, 4–5 mm in length with only one or two turns. Ends of cells are usually not hooked. Periplasmic flagella overlap in the central region of the cell. The diamino acid in the peptidoglycan is not known. Microaerophilic. Peptones are required for growth. Host-associated.   → Family III. Brevinemataceae 4. Cell diameter 0.1–0.3 mm. Ends of cells are usually hooked. The diamino acid in the peptidoglycan is diaminopimelic acid. Periplasmic flagella do not appear to overlap in the central region of the cell. Obligately aerobic, or microaerophilic. Use long-chain fatty acids or long-chain fatty alcohols as carbon and energy sources. Do not use carbohydrates or amino acids. Free-living and host-associated.   → Family IV. Leptospiraceae

Cechová, L., E. Durnová, S. Sikutová, J. Halouzka and M. Nemec. 2004. Characterization of spirochetal isolates from arthropods collected in South Moravia, Czech Republic, using fatty acid methyl esters analysis. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 808: 249–254. Charon, N.W., S.F. Goldstein, S.M. Block, K. Curci, J.D. Ruby, J.A. Kreiling and R.J. Limberger. 1992. Morphology and dynamics of protruding spirochete periplasmic flagella. J. Bacteriol. 174: 832–840.

Genus I. Spirochaeta Dröge, S., J. Fröhlich, R. Radek and H. König. 2006. Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite Neotermes castaneus. Appl. Environ. Microbiol. 72: 392–397. Ehrenberg, C.G. 1835. Dritter Beitrag zur Erkemtiss grosser Organisation in der Richtung des kleinsten Raumes. Abh. Preuss. Akad. Wiss. Phys. K1 Berlin aus den Jahre 1833–1835: 143–336. Paster, B.J. and E. Canale-Parola. 1980. Involvement of periplasmic fibrils in motility of spirochetes. J. Bacteriol. 141: 359–364.

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Paster, B.J., F.E. Dewhirst, W.G. Weisburg, L.A. Tordoff, G.J. Fraser, R.B. Hespell, T.B. Stanton, L. Zablen, L. Mandelco and C.R. Woese. 1991. Phylogenetic analysis of the spirochetes. J. Bacteriol. 173: 6101– 6109. Ritalahti, K.M. and F.E. Löffler. 2004. Characterization of novel free-living pleomorphic spirochetes (FLiPS), Abstract 539. Presented at the 10th International Symposium on Microbial Ecology. International Society for Microbial Ecology, Geneva, Switzerland.

Family I. Spirochaetaceae Swellengrebel 1907, 581AL Bruce J. Paster Spi.ro.chae.ta.ce¢ae. N.L. fem. n. Spirochaeta type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Spirochaetaceae the Spirochaeta family. Helical cells, 0.1–3.0  mm in diameter and 3.5–250  mm in length. Cells do not have hooked ends as do members of the family Leptospiraceae. Periplasmic flagella are inserted subterminally at each end of the cell and extend along most of the length of the cell overlapping in the central region (Figure 82). The diamino acid in the peptidoglycan is l-ornithine. Motile. Anaerobic, facultatively anaerobic, or microaerophilic. Chemo-organotrophic. Utilize carbohydrates and/or amino acids as carbon and energy sources. Do not use long-chain fatty acids or long-chain fatty alcohols as energy sources. Free-living or in association with animal, insect and human hosts. Some species are pathogenic. The DNA G+C content is 36–66 mol% (Tm, Bd, and genetic sequence analysis). Species examined by 16S rRNA sequence analysis are distinct from members of the families Brachyspiraceae, Brevinemataceae, and Leptospiraceae (Figure 83). Type genus: Spirochaeta Ehrenberg 1835, 313AL.

Key to the genera of the family Spirochaetaceae 1. Cells are 0.2–75  mm in diameter and 5–250  mm in length. Obligately anaerobic and facultatively anaerobic. Carbohydrates serve as energy and carbon sources. Amino acids are not used as growth substrates. Free-living in fresh water and marine environments, including mud, sediments and water of ponds, lakes, streams, and marshes. Many not-yet-cultivated species identified as based on 16S rRNA gene sequence

comparisons. Not considered as pathogenic. The DNA G+C content is 45–66 mol% (Tm, Bd, and HPLC).   → Genus I. Spirochaeta 2. Cells are 0.2–0.3  mm in diameter and 3–20  mm in length. Microaerophilic. Arthropod-borne pathogens of man, other mammals, and birds. The causative agents of tick-borne Lyme disease and relapsing fever and louse-borne relapsing fever in man. The DNA G+G content of a limited number of species is 27–32 mol% (Tm, HPLC, and genome ­sequencing).   → Genus II. Borrelia 3. Cells are 0.5–3 mm in diameter and 30–180  mm in length. Hundreds of periplasmic flagella present as a bundle forming a ridge called crista. Inhabit the crystalline style of the digestive tract of aquatic mollusks. Not grown in pure culture. The DNA G+C content is not known.   → Genus III. Cristispira 4. Cells are 0.1–0.4  mm in diameter and 5–20  mm in length. Obligately anaerobic. Carbohydrates and amino acids serve as energy and carbon sources. Found in the oral cavity, intestinal tract, and genital areas of humans and animals. Some species are pathogenic. Also found in the hindgut of termites. Many not-yet-cultivated species identified as based on 16S rRNA gene sequence comparisons. The DNA G+C content is 36–54 mol% (Tm, HPLC, genome sequence).   → Genus IV. Treponema

Genus I. Spirochaeta Ehrenberg 1835, 313AL (Spirochoeta Dujardin 1841, 225, and Spirochaete Cohn 1872, 180 (orthographic variants of Spirochaeta); Ehrenbergia Gieszczkiewicz 1939, 24) Susan Leschine and Bruce J. Paster Spi.ro.chae¢ta. Gr. n. speira (L. transliteration spira) a coil; Gr. fem. n. chaitê (L. transliteration chaete) hair; N.L. fem. n. Spirochaeta coiled hair.

Flexible helical cells 0.2–0.75 mm in diameter and 5–250 mm in length. All species have two periplasmic flagella per cell except Spirochaeta plicatilis, which has many periplasmic flagella. Under unfavorable conditions, spherical cells or structures 0.5–2.0 mm (occasionally up to 10 mm) in diameter are formed. Cells translocate when suspended in liquids and crawl or creep when in contact with solid surfaces. Obligately anaerobic or facultatively anaerobic. Under aerobic growth conditions the facultatively anaerobic species usually produce carotenoid pigments that give a yellow, yellow-orange, or red coloration to colonies or cells in liquid media. Thermophilic species are known. Optimum temperature range, 25–68°C. Chemo-organotrophic,

using a variety of carbohydrates as carbon and energy sources. The main products of anaerobic carbohydrate metabolism are ethanol, acetate, CO2, and H2. Under aerobic conditions, facultatively anaerobic ­species oxidize carbohydrates yielding primarily CO2 and ­acetate. Indigenous to aquatic freshwater and marine environments such as the sediments, mud and water of ponds, marshes, swamps, lakes, rivers, and hot springs. Occur commonly in H2S-­containing environments. Free-living. None reported to be pathogenic. DNA G+C content (mol%): 50–65 (Bd), 45–66 (Tm), 50–58.5 (HPLC). Type species: Spirochaeta plicatilis Ehrenberg 1835, 313AL.

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Family I. Spirochaetaceae

Further descriptive information Cells of all species are helical in shape and possess the typical ultrastructural features of spirochetes (Canale-Parola, 1984a). The outermost structure of the cells is an “outer membrane”, or “outer sheath”, which encloses the coiled cell body (“protoplasmic cylinder”) consisting of the cytoplasm, the nuclear region, and the peptidoglycan-cytoplasmic membrane complex. Organelles ultrastructurally similar to bacterial flagella are located in the area between the outer membrane and the protoplasmic cylinder. These organelles are essential components of the motility apparatus of spirochetes (Paster and Canale-Parola, 1980) and are usually called “periplasmic flagella”. One end of each periplasmic flagellum is inserted near a pole of the protoplasmic cylinder, while the other end is not inserted. Individual periplasmic flagella extend for most of the length of Spirochaeta cells so that those inserted near opposite ends overlap in the central region of the organism. The nature of the spherical structures, called “spherical ­bodies”, that are formed under unfavorable growth conditions has not been determined. Spherical bodies occur either in physical association with helical cells or free. When suspended in liquids, cells translocate “in straight lines or nearly straight lines, and they appear to spin rapidly about their longitudinal axis (Berg, 1976). The motility of a strain of Spirochaeta aurantia in liquid environments has been described (Greenberg et al., 1985, 1977c). Occasionally a cell stops momentarily and flexes, and then resumes spinning and translational motility. However, when translation resumes, the direction of movement is usually altered…” and frequently the previously leading cell end becomes the trailing end (Greenberg and Canale-Parola, 1977c). During runs, Spirochaeta aurantia cells have an average linear speed of approximately 16/s (Fosnaugh and Greenberg, 1988). Flexes last from a fraction of a second to several seconds. The average frequency of reversals in cell populations is approximately 0.31 reversals/ 5 s (Fosnaugh and Greenberg, 1988). Cells in motion usually retain their basic helical configuration, but they assume a variety of shapes as a result of flexing, undulating, and contracting, as well as wave propagation. Broad secondary coils or waves superimposed on the smaller primary coils are formed frequently (Canale-Parola, 1977, 1978). During creeping movements of Spirochaeta plicatilis on solid surfaces (Blakemore and Canale-Parola, 1973), “… the rear coils follow the tortuous path of the anterior cell end almost exactly” (Canale-Parola, 1978). Cells retain translational motility in environments of relatively high viscosity, usually becoming immotile at viscosities of 300–1000 centipoise, depending on the strain (Greenberg and Canale-Parola, 1977a, b). Strains of Spirochaeta aurantia exhibit chemotaxis toward ­carbohydrates, but not toward amino acids (Breznak and Canale-Parola, 1975; Greenberg and Canale-Parola, 1977c). Effective attractants for Spirochaeta aurantia strain M1 are as follows: d-glucose, 2-deoxy-d-glucose, a-methyl-d-glucoside, d-galactose, d-fucose, d-mannose, d-fructose, d-xylose, maltose, cellobiose, and d-glucosamine (Greenberg and Canale-Parola, 1977c). Taxis toward d-galactose and d-fucose is induced by the presence of d-galactose in the growth medium. The helical shape of the cells is maintained by the peptidoglycan layer ( Joseph and Canale-Parola, 1972). l-Ornithine is the only diaminoamino acid in the peptidoglycan of Spirochaeta

stenostrepta, Spirochaeta litoralis, Spirochaeta aurantia, and Spirochaeta halophila ( Joseph et al., 1973; B.J. Paster and E. CanaleParola, unpublished data). The peptidoglycans of Spirochaeta zuelzerae and Spirochaeta plicatilis have not been tested for the presence of l-ornithine. The peptidoglycan of Spirochaeta stenostrepta is composed of acylglucosamine, acylmuramic acid, l-alanine, d-glutamic acid, l-ornithine, and d-alanine ( Joseph et  al., 1973; Schleifer and Joseph, 1973). Peptidoglycan of similar composition is present in Spirochaeta litoralis. At least 50% of the peptide subunits of the peptidoglycan of Spirochaeta stenostrepta contain the tripeptide N-acyl-muramyl-l-alanyl-a-d-glutamyl-l-ornithine. Cross-linkage (30%) between the 6-amino group of l-ornithine and the carboxyl group of d-alanine occurs in the remaining peptide subunits of sequence N-acyl-muramyl-l-alanyl-a-d-glutamyl-lornithyl-d-alanine (Schleifer and Joseph, 1973). A lipoprotein layer, adjacent and external to the peptidoglycan, has been detected in Spirochaeta stenostrepta ( Joseph et al., 1970). This layer consists of a fine array of tightly packed, longitudinally oriented helices measuring 2.5 nm in diameter (Holt and Canale-Parola, 1968). Colonies of Spirochaeta diffuse or spread through the agar medium in which they are growing. This phenomenon is especially apparent in agar media containing low substrate concentrations and 1% or less agar. Diffusion of colonies is due to migration of the growing cells through the agar medium. Migration of the cells is the result of chemotaxis toward the growth substrate and of the ability of spirochetes to locomote through agar gels (Canale-Parola, 1977, 1978). The growth of obligately anaerobic species of Spirochaeta is abundant in media with 1.0 or 1.5 g of agar/100 ml, whereas the growth of some strains of facultatively anaerobic species of Spirochaeta is inhibited in media containing more than 1.0% agar. However, these strains grow abundantly when the agar concentration in the medium is 1% or lower. Under anaerobic conditions, Spirochaeta stenostrepta, Spirochaeta litoralis, Spirochaeta aurantia, and Spirochaeta halophila ferment carbohydrates to pyruvate via the Embden–Meyerhof pathway (Canale-Parola, 1977; Greenberg and Canale-Parola, 1976). Pyruvate is metabolized to acetyl-CoA, CO2, and H2 by means of a clostridial-type clastic reaction. Acetyl-CoA is converted to acetate in reactions catalyzed by phosphotransacetylase and acetate kinase, and to ethanol through a double reduction involving aldehyde and alcohol dehydrogenase activities (Canale-Parola, 1977). These pathways constitute the major anaerobic energy-yielding mechanisms utilized by the four Spirochaeta species mentioned above (Canale-Parola, 1977). The pathways of carbohydrate catabolism utilized by Spirochaeta zuelzerae have not been elucidated. This spirochete does not form ethanol, but produces succinate and larger amounts of lactate than do other species. In addition to carbohydrates, Spirochaeta isovalerica ferments l-leucine, l-isoleucine, and l-valine, forming isovaleric, 2-methylbutyric, and isobutyric acids, respectively, as end products (Harwood and Canale-Parola, 1983). Fermentation of the amino acids in the absence of glucose does not support measurable growth of Spirochaeta isovalerica, but serves to generate ATP, which is utilized as a source of maintenance energy by the  spirochete when fermentable carbohydrates are not available (Harwood and Canale-Parola, 1981a, 1983). In addition to the branched-chain fatty acids, amino acid catabolism by

Genus I. Spirochaeta

Spirochaeta isovalerica yields small quantities of isobutanol and isoamyl ­alcohol (Harwood and Canale-Parola, 1981b, 1983). When growing aerobically, Spirochaeta aurantia and Spirochaeta halophila derive energy by performing an incomplete oxidation of carbohydrates, with CO2 and acetate being the main dissimilatory products. The tricarboxylic acid cycle either is not present or serves in a minor catabolic capacity in these two species. Determinations of molar growth yields and other studies indicate that, when growing aerobically, Spirochaeta aurantia and Spirochaeta halophila generate ATP via oxidative phosphorylation as well as by substrate level phosphorylation (Breznak and Canale-Parola, 1972a; Greenberg and Canale-Parola, 1976). Cytochromes b558 and cytochrome o are present in Spirochaeta aurantia (Breznak and Canale-Parola, 1972a). Spirochaeta species are able to synthesize all of their cell lipids de novo. The chain length of cellular fatty acids varies from 12 to 18 carbons (Livermore and Johnson, 1974). Spirochaeta aurantia and Spirochaeta zuelzerae but not Spirochaeta litoralis and Spirochaeta stenostrepta synthesize unsaturated fatty acids. Spirochaeta stenostrepta and Spirochaeta zuelzerae but not Spirochaeta litoralis and Spirochaeta aurantia synthesize anteiso branched-chain fatty acids (Livermore and Johnson, 1974). Spirochaeta species are resistant to the antibiotic rifampicin (rifampin) at concentrations of 1–50 µg rifampicin/ml. Resistance to rifampicin may be due to low affinity of the spirochetal RNA polymerase for the antibiotic.

Enrichment and isolation procedures Anaerobic and facultatively anaerobic spirochetes occur commonly in aquatic freshwater and marine environments such as the sediments, mud and water of ponds, marshes, swamps, lakes, rivers, and hot springs, often in association with decomposing plant biomass (Harwood and Canale-Parola, 1984; Leschine, 1995). Spirochaeta strains are readily isolated from natural environments by means of selective procedures and usually grow abundantly in ordinary laboratory media. Anaerobic growth yields of the isolates range from 2 to 108 to approximately 109 cells/ml, but commonly are 6–8×108 cells/ml (Breznak and Canale-Parola, 1975; Canale-Parola, 1973; Greenberg and Canale-Parola, 1976; Harwood and Canale-Parola, 1984; Hespell and Canale-Parola, 1970a, b). Cell population doubling times in anaerobic cultures are 2.2–12 h, depending on the species and the growth conditions. Aerobically grown cultures yield 0.7–1.2×109 spirochetes/ml, with doubling times of 2–4 h (Breznak and Canale-Parola, 1975; Canale-Parola, 1973; Greenberg and Canale-Parola, 1976). A procedure in which the antibiotic rifampicin (rifamycin) serves as a selective agent is quite effective for the isolation of free-living (genus Spirochaeta) and host-associated (genus Treponema) spirochetes from natural environments (Harwood et al., 1982; Patel et al., 1985; Stanton and Canale-Parola, 1979; Weber and Greenberg, 1981). This procedure (described below) is based on the observation that spirochetes in general are naturally resistant to rifampicin (Leschine and Canale-Parola, 1986; Stanton and Canale-Parola, 1979). Thus, spirochetes such as Spirochaeta stenostrepta and Spirochaeta aurantia grow in the presence of as much as 100–200 µg of rifampicin per ml of medium (Leschine and Canale-Parola, 1986), whereas the growth of many other bacteria is inhibited. The resistance of spirochetes to rifampicin is probably due to the low affinity of their RNA

475

polymerase for the antibiotic (Allan et al., 1986; Leschine and Canale-Parola, 1986). Enrichment procedures used in the isolation of Spirochaeta species are based on one or more of the following selective factors: (1) resistance to rifampicin (mentioned above), (2) the ability of spirochetes to pass through filters that retain most other bacteria, (3) the migratory movement of spirochetes through agar media (Canale-Parola, 1973, 1984b). The latter two procedures will enrich for species of Spirochaeta measuring less than 0.5 mm in diameter. In the enrichment-by-filtration procedure (described below), separation of Spirochaeta species from most of the micro-organisms present in mud or water is achieved by techniques involving filtration through cellulose ester filter discs (e.g., Millipore filters) having a mean pore diameter of 0.3 or 0.45 mm. Spirochetes pass through these filter discs because their cell diameter is relatively small, and probably also because their motility apparatus enables them to swim freely in liquids as well as to move in contact with solid surfaces. The enrichment-by-migration procedure uses the ability of spirochetes to move through agar gels or media containing as much as 1–2% (w/v) agar. This movement or migration occurs primarily within the agar gel, i.e., below the surface of the agar medium. In contrast, flagellated bacteria usually cannot carry out translational movement through gels or media containing agar at the above-mentioned concentrations, although several exceptions have been reported (Greenberg and Canale-Parola, 1977a). Apparently, the cell coiling of spirochetes is important for their translational motion through agar gels, inasmuch as this type of movement is impaired in mutant spirochetes lacking the cell-coiling characteristic of the parental strain (Greenberg and Canale-Parola, 1977b). Migration of spirochetes through agar media results from the unique motility mechanism of these bacteria (Canale-Parola, 1977, 1978), as well as from chemotaxis toward the energy and carbon source (Breznak and Canale-Parola, 1975; Greenberg and Canale-Parola, 1977c; Terracciano and Canale-Parola, 1984). The role of chemotaxis in the migration of saccharolytic spirochetes through agar media has been studied (Breznak and Canale-Parola, 1975). When these spirochetes are inoculated in the center of glucose-containing agar medium plates, they grow using this sugar as their energy source. Utilization of the sugar by the spirochetes gives rise to a glucose concentration gradient that moves away from the center of the plate as more of this carbohydrate is metabolized by the spirochetes. Because the spirochetes exhibit chemotaxis toward glucose and are able to move through the agar gel, they migrate into the areas of higher glucose concentration within the gradient. Thus, the spirochetal population, by following the outward movement of the gradient, migrates toward the periphery of the plate. This behavior results in the formation of a growth “veil” or “ring” of spirochetes for which glucose serves both as the energy source for growth and as the chemoattractant (Breznak and CanaleParola, 1975; Canale-Parola, 1973). The veil or ring increases continuously in diameter during incubation and may reach the outer edge of the plate. The migration rate of the spirochetal population is greatest in agar media containing low substrate concentrations (e.g., 0.02% glucose). In these media the substrate becomes rapidly depleted in the region where spirochetes are growing, and the spirochetal population moves toward the

476

Family I. Spirochaetaceae

outer zone of higher substrate concentrations at a relatively fast rate (Breznak and Canale-Parola, 1975). In procedures for the isolation of Spirochaeta species from natural environments, the chemotactic behavior and the ability of these bacteria to move through agar gels have important selective functions. In a typical isolation procedure, a small, shallow cylindrical hole is made through the surface of an agar medium containing a low concentration of carbohydrate. Rifampicin may be included in the medium as an additional selective agent for spirochetes. The medium may be in a Petri dish or a small bottle. A tiny drop of pond water, or of any other material in which spirochetes have been observed, is placed within the hole. The chemotactic, saccharolytic spirochetes in the inoculum multiply and form a growth veil that extends outwardly through the agar medium. Thus, the spirochetes in the veil move away from contaminants, which remain mainly in the vicinity of the inoculation site. Spirochetal cells from the outermost edge of the veil are used to obtain pure cultures by conventional methods, such as streaking on agar medium plates. Isolation procedures involving chemotaxis and movement through agar gels are described below. Selective isolation techniques have not been developed for the large Spirochaeta species, such as Spirochaeta plicatilis.

stenostrepta, ­Spirochaeta zuelzerae, and Spirochaeta caldaria, do not belong with other free-living Spirochaeta (shown in Figure 84), but are more closely related to members of the genus Treponema. The phylogenetic clustering of these species has also been confirmed by ­single-base signature analysis, i.e., the sequences of these species have more signature bases found in the sequences of the treponemes than in the sequences of Spirochaeta ­species. Con­sequently, these species should be classified as species of Treponema, but since there has been no formal renaming, they remain Spirochaeta spp. in this chapter. These “free-living”

5%

Spirochaeta litoralis Spirochaeta isovaleric a Spirochaeta halophil a Spirochaeta americana Spirochaeta alkalica Spirochaeta africana Spirochaeta asiatic a

Spirochaeta

Spirochaeta thermophil a

Maintenance procedures

Spirochaeta coccoides Spirochaeta smaragdinae

Species of Spirochaeta remain viable for many years when stored in the frozen state at the temperature of liquid nitrogen. Methods for liquid nitrogen storage of Spirochaeta species and for the preparation of other types of stock cultures of these bacteria have been described (Canale-Parola, 1973).

Spirochaeta bajacaliforniensis Spirochaeta aurantia [Spirochaeta ] caldaria [Spirochaeta ] xylanolyticus

Differentiation of the genus Spirochaeta from other genera

[Spirochaeta ] taiwanensis

Members of the genus Spirochaeta are readily differentiated from other genera of spirochetes as shown in Table 118.

[Spirochaeta ] zuelzerae

[Spirochaeta ] stenostrept a

Treponema

Treponema denticola

Taxonomic comments

Treponema pallidum

Based on 16S rRNA gene sequence comparisons (Paster et al., 1984, 1991), members of the genus Spirochaeta belong within the family Spirochaetaceae and are clearly distinct from the other genera of spirochetes (Figure 84). These data also suggest that Spirochaeta aurantia warrants separate genus designation, although it would still fall within the family Spirochaetaceae (­Figure 84). However, several species of Spirochaeta, including Spirochaeta

Figure 84.  Phylogenetic tree of the genus Spirochaeta and related organisms, based on 16S ribosomal RNA gene (rRNA) sequences. Some of the named Spirochaeta species shown in brackets, namely [Spirochaeta] zuelzerae, [Spirochaeta] caldaria, [Spirochaeta] stenostrepta, [Spirochaeta] xlanolyticus and [Spirochaeta] taiwanensis, are phylogenetically more related to members of the genus Treponema. The scale bar represents a 5% difference in nucleotide sequence.

Table 118.  Differentiation of the genus Spirochaeta from other genera of spirochetes

Characteristic Free-living Host-associated Obligate aerobes Obligate anaerobes Facultative anaerobes Microaerophiles Energy and carbon sources:   Carbohydrates   Amino acids   Long-chain fatty acids DNA G+C content (mol%)

Spirochaeta

Cristispira

Treponema

Borrelia

Brevinema

Leptospira and related genera

Brachyspira

+ − − + + −

− + − − − +

− + − + − −

− + − − − +

− + − + − −

+ + + − − nr

− + − − − +

+ − − 41–65

nr nr nr nr

+ + − 36–54

+ − − nr

nr nr nr nr

− − + 35–53

nr nr nr 26

b

nr, Not reported or not determined.

a

Some species of the free-living Spirochaeta are more closely related to species of Treponema based on 16S rRNA sequence comparisons.

b

Genus I. Spirochaeta

s­ pirochetes might represent transitional species, i.e., they could be descendants of precursors of host-associated treponemes. Alternatively, these species may have been from a mammalian host and were disseminated via fecal contamination. Two other purported species of Spirochaeta that belong more with the treponemes have been described. Two thermophilic anaerobic, xylan-degrading spirochetal strains were isolated from a hot spring in Taiwan. They have been provisionally named (unpublished) as Spirochaeta taiwanensis (AY735103) and Spirochaeta xylanolyticus (AY735097), but are likely the same species with nearly identical 16S rRNA sequences. However, these thermophilic spirochetes are also close relatives of Spirochaeta caldaria and consequently are likely members of the genus Treponema. 16S rRNA sequences of these Spirochaeta species possess a 20–30-base extension at the 5¢ end, which is typical of 16S rRNA sequences of species of Spirochaeta, Treponema, Leptospira, and Leptonema (Defosse et al., 1995; Paster et al., 1991).

Differentiation of the species of the genus Spirochaeta Sixteen species of Spirochaeta are presently known and listed below. Characteristics that differentiate these species are shown in Table 119. Spirochaeta plicatilis has not been grown in pure culture, but its ultrastructure and some of its ecological characteristics have been described (Blakemore and Canale-Parola, 1973). Ten species (Spirochaeta stenostrepta, Spirochaeta litoralis, Spirochaeta zuelerae, Spirochaeta isovalerica, Spirochaeta bajacaliforniensis, Spirochaeta thermophila, Spirochaeta caldaria, Spirochaeta smargdinae, Spirochaeta asiatica, and Spirochaeta americana) are obligate anaerobes, and two species (Spirochaeta alkalica and Spirochaeta africana) are aerotolerant anaerobes. Three other species, Spirochaeta aurantia, Spirochaeta halophila, and Spirochaeta cellobiosiphila are facultative anaerobes. When grown aerobically, Spirochaeta aurantia and Spirochaeta halophila but not Spirochaeta

477

cellobiosiphila characteristically produce carotenoid pigments (Breznak and Warnecke, 2008; Greenberg and Canale-Parola, 1975). Most species of Spirochaeta are mesophilic, growing at optimum temperatures in the range 15–40°C. However, the thermophilic species, Spirochaeta thermophila and Spirochaeta caldaria, both from thermal springs, have optimum growth temperature of 66–68 and 48–52°C, respectively. Two subspecies of Spirochaeta aurantia are known. One of these (Spirochaeta aurantia subsp. stricta) is characterized by significantly narrower coils than the other (Spirochaeta aurantia subsp. aurantia), and its DNA possesses a slightly lower G+C content (Breznak and Canale-Parola, 1975; Canale-Parola, 1984b). Spirochaeta stenostrepta, Spirochaeta zuelerae, Spirochaeta ­caldaria, and Spirochaeta aurantia are freshwater species, whereas Spirochaeta litoralis, Spirochaeta isovalerica, Spirochaeta bajacaliforniensis, Spirochaeta thermophila, and Spirochaeta cellobiosiphila are marine species and require sodium ion (Na+) concentrations in the range 200–480 mM for optimal growth (Aksenova et al., 1990, 1992; Breznak and Warnecke, 2008; Fracek and Stolz, 1985; Harwood and Canale-Parola, 1983; Hespell and Canale-Parola, 1970b). Spirochaeta halophila was isolated from a high-salinity pond and grows optimally when 750 mM NaCl, 200 mM MgSO4, and 10 mM CaCl2 are present in the medium (Greenberg and Canale-Parola, 1976). Other halophilic species include Spirochaeta americana, Spirochaeta asiatica, Spirochaeta alkalica, and Spirochaeta africana, which were isolated from the sediments of hypersaline lakes and require Na+ concentrations in the range 850–1200 mM for optimal growth (Hoover et al., 2003; Zhilina et al., 1996). The latter three species are also alkaliphilic and growth does not occur below pH 8. Spirochaeta smargdinae, isolated from a production water sample collected from an offshore oilfield, requires at least 170 mM NaCl and grows ­optimally with 850 mM NaCl (Magot et al., 1997).

List of species of the genus Spirochaeta 1. Spirochaeta plicatilis Ehrenberg 1835, 313AL pli.ca¢ti.lis. L. fem. adj. plicatilis flexible. Helical cells, 0.75 mm in diameter and usually 80–250 mm in length. Cells have regular primary coils, which are stable (persist both in the presence and absence of movement). Cells in motion may exhibit broad secondary coils superimposed on the smaller primary coils and when suspended in liquids, they display rotation about the longitudinal axis and wide waves traveling along the length of the organism. Cells creep in contact with solid surfaces (Blakemore and Canale-Parola, 1973). Regularly spaced cross-walls or transverse septa are present (Blakemore and Canale-Parola, 1973). Long specimens may consist of chains of multicellular spirochetes. Many periplasmic flagella are present, occurring as a bundle wound around the protoplasmic cylinder. Phase-contrast photomicrographs and electron micrographs of the cells have been published (Blakemore and Canale-Parola, 1973). Not cultivated in pure culture. Presumed to be either a microaerophile or an anaerobe that can tolerate low O2 tensions. Present in H2S-containing freshwater, brackish and marine mud, frequently in association with Beggiatoa trichomes.

DNA G+C content (mol%): not determined. Type strain: not yet grown in pure culture. Sequence accession no. (16S rRNA gene): none. 2. Spirochaeta africana Zhilina 1996, 310VP a.fri.ca¢na. L. fem. adj. africana of African continent, found in African alkaline Lake Magadi. Motile, helical cells, 0.25–0.3 mm in diameter and 15–30 mm in length, with shorter (7.5 mm) and longer (up to 40 mm) cells occurring in culture. Outermost structure is an outer membrane enclosing periplasmic flagella and a protoplasmic cylinder. Cells have regular, stable primary coils. Cell mass is orange. Anaerobic, aerotolerant, fermentative; utilizes carbohydrates, mainly mono- and disaccharides, as carbon and energy sources. Preferred substrates: fructose > maltose = trehalose = sucrose > cellobiose > glucose > glycogen > starch; poor growth with mannose or xylose; no growth with galactose, N-acetylglucosamine, or ribose (the optical isomers of the sugars were not described). Amino acids do not serve as fermentable substrates. A supplement of vitamins is required; yeast extract can be omitted from culture media. Aerotolerant; develops under a cotton plug in liquid medium.

16. S. zuelzerae

15. S. thermophila 14. S. stenostrepta

13. S. smaragdinae 12. S. litoralis

11. S. isovalerica

10. S. halophila

9. S. cellobiosiphila

8. S. caldaria

7. S. bajacaliforniensis

6. S. aurantia

5. S. asiatica

2. S. africana

1. S. plicatilis

a 

Abbreviations: ObAn, obligate anaerobe; FAn, facultative anaerobe; O2An, oxygen-tolerant anaerobe; A, acetic acid; E, ethanol; H, hydrogen; C, carbon dioxide; L, lactic acid; F, formate; nd, not determined.

Relationship Unknown O2An O2An ObAn ObAn FAn ObAn ObAn FAn FAn ObAn ObAn ObAn ObAn ObAn ObAn to O2 DNA G+C Unknown 57 57 58.5 49 61–65 50 45 41 62 64.5 51 50 60 52 56 content (mol%) Optimum temp. Unknown 30–37 33–37 37 33–37 25–30 36 48–52 37 35–40 15–35 30 37 35–37 66–68 37–40 (°C) Optimum pH Unknown 8.8–9.8 8.7–9.6 9.5 8.4–9.4 7.0–7.3 7.5 nd 7.5 7.5 7.5 7.0–7.5 7.0 7.0–7.5 7.5 7.0–8.0 Optimum NaCl Unknown 0.8–1.4 0.8–1.4 0.5 0.5–1.4 nd 0.12 nd 0.3–0.4 0.75 0.3 0.35 0.85 nd 0.25 nd (M) Products from Unknown A, E, H A, C, H A, E, H, F A, E, L A, E, C, H A, E, C, H A, L, C, H A, E, C, H A, E, C, H A, E, C, H A, E, C, H A, L, C, H A, E, H, C A, L, C, H A, L, H, C glucose Environment Fresh water, Alkaline, Alkaline, Alkaline, Alkaline, Fresh Marine Fresh Marine High salinity Marine Marine Oil field Fresh Marine Fresh marine hyper-saline hyper-saline hyperhyper-saline water water hot pond water hot spring water lake lake saline lake lake spring Treponema 16S Unknown − − − − − − + − − − − − + − + rRNA signature

Characteristic

3. S. alkalica

Table 119.  Comparison of species of the genus Spirochaeta a

4. S. americana

478 Family I. Spirochaetaceae

Genus I. Spirochaeta

Primary products of glucose fermentation are acetate, ethanol, and H2. Lactate is a minor product in stationary phase. Halophilic, growing in sodium carbonate medium, but not requiring it. Depends on sodium; no growth below 3% (w/v) or above 10% (w/v) NaCl. Optimal growth at pH 8.8–9.75. No growth at pH 8.0–10.8. Optimum temperature for growth, 30–37°C; range, 15–47°C; slow growth at 6°C after a long lag phase. Source: a bacterial bloom in the brine under trona (e.g., a sedimentary deposit that results from the evaporation of seawater) from alkaline equatorial Lake Magadi. DNA G+C content (mol%): 57.1 (Tm). Type strain: strain Z-7692, ATCC 700263, DSM 8902. Sequence accession no. (16S rRNA gene): X93928. 3. Spirochaeta alkalica Zhilina 1996, 309VP al.ka.li¢ca. N.L. n. alkali (from Arabic al-qalyi the ashes of saltwort), soda ash; L. fem. suff. -ica suffix used with the sense of pertaining to; N.L. fem. adj. alkalica intended to mean alkaline, developing in the alkaline medium. Motile, helical cells, 0.4–0.5 mm in diameter and 9–18 mm in length, with shorter (6 mm) and longer (up to 35 mm) cells occurring in culture. Outermost structure is an outer membrane enclosing periplasmic flagella and a protoplasmic cylinder. Cells have regular, stable primary coils. Cell mass is orange. Anaerobic, aerotolerant, fermentative; utilizes carbohydrates, mainly mono- and disaccharides, as carbon and energy sources. Preferred substrates: sucrose > trehalose > cellobiose > glucose = maltose > xylose > starch; poor growth with fructose, galactose, ribose, or N-acetylglucosamine (the optical isomers of the sugars were not described); no growth with mannose or glycogen. Amylolytic and agarolytic. Amino acids do not serve as fermentable substrates. A supplement of vitamins and yeast extract is required. Aerotolerant; growth develops under a cotton plug in liquid medium. Primary products of glucose fermentation are acetate, H2, and CO2. Minor products in stationary phase are ethanol and lactate. Alkaliphilic; growth in sodium carbonate medium optimally at pH 8.7–9.6. No growth at pH 8.3 or 10.8. Dependent on sodium; no growth below 3% (w/v) or above 10% (w/v) NaCl. Growth is possible when NaCl is substituted by equimolar Na2CO3+NaHCO3. Requires carbonate anion. Optimum temperature for growth, 33–37°C; range, 15–44°C; slow growth at 6°C after a long lag phase. Source: a cyanobacterial mat in a warm spring from under the horst in the equatorial alkaline Lake Magadi. DNA G+C content (mol%): 57.1 (Tm). Type strain: strain Z-7491, ATCC 700262, DSM 8900. Sequence accession no. (16S rRNA gene): X93927. 4. Spirochaeta americana Hoover, Pikuta, Bej, Marsic, Whitman, Tang and Krader 2003, 820VP a.me.ri.ca¢na. N.L. fem. adj. americana of American continent, isolated from soda Mono Lake, California, USA. Cells are motile and helix-shaped. Flagellum present in periplasmic space. Gram-stain-negative. Cells have regular,

479

unstable primary coils. Sphaeroplasts are formed at the end of the growth phase. Strictly anaerobic, catalase-negative chemoheterotroph with fermentative type of metabolism. Preferred substrates are d-glucose, fructose, maltose, sucrose, starch, and d-­mannitol. Requires vitamins and yeast extract for growth. Primary end products of glucose fermentation is H2, acetate, ethanol, and formate. Resistant to kanamycin and rifampicin, but sensitive to gentamicin, tetracycline, and chloramphenicol. Haloalkaliphile that cannot grow at pH 7.0. Growth is dependent upon the presence of carbonate and sodium ions in the medium. No growth occurs below 2% (w/v) NaCl or above 12% (w/v) NaCl. Mesophilic. Cells can be stored frozen in a liquid medium. Source: mud sediments of the alkaline, hypersaline, meromictic, soda Mono Lake in Northern California, USA. DNA G+C content (mol%): 58.5 (HPLC). Type strain: strain ASpG1, ATCC BAA-392, DSM 14872. Sequence accession no. (16S rRNA gene): AF373921. 5. Spirochaeta asiatica Zhilina 1996, 311VP a.si.a¢ti.ca. L. fem. adj. asiatica from the Asian continent, in the central part of which the organism was found. Motile, helical cells, 0.2–0.25 mm in diameter and 15–22.5 mm in length, with shorter (7.5 mm) and longer (up to 40 mm) cells occurring in culture. Outermost structure is an outer membrane enclosing periplasmic flagella and a protoplasmic cylinder. Cells have regular, nonstable primary coils. Round bodies, usually nonviable, are formed at the end of the growth period. Nonpigmented. Strictly anaerobic, fermentative, and utilizes simple and complex carbohydrates. Preferred substrates: glucose > maltose > glycogen > mannose > trehalose > cellobiose > sucrose > starch > galactose > pectin > xylan; poor growth with xylose or arabinose; no growth with fructose, ribose, lactose, agar, or N-acetylglucosamine (the optical isomers of the sugars were not described). Amino acids are not fermented. A supplement of vitamins is required; yeast extract enhances growth. Fermentation products from glucose include acetate, ethanol, and lactate; H2 not produced. Haloalkaliphilic; growth in soda solution at optimal pH 8.4–9.4 with limits pH 7.9–9.7. Growth is Na-dependent. No growth below 2% (w/v) NaCl or above 8% (w/v) NaCl. Optimum NaCl concentration, 3–6% (w/v). Requires carbonate anion. Optimum temperature for growth, 33–37°C; range, 20–43°C; broad thermal adaption with prolonged lag phase. Source: mud of alkaline Lake Khatyn in Tuva, Central Asia. DNA G+C content (mol%): 49.2 (Tm). Type strain: strain Z-7591, ATCC 700261, DSM 8901. Sequence accession no. (16S rRNA gene): X93926. 6. Spirochaeta aurantia Canale-Parola 1980, 594VP au.ran¢tia. N.L. fem. adj. aurantia orange-colored. Helical cells, 0.3 mm in diameter and 5–50 mm in length. Most cells in cultures measure 10–20 mm in length during exponential growth. Spherical bodies 0.5–2.0 mm in diameter are present, especially in the stationary phase of growth

480

Family I. Spirochaetaceae

or when the cells are incubated at temperatures unfavorable for growth (e.g., 37°C). The spherical bodies are either in association with cells or free. Each cell has two subterminally inserted periplasmic flagella in a 1:2:1 arrangement. Phasecontrast photomicrographs and electron micrographs of the cells have been published (Breznak and Canale-Parola, 1969, 1975; Canale-Parola et al., 1968). Colonies on aerobic plates (in media containing 1 g of agar/100 ml; see Breznak and Canale-Parola, 1975) are 1–4 mm in diameter, yellow-orange to orange, round with slightly irregular edges, growing primarily within the agar medium just under the surface, sometimes with a slightly raised center. At low carbohydrate concentrations (see Breznak and Canale-Parola, 1975), the colonies are larger, and they diffuse through the agar medium in the shape of almost perfect circles. Under these growth conditions, the colonies have a lower cell density and their pigmentation is not readily apparent. Anaerobically grown colonies are white. Subsurface anaerobic colonies are spherical, fluffy, 1–3 mm in diameter. Facultatively anaerobic, having both fermentative and respiratory types of metabolism. Carbohydrates, but not amino acids, are utilized as energy sources for growth (Breznak and Canale-Parola, 1969, 1975). Amino acids but usually not inorganic ammonium salts or nitrates serve as sole nitrogen sources. Exogenous thiamine is required by all strains tested, and riboflavin is required by most strains. Exogenous biotin is required for growth of the type strain and is stimulatory to the growth of other strains (Breznak and Canale-Parola, 1975). Nitrate is reduced to nitrite anaerobically. Oxidase-negative. Weakly catalase-positive. Superoxide dismutase (SOD) is present, and levels of SOD are higher in aerobically grown cells than anaerobically grown cells. Optimum growth occurs between 25 and 30°C. Slow growth occurs at 15°C and usually no growth occurs at 5°C. There is poor or no growth at 37°C. Optimum growth yields result when the initial pH of the medium is 7.0–7.3. Cells grown anaerobically, ferment glucose primarily to ethanol, acetic acid, CO2, and H2 (Breznak and CanaleParola, 1969, 1972b). Under aerobic conditions, growing cells oxidize glucose mainly to CO2 and acetic acid (Breznak and Canale-Parola, 1972a). Cells growing aerobically produce carotenoid pigments responsible for the yellow-orange to orange color of colonies. The major carotenoid pigment is 1¢,2¢-dihydro-l¢-hydro­ xytorulene (Greenberg and Canale-Parola, 1975). Nonpigmented mutants have been isolated. Chemotactic toward carbohydrates, but not toward amino acids. Based on 16S rRNA sequence comparisons, Spirochaeta aurantia branches deeply within the family Spirochaetaceae and may warrant separate genus designation (Figure 84). Source: Water and mud of freshwater ponds and swamps. DNA G+C content (mol%): 61–65 (Bd). Type strain: strain J1, ATCC 25082, DSM 1902. Sequence accession no. (16S rRNA gene): M57740. 6a. Spirochaeta aurantia subsp. aurantia Canale-Parola 1980, 594VP The characteristics are as described for the species. ­Distinguished from the subspecies stricta by having a cell

wavelength of 2.0–2.8 mm, a wave amplitude of 0.5 mm (the cells have loose coils), and a mol% G+C of 62–65 (Bd). Type strain: ATCC 25082. 6b. Spirochaeta aurantia subsp. stricta subsp. nov. stric¢ta. L. v. stringere to draw tight, compress; L. fem. part. adj. stricta drawn tight. The characteristics are as described for the species. Distinguished from the subspecies aurantia by having a cell wavelength of 1.1–1.5 mm, a wave amplitude of 0.35 mm (cells have tight coils), and a mol% G+C of 61 (Bd). Type strain: J4T (Breznak and Canale-Parola, 1975). 7. Spirochaeta bajacaliforniensis Fracek and Stolz 2004, 631VP (Effective publication: Fracek and Stolz 1985, 324.) ba.ja.ca.li.for.ni.en¢sis. N.L. fem. adj. bajacaliforniensis of or belonging to Baja California for the geographical location from where it was isolated. Helical cells, 0.2–0.3 mm by 15–45 mm. Shorter (10 mm) and longer (up to 300 mm) cells may occur. Highly motile. The amplitude is 0.5 mm and the wavelength is 1.0–1.5 mm. Two subterminally inserted periplasmic flagella are present in a 1:2:1 arrangement. The ratio of protoplasmic cylinder diameter to the cell diameter is 2:3. The surface of the protoplasmic cylinder has a characteristic polygonal pattern. Subsurface colonies are fluffy, white, and spherical. Strictly anaerobic. Arabinose, cellobiose, galactitol, fructose, galactose, gluconate, glucose, inulin, lactose, malate, maltose, mannitol, pyruvate, rhamnose, sorbose, and trehalose are fermented (the optical isomers of the sugars were not described (Fracek and Stolz, 1985). Products of glucose fermentation are acetate, ethanol, CO2, and H2. No test for formate was performed. Catalase-negative. Grow in media containing at least 20% seawater and 0.12 M NaCl. Reducing agent required in liquid medium. Growth on solid medium occurs in an 80% N2, 17% CO2, and 3% H2 atmosphere. Temperature optimum: 36°C. Do not grow below 25°C or above 44°C. Optimum growth at pH 7.5. Source: anaerobic sulfide-rich mud underlying the laminated sediment of the microbial mats at North Pond, Laguna Figueroa, Baja California Norte, Mexico. DNA G+C content (mol%): 50.1 (Bd). Type strain: strain BA-2, ATCC 35968, DSM 16054. Sequence accession no. (16S rRNA gene): M71239. 8. Spirochaeta caldaria Pohlschroeder 1994, 21VP cal.da¢ri.a. L. fem. adj. caldaria of warm water, inhabiting warm water. Motile, helical cells, 0.2–0.3 mm in diameter and mostly 15–45 mm in length, the outermost structure being an outer membrane (outer sheath) enclosing the periplasmic flagella in a 1:2:1 arrangement and the protoplasmic cylinder. Cells have regular, stable primary coils. Broader secondary coils occasionally are present in cells in motion. Subsurface colonies (in media containing 1 g agar per 100 ml) are white, fluffy, cotton ball-like, approximately 2–3 mm in diameter. Obligately anaerobic, fermentative, utilizes carbohydrates as carbon and energy sources. Amino acids do not serve as fermentable substrates for growth. Fermentable compounds

Genus I. Spirochaeta

include l-arabinose, d-galactose, d-glucose, d-mannose, d-fructose, d-xylose, cellobiose, cellotriose, cellotetraose, lactose, maltose, sucrose, and starch. The following substances are not fermented: d-ribose, mannitol, cellulose, xylan, glycerol, peptone, casein hydrolysate, and sodium acetate. Exogenous fatty acids, reported to be needed by Treponema species for cellular lipid synthesis and growth (Livermore and Johnson, 1974; Miller et  al., 1991), are not required. A supplement of vitamins is required. Specific vitamin requirements have not been determined. Products of d-glucose fermentation are H2, CO2, acetate, and lactate. Grows in the presence of rifampicin (100 µg/ml of medium). Growth is inhibited by penicillin G, neomycin, chloramphenicol, or tetracycline (10 µg/ml of medium each). Thermophilic. Grows optimally between 48 and 52°C. No growth at 25 or 60°C. No growth in the presence of 0.4% NaCl or higher NaCl concentrations. Source: freshwater hot springs. DNA G+C content (mol%): 45 (Tm). Type strain: strain H1, ATCC 51460, DSM 7334. Sequence accession no. (16S rRNA gene): M71240, EU580141. 9. Spirochaeta cellobiosiphila Breznak and Warnecke 2008, 2762VP cel.lo.bi.o.si¢phi.la. N.L. neut. n. cellobiosum cellobiose; N.L. fem. adj. phila from Gr. fem. adj. philê friendly to, loving; N.L. fem. adj. cellobiosiphila loving cellobiose, isolated from a microbial mat, Little Sippewissett salt marsh, Woods Hole, MA, USA. Cells are pale yellow, 0.3–0.4×10–12 mm in size and helical, with a body pitch of 1.4 mm. Motile by means of two (occasionally four) periplasmic flagella, of which one (or two) is inserted near each end of the cell. Facultatively anaerobic and catalase-negative. Growth occurs at 9–37°C (optimally at or near 37°C), at initial pH 5–8 (optimally at initial pH 7.5) and in media prepared with 20–100% (v/v) seawater (optimally at 60–80%) or containing 0.10–1.00 M NaCl (optimally at 0.30–0.40 M). A variety of monosaccharides and disaccharides and pectin (but not cellulose or arabinoxylan) are used as energy sources; the most rapid growth occurs on cellobiose. Neither organic acids nor amino acids are utilized as energy sources. One or more amino acids in tryptone and one or more components of yeast extract are required for growth. The products of cellobiose fermentation are acetate, ethanol, CO2, H2, and small amounts of formate. Aerated cultures oxidize cellobiose incompletely to acetate (and, presumably, CO2) plus small amounts of ethanol and formate; they exhibit a Ycellobiose value that is 1.2fold greater than that of cellobiose-fermenting cultures. Source: interstitial water of a cyanobacteria-containing microbial mat collected from Little Sippewissett salt marsh, Woods Hole, MA, USA. DNA G+C content (mol%): 41.4 (HPLC). Type strain: strain SIP1, ATCC BAA-1285, DSM 17781. Sequence accession no. (16S rRNA gene): EU448140. 10. Spirochaeta halophila Greenberg and Canale-Parola 1976, 193AL ha.lo.phi¢la. Gr. n. hals, halos salt; Gr. adj. philus -ê -on loving; N.L. fem. adj. halophila salt-loving.

481

Helical cells, 0.4 mm in diameter and 15–30 mm in length. Some of the cells in cultures are as short as 5 mm and as long as 60 mm. Cells have regular, stable primary coils. Spherical bodies 1–2 mm in diameter occur in cultures, especially in the stationary phase of growth or during growth at unfavorable temperatures (e.g., 45°C). Each cell has two subterminally inserted periplasmic flagella that overlap in the central region of the cell (1:2:1 arrangement). Phase-contrast micrographs and electron micrographs of the cells have been published (Greenberg and Canale-Parola, 1976). Colonies growing aerobically on ISM plates (0.75 g of agar/100 ml of medium) are red, round, with areas of diffuse growth at their periphery, and usually 2–6 mm in dia­ meter (after 5 d at 35°C). Each colony grows partially above and partially below the surface of the agar medium. Anaerobically grown colonies are white. When cells are streaked onto agar medium plates and incubated anaerobically, the colonies grow below the surface of the medium and are spherical, diffuse and white. Facultatively anaerobic, having both respiratory and fermentative types of metabolism. Carbohydrates, but not amino acids, are utilized as energy sources for growth (Greenberg and Canale-Parola, 1976). Cells have specific growth requirements for relatively high concentrations of Na+, Cl−, Ca2+, and Mg2+ (Greenberg and Canale-Parola, 1976). Optimum cell yields result when 0.75 M NaCl, 0.2 M MgSO4, and 0.01 M CaCl2 are included in growth media containing (in g/100 ml) a carbohydrate (0.5), peptone (0.2), and yeast extract (0.4). No growth occurs when any one of the three inorganic salts is omitted from the medium (e.g., ISM medium). Nitrate is reduced to nitrite anaerobically. Catalase-negative. Cells growing anaerobically ferment glucose primarily to ethanol, acetic acid, CO2, and H2 (Greenberg and Canale-Parola, 1976). Under aerobic conditions, growing cells oxidize glucose mainly to CO2 and acetic acid (Greenberg and Canale-Parola, 1976). Optimum temperature, 35–40°C. Poor growth occurs at 45°C and no growth occurs at 22°C. Cells growing aerobically produce carotenoid pigments responsible for the red color of the colonies. The major carotenoid pigment is 4-keto-l¢,2¢-dihydro-l¢-hydroxytorulene (Greenberg and Canale-Parola, 1975). Nonpigmented mutants, occurring spontaneously in cultures, have been isolated. Source: H2S-containing mud of a high salinity pond (Solar Lake) located on the Sinai shore of the Gulf of Elat. DNA G+C content (mol%): 62 (Tm, Bd). Type strain: strain RS1, ATCC 29478, DSM 10522. Sequence accession no. (16S rRNA gene): M88722. 11. Spirochaeta isovalerica Harwood and Canale-Parola 1983, 578VP i.so.va.le.ri¢ca. N.L. n. acidum isovalericum isovaleric acid; N.L. fem. adj. isovalerica intended to mean pertaining to isovaleric acid, the major acid formed as a fermentation product from branched-chain amino acids. Helical cells, 0.4 mm in diameter and 10–15 mm in length. Shorter (6 mm) and longer (up to 50 mm) cells occur in cultures. Motile. Two subterminally inserted periplasmic (axial) fibrils are present in a 1:2:1 arrangement. The protoplasmic cylinder and periplasmic fibrils are enclosed in an outer sheath. Gram-stain-negative.

482

Family I. Spirochaetaceae

Chemo-organotrophic. Grows (final yield, 6–9×108 cells per ml) in medium containing yeast extract, trypticase, peptone, a carbohydrate, and inorganic salts under anaerobic conditions. No growth occurs aerobically. Subsurface colonies in medium containing 0.8% agar (Difco) are spherical and white, resembling cotton balls in appearance. Growth (final yield, 3–6×108 cells per ml) occurs in a chemically defined medium containing glucose, cysteine or sulfide, asparagine, vitamins, 0.3 M NaCl, 0.05 M MgSO4, 0.01 M KCl, 0.01 M CaC12, and trace elements. Exogenously supplied vitamins are not required for growth, but a mixture of vitamins stimulates growth. Fails to grow when NaCl is omitted from the medium. Growth is stimulated by MgSO4 and CaCl2. Inorganic ammonium salts or amino acids serve as nitrogen sources. Carbohydrates, but not amino acids, serve as carbon and energy sources for growth. The major products of glucose fermentation are CO2, H2, acetate, and ethanol. Cells have the ability to generate ATP by catabolizing l-leucine, l-isoleucine, and l-valine to form isovalerate, 2-methylbutyrate, and isobutyrate, respectively, as end products. ATP formed in this way is utilized by cells to prolong survival during periods of growth substrate starvation. Smaller amounts of isobutanol and isoamyl alcohol are also formed by cells from valine, isoleucine, and leucine. Catalase-negative. Nitrate not reduced to nitrite. Optimum temperature range, 15–35°C. Poor growth at 5 and 39°C. Source: anoxic marine marsh mud. DNA G+C content (mol%): 63.6–65.6 (Tm). Type strain: strain MA-2, ATCC 33939, DSM 2461. Sequence accession no. (16S rRNA gene): M88720. 12. Spirochaeta litoralis Canale-Parola 1980, 594VP li.to.ra¢lis. L. fem. adj. litoralis of the shore. Helical cells, 0.4–0.5 mm in diameter and 5.5–7.0 mm in length. The cells are regularly and tightly coiled during the exponential phase of growth. Spherical bodies (2.0–3.5 mm in diameter) are present in the stationary growth phase under unfavorable growth conditions (e.g., in the presence of O2). Two subterminally inserted periplasmic flagella are present in a 1:2:1 arrangement. Phase-contrast photomicrographs and electron micrographs of the cells have been published (Hespell and Canale-Parola, 1970b; Joseph and Canale-Parola, 1972). Subsurface colonies in agar media are spherical, fluffy, cream colored, 1–5 mm in diameter. Surface colonies (anaerobic) are round, growing partially within the agar medium, cream colored, and 2–5 mm in diameter. Obligately anaerobic, having a fermentative type of metabolism. Various carbohydrates are fermented (Hespell and Canale-Parola, 1970b). Main products of glucose fermentation are ethanol, acetic acid, CO2, H2, and trace amounts of lactic, formic, and pyruvic acids (Hespell and Canale-Parola, 1970b, 1973). Nitrite is not accumulated in the medium by cells growing in the presence of nitrate. Catalase-negative. Cells grow in media prepared with seawater, but do not grow in media prepared with freshwater unless NaCl is added (minimum concentration, 0.05 M; optimum, 0.35 M). Cells have specific requirements for Na+ and Cl−.

Exogenous ­supplements of biotin, niacin, and coenzyme A are required for growth. Coenzyme A may be replaced by pantothenate, but the resulting cell yields are low. Added thiamine is stimulatory for growth. A reducing agent (e.g., sulfide or cysteine) is required for growth in laboratory media. Cells grow in chemically defined media containing glucose, (NH4)2SO4 or amino acids, sulfide, NaCl, vitamins, coenzyme A, and inorganic salts (Hespell and CanaleParola, 1970b). Optimum temperature, near 30°C. Growth occurs slowly at 15°C and not at all at 5°C or 40°C. Optimum growth yields result when the initial pH of the medium is between 7.0 and 7.5. Source: sulfide-containing marine mud. DNA G+C content (mol%): 51 (Bd). Type strain: strain RI, ATCC 27000, DSM 2029. Sequence accession no. (16S rRNA gene): M88723. 13. Spirochaeta smaragdinae Magot, Fardeau, Arnauld, Lanau, Ollivier, Thomas and Patel 1997, 190VP sma.rag.di¢nae. L. masc. n. smaragdus emerald; N.L. gen. n. smaragdinae intended to mean from Emerald, the name of the oilfield in Congo, Central Africa. Spirochetes with corkscrew-like motility. Spiral cells are 0.3–0.5 mm in diameter and 5–30 mm in length with two periplasmic flagella in a 1:2:1 arrangement. Produce translucent colonies with regular edges and a diameter of 0.5 mm after 2 weeks at 37°C. Obligately anaerobic chemo-organotroph. Growth occurs in the presence of fructose, galactose, d-xylose, d-glucose, ribose, d-mannose, mannitol, glycerol, yeast extract, biotrypticase, and fumarate, but not with d-arabinose, rhamnose, sorbose, l-xylose, sucrose, maltose, acetate, butyrate, propionate, pyruvate, lactate, or Casamino acids. Yeast extract is required for growth and cannot be replaced by a vitamin mixture. Thiosulfate and elemental sulfur are reduced to sulfide. Glucose is oxidized to lactate, acetate, CO2, and H2S in the presence of thiosulfate, and to lactate, ethanol, CO2, and H2 in its absence. Fumarate is fermented to acetate and succinate. Obligately halophilic. Optimum NaCl concentration for growth is 5% and the NaCl concentration range for growth is 1.0–10%. Optimum temperature for growth is 37°C with growth occurring between 20 and 40°C. Optimum pH is 7.0 with growth occurring between pH 5.5 and 8.0. Doubling time in the presence of glucose and thiosulfate under optimal conditions is about 25 h. Source: oil-injection water from Emerald oilfield in Congo, Central Africa. DNA G+C content (mol%): 50 (HPLC). Type strain: strain SEBR 4228, DSM 11293, JCM 15392. Sequence accession no. (16S rRNA gene): U80597. 14. Spirochaeta stenostrepta Zuelzer 1912, 17AL ste.no.strep¢ta. Gr. adj. stenos narrow; Gr. adj. streptos -ê -on pliant, easily bent; N.L. fem. adj. stenostrepta tightly coiled. Helical cells, 0.2–0.3 mm in diameter and 15–45 mm in length. Some of the cells in cultures are shorter than 15 mm. In the late exponential and stationary phases the organisms increase in length (up to 300 mm). Long organisms

Genus I. Spirochaeta

­ ccasionally pair and become entwined, or a single organism o becomes partially wrapped around itself. The cells have regular, stable primary coils. Cells in motion occasionally exhibit broader secondary coils or waves superimposed on the smaller primary coils. Spherical bodies generally 1–3 mm in diameter are occasionally observed in cultures. The spherical bodies occur either free or in association with helical cells. Each cell has two subterminally inserted periplasmic flagella that overlap in the central region of the cell (1:2:1 arrangement). Phase-contrast photomicrographs and electron micrographs of the cells have been published (CanaleParola et al., 1967, 1968; Holt and Canale-Parola, 1968). Subsurface colonies (in GYPT medium containing 1.5 g agar/100 ml) are white, spherical, fluffy, and approximately 2–3 mm in diameter when fully developed. Smaller spherical colonies may occasionally lack the characteristic fluffiness. Obligately anaerobic, having a fermentative type of metabolism. Various carbohydrates are fermented (Hespell and Canale-Parola, 1970a). The main products of glucose fermentation are ethanol, acetic acid, CO2, H2, and smaller amounts of lactic acid (Canale-Parola et  al., 1967, 1968; Hespell and Canale-Parola, 1970a). Catalase-negative. Growth reported only on complex media. Minimal growth requirements are unknown. Growth occurs between 15 and 40°C; optimum temperature is 35–37°C. Optimum growth yields result when the initial pH of the medium is between 7.0 and 7.5. Based on 16S rRNA sequence comparisons, Spirochaeta stenostrepta does not belong with other free-living Spirochaeta, but is more closely related with members of the genus Treponema (Figure 84). Source: H2S-containing mud of a freshwater pond (CanaleParola et al., 1967, 1968). DNA G+C content (mol%): 60 (Bd). Type strain: strain Zl, ATCC 25083, DSM 2028. Sequence accession no. (16S rRNA gene): M88724. 15. Spirochaeta thermophila Aksenova, Rainey, Janssen, Zavarzin and Morgan 1992, 176VP ther.mo¢phi.la. Gr. n. thermê heat; Gr. fem. adj. philê loving; N.L. fem. adj. thermophila heat-loving. Helical cells, 0.2–0.25 mm in diameter and 16–50 mm in length. An outer sheath encloses a protoplasmic cylinder; two periplasmic flagella in a 1:2:1 arrangement are subterminally anchored by an insertion disc. No cell lysis in 3% KOH. Strictly anaerobic chemo-organotroph. Utilizes various mono-, di-, and polysaccharides but not sugar alcohols, organic acids, or amino acids. Glucose is fermented via the Embden–Meyerhof–Parnas pathway, involving a pyrophosphate-dependent phosphofructokinase. Fermentation end products from glucose are acetate, CO2, H2, and lactate. Ethanol and succinate not produced. No reduction of fumarate, nitrate, oxygen, sulfate, or sulfur. Indole not formed; urea not hydrolyzed. Sulfide not produced from cysteine; esculin hydrolyzed. Inhibited by penicillin, neomycin, erythromycin, tetracycline, polymyxin B, and novobiocin but resistant to rifampicin and streptomycin. Temperature range for the type strain, 40–73°C (optimum, 66–68°C). pH range for the type strain, 5.9–7.7

483

(optimum, 7.5). NaCl concentration range for the type strain, 0.5–4.5% (optimum, 1.5%). Doubling time, 70 min. Temperature, pH, and salinity parameters vary for different strains, reflecting the environmental conditions prevailing at the sites of isolation. Source: marine hot spring near the beach on Shiashkotan Island, Soviet Far East, USSR. DNA G+C content (mol%): 52 (Tm). Type strain: strain Z-1203, ATCC 700085, DSM 6578. Sequence accession no. (16S rRNA gene): X62809, L09180. 16. Spirochaeta zuelzerae Canale-Parola 1980, 594VP (Treponema zuelzerae Veldkamp 1960, 122) zu.el.ze¢rae. N.L. fem. gen. n. zuelzerae of Zuelzer, named after Margarete Zuelzer, who described the occurrence of morphologically diverse spirochetes in sulfide-containing environments. Helical cells, 0.2–0.35 mm in diameter and 8–16 mm in length. Shorter cells (as short as 2–3 mm) are occasionally observed in cultures. Long organisms (up to 80 mm) are present in old cultures. Exponentially growing cells have fairly regular, stable primary coils. Secondary coils or waves are present infrequently. Spherical bodies, generally not exceeding 3–4 mm in diameter, are formed usually at the ends of the cells in the stationary phase of growth. Two subterminally inserted periplasmic flagella are present in a 1:2:1 arrangement. Phase-contrast photomicrographs and electron micrographs of the cells have been published (Canale-Parola et  al., 1968; Joseph and Canale-Parola, 1972). The type strain was originally isolated from an enrichment culture for green photosynthetic bacteria that had been inoculated with sulfide-containing mud from a freshwater pond (Veldkamp, 1960). Subsurface colonies in agar media (for composition of medium, see Veldkamp (1960), and also SZ medium in table) are white, fluffy, spherical, with a tendency to diffuse in the agar medium. Disc-shaped colonies are present occasionally. Obligately anaerobic, having a fermentative type of metabolism. Various carbohydrates are fermented (Veldkamp, 1960). Cells growing in media containing 0.05% NaHCO3 ferment glucose mainly to acetic, lactic, and succinic acids, CO2, and H2 (Veldkamp, 1960). Catalase-negative. Growth occurs at 20°C but not at 45°C. The optimum temperature range is 37–40°C. Growth is optimum when the initial pH of the medium is between 7 and 8. Inorganic ammonium salts or nitrates are not utilized as sole nitrogen sources. Added CO2 is an absolute requirement for growth (Veldkamp, 1960). Growth has been reported only on complex media. The minimal growth requirements are unknown. Cells have a protein antigen that gives a positive complement fixation reaction with syphilitic serum. Based on 16S rRNA sequence comparisons, Spirochaeta zuelzerae does not belong with other free-living Spirochaeta, but is more closely related with members of the genus Treponema (Figure 84). DNA G+C content (mol%): 56 (Bd). Type strain: ATCC 19044, DSM 1903. Sequence accession no. (16S rRNA gene): M88725.

484

Family I. Spirochaetaceae

Other organisms   1. Spirochaeta coccoides Dröge, Fröhlich, Radek and König 2006, 1460VP (Effective publication: Dröge, Fröhlich, Radek and König 2006, 396.) coc.co¢i.des. Gr. n. kokkos a berry; L. fem. suff. -oides [from Gr. suff. -eides (from Gr. n. eidos that which is seen, form, shape, figure)], resembling, similar. N.L. fem. adj. coccoides berryshaped. Placement within the genus Spirochaeta is questionable owing to the absence of defining morphological, ultrastructural, and behavioral features (i.e., helically shaped motile cells with protoplasmic cylinder and periplasmic fibrils enclosed in an outer sheath). In this regard, this species resembles isolates from freshwater sediments referred to as “free-living pleomorphic spirochetes” or FLiPS (Ritalahti and Löffler, 2004). 16S rRNA sequence analysis indicates that these microbes form a cluster within the Spirochaeta– Treponema branch of the Spirochaetaceae tree, and should be considered for separate taxonomic status. Type strain: strain SPN1, DSM 17374, ATCC BAA-1237. Sequence accession no. (16S rRNA gene): AJ698092. It has long been known that greater diversity exists among the free-living spirochetes capable of anaerobic growth than is reflected by the species presently recognized in the genus Spirochaeta. For example, a free living, strictly anaerobic ­spirochete (strain Z4), resembling Spirochaeta zuelzerae ­morphologically, but differing in certain physiological properties, has been isolated from freshwater mud (Canale-Parola et al., 1968). The spirochete ferments glucose to acetic, lactic, succinic, and formic acids, ethanol, CO2, and H2. Unlike Spirochaeta zuelzerae, it does not require an exogenous source of CO2 and the mol% G+C of its DNA is 59.2 (Bd). Additionally, diverse obligate anaerobic thermophilic strains of Spirochaeta have been isolated from thermal springs and other high temperature environments. For example, a strain isolated from a hydrothermal spring on Raoul Island of the Kermadec archipelago of New Zealand exhibited maximum growth at 73°C (Rainey et  al., 1991). This isolate fermented a range of mono-, di-, and polysaccharides, including cellulose. Various strains of facultatively and obligately anaerobic spirochetes have been isolated from marine environments, including intertidal muds and water. An obligately

a­ naerobic strain was isolated from water collected at a depth of 2550 m near the Galápagos hydrothermal vents in the Pacific Ocean (Harwood et  al., 1982). These isolates are indigenous to marine environments inasmuch as they have Na+ requirements typical of marine bacteria. The facultatively anaerobic marine isolates form either white or yellow colonies. Thus, they differ both in salt requirements and in pigmentation from the facultatively anaerobic species Spirochaeta aurantia, which is a freshwater species and forms orange colonies, and Spirochaeta halophila, which requires high concentrations of Ca+ and Mg+ and forms red colonies. However, 16S rRNA sequences are not available for these strains to determine their phylogenetic relationships with other species of Spirochaeta. Diversity of not-yet-cultivated species.  The breadth of diversity of species of Spirochaeta is impressive. By cloning and sequencing of 16S rRNA genes amplified from DNA isolated from a wide variety of environments, over 100 phylotypes of not-yet-cultivated species of Spirochaeta have been identified. These environments include the following: (1) microbial mats from deltas in France and Spain (Berlanga et al., 2003), Puerto Rico, and California; (2) hypersaline soda lakes in Asia, Africa, Egypt, and California; (3) volcano seep sediments in Greece and Japan; (4) marine sediments in China and Japan; (5) gray whale bone from deepsea; (6) Alvinella pompeiana white tube worms and other gutless worms from deep-sea vents; (7) forested wetlands; (8) fresh water ponds or lakes in China, Japan, Greece, and Massachusetts, USA; (9) karyomastigonts (protozoa); and (10) termite hindguts. Phylogenetic analysis of representatives of these taxa and their GenBank accession numbers are shown in Figure 85. Notably, some of these not-yet-cultivated species of Spirochaeta are “host-associated”, i.e., found in termite hindguts (Figure 85). In contrast, most termite spirochetes fall within the genus Treponema (see chapter on Treponema). Conversely, as discussed above, there are several “free-living” species of Spirochaeta, e.g., Spirochaeta zuelzerae, Spirochaeta stenostrepta, and Spirochaeta caldaria, that are more closely related to “host-associated” members of the genus Treponema than to other “free-living” members of the genus Spirochaeta. Consequently, these data suggest that “free-living” vs “host-associated” designations may not be valid taxonomic criteria to differentiate species of Spirochaeta and Treponema.

Genus II. Borrelia Swellengrebel 1907, 582AL Guiqing Wang and Ira Schwartz Bor.re¢li.a. N.L. fem. n. Borrelia named after Amedée Borrel (1867–1936).

Helical cells are 0.2–0.5 mm by 3–30 mm, composed of 3–10 loose coils. The cells are surrounded by a surface layer, an outer membrane, periplasmic flagella, and a protoplasmic cylinder (Figure 86). Typically, 15–20 periplasmic flagella (which also have been termed endoflagella, axial fibrils, or periplasmic fibrils) originate at each end of the cell and wind about the protoplasmic cylinder to overlap in the middle of the cell. The protoplasmic cylinder consists of a peptidoglycan layer and an inner

membrane which encloses the internal components of the cells. The cells are actively motile with frequent reversal of the direction of translational movement. Gram-stain-negative. Stain well with Giemsa stain. Species which have been grown in vitro are microaerophilic. Nutritional requirements for in  vitro growth are complex. Arthropod-borne pathogens of man, other mammals, and birds. The causative agents of tick-borne Lyme disease and relapsing fever and louse-borne relapsing fever in man.

Genus II. Borrelia

5%

485

Microbial mats, Puerto Rico & California; DQ329698 (6) Hypersaline microbial mat; DQ154846 Microbial mats, Spain & California; AY605135 (4) Microbial mat, California; DQ329733 Microbial mat, Puerto Rico; EU245250 (6) Microbial mat, France; AY605150 Microbial mats, Puerto Rico & Spain; AY605174 (3) Microbial mats; Puerto Rico, California & France; AY605154 (4) Microbial mat, Puerto Rico; EU246240 Microbial mat, Puerto Rico; EU245118 (2) Hypersaline lake, Egypt; DQ432375 Microbial mat, Spain; AY605127 Microbial mats, France & California; AY605164 (2) Microbial mat, Spain; AY605171 Microbial mat, Puerto Rico; EU245339 Microbial mat, Puerto Rico; EU245691 (4) Microbial mats, Puerto Rico & California; EU245678 (6) Microbial mat, California; DQ329718 Microbial mat, California; DQ329699 Microbial mat, Puerto Rico; EU245461 (3) Alkaliphilic, Mono Lake, California; AF454308 Spirochaeta african a Spirochaeta asiatica Alkaliphilic, Mono Lake, California; AF507855 Spirochaeta alkalica Spirochaeta halophila Microbial mat, Spain; AY605175 Microbial mat, Spain; AY605159 Microbial mats, Spain & Puerto Rico; AY605132 (2) Grey whale bone, deep sea; AY922180 Hydrothermal vent sediment, Guaymas Basin; AY197374 Termite hindgut, Nasutitermes sp.; EF454901 Termite hindgut, Reticulitermes speratus ; AB088873 Deep sea sediment, Japan Sea; AB121108 Lucinid bivalve endosymbiont; AM236337 Amsterdam mud volcano; AY592404 Spirochaeta coccoides Nautilus macromphalus endosymbiont; AM399077 Deep sea sediment, Japan Sea; AB121100 Gray whale bone, deep sea; AY922204 Microbial mat, Spain; AY605142 Microbial mat, California; DQ329738 Spirochaeta smaragdinae Spirochaeta bajacaliforniensis Microbial mat, Spain; AY605167 Microbial mats; Spain, France & Puerto Rico; AY605176 (8) Microbial mats, Spain & California; AY605136 (6) Microbial mat, Puerto Rico; EU245577 Microbial mat, Spain; AY605134 Microbial mat, France; AY605155 Microbial mat, Spain; AY605133 Seep sediment, Gulf of Cadiz; DQ004672 Microbial mats, Spain & California; AY605138 (4) Microbial mat, France; AY605165 Microbial mats, Puerto Rico & California; EU245551 (2)

Figure 85.  Phylogenetic tree illustrating the diversity of cultivable and not-yet-cultivated species of the genus Spirochaeta based on 16S rRNA

sequence comparisons. Not-yet-cultivated species are noted by their environmental source, location, and GenBank accession numbers. Numbers in parentheses indicate the number of sequences available for a given phylotype. The scale bar represents a 5% difference in nucleotide sequence.

DNA G+C content (mol%): 27–32. Type species: Borrelia anserina (Sakharoff 1891) Bergey, ­Harrison, Breed, Hammer and Huntoon 1925, 435AL.

Further descriptive information DNA sequence analysis of rRNA and other conserved genes (e.g., fla, hbb) has established that Borrelia spp. fall into two major groups (Figure 87). The first major group contains the agents of Lyme borreliosis that were first isolated from Ixodes scapularis ticks (Burgdorfer et  al., 1982). These include the three human-pathogenic species Borrelia burgdorferi (Johnson et  al., 1984), Borrelia afzelii (Canica et  al., 1993), and Borrelia

garinii (­Baranton et  al., 1992) and seven other species that are minimally pathogenic or nonpathogenic: Borrelia japonica (Kawabata et  al., 1993), Borrelia lusitaniae (Le Fleche et  al., 1997), Borrelia tanukii, Borrelia turdi (Fukunaga et  al., 1996a), Borrelia sinica (Masuzawa et al., 2001), Borrelia spielmanii (Richter et al., 2006), and Borrelia valaisiana (Wang et al., 1997). Spirochetes in the first major group are all transmitted by hard ticks in the Ixodes ricinus complex, and are referred to collectively as “Borrelia burgdorferi sensu lato” (Baranton et  al., 1992; Wang et al., 1999). The second major group includes more than 20 Borrelia species associated with relapsing fever that are mainly transmitted by soft-bodied or argasid ticks, with the exception

486

Family I. Spirochaetaceae Microbial mats, Spain & Puerto Rico; AY605137 (2) Deep sea sediment, Pacific; AB177323 Microbial mat, Spain & Puerto Rico; AY605144 (4) Hypersaline lake, La Macha, Spain; EF031095 Microbial mat, Puerto Rico; EU245482 (4) Microbial mat, Puerto Rico; EU245142 Microbial mats, Spain & California; AY605141 (2) Microbial mat, France; AY605152 Microbial mats, France & Puerto Rico; AY605180 (3) Microbial mats; Spain, Puerto Rico, and California; AY605140 (8) Microbial mat, Spain; AY605126 Microbial mat, Spain; AY605169 Microbial mat, Puerto Rico; EU245523 (2) Termite hindgut, Reticulitermes speratus ; AB088910 Termite hindgut, Reticulitermes speratus ; AB088897 Termite hindgut, Reticulitermes speratus ; AB192150 Termite hindgut, Neotermes koshunensis ; AB084961 Bioreactor sludge; EF515492 Anaerobic phenol-degrader; EF198027 Spirochaeta thermophila Microbial mat, Spain; AY605173 Microbial mat, Spain; AY605139 (2) Microbial mats, Spain & California; AY605128 (2) Arctic sediment; EU287233 Microbial mat, France; AY605157 Mud volcano sediment, Mediterranean Sea; DQ103600 Sponge spirochete; AJ347045 Sponge spirochete; EF076172 Lake Suwa sediment, Japan; AB234282 Spirochaeta strain TM3; X97096 Lake Kastoria sediment, Greece; EF203193 Microbial mat, Spain; AY605177 Microbial mat, Spain; AY605146 Microbial mat, California; DQ329754 Surface slope sediment, China Sea; EU048676 Microbial mat, Spain; AY605130 Microbial mat, Spain; AY605143 Microbial mats, Spain & Puerto Rico; AY605131 (4) Karyomastigont and microbial mat, Spain; AY337318 (3) Spirochaeta litoralis Spirochaeta isovalerica Spirochaeta cellobiosiphila ; EU448140 Olavius loisae endosymbiont 4; AF104475 Oiavius crassitunicatus endosymbiont; AJ620512 Alvinella pompeiana endosymbiont; AJ431239 Volcano sediment, Gulf of Cadiz; DQ004680 Alvinella pompeiana endosymbiont; AJ432238 Forrested wetlands, Georgia; AF523927 Reed bed reactor, Wetlands grass; AB240336 Freshwater isolate, China Pond; AJ565434 Freshwater river, Massachusetts; AY947932 Freshwater isolate, China Pond; AJ565433 Spirochaeta auranti a [Spirochaeta ] stenostrept a Treponema pallidum [Spirochaeta ] zuelzerae Treponema denticol a

Figure 85.  (continued)

of louse-borne Borrelia recurrentis (Table 121). Additional novel Borrelia species or strains closely related to the Lyme disease or relapsing fever borreliae have been described. Their taxonomic status and pathogenicity in humans remain to be determined. Cells of all species are helically shaped with similar cell morphology and ultrastructure (Barbour and Hayes, 1986; Cutler, 2001; Wang et al., 2001). Among these, the Lyme disease borreliae are the longest (20–30 mm) and narrowest (0.2–0.3 mm). Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions. Inactivation of the gene encoding the major periplasmic flagellar filament protein FlaB results in nonmotile cells that are also rod-shaped rather than helical (Motaleb et al.,

2000). Further, mutants may show only asymmetrical flagellar rotation in the nonchemotactic mutants (Li et al., 2002). The lipid compositions of the outer membrane and whole cell are highly similar, suggesting that bulk transfer of lipid occurs between the cytoplasmic and outer membranes. The isolated outer membrane of Borrelia burgdorferi has a specific gravity of 1.12–1.19 g/cm2, depending on the purification procedure used (Bledsoe et al., 1994; Radolf et al., 1995); this comprises approximately 16.5% of the whole spirochete by dry weight. Chemical analysis of the outer envelope of Borrelia burgdorferi revealed a composition of 45.9% protein, 50.8% lipid, and 3.3% carbohydrate (Coleman et al., 1986). The ­purified periplasmic

487

Genus II. Borrelia

Figure 86.  Micrographs of Borrelia burgdorferi by light microscopy (a) and high voltage electron microscopy (b). The

cell diameter of Borrelia burgdorferi strain 297 shown in electron microscopy is 0.33 µm. The bundle of periplasmic flagella is clearly visible in the cell on the right (the EM micrograph was provided by K. Buttle, S.F. Goldstein and N.W. Charon). (c) Cross-section of Borrelia burgdorferi strain B31. The periplasmic flagella are visible in the lower right of the cell (micrograph provided by N.W. Charon, West Virginia University). Reprinted from Wang et al. (2001), with permission. Borrelia miyamotoi HT31T (D45192) 91 0.02

Borrelia theileri MT46 (DQ872186) “Borrelia lonestari ” sp. nov. Texas20 (U23211) Borrelia coriaceae Co53T (U42286)

Borrelia hermsii HS1T (U42292) 83 Borrelia parkeri M3001 (U42296) Borrelia turicatae M2007 (U42299)

92

Borrelia anserina ES-1 (U42284) Borrelia persica UESV/340 (U42297) Borrelia hispanica UESV/246 (U42294) 98 91

Borrelia crocidurae UESV/ACH (U42283) Borrelia recurrentis A1 (U42300) Borrelia duttonii UESV/117DUTT (U42288) Borrelia afzelii PKo (CP000395) Borrelia spielmanii A14S (AF102056) Borrelia turdi Ya501T (D67022) Borrelia tanukii Hk501T (D67023) Borrelia valaisiana VS116T (X98232) “Borrelia andersonii ” sp. nov. 21038 (L46701)

100 87

Borrelia japonica HO14T (L40597) Borrelia sinica CMN3T (AB022101) Borrelia burgdorferi B31T (U03396) “Borrelia bissettii ” sp. nov. DN127 (AJ224141) Borrelia garinii 20047T (D67018) Borrelia lusitaniae PotiB2T (X98228)

Borrelia turcica IST7T (AB111849) Treponema pallidum Nichols (AE000520)

Figure 87.  Phylogenetic tree based on 16S rRNA gene sequences of Borrelia species. A neighbor-joining phylogenetic

tree was constructed based on Kimura’s two-parameter distance estimation method using MEGA 2.1 program. Accession numbers are shown in parentheses. The numbers at the branch nodes indicate the results of the bootstrap analysis. The bar represents 2% sequence divergence. Treponema pallidum subsp. pallidum strain Nichols was used as an outgroup.

488

Family I. Spirochaetaceae

Table 120.  Barbour–Stoenner–Kelly (BSK) II mediuma

After detergent cleaning, all glassware is rinsed thoroughly with ­ glass-distilled water and then autoclaved. To 900 ml of glass-distilled water are added 100 ml of 10× concentrate of CMRL 1066 without glutamine. Add to the 1× CMRL 1066 in the following order:   5 g Neopeptone   50 g Bovine serum albumin, Fraction V   2 g Yeastolate   6 g N-2-Hydroxyethylpiperazine-N¢-2-ethanesulfonic acid   5 g Glucose   0.7 g Sodium citrate   0.8 g Sodium pyruvate   0.4 g N-Acetylglucosamine   2.2 g Sodium bicarbonate Adjust the pH of medium at 20–25°C to 7.6 with 1 N NaOH. Add 200 ml of 7% gelatin which had been dissolved in boiling water. Sterilize by filtration with air pressure (0.2 mm nitrocellulose). Store medium at 4°C. Before use, add unheated rabbit serum to a final concentration of 6%. Dispense to glass or polystyrene tubes or bottles. Fill containers to 50–90% capacity and cap tightly. Incubate at 34–37°C. a  Reprinted from Barbour (1984) with permission. Modifications of BSK II medium include BSK-H with removal of gelatin and different proportions of certain ingredients compared to BSK II (Pollack et al., 1993), and Kelly’s medium Preac-Mursic (MKP) (Preac-Mursic et al., 1986).

membrane possesses a lower specific gravity (1.12 g/cm2) but a higher percentage of proteins (56%) by dry weight of the membrane. Since Borrelia burgdorferi lacks the ability to elongate long-chain fatty acids, the fatty acid composition of the cells reflects that present in the growth medium. Chemical analysis of Borrelia hermsii cells that were cultivated in  vitro has demonstrated the presence of muramic acid and ornithine in the whole cells and in the protoplasmic cylinders, but not in the outer envelope preparations (Klaviter and Johnson, 1979). These data suggest that ornithine is a component of the cell wall. Borrelia hermsii contains cholesterol glucoside and its acylated derivatives (Livermore et  al., 1978), and the lipid composition and metabolism of this species of Borrelia is remarkably similar to that of several species of mycoplasmas. Spirochetes of the genus Borrelia do not have lipopolysaccharides containing lipid A (Takayama et  al., 1987), and potent exotoxins are not evident. Borrelia species are routinely cultured in liquid Barbour– Stoenner–Kelly (BSK) II medium under microaerophilic conditions. Borrelia burgdorferi isolates can form colonies when plated onto BSK medium solidified with 1.5% agarose (Kurtti et  al., 1987). Colonies are typically observed after 2–3 weeks of incubation and may differ in morphology among different strains, e.g., compact, round colonies (mean diameter, 0.43 mm) restricted to the surface of the agarose medium, diffuse colonies (mean diameter, 1.80 mm) penetrating into the solid medium, or colonies with a raised center surrounded by a diffuse ring of spirochetes. Borrelia burgdorferi lacks genes encoding enzymes required for the synthesis of most amino acids, fatty acids, enzyme cofactors, and nucleotides. Instead, there are 52 open reading frames (ORFs) that encode transport and binding proteins which would contribute to 16 distinct membrane transport systems for amino acids, carbohydrates, anions, and cations (Fraser et al.,

1997). Borrelia burgdorferi also shows limited metabolic capacity. Growth of Borrelia burgdorferi depends largely on the availability of nutrients provided in the culture medium or from the host (mammal or tick). Analysis of the genome and reconstruction of metabolic pathways suggests that Borrelia burgdorferi uses glucose as a primary carbon and energy source, although other carbohydrates such as glycerol, glucosamine, fructose, and maltose may be used in glycolysis. Borrelia burgdorferi does not contain genes encoding enzymes of the tricarboxylic acid cycle or components of the electron transport system (Fraser et al., 1997). Thus, it is assumed that lactic acid is the main end product of glycolysis, which is consistent with the microaerophilic nature of this spirochete. Since genes for the respiratory electron transport chain were not identified, ATP production must be accomplished by substrate-level phosphorylation. Borrelia hermsii and Borrelia parkeri ferment glucose, maltose, trehalose, starch, dextrin, and glycogen, but not raffinose. In contrast, Borrelia turicatae is able to ferment only glucose, raffinose, and dextrin. The genome of the borreliae is composed of a small linear chromosome of approximately 1000 kb and a collection of linear and circular plasmids that are variable in number and size among species and strains. The DNA–DNA homology of Lyme disease borreliae is 76–100% among strains within species and 46–74% between different species (Baranton et al., 1992; Hyde and Johnson, 1984). For the relapsing fever spirochetes, there is >70% DNA homology between Borrelia hermsii and Borrelia turicatae (86%) or Borrelia parkeri (77%) that are endemic in North America, whereas 17–63% DNA relatedness was demonstrated between Borrelia hermsii and other relapsing fever agents (Barbour and Hayes, 1986). All Borrelia species studied to date have DNA G+C contents of approximately 30 mol%. The complete genome of Borrelia burgdorferi has been determined (Casjens et  al., 2000; Fraser et  al., 1997). The genome size of the type strain Borrelia burgdorferi sensu stricto B31T is 1,521,419  bp. This consists of a linear chromosome of 910,725  bp, with a DNA G+C content of 28.6 mol%, and 21 plasmids (9 circular and 12 linear) with a combined size of 610,694  bp (Casjens et  al., 2000; Fraser et  al., 1997). The genomes of two other pathogenic Lyme disease borreliae, Borrelia garinii strain PBi and Borrelia afzelii strain PKo, have collinear chromosomes of comparable size to Borrelia burgdorferi B31T with only minor insertions and deletions. The plasmid fraction may vary significantly between different species. About 40% of Borrelia burgdorferi B31T genomic DNA is of plasmid origin, whereas plasmid DNA constitutes only 29 and 36% of the analyzed genomes of Borrelia garinii PBi and Borrelia afzelii PKo, respectively (Glockner et al., 2004, 2006). Genome analysis revealed that the Lyme disease borreliae possess some genetic structures that are unique among prokaryotes (Casjens et al., 2000; Fraser et al., 1997). These include: (1) the presence of a linear chromosome and multiple linear and circular plasmids in a single bacterium; (2) unique organization of the rRNA gene cluster, consisting of a single 16S rRNA gene and tandemly repeated 23S and 5S rRNA genes; (3) significantly higher frequency of lipoprotein-encoding genes (4.9% of the chromosomal genes and 14.5% of the plasmid genes); (4) a substantial fraction of plasmid DNA that appears to be in a state of evolutionary decay; and (5) evidence for ­numerous, and potentially recent, DNA rearrangements among the plasmid genes.

a 

nd, Not determined.

  B. tillae   B. turicatae   B. venezuelensis B. burgdorferi sensu lato   B. burgdorferi         B. afzelii     B. garinii     B. japonica   B. lusitaniae   B. sinica     B. spielmanii   B. tanukii     B. turdi   B. valaisiana   Other borreliae   B. turcica

nd nd nd 100

48 55 50–53 44–53 58 nd 50 58 51–65 85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive; nr, not reported.

pallidum repeat (tpr) genes tprC and tprI could be detected by RFLP analysis and used to distinguish each of the Treponema pallidum subspecies and a simian isolate, Treponema strain Fribourg-Blanc. These are currently the only systematic means for distinguishing the pathogenic Treponema species and subspecies. Additional approaches have been developed for the molecular subtyping of Treponema pallidum subsp. pallidum strains (Pillay et  al., 1998), and have been used in epidemiologic studies of

syphilis transmission (Diclemente et  al., 2004; Molepo et  al., 2007; Pillay et al., 2002; Sutton et al., 2001). Distinction of the cultivatable Treponema species is dependent on 16S rRNA gene sequence heterogeneity (Figure 93), phenotypic characteristics, some morphologic properties (e.g., cellular dimensions and number of flagella), and natural habitat. Little DNA–DNA reassociation data are available (Maio and Fieldsteel, 1978). 16S rRNA comparisons (Paster

508

Family I. Spirochaetaceae

4. T. berlinense

5. T. brennaborense

8. T. denticolab

9. T. lecithinolyticum

10. T. maltophilum

14. T. parvum

15. T. pectinovorumb

17. T. porcinum

19. T. putidum

23. T. socranskii subsp. buccale b

23. T. socranskii subsp. paredisb

23. T. socranskii subsp. socranskii b

25. T. vincentiib

Number of strains examined   1. Alkaline phosphatase   2. Esterase (C 4)   3. Esterase Lipase (C 8)   4. Lipase (C 14)   5. Leucine arylamidase   6. Valine arylamidase   7. Cystine arylamidase   8. Trypsin   9. a-Chymotrypsin 10. Acid phosphatase 11. Naphthol-AS-BIphosphohydrolase 12. a-Galactosidase 13. b-Galactosidase 14. b-Glucuronidase 15. a-Glucosidase 16. b-Glucosidase 17. N-acetyl-b-glucosaminidase 18. a-Mannosidase 19. a-Fucosidase

2. T. amylovorum

Table 129.  Enzymic activities of Treponema species, as determined by API ZYM analysisa

1 + + − − − − − − − + +

6 − − − − − − − − − + +

1 + + + − − − − − − + +

2 + + + − + − d + + + +

8 + + + − − − − − − + +

3 + + + − − − − − − + +

4 + + + − − − − − − + +

1 − + + − − − − − − + +

1 − + − − − − − − − + +

7 d + + − + − d + − + +

1 + + + − − − − − − + +

1 + + + − − − − − − + +

1 + + − − − − − − − + +

2 − d d − + − − − − + +

− − − − − − − +

− − − − − − − −

− + − + − + − −

+ d − − + − − −

− + + − − + − +

d d d + d − − d

− − + − − − − −

− − − − − − − −

− − − + − − − −

d + − − + − − −

− − + − − − − −

− − − − − − − −

− − − − − − − −

− + − − − + − −

Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive.

a 

Results from Wyss et al. (1996). Other results are from the defining publications.

b 

and Dewhirst, 2000) and the available mol% G+C data indicate that the genus Treponema is an ancient, diverse phylogenetic group. There are instances where Treponema with high 16S rRNA similarity are from the same environment (e.g., Treponema primitia and Treponema azotonutricum from termite guts), and others where organisms with 16S rRNA relatedness are from different environments (e.g., Treponema berlinense from porcine intestinal tract and Treponema pectinovorum from human gingival crevices) (Figure 93). These results suggest that some Treponema strains may have “jumped” from one environment to another during evolution. Analyses of oral treponeme strains (Paster et al., 1998) from the Smibert collection indicate the difficulty in reconciling 16S rRNA and phenotypic data (such as fatty acid content), and the importance of using more global genotyping approaches, including DNA–DNA reassociation studies, for distinguishing phylotypes with ³98% 16S rRNA sequence identity. As noted in the chapter on Spirochaeta, three species of Spirochaeta, namely Spirochaeta stenostrepta, Spirochaeta zuelzerae, and Spirochaeta caldaria, belong with members of the genus Treponema rather than with other “free-living” Spirochaeta as indicated by 16S rRNA phylogeny (Figure 93) and single-base signature analysis (Paster et al., 1991). There has been no formal renaming, so they are described in the Spirochaeta chapter. As previously discussed, these “free-living” spirochetes may be descendants of precursors of host-associated treponemes or

may have been disseminated via fecal contamination (Paster et al., 1991). Intestinal spirochetes previously classified as Treponema ­hyodysenteriae and Treponema innocens have been reclassified as species of Brachyspira.

Further reading Additional information regarding the genus Treponema is available in several recent books (Cabello et al., 2006; Radolf and Lukehart, 2006; Saier and Garcia-Lara, 2001), book chapters (Norris et  al., 2003; Pope et  al., 2006), and reviews (Antal et al., 2002; Cullen and Cameron, 2006; LaFond and Lukehart, 2006; Norris et al., 2001; Radolf et al., 1999).

Differentiation of the genus Treponema from other closely related genera Characteristics useful for distinguishing Treponema from the other members of the family Spirochaetaceae are indicated in the key to the family. In terms of the pathogenic genera, Borrelia species are also host-dependent, but utilize arthropods (ticks or lice) as intermediate hosts and transmission agents. They are also somewhat larger in diameter and more loosely coiled than Treponema, and are facultative anaerobes. Leptospira are also associated with mammals, but are capable of surviving for prolonged periods in water or soil. They are more tightly coiled than either Borrelia or Treponema, and are obligate aerobes.

Genus IV. Treponema

509

List of species of the genus Treponema*† 1. Treponema pallidum (Schaudinn and Hoffman 1905) Schaudinn 1905, 1728AL (Spirochaeta pallida Schaudinn and Hoffman 1905, 528) pal¢li.dum. L. neut. adj. pallidum pale, pallid. Tightly coiled, ~0.18 mm in diameter by 6–20 mm in length. The wavelength of coils is 1.1 mm and the amplitude is 0.2–0.3 mm. The ends of the cells are pointed, and a protrusion of the outer membrane at the end is often visible in well-preserved specimens by electron microscopy with negative staining (Figure 94). Two to four periplasmic flagella are inserted into each end of the cell, and overlap in the middle of the cell. Motile with graceful flexuous movements. Microaerophilic, with an optimal O2 concentration in the range of 1–5%. Obligate pathogens of humans. Can cause experimental infection and skin lesions in rabbits, guinea pigs, hamsters, and primates (Table 126). Sequence comparisons and DNA/DNA hybridization indicates that close to 100% DNA homology exists between the Treponema strains that cause syphilis, yaws, and endemic syphilis (Maio and Fieldsteel, 1980). Therefore, these organisms are considered subspecies of Treponema pallidum with distinctive clinical symptoms in humans and different patterns of infection in laboratory animals (Table 126). The subspecies can be distinguished by PCR and RFLP analysis of the tpp15, tprC, and tprI genes (Centurion-Lara et al., 1998, 2006). Source: humans. DNA G+C content (mol%): 52.4–53.7 (Tm). Type strain: none designated. 1a. Treponema pallidum subsp. pallidum (Schaudinn and Hoffman 1905) Schaudinn 1905, 1728, subsp. nov. The morphology and characteristics are as described for the species and as listed in Table 126. Treponema pallidum subsp. pallidum is the cause of venereal and congenital syphilis in humans. It has not been cultivated continuously in artificial media or in tissue culture. Propagated by intratesticular inoculation of rabbits. Successful replication of Treponema pallidum subsp. pallidum (virulent Nichols strain) has been reported to occur on the surface of tissue culture cells of cottontail rabbit epithelium (Sf1EP) growing in a monolayer in an atmosphere of 1.5% O2. A 49-fold increase (mean value) in cell numbers was reported in primary cultures of Treponema pallidum, but increased or prolonged multiplication with subculture has not been obtained to date (Cox, 1994; Fieldsteel et al., 1981; Norris and Edmondson, 1987). Treponema pallidum cells show rapid attachment to cultured mammalian cells (Fitzgerald et al., 1977a; Hayes et al., 1977). Proteins that bind to the host extracellular matrix proteins laminin and fibronectin have been identified ­(Cameron, 2003, 2004, 2005).

*The 16S rRNA sequences of Spirochaeta stenostrepta, Spirochaeta zuelzerae, and Spirochaeta caldaria (described in the section on Spirochaeta) are most similar to those of members of the genus Treponema (Figure 93). †Treponema pallidum, Treponema carateum, and Treponema paraluiscuniculi have not been cultivated continuously in vitro. The other species are anaerobes and have been cultivated; their characteristics are provided in Table 127, Table 128, and Table 129.

Obligate pathogen of humans. Strains such as the Nichols pathogenic strain are propagated by intratesticular inoculation of rabbits. Cutaneous inoculation of rabbits produces skin lesions. Cutaneous inoculation of hamsters, mice, and guinea pigs produces no apparent infection or visible lesions. A slight lesion is occasionally seen at the point of injection of guinea pigs. Microaerophilic. Survives in artificial media or tissue culture longest when incubated in an atmosphere of 1–5% O2 (Cox, 1994; Fieldsteel et al., 1977; Fitzgerald et al., 1977b, 1980; Norris et  al., 1978; Sandok et  al., 1978). Glucose is metabolized by way of the Embden–Meyerhof–Parnas and hexose monophosphate pathways (Schiller and Cox, 1977). Oxygen uptake by Treponema pallidum has been reported and is glucose-dependent (Barbieri and Cox, 1981; Cox and Barber, 1974). Oxidation of pyruvate occurs only when oxygen is present (Barbieri and Cox, 1979). Major fermentation products of glucose are acetate and CO2 (Nichols and Baseman, 1975). Isolated from human patients with syphilis. The reference Nichols pathogenic strain was isolated from the cerebrospinal fluid of a patient with neurosyphilis (Nichols and Hough, 1913). Shows 100% DNA/DNA homology by saturation reassociation with Treponema pallidum subsp. pertenue (Gauthier strain) but no significant DNA–DNA reassociation with Treponema phagedenis or Treponema refringens (Maio and Fieldsteel, 1978, 1980). Source: human patients with syphilis. DNA G+C content (mol%): 52.8 (genome sequence); ­52.4–53.7 (Tm). Type strain: none designated. Reference strain: Nichols pathogenic. Sequence accession no. (16S rRNA gene): M88726. 1b. Treponema pallidum subsp. pertenue (Castellani 1905) subsp. nov. (Spirochaeta pertenius Castellani 1905, 54) per.te.nu¢e. L. neut. adj. pertenue very thin, slender. The morphology and characteristics as described for the species and as listed in Table 126. Pathogenic to humans. Causes yaws in humans, a contagious disease that is spread by skin-to-skin contact. Treponema pallidum subsp. pertenue has not been cultivated in artificial media or in tissue culture. Cutaneous lesions are produced at the point of inoculation in rabbits and Syrian hamsters, but not in guinea pigs. Sera from patients with yaws give positive results with serologic tests for syphilis. Attachment of Treponema pallidum subsp. pertenue to five different mammalian cell lines was compared to that of Treponema pallidum subsp. pallidum (Fieldsteel et al., 1979). Treponema pallidum subsp. pertenue attached to all five cell lines, as did Treponema pallidum subsp. pallidum. No preferential attachment was found with Treponema pallidum subsp. pertenue for nude mouse ear and cottontail rabbit epithelial (Sf1Ep) cells, but preferential attachment did occur with Treponema pallidum subsp. pallidum. Inbred hamsters (LSH/Ss LAK) infected with Treponema pallidum subsp. endemicum (Bosnia A strain) were resistant to reinfection with both Treponema pallidum subsp. pertenue and Treponema pallidum subsp. pallidum (Schell et al., 1980). Treponema pallidum subsp. pertenue (Gauthier strain) shows 100% DNA homology to Treponema pallidum (Nichols

510

Family I. Spirochaetaceae

and KKJ strains) and no homology to Treponema phagedenis and Treponema refringens by DNA–DNA hybridization (Maio and Fieldsteel, 1980). Source: Lesions from cases of yaws. Present in tropical areas of Africa, Southeast Asia, the Western Pacific Islands, and South and Central America (Antal et al., 2002). DNA G+C content (mol%): 52–53.7 (Tm). Type strain: none designated. Reference strains: Gauthier or Haiti B. Sequence accession no. (16S rRNA gene): AF42610. 1c. Treponema pallidum subsp. endemicum subsp. nov. en.de¢mi.cum. N.L. neut. adj endemicum (from Gr. adj. endêmos -on native, dwelling in place), endemic. The morphology and characteristics are as described for the species and as listed in Table 126. Pathogenic to humans. The cause of nonvenereal endemic syphilis in humans, a contagious disease spread in pre-pubertal years by contact with infected individuals or shared use of contaminated utensils. Treponema pallidum subsp. endemicum has not been successfully cultivated in artificial media or tissue culture. Propagated by intratesticular inoculation of rabbits or by intradermal inoculation of hamsters. The organisms can be isolated from inguinal lymph nodes 3–4 weeks after intradermal infection. Inbred hamsters (e.g., LSH/Ss LAK) are particularly useful for study of this organism (Schell et al., 1980). Produces cutaneous lesions in rabbits, hamsters, and guinea pigs but not in mice. Sera from patients with nonvenereal epidemic syphilis give positive results with serologic tests for syphilis. This subspecies was created (Smibert, 1984) because the organism is considered a variant of Treponema pallidum and has its own clinical symptoms in human infection as well as the ability to infect and produce skin lesions in different laboratory animals, as does the organism of venereal syphilis (Table 126). Source: lesions from patients with nonvenereal endemic syphilis. Found in semi-arid areas of Africa, the Middle East, and some areas of Southeast Asia. DNA G+C content (mol%): not determined. Type strain: none designated. Reference strain: Bosnia A. Sequence accession no. (16S rRNA gene): not determined. 2. Treponema amylovorum Wyss, Choi, Schüpbach, Guggenheim and Göbel 1997, 844VP a.my.lo.vo¢rum. Gr. n. amylum starch; N.L. neut. adj. vorum (from L. v. voro to devour), devouring; N.L. neut. adj. amylovorum starch-devouring. Treponema amylovorum is an intermediate-sized, obligately anaerobic, helically coiled, motile treponeme (Wyss et al., 1997). The type strain HA2PT was isolated from subgingival plaque of a deep human periodontal lesion. The cells are approximately 0.25 mm in diameter and 7 mm in length, with a wavelength of ~1.2 mm and an amplitude of ~0.3 mm. They have six periplasmic flagella (three at each end of the cell) that overlap in the center of the cell (i.e., flagellar arrangement of 3:6:3). In liquid media of low viscosity, cells exhibit active cellular rotation and jerky flexing but no directional motility. However, in media of higher ­viscosity or

when cells creep along the surface, they exhibit slow translational movement. Cells can be stored at temperatures below −70°C in medium supplemented with 10–20% glycerol. When streaked onto OMIZ-Pat/HuS agarose plates (Wyss et al., 1997), Treponema amylovorum forms dense, offwhite, subsurface colonies up to 3 mm in diameter within 5 d of inoculation. Treponema amylovorum does not grow in chemically defined OMIZ-W1 medium and requires the addition of yeast extract and/or Neopeptone (or fractions thereof). Addition of 1% human serum is highly stimulatory, whereas fetal bovine serum or higher concentrations of human serum are inhibitory. Growth of strain HA2PT is accompanied by acid production as detected by phenol red indicator in the medium, and strictly depends on the presence of at least one of the following carbohydrates: d-glucose, maltose, starch, or glycogen. Acid is produced from these carbohydrates. None of the other carbohydrates tested (at a concentration of 2 g/l) support growth; these carbohydrates include d-arabinose, d-cellobiose, d-fructose, d-fucose, d-galactose, d-galacturonic acid, d-glucuronic acid, d-lactose, d-mannitol, d-mannose, d-melibiose, d-ribose, d-sucrose, d-trehalose, d-xylose, l-arabinose, l-fucose, l-rhamnose, l-sorbose, and l-xylose. Following the exponential growth phase, with an estimated doubling time of less than 4 h, cells of HA2PT rapidly lose viability and disintegrate into small vesicles. Catalase-negative. With API ZYM strips, the only (weak) enzyme activities detected in cells grown on glucose are alkaline and acid phosphatases, naphtholphosphohydrolase, C4 esterase, and a-fucosidase. In cells grown on either maltose, starch, or glycogen, weak a-glucosidase activity is also detected. Strain HA2PT is resistant to 1 mg/l rifampicin and 100 mg/l phosphomycin. On the basis of a phylogenetic comparison of 16S rRNA sequences, Treponema amylovorum is a species that is genetically distinct from previously described treponemes. Its SDS-PAGE protein and antigen profiles and its Western blot profile are readily distinguished from those of other cultivable, oral Treponema species. Furthermore, size and flagellation, as well as rapid flexing motility, clearly distinguish Treponema amylovorum from other treponeme species, such as Treponema denticola, Treponema maltophilum, Treponema pectinovorum, and Treponema socranskii. In contrast to the growth of the two asaccharolytic oral species, Treponema denticola and Treponema vincentii, growth of strain HA2PT is strictly carbohydrate-dependent, and the range of carbohydrates utilized is clearly distinct from the range of carbohydrates utilized by Treponema maltophilum, Treponema pectinovorum, and Treponema socranskii. Source: subgingival crevice of humans. DNA G+C content (mol%): not determined. Type strain: HA2P, ATCC 700288. Sequence accession no. (16S rRNA gene): Y09959. 3. Treponema azotonutricium Graber, Leadbetter and Breznak 2004, 1319VP a.zo.to.nu.tri¢ci.um. N.L. neut. n. azotum (from Fr. azote), nitrogen; L. neut. adj. nutricium nourishing; N.L. neut. adj. azotonutricium nourishing with nitrogen (symbiotic dinitrogen fixation). Cells 0.2–0.3 mm in diameter by 10–12 mm in length, with a wavelength or body pitch of 1.2 mm. Motile by

Genus IV. Treponema

two ­periplasmic flagella, inserted at opposite ends of the ­protoplasmic cylinder. Anaerobe. Catalase-negative. Yeast autolysate required for growth. Optimum temperature for growth is 30°C. Energy sources utilized for fermentative growth include d-glucose, d-fructose, d-ribose, d-xylose, d-maltose, and cellobiose. Maltose is fermented to acetate, ethanol, CO2, and H2 as major products. d-Mannitol, d- and l-arabinose, d-sucrose, d-trehalose, glycine, lactate, pyruvate, uric acid, and H2 (plus CO2) are not utilized. Exhibits nitrogenase activity and N2-dependent growth in media low in combined N. Genome is 3901 kb and contains 50.0 mol% G+C and two rrs gene copies. Nucleotide sequence of the 16S rRNA places this spirochete within the “termite cluster” of the genus Treponema. Source: hindgut contents of the Pacific dampwood termite Zootermopsis angusticollis (Hagen) (Isoptera: Termopsidae). DNA G+C content (mol%): 50 (Tm). Type strain: ZAS-9, ATCC BAA-888, DSM 13862. Sequence accession no. (16S rRNA gene): AF320287. 4. Treponema berlinense Nordhoff, Taras, Macha, Tedin, Busse and Wieler 2005, 1678VP ber.li.nen¢se. N.L. neut. adj. berlinense pertaining to Berlin, Germany, where the type strain was isolated. Cells show typical spirochete morphology exhibiting two to three windings with two periplasmic, subterminally inserted flagella (Nordhoff et al., 2005). Cells are approximately 0.3 mm in width and 6 mm in length. Strictly anaerobic. Good growth is observed in liquid OMIZ-Pat medium at 37°C supplemented with 10% (v/v) BHI and 10% (v/v) TSYE. On OMIZ-Pat agar plates (1–3%, w/v) supplemented with 5% (v/v) sheep blood, 10% (v/v) BHI, and 10% (v/v) TSYE, species form small, irregular, grayish swarms up to 1–2 mm in diameter, visible after 3–4 d. Addition of galacturonic or glucuronic acid promotes growth, which is enhanced further by addition of any of the following carbohydrates: d-glucose, d-fructose, maltose, d-mannitol, d-mannose, d-arabinose, l-fucose, d-trehalose, d-sucrose, and l-rhamnose. No visible growth is observed with pectin as the sole carbon source. Using the API ZYM and Rapid ID 32A systems, positive enzyme reactions are obtained only for acid phosphatase and naphthol-AS-BI-phosphohydrolase. Negative in tests for alkaline phosphatase, esterase C4, esterase lipase C8, leucine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-bglucosaminidase, a-fucosidase, urease, arginine dihydrolase, a-arabinosidase, mannose, and raffinose, glutamic acid decarboxylase, a-fucosidase, arginine arylamidase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, and serine arylamidase. Reduction of nitrates and indole production are not detected. The polar lipid profile contains diphosphatidylglycerol, phosphatidylethanolamine, an unknown aminophospholipid, and an unknown highly hydrophobic compound as major ­components. Moderate or minor amounts of phosphatidylglycerol, several unknown aminophospholipids,

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­ hospholipids, amino lipids, polar lipids, and a glycolipid p are also present. Source: swine feces in Berlin, Germany. DNA G+C content (mol%): not determined. Type strain: 7CPL208, ATCC BAA-909, CIP 108244, JCM 12341. Sequence accession no. (16S rRNA gene): AY230217. 5. Treponema brennaborense Schrank, Choi, Grund, Moter, Heuner, Nattermann and Göbel 1999, 49VP bren.na.bo.ren¢se. N.L. neut. adj. brennaborense of or belonging to Brennabor, where the cow was raised from which the organism was first isolated. Treponema brennaborense is an anaerobic, Gram-stain­ egative, helically coiled, motile treponeme that was ison lated initially from a digital dermatitis biopsy of a dairy cow. Bacterial cells are 5–8 mm long and 0.25–0.55 mm wide. One periplasmic flagellum originates subterminally at each cell pole, and the flagella overlap in the middle of the cell (i.e., have a 1:2:1 arrangement). In stationary-phase liquid cultures, the bacteria develop spherical forms. In liquid culture, the bacteria exhibit rotational movement. Growth of strain DD5/3T is accompanied by acid production. The optimum growth temperature is 37°C and maximum cell density of approximately 8×108 bacteria per ml is reached after 21 h incubation. Cells can be stored frozen (−80°C) in OMIZ-Pat medium (Wyss et  al., 1996) supplemented with 15% (v/v) glycerol. On semi-solid agarose plates, Treponema brennaborense forms diffuse, submersed white colonies up to 3 mm in diameter within 5-d incubation. Strain DD5/3T ferments raffinose and mannose and exhibits the enzyme activities alkaline phosphatase, C4 esterase, C8 esterase lipase, acid phosphatase, naphtholphosphohydrolase, b-galactosidase, a-glucosidase, N-acetyl-b-glucosaminidase, and arginine arylamidase, as determined by the API ZYM and Rapid ID 32A systems. Catalase-negative. The addition of 2–10% (v/v) rabbit serum leads to decreased growth rate. The strain is resistant to rifampicin (1 mg/l) and phosphomycin (100 mg/l). All previously described treponemes are genetically distinct from Treponema brennaborense as determined by comparative 16S rRNA sequencing. Treponema brennaborense is clearly distinguished by its morphology, protein pattern, and enzyme activities from the other cultivable Treponema species. Furthermore Treponema brennaborense is distinguishable from Treponema maltophilum by the presence of N-acetylb-glucosaminidase activity and its lack of a-galactosidase activity. Treponema brennaborense is clearly distinguishable from veterinary isolate 1-9185MED by its lack of trypsin and chymotrypsin activities. Source: a digital dermatitis biopsy of a dairy cow in ­Brandenburg, Germany. DNA G+C content (mol%): not determined. Type strain: DD5/3, CIP 105900, DSM 12168. Sequence accession no. (16S rRNA gene): Y16568. 6. Treponema bryantii Stanton and Canale-Parola 1981, 676VP (Effective publication: Stanton and Canale-Parola 1980, 145). bry.an¢ti.i. N.L. masc. gen. n. bryantii of Bryant, named after Marvin P. Bryant.

512

Family I. Spirochaetaceae

Helical obligate anaerobe, 3–8 mm long and 0.3 mm wide. One periplasmic flagellum is inserted at each end of the cell. No translational motility occurs at 22°C. Motile at 37°C. Requires CO2. Grows in chemically defined reduced medium containing isobutyrate, dl-2-methyl butyrate, pyridoxal, folic acid, niacinamide, biotin, thiamine, glucose, CO2, salts, and ammonium sulfate (Stanton and Canale-Parola, 1981). Riboflavin is stimulatory. In 0.7% Noble agar (Difco), cells form colonies in agar deeps, which are spherical and white (resembling cotton balls), and 0.5–1.0 mm in diameter after 24–36 h incubation, and eventually reach 2–3 mm in diameter. Growth in broth medium containing rumen fluid, glucose, and sodium bicarbonate yields 1.9×109 cells/ml. Does not utilize gluconate, succinate, acetate, formate, fumarate, sugar alcohols, or Tween 80. Grows in a medium containing cellulose and a cellulolytic bacterium such as Bacteroides succinogenes or Ruminococcus albus. No growth at 22 or 45°C. End products of glucose fermentation (mmol/100 mmol of glucose and 84 mmol of CO2 utilized) are acetate, 100; formate, 119; and succinate, 53. About 15% of glucose carbon is assimilated. Growth is inhibited by penicillin (10 U disk), ­cephalothin (30 µg disk), tetracycline (30 µg disk), chloramphenicol (30  µg disk), erythromycin (15 µg disk), and vancomycin (30 µg disk), slightly inhibited by polymyxin B (100 U/ disk), and not inhibited by rifampicin (5 µg disk or up to 10 mg/l in broth medium). Source: bovine rumen contents. DNA G+C content (mol%): 36±1 (Tm). Type strain: RUS-1, ATCC 33254, DSM 1788. Sequence accession no. (16S rRNA gene): M57737. 7. Treponema carateum (ex Brumpt 1939) sp. nov., nom. rev. ca.ra¢te.um. N.L. n. carate name of a South American disease, pinta; N.L. neut. adj. carateum of carate. The cause of pinta or carate, a contagious disease of man transmitted by skin-to-skin contact. Morphologically similar to Treponema pallidum. Virulent strains have not been grown in vitro. Experimental transmission of the disease has been accomplished in man as well as in chimpanzees by intradermal inoculation and by direct exposure of scarified areas of skin to abraded human lesions. Has not been propagated successfully in rabbits, hamsters, or guinea pigs. Source: exudate of cutaneous lesions of pinta. Occurs only in Mexico, Central America and parts of subtropical South America, the West Indies, and Cuba. DNA G+C content (mol%): not determined. Type strain: none designated. Sequence accession no. (16S rRNA gene): not determined. 8. Treponema denticola (ex Brumpt 1922a) Chan, Siboo, Keng, Psarra, Hurley, Cheng and Iugovaz 1993, 201VP den.ti¢co.la. L. masc. n. dens, dentis tooth; L. suff. cola from L. n. incola inhabitant dweller; N.L. n. denticola tooth-dweller. A small to intermediate-sized spirochete. Many characteristics are listed in Tables 127 and 129. A 2:4:2 periplasmic flagellar arrangement is common, but Treponema denticola strains with higher numbers of flagella have been found. Cells are motile with a jerky, but fairly rapid motion. Cells are typically 7.74±0.94 mm in length, 0.20±0.02 mm in

­ ia­meter, with a wavelength and amplitude of 1.23±0.15 mm d and 0.50±0.05 mm, respectively. The organism grows well in a peptone-yeast extractserum medium [e.g., New Oral Spirochete (NOS) medium; Leschine and Canale-Parola, 1980b) under anaerobic conditions. Surface and subsurface colonies are 0.3–1.0 mm in diameter, white, diffuse, and visible after 2 weeks incubation. Treponema denticola is primarily an amino acid fermenter and does not use the glycolytic pathway as a major source of energy, although it possesses genes encoding all of its enzymes (Seshadri et al., 2004). Amino acids in peptone-yeast extract-serum medium are fermented mainly to acetic acid, and to a lesser extent, lactic acid, succinic acid, and formic acid. Trace amounts of propionic acid, n-butyric acid, ethanol, n-propanol, and n-butanol may occasionally be found. Only 10% of the end products are from glucose. Alanine, cysteine, glycine, and serine are fermented. Arginine is metabolized to citrulline, NH3, CO2, proline, and small amounts of ornithine. Arginine iminohydrolase and ornithine carbamoyltransferase activity have been reported (Blakemore and Canale-Parola, 1976). Arginine can be an energy source and ornithine can be converted to putrescine and proline (Leschine and Canale-Parola, 1980a). Treponema denticola ATCC 35405T (Cheng et  al., 1985) exhibits 76% DNA homology with Treponema denticola 33520 and 82% DNA homology with Treponema denticola ATCC 35404 (Chan et al., 1993). The genome sequence of Treponema denticola 35405T has been determined (Seshadri et al., 2004). Treponema denticola ATCC 35405T is susceptible to the antimicrobial agents spiramycin, metronidazole, tetracycline, penicillin G, and streptomycin but highly resistant to rifampicin (minimum inhibitory concentration, 50 mg/l). Subdivided into biovar denticola (indole-positive) and biovar comondonii (indole-negative) (Smibert, 1984). Methyl red-negative. Chopped meat-serum medium is neither blackened nor digested. No action on milk. Growth occurs at pH 6.5–8.0 but not at pH 6.0 or 9.6, and at 30–42°C; multiplication is minimal or absent at 25 and 45°C. Source: oral cavity of humans and, perhaps, chimpanzees, typically from subgingival plaque. DNA G+C content (mol%): 37.9 (genome sequence). Type strain: ATCC 35405, CIP 103919, DSM 14222, JCM 8153. Reference strains: ATCC 33520, ATCC 35404. Sequence accession nos (16S rRNA gene): AE017226 (nt 610211–611726 and 1219839–1221354). 9. Treponema lecithinolyticum Wyss, Choi, Schüpbach, Moter, Guggenheim and Göbel 1999, 1337VP le.ci.thi.no.ly¢ti.cum. Gr. n. lekithos egg yolk; Gr. adj. lutikos ê -on able to loosen, dissolve; N.L. neut. adj. lecithinolyticum effecting the breakdown of egg yolk. An obligately anaerobic, helically coiled, motile treponeme. Cells are approximately 5×0.15 mm, with a wavelength of 0.7 mm and an amplitude of 0.3 mm. They contain two periplasmic flagella, one originating at each end and overlapping in the center of the cell. In liquid media, the cells flex and rotate but motility is not directional. However, in media of higher viscosity, or when cells creep along a surface, motility is directional.

Genus IV. Treponema

Since dipalmitoyl phosphatidylcholine (lecithin) inhibits growth, it is omitted from OMIZ-Pat medium (Wyss et al., 1996) (yielding OMIZ-Pat-w/oPC). When streaked onto OMIZ-Pat-w/oPC agarose, Treponema lecithinolyticum forms off-white, diffuse subsurface colonies up to 3 mm in diameter within 7 d incubation at 37°C. Does not grow in the chemically defined medium OMIZ-W1 (Wyss, 1992) but requires addition of yeast extract and/or Neopeptone (or fractions thereof). Cells can be stored frozen (liquid nitrogen or mechanical freezer) in OMIZ-Pat-w/oPC medium supplemented with 10–20% glycerol. Growth is strictly dependent on N-acetylglucosamine, strongly enhanced by further addition of d-arabinose, l-fucose, or d-ribose, d-fructose (some strains excluding OMZ 684T), and/or d-xylose (some strains including OMZ 684T), and not influenced by l-arabinose, d-cellobiose, d-fucose, d-galactose, d-galacturonic acid, d-glucose, d-glucuronic acid, d-lactose, maltose, d-mannitol, d-melibiose, l-rhamnose, l-sorbose, sucrose, d-trehalose, or d-xylose. Heat-inactivated human serum (1% v/v) is tolerated or stimulatory, whereas 1% fetal bovine serum is completely inhibitory. All strains are resistant to rifampicin (1 mg/l) and phosphomycin (100 mg/l). In all eight isolates examined (Wyss et al., 1999), activities of alkaline phosphatase, acid phosphatase, b-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, phospholipase A, and phospholipase C are prominent, whereas only intermediate activities of C4-esterase, C8-esterase, naphthol phosphohydrolase, and a-fucosidase are expressed. Catalase-negative. OMZ 684T, OMZ 685, and BL2B have strong sialidase activity (the other five strains were not tested). Phylogenetically distinct from other cultivable treponemes on the basis of its 16S rRNA sequence. Protein and antigen patterns (SDS-PAGE) are also readily distinguished from those of other cultivable treponemes, though more conventional criteria may suffice to distinguish it from the other characterized oral spirochetes. Simultaneous expression of strong activities of phospholipase C, phospholipase A, alkaline phosphatase, acid phosphatase, b-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, and sialidase and intermediate activities of C4-esterase, C8-esterase, naphthol phosphohydrolase, and a-fucosidase distinguish Treponema lecithinolyticum from all other oral spirochetes. Size, flagellation, and growth characteristics additionally distinguish it from Treponema amylovorum, Treponema denticola, Treponema medium, and Treponema vincentii. Finally, Treponema lecithinolyticum is phenotypically distinguished from the two other lecithinolytic isolates described by Wyss et  al. (1999) (i.e., OMZ 702 and BL2A, which are phylogenetically classified as Treponema maltophilum) by its SDSPAGE protein profile, a ~30-kDa antigen, and activities of phospholipase A, sialidase, b-glucuronidase, and N-acetylb-glucosaminidase. Source: Only in human subgingival plaque, with a strong association suggested for diseased versus control sites in patients with adult periodontitis and rapidly progressive periodontitis. Strains OMZ 684T and OMZ 685 were isolated from subgingival plaque of human deep periodontal lesions. DNA G+C content (mol%): not determined. Type strain: OMZ 684, ATCC 700332, CIP 107075.

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Reference strains: OMZ 685, ATCC 700333. Sequence accession no. (16S rRNA gene): AJ131282. 10. Treponema maltophilum Wyss, Choi, Schüpbach, Guggenheim and Göbel 1996, 751VP mal.to¢phi.lum. N.L. n. maltosum maltose; Gr. adj. philos ê -on loving, friendly to; N.L. neut. adj. maltophilum intended to mean maltose-loving. An obligately anaerobic, helically coiled, motile treponeme (5 mm long, 0.2 mm wide with a wavelength of 0.7 mm and an amplitude of 0.3 mm) isolated from human subgingival plaque (Wyss et  al., 1996). One periplasmic flagellum originates at each end, overlapping in the center of each cell in a 1:2:1 arrangement. In low-viscosity liquid media, cellular rotation produces standing waves with amplitudes of up to 2 mm, but this results in no directional motility. Translational movement, however, occurs in higher-viscosity media or when cells creep along a surface. Cells can be stored frozen in liquid nitrogen or in a mechanical freezer in OMIZ-Pat (Wyss et al., 1996) supplemented with 10–20% glycerol. On OMIZ-Pat agarose, off-white diffuse subsurface colonies (up to 3 mm in diameter) form within 5 d. Treponema maltophilum does not grow in OMIZ-W1 and requires yeast extract and/or Neopeptone. Growth of most strains is strictly dependent on N-acetyl-b-glucosamine and at least one additional sugar. The most commonly used second sugars are d-arabinose, l-fucose, d-maltose, l-rhamnose, d-ribose, d-sucrose, and d-trehalose, but not d-glucose, which is totally ineffective. Some strains without a-fucosidase activity may not depend on N-acetyl-b-glucosamine. Growth is not influenced by d-cellobiose, d-fucose, d-lactose, d-mannitol, l-sorbose, or l-xylose. Fetal bovine serum at concentrations as low as 0.1% (v/v) prevents growth in OMIZ-Pat. Catalasenegative. API ZYM strips detected alkaline phosphatase, acid phosphatase, naphtholphosphohydrolase, C4 esterase, C8 esterase, and a-glucosidase activities in all strains, aand b-galactosidase, b-glucosidase, and a-fucosidase activities in most strains, and a-fucosidase and b-glucuronidase ­activities in some strains. Strains with b-glucuronidase activity can grow on glucuronic acid. Immunoblotting with patient sera revealed an antigen only in strains with a-fucosidase activity. All strains examined are resistant to rifampicin (1  mg/l) and phosphomycin (100  mg/l). The type strain BRT (a-fucosidase activity but no b-glucuronidase activity) differs markedly from reference strains HO2A (b-glucuronidase activity but no a-fucosidase activity) and PNA1 (neither a-fucosidase activity nor b-glucuronidase activity) (Wyss et al., 1996). Phylogenetically distinct from previously described treponemes as determined by comparison of 16S rRNA sequences. Its protein and antigen patterns on SDS-PAGE differ from those of other cultivable treponemes. clearly distinguish Treponema maltophilum differs from Treponema vincentii on the basis of size, morphology, enzyme activities, and growth characteristics, from the asaccharolytic organism Treponema denticola by its lack of trypsin activity, from Treponema pectinovorum by its lack of a requirement for either glucuronic acid or galacturonic acid, and from Treponema socranskii by synthesis of a wide spectrum of glycosidases and

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Family I. Spirochaetaceae

utilization of a wide spectrum of carbohydrates. a-Glucosidase activity is characteristic of Treponema maltophilum and has never been observed in Treponema socranskii, although many strains of this species can ferment maltose. Source: only in subgingival plaque samples of patients with periodontal disease. DNA G+C content (mol%): not determined. Type strain: BR, ATCC 51939, CIP 105146. Reference strains: HO2A (ATCC 51940), PNA1 (ATCC 51941). Sequence accession no. (16S rRNA gene): X87140. 11. Treponema medium Umemoto, Nakazawa, Hoshino, Okada, Fukunaga and Namikawa 1997, 71VP me¢di.um. L. neut. adj. medium not very great or small, medium, referring to the cell size. A Gram-stain-negative, anaerobic, motile, helically coiled, medium-sized treponeme. The cells (5–16 mm long and 0.2–0.3 mm wide) have cytoplasmic filaments and 5–7 periplasmic flagella (axial flagella) that originate subterminally at each end and in broth cultures exhibit rotational and translational movement. The optimum growth temperature is 37°C, and colonies on agar plates are white and translucent. Ferment d-glucose, d-fructose, maltose, d-mannose, d-galactose, sucrose, d-ribose, trehalose, inulin, salicin, and d-raffinose. Produce ammonia and hydrogen sulfate and hydrolyze esculin and hippuric acid. The major acid products of strain G7201T grown in tryptone-yeast extract-gelatin-volatile fatty acids-serum (TYGVS) medium (Ohta et al., 1986) containing 0.1% glucose are acetic acid, n-butyric acid, and a trace of n-valeric acid. Phenotypic characteristics, DNA–DNA hybridization data, G+C content of the DNA, and 16S rRNA gene sequence data indicate that human oral spirochete strain G7201T is a member of a novel species. Source: subgingival plaque of patients with adult periodontitis. DNA G+C content (mol%): 51 (HPLC). Type strain: G7201. Sequence accession no. (16S rRNA gene): D85437. 12. Treponema minutum Dobell 1912, 117AL mi.nu¢tum. L. neut adj. minutum small, tiny. Many characteristics are listed in Table 127. Two to three periplasmic flagella are inserted into each end of the cell. Motile with sluggish movement. Colonies on prereduced peptone-yeast extract-serum agar (1.4%) are visible in 9–15 d, and are 0.5–1 mm in diameter, white, and round on the agar surface. Some colonies after longer incubation are white, fluffy, and up to 1.5 mm in diameter. Colonies grow on and below the surface of the medium. Size and texture of colonies will vary with the concentration of agar in the medium. Grow well in peptone-yeast extract-serum medium under anaerobic conditions. Require animal serum (inactivated at 56–60°C for 1 h) for growth. Amino acids in peptone-yeast extract-serum medium are fermented to a large amount of acetic acid, moderate amount of succinic acid, smaller amount of lactic acid, and trace amounts of propionic, n-butyric, and formic acids (most strains). Trace amounts of ethanol, n-propanol, and

n-butanol are also produced by most strains. There are no additional end products in the presence of glucose. Methyl red-negative. Skim milk is only slightly curdled. Ammonia produced by most strains. Grows at pH 6.5–8.0 but not at pH 6.0 or 9.6. Grows at 34–40°C. Chopped meat serum medium neither blackened nor digested. Slight putrid odor. Dupouey (1963) reported that Treponema minutum was antigenically only slightly related to Treponema refringens. Not pathogenic. Source: epidermal surfaces of male and female genitoperianal regions. DNA G+C content (mol%): 37 (Tm). Type strain: CIP 5162. Sequence accession no. (16S rRNA gene): not available. 13. Treponema paraluiscuniculi (Jacobsthal 1920) Smibert 1974, 177AL (Spirochaeta paraluis-cuniculi Jacobsthal 1920, 571) pa.ra.lu.is.cu.ni¢cu.1i. Gr. pref. para resembling; L. n. lues -is pestilences, plague, infection (here syphilis); L. n. cuniculus -i a rabbit; N.L. gen. n. paraluiscuniculi of a syphilis-like (disease) of rabbits. Produces venereal spirochetosis (rabbit spirochetosis or rabbit syphilis) in rabbits. Morphologically similar to Treponema pallidum. Transmitted by sexual contact. Has not been cultivated in  vitro. The organism can be propagated by intratesticular inoculation of rabbits. Causes a latent infection of mice, guinea pigs, and hamsters. Treponemes are found in the lymph nodes of these animals. Cutaneous lesions are found only in guinea pigs and rabbits. Nonpathogenic to humans (Graves and Downes, 1981). The cuniculi A strain has been shown to possess homologs of tpr genes found in Treponema pallidum, but many are predicted to be nonfunctional (Giacani et al., 2004). For additional information see Smith and Persetsky (1967). Source: lesions in the genital area of rabbits. Primarily involves the genitalia, although cutaneous lesions often occur around the face, eyes, ears, and nose. DNA G+C content (mol%): not determined. Type strain: none has been designated. Reference strain: Cuniculi A. Sequence accession no. (16S rRNA gene): not determined. 14. Treponema parvum Wyss, Dewhirst, Gmür, Thurnheer, Xue, Schüpbach, Guggenheim and Paster 2001, 960VP par¢vum. L. neut. adj. parvum small. Small, obligately anaerobic, helically coiled, motile treponeme. Approximately 1  mm long and 0.18  mm wide, with a wavelength of 0.8µm and an amplitude of 0.3 mm. In rapidly growing cultures, cells may be shorter than 1 wavelength, but chains of more than 10 wavelengths are also common. Cells contain two periplasmic flagella, one originating at each pole and overlapping in the center of the cell in a 1:2:1 arrangement. Although undulation, flexing, and rotation of cells occurs in liquid medium, motility does not appear to be directional. However, in media of higher viscosity or when cells creep along a surface, movement is translational. Cells can be stored frozen (liquid nitrogen or mechanical freezer) in OMIZ-Pat/HuS medium supplemented with

Genus IV. Treponema

10–20% glycerol. The four isolates OMZ 832, 833T, 842, and 843 are strictly carbohydrate-dependent; either N-acetyl-bglucosamine or N-acetyl-b-galactosamine is sufficient for growth, though growth is strongly promoted by addition of l-arabinose, d-galactose, d-glucose, d-fructose, d-­mannitol, d-mannose, pectin, d-ribose, or d-xylose but not by d-­arabinose, d-cellobiose, d-fucose, l-fucose, d-galacturonic acid, d-glucuronic acid, d-lactose, d-maltose, d-melibiose, l-rhamnose, l-sorbose, d-sucrose, d-trehalose, or l-xylose. The chemically complex (undefined) components of OMIZ-Pat, i.e., YEM (fractionated yeast extract) and DANP (fractionated peptone) (Wyss and Ermert, 1996), are not strictly required for growth but are strongly stimulatory. Similarly, human serum or FBS are not required but are growth-promoting at 1% (v/v). FBS is not inhibitory even at 10% (v/v). On OMIZPat/HuS agarose, Treponema parvum forms off-white diffuse subsurface colonies up to 3 mm in diameter after 5  d of anaerobic incubation at 37°C. The four OMZ isolates on API ZYM tests had weak alkaline phosphatase and esterase C4 and C8 activities, intermediate acid phosphatase and naphthol phosphohydrolase activities, and strong b-glucuronidase activity. Although Treponema parvum and Treponema pectinovorum are similar, the former is clearly distinguishable phenotypically by the presence of a strong b-glucuronidase activity, inability to utilize pectin as sole source of carbohydrate, and marked differences in protein and antigen patterns revealed by SDS-PAGE. Very short and thin cells, though this is not strongly diagnostic, since all cultured treponemes show variation in morphology under different growth conditions. The “Smibert-2” isolates also differ from the 1:2:1 flagellated pectinolytic treponemes isolated from nonhuman primates by Sela et  al. (1987) by virtue of their inability to grow on pectin. There are 16S rRNA sequence differences between Treponema parvum and other species of Treponema, including Treponema pectinovorum. Treponema parvum is the sole representative of Group 7 of oral Treponema as defined by Paster et  al. (1998). Its closest relatives are Treponema pectinovorum (88% similarity) and Treponema amylovorum (90% similarity). Source: OMZ 833T was isolated from subgingival plaque of a human deep periodontal lesion. Strain OMZ 842 was isolated from acute necrotizing ulcerative gingivitis (ANUG) lesions of a patient in China. DNA G+C content (mol%): not determined. Type strain: OMZ 833, ATCC 700770, DSM 16260. Reference strain: OMZ 842, ATCC 700773. Sequence accession no. (16S rRNA gene): AF302937. 15. Treponema pectinovorum Smibert and Burmeister 1983, 853VP pec.ti.no¢vo.rum. N.L. n. pectinum pectin; N.L. neut. adj. vorum (from L. v. voro to devour) devouring; N.L. neut. adj. pectinovorum pectin destroying and devouring. Obligately anaerobic, motile helically coiled treponeme. The cells are 7–15 mm long and 0.28–0.30 mm wide. They are coiled and usually have straight, slightly pointed ends. The periplasmic flagella overlap in the center of the cell in a 2:4:2 arrangement. Secondary coils are observed in motile cultures. Movement is both rotational and ­translational.

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Serpentine movement can be observed in a semisolid medium. In Oral Treponeme Isolation (OTI) medium (Smibert and Burmeister, 1983) in bottle plates, colonies usually appear in the agar after 4–5 d. Colonies grow into the agar and are white and transluscent with slightly denser centers and entire edges. The colonies spread out and become larger after additional incubation. These organisms grow in PY-pectin broth containing either rumen fluid or a short-chain fatty acid-heme supplement. Serum and thiamine pyrophosphate are not required. Growth only occurs in the presence of a fermentable energy source, such as pectin, polygalacturonic acid, galacturonic acid, or glucuronic acid and is greatly stimulated by the addition of a fresh filter-sterilized yeast autolysate to the medium. Growth occurs at 37°C (optimum) but not at 25 or 42°C. Broth cultures become turbid with a granular sediment that can be seen after 4–5 d of incubation. Cultures can be stored frozen in liquid nitrogen or at −85°C in a mechanical freezer. Pectin (final pH, 5.3–5.9) is utilized. Polygalacturonic acid, galacturonic acid, and glucuronic acid are also fermented and may be substituted for pectin in PY-rumen fluid broth. Growth and acid production occur in PY-rumen fluid broth supplemented with either autoclaved or filter-sterilized pectin. Growth and acid production also occur in PY-rumen fluid broth containing 0.5% polygalacturonic acid. The pH of this medium is 5.0 after 5 d of incubation at 37°C. ­Adonitol, amygdalin, arabinose, cellobiose, dextrin, starch, dulcitol, erythritol, esculin, fructose, galactose, glycerol, glycogen, inositol, inulin, lactose, glycerol, glycogen, inositol, inulin, lactose, maltose, mannitol, mannose, melezitose, melibiose, mucin, raffinose, rhamnose, ribose, salicin, sorbose, sorbitol, sucrose, trehalose, xylose, and glucose are not fermented; no growth occurs in PY-rumen fluid broth containing any of these substrates. Negative for catalase and hydrogen sulfide production. Hydrogen gas was not detected by gas chromatography of the atmospheric phase of cultures in rubber-stopper sealed tubes; gas was not detected in agar deep cultures. Gelatin, esculin, glycogen, or starch is not hydrolyzed. Indole and acetylmethylcarbinol are not produced. The major fermentation products from PY-pectinrumen fluid broth are acetic acid (27.9 mM) and formic acid (8.5 mM). Only traces of pyruvic and lactic acids are detected. The products from polygalacturonic acid are acetic acid (39.5  mM) and formic acid (16.7  mM) with only traces of lactic and pyruvic acids. Distinction from other Treponema species is based on 16S rRNA sequence, mol% G+C content, ability to ferment pectin, polygalacturonic acid, galacturonic acid, and glucuronic acid but not other carbohydrates, cellular dimensions, and the origin of the two periplasmic flagella attached at each end of the cell (Smibert and Burmeister, 1983). Source: human supragingival and subgingival plaque specimens but not from adults with normal, healthy gingivae and no signs of gingivitis or periodontitis. DNA G+C content (mol%): 39 (Tm). Type strain: ATCC 33768T, VPI D-36DR-2T. Sequence accession no. (16S rRNA gene): M71237.

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Family I. Spirochaetaceae

16. Treponema phagedenis (ex Brumpt 1922a) sp. nov., nom. rev. pha.ge.de¢nis. Gr. gen. n. phagedenis of a cancerous sore. Many characteristics are listed in Tables 127 and 128. Widest cells show double contours with darkfield microscopy. Ends of the cells are blunt with no covering sheath. Three-to-eight periplasmic flagella are inserted into each end of the cell. In old cultures, the flagella may be seen trailing from the ends of the cells. Motility in culture media is jerky with slow rotational movement. Colonies in prereduced anaerobic peptoneyeast extract-serum medium containing 1.3–1.4% agar are white, annular, 0.5–1 mm in diameter with a dense center after incubation for 2–5 d at 37°C. Colonies can grow on the surface but mainly in the agar. Requires animal serum (heat inactivated at 56–60°C for 0.5–1 h) for growth. Bovine serum albumin supplemented with a pair of fatty acids can substitute for serum (Johnson and Eggebraten, 1971). The pair includes (a) an unsaturated fatty acid such as oleic acid and (b) a saturated fatty acid such as palmitic acid. No growth with short-chain fatty acids or a-, b-, g-globulins. Fermentation of glucose is by the Embden–Meyerhof– Parnas pathway. Contains ferredoxin. End products of fermentation in a serum medium without glucose are mainly acetic and n-butyric acids with moderate-to-small amounts of propionic and formic acids and usually small-to-trace amounts of lactic and succinic acids. Trace amounts of alcohols are also produced. In a medium containing glucose, large amounts of ethanol and n-butanol and smaller amounts of n-propanol are produced. A very slight curd is formed in skim milk. Weakly methylred-positive. Does not grow at a pH of 6.0 or 9.6. Grows at 30–42°C but not or only slightly at 25 and 45°C. Reduces neutral red. Chopped meat serum medium is neither blackened nor digested. A slight fetid odor is produced in ­cultures. Subdivided into reiter and kazan biovars. Biovar reiter does not hydrolyze esculin, whereas biovar kazan does. Meyer and Hunter (1967) showed that the Reiter, Kazan, and English Reiter strains are antigenically closely related. Reiter and English Reiter contained the same antigens while the Kazan strain contained an antigen not shared by the Reiter treponemes. The Nichols strain of Treponema refringens was antigenically unrelated. Christiansen (1964) also reported that the Reiter and Kazan 11 strains were closely related but not identical. Dupouey (1963) reported that Treponema phagedenis and Reiter strain were closely related antigenically, sharing at least six common antigens. The Reiter strain has a large amount of an antigen that may be shared with a number of other species including Treponema pallidum. More than 40 water-soluble antigens have been demonstrated in the Reiter treponeme by crossed immunoelectrophoresis. Five antigens cross-reacted with antibodies in syphilitic sera (Strandberg-Pedersen et al., 1980, 1981). Reiter and Kazan strains have high DNA/DNA homology to each other and no homology to Treponema refringens (Miao and Fieldsteel, 1978; Smibert, 1974). There is no DNA/DNA homology to Treponema denticola (Smibert,

1974). There is no detectable DNA homology between Treponema phagedenis (Reiter and Kazan 5) and pathogenic Nichols strain of Treponema pallidum subsp. pallidum (Maio and Fieldsteel, 1978). Additional information on Treponema phagedenis Reiter can be found in an excellent review by Wallace and Harris (1967). Source: nonpathogenic. Phagedenic ulcer on human external genitalia. Reiter treponeme from a case of primary syphilis in man and also as normal flora in the anal and genital areas of normal male and female chimpanzees. DNA G+C content (mol%): 38–39 (Tm). Type strain: none designated. Reference strain: Reiter. Sequence accession no. (16S rRNA gene): M57739. 17. Treponema porcinum Nordhoff, Taras, Macha, Tedin, Busse and Wieler 2005, 1678VP por.ci¢num. L. neut. adj. porcinum pertaining to swine, from which the type strain was isolated. Cells exhibit typical spirochete morphology and are approximately 6–8 mm in length and 0.3 mm in width with 2–3 windings and 2:4:2 flagella arrangement. Strictly anaerobic. Best growth is obtained in liquid OMIZ-Pat medium at 37°C supplemented with 10% (v/v) BHI and 10% (v/v) TSYE. Growth is independent of glucuronic or galacturonic acid. d-Maltose is essential for growth, whereas any of the following carbohydrates (as the sole carbohydrate source) do not support growth: d-glucose, d-fructose, d-mannitol, d-mannose, d-arabinose, l-fucose, trehalose, sucrose, and l-rhamnose. Does not grow with pectin as a sole carbon source. On OMIZ-Pat (1–3% w/v) agar supplemented with 5% egg yolk, 10% BHI, and 10% TSYE, the species forms grayish, irregular swarms up to 2 mm in diameter, visible after 3–4 d. Reactions using the API ZYM and Rapid ID 32A system are positive for acid phosphatase, esterase C4, naphthol- AS-BI-phosphohydrolase, and a-glucosidase, and negative for alkaline phosphatase, esterase lipase C8, leucine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-fucosidase, urease, arginine dihydrolase, a-arabinosidase, mannose, and raffinose fermentation, glutamic acid decarboxylase, a-fucosidase, arginine arylamidase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, and serine arylamidase. Reduction of nitrates and indole production are not detected. In the polar lipid profile, three unknown phospholipids and a highly hydrophobic compound predominate. Diphosphatidylglycerol, phosphatidylglycerol as well as phospholipids are present in moderate amounts. Additionally, a glycolipid and several phospholipids are present in minor amounts. Source: swine feces in Berlin, Germany. DNA G+C content (mol%): 38–39 (Tm). Type strain: 14V28, ATCC BAA-908, CIP 108245, JCM 12342. Sequence accession no. (16S rRNA gene): AY518274.

Genus IV. Treponema

18. Treponema primitia Graber, Leadbetter and Breznak 2004, 1319VP pri.mi¢ti.a. N.L. fem. sing. n. primitia (nominative in apposition), the first fruit (of isolation after long work). Cells 0.2 mm in diameter by 3–7 mm long, with a wavelength or body pitch of 2.3 mm. Motile by two periplasmic flagella, inserted at opposite ends of the protoplasmic cylinder. Anaerobe. Possesses NADH and NADPH peroxidases but neither catalase nor superoxide dismutase. Optimum temperature for growth is 30°C. Optimum pH for growth is 7.2 (range, 6.5–7.8). Homoacetogen. Energy sources used for growth include glucose, maltose, mannitol, xylose, and H2 (plus CO2), which are fermented to acetate as the sole product. Strain ZAS-1 also uses arabinose and cellobiose, whereas strain ZAS-2 can grow slowly by acetogenic demethylation of methoxylated benzenoids (syringate, ferulate, vanillate, and trimethoxybenzoate). Ribose, methanol, formate, CO, lactate, pyruvate, glycine, betaine, and choline are not utilized. Growth by mixotrophy (i.e., simultaneous use of H2 and organic substrates) has been demonstrated. Laboratory-prepared yeast autolysate or certain commercial yeast extracts are required for growth. Folinate (formyltetrahydrofolate) is required for growth of strain ZAS-1, whereas folic acid or folinate is required for growth of strain ZAS2. Cells possess homologs of the dinitrogenase reductase gene nifH and exhibit low levels of nitrogenase activity, but unambiguous N2-dependent growth has not been demonstrated. Genome sizes are 3461 kb (ZAS-1) and 3835 kb (ZAS-2); G+C contents of DNA are 51.0 mol% (ZAS-1) and 50.9 mol% (ZAS-2) (by HPLC); each strain possesses 2 rrs gene copies. The 16S rRNA nucleotide sequences of strains ZAS-1 and ZAS-2 place them within the “termite cluster” of the genus Treponema. Source: hindgut contents of the Pacific dampwood termite Zootermopsis angusticollis (Hagen) (Isoptera: Termopsidae). DNA G+C content (mol%): 50.9–51.0 (HPLC). Type strain: strain ZAS-2, ATCC BAA-887, DSM 12427. Sequence accession no. (16S rRNA gene): AF093252 (ZAS-2T), AF093251 (ZAS-1). 19. Treponema putidum Wyss, Moter, Choi, Dewhirst, Xue, Schüpbach, Göbel, Paster and Guggenheim 2004, 1121VP pu¢ti.dum. L. neut. adj. putidum stinking, fetid. Obligately anaerobic, helically coiled, motile, asaccharolytic, and proteolytic. The human oral cavity is so far its only known habitat. Approximately 0.25 mm in diameter and approximately 10 mm long, with a wavelength of approximately 3 mm and amplitude of approximately 1.5 mm. They contain four periplasmic flagella, two originating at each cell end and overlapping in the center of the cell in an arrangement of 2:4:2. In liquid media of low viscosity, cells appear highly active with cellular rotation and jerky flexing but no directional motility. Translational movement, however, is seen in media of higher viscosity or when cells creep along a surface. Cells can be stored at temperatures below −70°C in medium supplemented with 10–20% glycerol. Within 5 d of anaerobic incubation at 37°C when streaked onto OMIZ-Pat agar (Wyss et al., 1996), dense, off-white subsurface colonies up to 3 mm in diameter are formed. Does not grow in the chemically defined OMIZ-W1 medium, but

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requires the addition of yeast extract and/or Neopeptone (or fractions thereof); addition of 1–10% human or fetal bovine serum is highly stimulatory. Growth is neither dependent on nor stimulated by any of the following carbohydrates, each tested at 2 g/l: d-arabinose, d-cellobiose, d-fructose, d-fucose, d-galactose, d-galacturonic acid, d-glucose, d-glucuronic acid, glycogen, d-lactose, d-maltose, d-mannitol, d-mannose, d-melibiose, d-ribose, starch, sucrose, d-trehalose, d-xylose, l-arabinose, l-fucose, l-rhamnose, l-sorbose, and l-xylose. Neuraminidase and dentilisin acti­vities are not detected. Using API ZYM strips (Table 129), the following enzyme activities are always detected: esterase C4, esterase C8, leucyl arylamidase, trypsin, acid phosphatase, naphtholphosphohydrolase, b-galactosidase, and b-glucosidase; the following activities are never detected: lipase C14, valine arylamidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase. Other enzyme activities detectable by API ZYM are present only in some strains. Growth is resistant to rifampicin (1 mg/l), phosphomycin (100 mg/l), nalidixic acid (30 mg/l), and polymyxin (5 mg/l). Source: subgingival plaque of a deep human periodontal lesion. DNA G+C content (mol%): not determined. Type strain: JZC3, OMZ 758, ATCC 700334, CIP 108088, OMZ 758. Sequence accession no. (16S rRNA gene): AJ543428. 20. Treponema refringens (ex Castellani and Chalmers 1919) sp. nov., nom. rev. re.frin¢gens. L. part. adj. refringens refringent, refractive. Many characteristics are listed in Tables 127 and 128. The average cells are 5–8 mm long and 0.24 mm wide. Some cells may appear loosely coiled. Two to four periplasmic fibrils are inserted at each end of the cell. Motile, with a slow, sluggish movement. Rotation of cells is rare, and when observed, usually slow. Colonies on prereduced anaerobic peptone-yeast extractserum agar (1.4%) are visible in 9–15 d. They are white, round, surface colonies 0.5–1 mm in diameter. Some colonies after longer incubation are white, fluffy, and up to 1.5 mm in diameter. Colonies grow on and below the surface of the medium. Size and texture of colonies varies with the concentration of agar in the medium. Grows well in peptone-yeast extract-serum medium under anaerobic conditions. Requires animal serum (inactivated at 56–60°C for 1 h) for growth. Amino acids in serum medium are fermented to mostly acetic acid, moderate amounts of succinic acid, and smaller amounts of lactic acid. Some strains produce trace amounts of propionic acid, n-butyric acid, formic acid, ethanol, n-propanol, and n-butanol. No additional end products are produced in the presence of d-glucose. Methyl red-negative. Skim milk is only slightly curdled. Ammonia produced by most strains. Growth occurs at pH 6.5–8.0 but not at pH 6.0 or 9.6. Grows at 30–42°C but only very slightly or not at all at 25 or 45°C. Chopped meat serum medium is neither blackened nor digested. Only a very slight putrid odor is detectable. Dupouey (1963) reported that strains labeled Treponema refringens and “Treponema calligyrum” were closely related

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antigenically, sharing 4–5 common antigens, but only slightly related to Treponema minutum. The three strains had only one antigen in common with Treponema pallidum. DNA from Treponema refringens shows a high homology with DNA from the avirulent Nichols strain, the Noguchi strain, and “Treponema calligyrum”, and a very low homology with DNA from strains of Treponema denticola (R. M. Smibert and J. Johnson, unpublished data). No detectable DNA homology by hybridization to Treponema phagedenis or Treponema pallidum subsp. pallidum (Nichols) and Treponema pallidum subsp. pertenue (Maio and Fieldsteel, 1978). On this basis, “Treponema calligyrum” was designated a biovar of Treponema refringens by Smibert (1984). Biovar refringens does not grow with 1% glycine, whereas biovar calligyrum does grow in 5–6 d. Source: condyloma acuminata lesions, occasionally from syphilitic lesions. Part of normal flora of male and female genitalia of man and animals. Not pathogenic. DNA G+C content (mol%): 39–43 (Tm). Type strain: none designated. Reference strain for biovar refringens: Treponema refringens, Institut Pasteur, Paris. Reference strain for biovar calligyrum: CIP 64.40. Sequence accession nos (16S rRNA gene): AF426101 (Treponema refringens biovar refringens); AF426100 (Treponema refringens biovar calligyrum). 21. Treponema saccharophilum Paster and Canale-Parola 1985, 218VP sac.cha.ro.phi¢lum. Gr. n. sacchar sugar; Gr. adj. philus -ê -on loving; N.L. neut. adj. saccharophilum sugar-loving. Helical cells, 0.6–0.7 mm by 12–20 mm. Cell coiling is regular except when cells are in contact with solid surfaces. A bundle of periplasmic flagella is wrapped around the cell body. At least 16 periplasmic flagella are inserted near each end of the cell. Cells swim at velocities in excess of 60 µm/s in liquid media at 37°C, but no translational motility is observed at 23°C. Translational motility ceases 1–2 min after the cells are exposed to air. Cells in contact with solid surfaces exhibit creeping motility. Obligate anaerobe. Optimum growth is at 37–39°C. At these temperatures, the final growth yield in rumen fluid-glucose-sodium bicarbonatesalts broth is 7×108 cells per ml and the population doubling time is 90 min. No growth at 23°C or 45°C. Subsurface colonies in agar media are spherical and opaque with diffuse edges. Utilizes as fermentable substrates for growth: l-arabinose, d-galactose, d-glucose, d-mannose, d-fructose, d-galacturonic acid, d-glucuronic acid, cellobiose, lactose, maltose, sucrose, d-raffinose, dextrin, inulin, starch, pectin, polygalacturonic acid, and arabinogalactan. Does not grow on: l-rhamnose, d-xylose, l-sorbose, d-ribose, cellulose, dextran, amino acids, d-arabitol, dulcitol, mannitol, ribitol, sorbitol, xylitol, glycerol, potassium galactonate, potassium gluconate, sodium acetate, sodium formate, sodium lactate, sodium succinate, potassium fumarate, Tween 80, glucosamine, and xylan. Exogenous isobutyric acid is required for growth, and valeric acid is stimulatory. Neither NaHCO3 in media nor a CO2-containing atmosphere is required for growth. Fermentation end products of growing cells (in micromoles per 100 pmol of glucose utilized):

formate, 150; acetate, 91.2; ethanol, 79.4. Approximately 15% of the ­glucose carbon consumed by growing cells is assimilated in cell material. Acetate and formate are major end products of pectin or glucuronic acid fermentation. Pyruvate is metabolized via a coliform-type clastic reaction. Isolated from bovine rumen fluid using an agar medium that contained rifampicin as a selective agent and pectin as a fermentable substrate. Source: bovine rumen contents. DNA G+C content (mol%): 54 (Tm). Type strain: PB, ATCC 43261, DSM 2985. Sequence accession no. (16S rRNA gene): M71238. 22. Treponema scoliodontus (ex Noguchi 1928) sp. nov., nom. rev. sco.li.o.don¢tus. Gr. adj. skolios crooked, bent; Gr. n. odous, odontos tooth; N.L. neut. n. scoliodontus crooked tooth. Many characteristics are listed in Tables 127 and 128. Very tightly coiled cells. Motile with a jerky but fairly rapid motion. Grows in peptone-yeast extract-serum medium under anaerobic conditions. Requires animal serum or ascitic fluid. Amino acids in a peptone-yeast extract-serum medium are fermented to moderate amounts of acetic acid and small amounts of formic, succinic, lactic, propionic, and n-butyric acids. No additional end products are produced in the presence of glucose. Methyl red-negative. Ammonia is not produced from amino acids. No action on milk. Chopped meat serum medium is neither blackened nor digested. Produces a slight fetid odor. Grows at pH 6.5–8.0 but not at pH 6.0 or 9.6. Grows at 30–42°C. Source: oral cavity of humans. DNA G+C content (mol%): not known. Type strain: none designated. Reference strain: Treponema scoliodontus Institut Pasteur, Paris. Sequence accession no. (16S rRNA gene): not determined. 23. Treponema socranskii (Noguchi 1928) Smibert, Johnson and Ranney 1984, 459VP so.crans¢ki.i. N.L. masc. gen. n. socranskii of Socransky, named for Sigmund S. Socransky, Forsyth Dental Center, Boston, USA. Cells are 6–15 mm long and 0.16–0.18 mm wide. They have tapered ends with a slight bend or “hook” at one or both ends of the cell. Obligately anaerobic, motile, helically coiled treponeme. The species is subdivided into three distinct subspecies based on DNA homology (by hybridization) and phenotypic characteristics: subspecies socranskii, buccale, and paredis (Smibert et al., 1984). The periplasmic flagella overlap in the center of the cell, in a 1:2:1 relationship. The cells form coccoid bodies in the late stationary growth phase. Cells in broth cultures have both rotational and translational movement. Serpentine movement of cells can be seen by darkfield microscopy of cultures grown in a semisolid medium. Colonies usually appear in Oral Treponeme Isolation (OTI) agar (Smibert and Burmeister, 1983) in 7–10 d. The colonies grow into the agar, are white and translucent, and often have slightly denser centers and edges that can be

Genus IV. Treponema

entire or irregular. T he colonies spread and become larger after extended incubation. Growth occurs only in media containing a fermentable carbohydrate and either rumen fluid (20–30%) or a mixture of short-chain fatty acids. Serum is not required. Growth is optimum at 37°C and only slight at 25 or 42°C. The phenotypic characteristics of representative strains (10–12 of each subspecies) were examined (Smibert et al., 1984). Fermentation of glucose by a majority of the strains leads to a pH ranging from 5.1 to 5.9. A few strains belonging to Treponema socranskii subsp. buccale do not reduce the pH below 6.0 when they are grown in glucose-containing broth. These cultures have a pH range of 6.2–6.7. Inulin, lactose, Melezitose, cellobiose, salicin, d-sorbitol, glycerol, amygdalin, adonitol, dulcitol, i-erythritol, inositol, and d-mannitol are not fermented, as indicated by only a slight change in the pH of the medium. Hydrogen sulfide is produced in SIM medium supplemented with rumen fluid by all but 1 of 32 strains tested. A distinctive phenotypic trait found in all strains of all subspecies of Treponema socranskii (but not in any other oral spirochete) is the formation of intensely yellow colonies (or cell pellets after growth in liquid medium) in OMIZ-Pat agarose. Furthermore, live cells of Treponema socranskii can be recognized microscopically, since cells that are rotating around their axes have both cell tips markedly deflected, which gives the appearance of propellers on both ends of a straight helix. None of the strains studied produces catalase, peroxidase, indole, or acetylmethylcarbinol. Esculin is not hydrolyzed. Hydrogen gas is not detected by gas chromatography of samples taken from the atmosphere above the broth medium in rubber-stoppered tubes. The major acid fermentation products of all strains grown in PY-glucose-rumen fluid broth are acetic, lactic, and succinic acids. Trace amounts of formic acid occasionally can be found. The strains produce a mean of 8 mM lactic acid (range, 3–16 mM), 6.5 mM acetic acid (range, 2.6–10.4 mM), and 3 mM succinic acid (range, 1–6.5 mM). Treponemes in homology group A1 are designated Trepo­ nema socranskii subsp. socranskii. The type strain of Treponema socranskii subsp. socranskii is ATCC 35536T (= VPI DR56BRIII6). Treponemes in homology group A2 are designated Treponema socranskii subsp. buccale (buc.ca¢le. L. n. bucca the mouth; L. neut. suff. -ale suffix denoting pertaining to; N.L. neut. adj. buccale buccal, pertaining to the mouth); the type strain is VPI D2B8T (=ATCC 35534). The phenotypic characteristics of these subspecies are described in detail in the defining publication (Smibert et al., 1984). Patterns of phenotypic reactions (Smibert et  al., 1984) show that Treponema socranskii subsp. socranskii (homology group A1) cannot be readily differentiated from Treponema socranskii subsp. buccale (homology group A2). However, when a slide agglutination test with washed cells as the antigen was used, antisera against the type strain (strain ATCC 35536) and strain VPI D43BR1 agglutinated 11 of 12 Treponema socranskii subsp. socranskii strains (Smibert et al., 1984), and no strain of Treponema socranskii subsp. buccale or Treponema socranskii subsp. paredis. Antisera against the type strain (strain ATCC 35534) and strains VPI D11A1 and VPI

519

D40DPEI of Treponema socranskii subsp. buccale agglutinated 7 of 10 Treponema socranskii subsp. buccale strains. Antiserum against the type strain of Treponema socranskii subsp. paredis (strain ATCC 35535) agglutinated 7 of 9 Treponema socranskii subsp. paredis strains but no Treponema socranskii subsp. socranskii or Treponema socranskii subsp. buccale strains. Treponemes in homology group K are designated Treponema socranskii subsp. paredis (pa.re¢dis. Gr. n. pareias cheek; N.L. gen. n. paredis of a cheek). l-Arabinose and rhamnose are not fermented. Other characteristics of this subspecies are the same as those of the species. The type strain of Treponema socranskii subsp. paredis is strain ATCC 35535 (= VPI D46CPE1), which was isolated from a supragingival sample from a patient with severe periodontal ­disease. Treponema socranskii subsp. paredis can be easily separated from Treponema socranskii subsp. socranskii and Treponema socranskii subsp. buccale by the inability of Treponema socranskii subsp. paredis to ferment l-arabinose and rhamnose. Source: subgingival sample from a patient with severe periodontal disease. It is the most frequently isolated treponeme and is usually the most numerous of the cultivable treponemes in either supragingival or subgingival samples. DNA G+C content (mol%): 50.5±2 (Tm). Type strains: Treponema socranskii subsp. socranskii ATCC 35536, JCM 8157, VPI D56BRIII6; Treponema socranskii subsp. buccale ATCC 35534, JCM 8155, VPI D2B8; Treponema socranskii subsp. paredis ATCC 35535, JCM 8156, VPI D46CPE1. Sequence accession nos (16S rRNA gene): AF033306 (Treponema socranskii subsp. socranskii); AF033305 (Treponema socranskii subsp. buccale); AF033307 (Treponema socranskii subsp. ­paredis). 24. Treponema succinifaciens Cwyk and Canale-Parola 1981, 383VP (Effective publication: Cwyk and Canale-Parola 1979, 231.) suc.ci.ni.fa¢ci.ens. N.L. n. acidum succinicum succinic acid; L. part. adj. faciens making, producing; N.L. part. adj, succinifaciens succinic acid-producing. Helical, anaerobic bacterium 0.3 mm wide by 4–8 mm long. Some cells may be up to 16 mm long. May form chains. Possesses a 2:4:2 flagellar arrangement. No transitional movement at 25°C. Motile at 37°C, requires carbon dioxide. Colonies in rumen fluid agar deeps are spheroid with an opaque center and diffuse peripheral growth. Colonies are 4–8 mm in diameter after 2 d growth at 37°C. In broth, cell yields are 1.5×109 cells/ml with a mean generation time of 3.5 h. The pH of a glucose culture after 48 h is about 6.0. ­Ferments glucose by the Embden–Meyerhof pathway. CO2/ bicarbonate and a carbohydrate are required for multiplication. Growth is supported by l-arabinose, d-xylose, d-­glucose, d-mannose, d-galactose, maltose, lactose, cellobiose, dextrin, and starch, but not by d-ribose, l-sorbose, raffinose, l-rhamnose, d-fructose, sucrose, dextran, inulin, ball-milled cellulose, trehalose, glycerol, d-mannitol, d-sorbitol, dulcitol, xylitol, sodium acetate, sodium formate, sodium succinate, potassium pyruvate, sodium lactate, and potassium gluconate. Does not ferment acetate, formate, succinate, xylitol,

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Family I. Spirochaetaceae

pyruvate, lactate, gluconate, and Tween 80. End products are (in mmol/100 mmol glucose and 51 mmol of CO2 utilized) acetate, 82; formate, 81; succinate, 58; lactate, 30; 2,3-butanediol, 5; pyruvate, 4; acetoin, 3. ­Catalase-negative. Poor growth occurs at 22 and 43°C. Inhibited by penicillin G (4000 U/l), cephalothin (4  mg/l), and chloramphenicol (4 mg/l), not by erythromycin (4 mg/l), oxytetracycline (4 mg/l), polymyxin B (40,000 U/l), rifampicin (4 mg/l), streptomycin (4 mg/l), tetracycline (4 mg/l), and vancomycin (4 mg/l). Source: colon of swine. DNA G+C content (mol%): 36 (Tm). Type strain: strain 6091, ATCC 33096, DSM 2489. Sequence accession no. (16S rRNA gene): M57738. 25. Treponema vincentii (ex Brumpt 1922a) sp. nov., nom. rev. vin.cen¢ti.i. N.L. masc. gen. n. vincentii of Vincent, named after Jean-Hyacinthe Vincent (1862–1950), a French military physician. Many characteristics are listed in Tables 127–129. Cells may have shallow and irregular spirals. Four to six periplasmic flagella are inserted at each end of cell. Motile with a rapid, jerky, vibratory motion. Colonies of strain N-9 are visible after incubation for 2 weeks. The colonies are white, 12–15 mm in diameter, appearing as a slight haze in the

agar. Unlike other Treponema species, Treponema vincentii is reported to produce LPS (Blanco et  al., 1994; Kurimoto et  al., 1990). Grows in a peptone-yeast extract medium under anaerobic conditions. Requires animal serum or ascitic fluid for growth. Amino acids are fermented to mainly acetic and n-butyric acids, moderate amounts of lactic acid, and smaller amounts of succinic and formic acids, and trace amounts of propionic acid, ethanol, n-propanol, and n-butanol. No additional end products are produced in the presence of glucose. Methyl red-negative. Skim milk is not changed. Ammonia is produced in cultures. Chopped meat is neither blackened nor digested. A slight fetid odor is produced in cultures. Grows at pH 6.5–7.5 and at 25–45°C. Meyer and Hunter (1967) reported that Treponema vincentii strain N-9 was antigenically distinct from Treponema denticola (FM) and the Nichols and Noguchi strains of Treponema refringens. Antigens were shared with [Spirochaeta] zuelerzae and Treponema phagedenis (Reiter and Kazan strains). Source: oral cavity of humans. DNA G+C content (mol%): not known. Type strain: none designated. Reference strain: N-9. Sequence accession no. (16S rRNA gene): AF033309.

Species Candidatus*   1. “Treponema suis” (Molbak et al., 2006) su¢is. L. n. sus suis a swine, hog, pig, boar, sow; L. gen. n. suis of a pig. This Candidatus was identified in paraffin-embedded biopsy specimens of porcine colon. Laser capture dissection and PCR amplification were used to determine the 16S rRNA sequence, and fluorescence in situ hybridization (FISH) was performed to show that the spirochetes were distributed within the colonic epithelium and lamina propria. The fact that the organism was identified by FISH in nearly equal proportions in pigs with colitis (60%) and in normal controls (43%) indicates that it is not a causative agent of colitis. The 16S rRNA sequence was most closely related to Treponema bryantii; however, the sequence identity was only 90.1%, making it likely that this organism, provisionally called “Treponema suis”, represents a separate species. By electron microscopy, “Treponema suis” is longer (6–11 mm) than other Treponema species identified in the porcine intestinal tract (Treponema succinifaciens, Treponema berlinense, and Treponema porcinum, 4–8 mm). In addition, “Treponema suis” has a total of 10–14 periplasmic flagella, as compared to two for the aforementioned species. The in vitro culture of this organism has not as yet been reported. Source: porcine colon. DNA G+C content (mol%): not known. Type strain: none designated. Sequence accession no. (16S rRNA gene): AM284386. *The following species have been described but can be found in the literature and are not yet classified formally. There are no known cultures of two of these species. They are listed so that if they are isolated again, the description can be used to aid in their identification. The names presently have no standing in nomenclature.

2. “Treponema macrodentium” Noguchi 1912, 82 [Spirochaeta macrodentium (Noguchi 1912) Pettit 1928, 182] mac.ro.den¢ti.um. Gr. adj. makros long; L. n. dens dentis tooth; N.L. gen. pl. n. macrodentium of large teeth. Slender helical rods, 5–16 mm long and 0.1–0.25 mm wide. The ends of the cell are pointed. One periplasmic flagellum is inserted into each end of the cell. Motile with a fairly rapid motion. Young cells rotate rapidly on their long axis. Grows in peptone-yeast extract-medium or PPLO medium (BBL) containing 10% serum or ascitic fluid with cocarboxylase (5  mg/l), glucose (1 g/l), and cysteine (1 g/l). Requires animal serum for growth. This requirement can be replaced by isobutyric acid (20 mg/l), spermine (150 mg/l), and nicotinamide (400 mg/l). Will also grow in a medium supplemented with rumen fluid and cocarboxylase. Requires a fermentable carbohydrate as an energy source. Carbohydrates are ­fermented. Acid but no gas is produced. The final pH in ­glucose broth is 5.0–5.4. Ferments fructose, glucose, maltose, ribose, and sucrose. May ferment cellobiose, galactose, and xylose. Does not ferment mannose, rhamnose, sorbose, lactose, arabinose, trehalose, mannitol, inulin, sorbitol, or salicin. Starch is not hydrolyzed. Glucose is fermented mainly to lactic acid, moderate amounts of acetic and formic acids, and a trace of succinic acid. Gelatin is hydrolyzed. Indole-negative. Hydrogen sulfide is produced. Lactate is not used. Ammonia is not produced. Optimum temperature, 37°C. Grows at pH 7.0. Source: subgingival crevice of humans. DNA G+C content (mol%): 39 (Tm). Type strain: no culture available. Sequence accession no. (16S rRNA gene): not known.

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Genus IV. Treponema

5%

Treponema vincentii (human) Treponema sp. oral taxon 237; AF061350 (human) Treponema sp. oral taxon 238; AF061347 (human) Treponema sp. oral taxon 262; AF182834 (human) Treponema sp. oral taxon 227; AF056339 (human) Treponema phagedenis (human) Treponema pedis ; EF061267 (bovine) Treponema sp. oral taxon 247; AF023032 (human) Treponema denticola (human) Treponema sp. oral taxon 251; AF056340 (human) Treponema sp. oral taxon 256; AF182835 (human) Treponema sp. oral taxon 252; AF056341 (human) Treponema sp. oral taxon 255; AF182833 human) Treponema pallidum (human) [Spirochaeta ] zuelzerae (free-living) Anaerobic digestor; AF275918 (free-living) Reticulitermes flavipes ; AF068429 (termite) Reticulitermes flavipes ; AF068432 (termite) Hodotermopsis sjoestedti ; AB032004 (termite) Reticulitermes flavipes ; AF068425 (termite) Reticulitermes flavipes ; AF068426 (termite) Reticulitermes flavipes ; AF068335 (termite) Reticulitermes flavipes ; AF068417 (termite) Reticulitermes flavipes ; AF068342 (termite) Reticulitermes flavipes ; AF068339 (termite) Reticulitermes flavipes ; AF068422 (termite) Mastotermes darwiniensis ; X89049 (termite) Treponema succinifaciens (swine) Treponema zioleckii ; DQ065758 (bovine) Treponema saccharophilum (bovine) Treponema socranskii ss socranskii (human) Treponema strain OMZ841; unpublished (canine) Treponema sp. oral taxon 260; AF023041 (human) Treponema maltophilum (human) Treponema sp. oral taxon 264; AF385536 (human) Treponema sp. oral taxon 263; AF182837 (human) Treponema amylovorum (human) Treponema parvum (human) Treponema porcinum AY518274 (swine) Treponema pectinovoru m (human) Treponema berlinense ; AY230217 (equine) Treponema fecal sp.; AY212749 (equine) Treponema fecal sp.; AY212774 (equine) “Treponema suis”; AM284386 (swine) Treponema sp.; AY178844 (bovine) Treponema sp.; AB009242 (bovine) Treponema bryanti (bovine) Treponema sp.; AF001693 (bovine) Reticulitermes flavipes ; AF068415 (termite) Reticulitermes flavipes ; AF068419 (termite)

Mammalian cluster 1

Termite cluster 1

Mammalian cluster 2

Figure 95.  Phylogenetic tree illustrating the diversity of cultivable and not-yet-cultivable species of the genus Treponema based on 16S rRNA sequence comparisons. Environmental source or host for each species is noted in parentheses. Several clusters were apparent, e.g., mammalian clusters 1 and 2, termite clusters 1 and 2, and a waste water cluster 1. GenBank accession numbers for the 16S rRNA sequences of not yet cultivated species, or phylotypes, tested are shown. The scale bar represents a 5% difference in nucleotide sequence

3. “Treponema orale” Socransky, Listgarten, Hubersak, Cotmore and Clark 1969, 881 [Treponema oralis (sic) Socransky, Listgarten, Hubersak, Cotmore and Clark 1969, 881] o.ra¢le. L. n. os, oris the mouth; L. neut. suff. -ale suffix denoting pertaining to; N.L. neut. adj. orale pertaining to the mouth, of the mouth. Slender helical cells, 6–16 mm long and 0.10–0.25 mm wide. Occasional chains are formed. One periplasmic flagellum is inserted into each end of the cell. Frequently end granules are seen in broth cultures. Motile with a jerky but fairly rapid motion. Grows in either PPLO medium without crystal violet (BBL) or peptone-yeast extract medium. Each medium contains glucose (1 g/l), cysteine (1 g/l), ­nicotinamide

(500  mg/l), cocarboxylase (5  mg/l), spermine tetrahydrochloride (150 mg/l), and sodium isobutyrate (20 mg/l), and each is further supplemented with 10% inactivated rabbit serum or ascitic fluid, or 0.05% a-globulin. Uniform turbidity occurs in liquid media. Does not grow well on surface cultivation. Does not require carbohydrates as an energy source. Carbohydrates not fermented. Amino acids are fermented. The final pH in glucose broth is 6.8–7.2. Amino acids are fermented to acetic and propionic acids. Hydrolyzes gelatin but not starch. Indole-positive. H2S produced. Utilizes lactate. Does not produce ammonia in cultures. Grows at pH 7.0 and at 37°C. Paster et al. (1998) reported that a strain labeled “Treponema oralis” in the Smibert collection had 16S rRNA sequence and

522

Family I. Spirochaetaceae Reticulitermes flavipes ; AF068420 (termite) Reticulitermes flavipes ; AF068427 (termite) Reticulitermes flavipes ; AF068421 (termite) Treponema primitia (termite) Reticulitermes flavipes ; AF068334 (termite) Reticulitermes flavipes ; AF068336 (termite) Neotermes koshunensi s; AB084973 (termite) Incisitermes tabogae ; AM182455 (termite) Zootermopsis angusticollis ; AF06840 (termite) Reticulitermes flavipes ; AF068341(termite) Reticulitermes santonensi s; AJ419822 (termite) Treponema azotonutricu m (termite) Mixotricha paradoxa ; AJ458946 (termite) Mastotermes darwiniensi s; X89048 (termite) Neotermes castaneu s; AJ419817 (termite) Terme s; AB189683 (termite) Macrotermes gilvu s; AB234359 (termite) Nasutitermes ; EF454213 (termite) Cubitermes orthognathus ; AY160872 (termite) Microcerotemes ; AB191828 (termite) Microcerotemes ; AB191912 (termite) Cryptotermes cavifron s; AB299521 (termite) Neotermes castaneu s; AJ419819 (termite) Neotermes koshunensi s; AB084968 (termite) Neotermes castaneu s; AJ419818 (termite) Neotermes koshunensi s; AB084956 (termite) Neotermes koshunensi s; AB084954 (termite) Reticulitermes santonensi s; AJ41982 (termite) Neotermes koshunensi s; AB085162 (termite) Neotermes koshunensi s; AB085167 (termite) Coptotermes formosanus ; AF068345 (termite) Reticulitermes speratus ; AB088889 (termite) Kalotermes flavicolli s; AJ418816 (termite) Kalotermes flavicolli s; AJ420234 (termite) Coptotermes formosanus ; AB062768 (termite) Reticulitermes flavipes ; AF068428 (termite) Reticulitermes santonensi s; AJ419822 (termite) Mastotermes darwiniensi s; X89047 (termite) Mixotrichia paradoxa ; AJ458945 (termite) Mastotermes darwiniensi s; X89044 (termite) Mixotrichia paradoxa ; AJ458947 (termite) Mastotermes darwiniensi s; X89046 (termite) Mastotermes darwiniensi s; X89042 (termite) Mastotermes darwiniensis ; X89050 (termite) Reticulitermes speratu s; AB088902 (termite) Reticulitermes flavipes ; AF068344 (termite) Reticulitermes flavipes ; AF068416 (termite) Reticulitermes speratus ; AB088863 (termite) Mastotermes darwiniensi s; X79548 (termite) Mastotermes darwiniensi s; X89043 (termite) Neotermes koshunensi s; AB085168 (termite) Reticulitermes flavipe s; AF068418 (termite) Coptotermes formosanu s; AF068346 (termite) Mastotermes darwiniensis ; X89045 (termite) Mastotermes darwiniensi s; X89051 (termite) Nasutitermes ; U40791 (termite) Microcerotemes ; AB243269 (termite) Nasutitermes ; EF454184 (termite) Coptotermes formosanus ; AF068347 (termite) Reticulitermes flavipes ; AF068424 (termite) Coptotermes formosanus ; AB062806 (termite) Nasutitermes ; EF454909 (termite) Microcerotemes ; AB191870 (termite) Nasutitermes ; EF454804 (termite) Nasutitermes ; EF453858 (termite) Nasutitermes ; EF453871 (termite) Nasutitermes ; EF454119 (termite) Nasutitermes ; EF454205 (termite) Nasutitermes ; EF454137 (termite) Reticulitermes flavipes ; AF068423 (termite) Kalotermes flavicolli s; AJ419824 (termite) [Spirochaeta ] caldaria (free-living) [Spirochaeta ] stenostrepta (free-living) Waste water; AY133086 Waste water; AY214182 Contaminated aquifer; AF050551 TCE waste water; AY133081 Waste water; AJ009481 SRB waste water; AY340825 TCB waste water; AJ009476 Spirochaeta isovalerica Spirochaeta litorali s

Figure 95.  (continued)

Termite cluster 2

Waste water cluster 1

Genus IV. Treponema

other characteristics closely resembling those of Treponema denticola. The mol% G+C (37) is close to that determined for Treponema denticola (37.9). Therefore, this Candidatus species appears to be Treponema denticola and should be eliminated. Source: subgingival crevice of humans. DNA G+C content (mol%): 37. Type strain: none. Sequence accession no. (16S rRNA gene): none available. 4. “Treponema zioleckii” (Ziolecki, 1979; Ziolecki and Wojciechowicz, 1980) zi.o.lec¢ki.i. N.L. gen. masc. n. zioleckii of Ziolecki in honor of Alexander Ziolecki, Polish Academy of Sciences, in recognition of his contribution to the microbiology of rumen treponemes. Cells are 0.5 mm in diameter by 5–11.5 mm in length. The number of periplasmic flagella per cell could not be estimated from electron microscopic observations, but it appears there are least four per cell (Piknova et al., 2008). Utilizes fructan, inulin, sucrose, and various plant mono- and disaccharides as fermentable substrates. Produces formate, acetate, and ethanol as endproducts of glucose fermentation. The name Treponema zioleckii was recently formally proposed (Piknova et al., 2008). The 16S rRNA gene sequence of Treponema zioleckii was > 99% similarity to strain CA, which was previously isolated from bovine rumen contents (Paster and CanaleParola, 1982). Cells of strain CA are similar in dimensions to Treponema zioleckii, have 16–20 periplasmic flagella per cell, are pectinolytic, have amylolytic activity, can grow on arabinogalactan, and have a G+C content of 42 mol% (Paster and Canale-Parola, 1982). Source: sheep rumen contents. DNA G+C content (mol%): not known for type strain. Type strain: kT.

References Abramson, I.J. and R.M. Smibert. 1971. Inhibition of growth of treponemes by antimicrobial agents. Br. J. Vener. Dis. 47: 407–412. Aksenova, E.Y., V.A. Svetlichnyi and G.A. Zavazin. 1990. Spirochaeta thermophila sp. nov., a thermophilic marine spirochete isolated from a littoral hydrotherm of Shiashkotan Island. Microbiology (En. transl. from Mikrobiologiya) 59: 735–741. Aksenova, H.Y., F.A. Rainey, P.H. Janssen, G.A. Zavarzin and H.W. Morgan. 1992. Spirochaeta thermophila sp. nov., an obligately anaerobic, polysaccharolytic, extremely thermophilic bacterium. Int. J. Syst. Bacteriol. 42: 175–177. Allan, B., E.P. Greenberg and A. Kropinski. 1986. DNA-dependent RNA-polymerase from Spirochaeta aurantia. FEMS Microbiol. Lett. 35: 205–210. Anda, P., W. Sanchez-Yebra, M. del Mar Vitutia, E. Perez Pastrana, I. Rodriguez, N.S. Miller, P.B. Backenson and J.L. Benach. 1996. A new Borrelia species isolated from patients with relapsing fever in Spain. Lancet 348: 162–165. Anderson, J.F. 1989. Epizootiology of Borrelia in Ixodes tick vectors and reservoir hosts. Rev. Infect. Dis. 11 (Suppl 6): S1451–1459. Antal, G.M., S.A. Lukehart and A.Z. Meheus. 2002. The endemic treponematoses. Microbes Infect. 4: 83–94. Baker-Zander, S.A. and S.A. Lukehart. 1983. Molecular basis of immunological cross-reactivity between Treponema pallidum and Treponema pertenue. Infect. Immun. 42: 634–638. Baker-Zander, S.A. and S.A. Lukehart. 1984. Antigenic cross-reactivity between Treponema pallidum and other pathogenic members of the family Spirochaetaceae. Infect. Immun. 46: 116–121.

523

Sequence accession no. (16S rRNA gene): DQ065758; strain CA: M59294.

Diversity of not-yet-cultivated species As with species of Spirochaeta, species of Treponema are broadly diverse. In addition to known species of Treponema, novel phylotypes, i.e., species that have not yet been cultivated in  vitro, have been identified by analysis of 16S rRNA genes of DNA isolated from host-associated sources, such as the human oral cavity (Choi et al., 1994; Dewhirst et al., 2000; Paster et al., 2001), termite hindguts (Lilburn et al., 1999) and the bovine rumen (Tajima et al., 1999). Representatives of treponemal phylotypes from each of these and other environments are included in Figure 95. In one study, Paster et  al. (2001) identified 49 not-yet-cultivated species of Treponema in human subgingival plaque (see expanded phylogenetic trees of phylotypes in Dewhirst et al., 2000, and Paster et al., 2001). The termite hindgut has an especially diverse array of treponemal phylotypes, with hundreds of potentially new species of Treponema identified (Berlanga et al., 2007; Lilburn et al., 1999; Ohkuma et al., 1999). The diversity of treponemes in termite hindguts is likely even more extensive considering that there are over 2,000 species of termites. Bovine and ovine digital dermatitis cases have also yielded several treponemal phylotypes (Demirkan et  al., 2006; Walker et  al., 1995). It is likely that other environments such as the intestinal tracts of most mammals, birds, and other insects will also contain many additional Treponema phylotypes. Figure 95 illustrates the phylogenetic diversity of representatives of treponemal species and not-yet-cultivable phylotypes from human, animal, insect, and waste water environments. Interestingly, several phylogenetic clusters based on host or environment are apparent, namely mammalian, termite, and waste water. Baranton, G., D. Postic, I. Saint Girons, P. Boerlin, J.C. Piffaretti, M. Assous and P.A. Grimont. 1992. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int. J. Syst. Bacteriol. 42: 378–383. Barbieri, J.T. and C.D. Cox. 1979. Pyruvate oxidation by Treponema pallidum. Infect. Immun. 25: 157–163. Barbieri, J.T. and C.D. Cox. 1981. Influence of oxygen on respiration and glucose catabolism by Treponema pallidum. Infect. Immun. 31: 992–997. Barbour, A.G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57: 521–525. Barbour, A.G. and S.F. Hayes. 1986. Biology of Borrelia species. Microbiol. Rev. 50: 381–400. Barbour, A.G., G.O. Maupin, G.J. Teltow, C.J. Carter and J. Piesman. 1996. Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: possible agent of a Lyme disease-like illness. J. Infect. Dis. 173: 403–409. Barbour, A.G. 2005. Relapsing fever. In Tick-borne Diseases of Humans (edited by Goodman, Dennis and Sonenshine). ASM Press, Washington, D.C., pp. 268–291. Bates, L.B. and J.H. St John. 1922. Suggestion of Spirochaeta neotropicalis as name for spirochaete of relapsing fever found in Panama. J. Am. Med. Assoc. 79: 575–576. Berg, H.C. 1976. How spirochetes may swim. J. Theor. Biol. 56: 269–273. Bergey, D.H., F.C. Harrison, R.S. Breed, B.W. Hammer and F.M. Huntoon. 1925. Bergey’s Manual of Determinative Bacteriology, 2nd edn. Williams & Wilkins, Baltimore.

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Family II. Brachyspiraceae Bruce J. Paster Bra.chy.spi.ra.ce¢ae. N.L. fem. n. Brachyspira type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Brachyspiraceae the Brachyspira family. The family Brachyspiraceae was circumscribed for this volume on the basis of phylogenetic analysis of 16S rRNA gene sequences. The family contains only one genus, Brachyspira. Description is the same as for the genus, Brachyspira.

Type genus: Brachyspira aalborgi Hovind-Hougen, BirchAndersen, Henrik-Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1983, 896VP (Effective publication: Hovind-Hougen, Birch-Andersen, Henrik-Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1982, 1135.).

Genus I. Brachyspira Hovind-Hougen, Birch-Andersen, Henrik-Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1983, 896VP (Effective publication: Hovind-Hougen, Birch-Andersen, Henrik-Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1982, 1135.) Thaddeus B. Stanton Bra.chy.spi¢ra. Gr. adj. brachys short; L. fem. n. spira a coil, spiral; N.L. fem. n. Brachyspira a short spiral, describing a bacterium that resembles a short spiral. Brachyspira spirochetes are helical shaped bacteria with ­regular coiling patterns. Cells measure 2–11 mm by 0.2–0.4 mm. Unicel­ lular, but dividing pairs and occasional chains of three or more cells can be observed in growing cultures. Under unfavorable growth conditions, spherical or round bodies are formed. Gram-stain negative. Obligately anaerobic, aerotolerant. Cell ends may be blunt or pointed. Cells have a typical spirochete cell ultrastructure, consisting of an outer sheath, helical protoplasmic cylinder, and internal flagella in the space between the protoplasmic cylinder and outer sheath. Brachyspire cells have 8–30 flagella per cell depending on the species (flagellar number usually correlates with cell size and species of smaller cells have fewer flagella). Flagella attach subterminally in equal numbers at each cell end, wrap around the protoplasmic cylinder, and their free ends overlap in the middle of the cells. Flexing and creeping motility at 22°C; translational movement in liquids at 37–42°C. Cultured anaerobically on commercially available media (trypticase soy or brain heart infusion broths) containing a carbohydrate growth substrate

and supplemented with defibrinated blood or animal (calf) serum. Grows at 36–42°C, optimally at 37–39°C. Population doubling times on glucose in broth cultures are 1–5 h (not reported for Brachyspira aalborgi). Chemoorganotrophic, using various carbohydrates for growth. Possess NADH ­oxidase for reducing molecular oxygen. Consume oxygen ­during growth in culture broth beneath a 1% oxygen atmosphere. Acetate, butyrate, H2, and CO2 are major endproducts of glucose metabolism. Higher amounts of H2 than CO2 are produced. Weakly hemolytic except for Brachyspira hyodysenteriae which exhibits b-hemolysis (strongly hemolytic). Associated with animal and human hosts. Some species are pathogenic. The genus Brachyspira is distinguished from other spirochete genera based on 16S rRNA gene sequences. Brachyspira species share high 16S rRNA sequence similarity with each other. Species can be differentiated by DNA–DNA relative reassociation and MLEE (multilocus enzyme electrophoresis) analyses. Similar to other spirochete genera, Brachyspira is insensitive to the antibiotic rifampin.

532

Family II. Brachyspiraceae

DNA G+C content (mol%): 24.5–27.1 (Tm). Type species: Brachyspira aalborgi Hovind-Hougen, BirchAndersen, Henrik-Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1983, 896VP (Effective publication: Hovind-Hougen, Birch-Andersen, Henrik-Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1982, 1135VP.).

Further descriptive information Cell morphology and ultrastructure.  By phase-contrast microscopy, Brachyspira species appear helical shaped with regular coils (Figure 96). They have a typical spirochete ultrastructure with protoplasmic cylinder, enclosed flagella winding around the protoplasmic cylinder, and outer sheath (membrane) (Sellwood and Bland, 1997). The species can be divided into two groups based on cell size and flagellar numbers (Table 130). Brachyspira species with larger cell size, such as Brachyspira ­hyodysenteriae, Brachyspira innocens, Brachyspira intermedia, Brachyspira murdochii, and Brachyspira alvinipulli, have 20–30 ­flagella per cell. Brachyspira pilosicoli and Brachyspira aalborgi cells are shorter in length and have 8–12 flagella per cell. As with other spirochetes, the flagella attach in roughly equal numbers at each end of the cell (Figure 97). The end shapes of cells of different species vary (Table 130). The pointed cell ends of Brachyspira pilosicoli and Brachyspira aalborgi may serve in the attachment of these spirochetes to

intestinal cells (Sellwood and Bland, 1997). Outer-membrane proteins of pathogenic Brachyspira species are frequently the targets of efforts to develop vaccines against intestinal spirochete diseases (Cullen et al., 2003; Cullen et al., 2004; La et al., 2004; McCaman et al., 2003; Trott et al., 2001, 2004). Nutrition and growth conditions.  Various anaerobic, nutritionally complex broth media have been described for Brachyspira species (Stanton, 1997). Incubation temperatures are 38–40°C. Shaking or stirring of broth cultures is important for optimum growth. BHIS broth and Kunkle’s broth are commonly used for routine growth of Brachyspira hyodysenteriae (Kunkle et al., 1986; Stanton and Lebo, 1988). Both media support high growth yields (1–4 × 109 cells/ml, direct microscope counts). In BHIS broth which contains glucose as a growth substrate, the population doubling times for most Brachyspira species are 1–5 h (Table 130). HS broth (Heart infusion broth supplemented with serum) requires added carbohydrates to support optimum growth yields of Brachyspira species. Consequently, HS broth is useful for identifying metabolic end products (Table 130) and growth substrates (Table 131). NT broth, a serum-free medium with low protein content, supports Brachyspira hyodysenteriae growth (Humphrey et al., 1997). Brachyspira species are generally aerotolerant and able to grow in sealed culture broth tubes beneath atmospheres containing 1–5% O2. They will not initiate growth, however, in oxidized media, i.e., in media containing resazurin indicator dye that has become colored by exposure to air. A too low redox potential appears to be a controlling factor for Brachyspira hyodysenteriae growth (Stanton, 1997). This species can be difficult to culture in stringently prepared anaerobic broth media unless a small amount of oxygen (1%, v/v) is introduced into the culture atmosphere (Stanton and Lebo, 1988). Brachyspira hyodysenteriae cells can be cultured in a fermenter (12 l) beneath an air atmosphere (Stanton and Jensen, 1993). Fermentation and growth substrates.  Fermentation substrates have been identified by measuring acid production (medium pH decreases) in cultures to which the substrates have been added (Jones et al., 1986; Kinyon and Harris, 1979; Ochiai et  al., 1998; Tompkins et  al., 1986). Alternatively, ­carbon/ energy sources of the spirochetes have been directly identified by measuring population density increases in HS broth, a culture medium that is growth limiting unless substrates are added (Table 131). The latter method is more sensitive for detecting substrates that support low growth yields. In HS broth, Brachyspira hyodysenteriae, Brachyspira innocens, Brachyspira intermedia, Brachyspira murdochii, Brachyspira pilosicoli, and Brachyspira alvinipulli cells use various monosaccharides, disaccharides, the trisaccharide trehalose, and amino sugars for growth (Table 131). The ability to ferment d-ribose distinguishes Brachyspira pilosicoli strains from other Brachyspira species (Fossi and Skrzypczak, 2006). Brachyspira strains do not use polysaccharides such as cellulose, hog gastric mucin, pectin, or glycogen. Brachyspira hyodysenteriae and Brachyspira innocens contain an inducible sucrase activity (Jensen and Stanton, 1994).

Figure 96.  (a) Brachyspira alvinipulli C1T cells. (b) Brachyspira hyo-

dysenteriae B78 cells. Phase-contrast photomicrographs of wet-mount preparations. Marker bars represent 10 mm. (Reproduced with permission from T.B. Stanton et al., 1998. Int. J. Syst. Bacteriol. 48: 669–676.) T

Cholesterol and phospholipids.  Brachyspira hyodysenteriae requires cholesterol and phospholipid for growth (Lemcke and Burrows, 1980; Stanton, 1987, 1997). Cholesterol is likely required for outer membrane biosynthesis (Lemcke and

Yes (chickens) Weak 3–5 h; 3–5 d 8–11 × 0.2–0.35 Blunt 22–30 24.6 16.8 0.8 19.4 22.8 2.2 No 95.7 98.4 − − + − +

Weak nr; 7–14 d 2–6 × 0.2 Tapered 8 27.1 nr nr nr nr nr No 100 96.5 nr − nr − −

Chickens

3. B. hyodysenteriae − +/− − − +

96.5 100

12.0 1.7 10.3 24.0 nd No

Strong 3–5 h; 3–4 d 7–12 × 0.3–0.4 Blunt 22–28 25.9

Yes (swine; rheas)

Swine, rheas, ducks

4. B. innocens − − − + +

96.5 99.5

18.0 1.8 13.1 22.0 0.9 No

Weak 3–5 h; 3–5 d 7–12 × 0.3–0.4 Blunt 20–26 26

No

Swine

5. B. intermedia − +/− − − +

96.0 99.1

14 10.3 7.2 10.7 1.4 No

Weak 2–4 h; 3–5 d 8–10 × 0.35–0.45 Blunt 24–28 25

Yes (chickens)

Swine, chickens

6. B. murdochii − − − − +

95.9 98.5

25.4 5.2 14.8 33 1 No

Weak 2–4 h; 3–5 d 5–8 × 0.35–0.4 Blunt 22–26 27

No

Swine, rats

+ +/− +/− +/− −

96.4 98.5

23.3 1.5 11.3 22.4 2.4 Yes

Weak 1–2 h; 3–4 d 5–7 × 0.23–0.3 Pointed 8–12 24.6

Swine, birds, dogs chickens, humans, nonhuman primates Yes (swine)

7. B. pilosicoli

Values represent population doubling time in hours for broth cultures and days for colony development on agar-containing media at 37–39°C.

Product yields of type strains after growth in HS broth containing glucose and beneath 1% O2 atmosphere. All strains consumed O2.

As reported by Fossi and Skrzypczak (2006).

h

g

Determined by using GenBank 16S rRNA gene sequences with accession numbers Z22781, U14930, U23033, U14920, U14927, U22838, and U23030. All sequences are for type strains except Brachyspira murdochii (strain 155-20).

f

A unique signature sequence of nucleotides was identified within the 16S rDNA of Brachyspira pilosicoli and has been used to design specific PCR tests for this species (Fellstrom et al., 1997; Park et al., 1995).

e

d

c

Pure cultures of either the type strain or another strain have been shown to cause disease when inoculated into normal, healthy, natural host animals. Brachyspira intermedia infections of chickens are mild to moderate “economic loss” diseases associated with “wet litter” and “unthriftiness”, in contrast to the severe pathology of swine dysentery.

b

Only one strain of Brachyspira aalborgii (513AT) and Brachyspira alvinipulli (strain C1T) have been extensively studied. Data from Harris et al. (1972a, b), Kinyon and Harris (1979), Hovind-Hougen et al. (1982), Stanton and Lebo (1988), Stanton et al. (1991, 1996, 1997, 1998,), Fellstrom et al. (1995, 1997, 1999), Park et al. (1995), Trott et al. (1996a, b, 1997a–c), McLaren et al. (1997), Sellwood and Bland (1997), De Smet et al. (1998), Duhamel et al. (1998a, b), Kraaz et al. (2000), Fossi et al. (2004), and Jansson et al. (2004).

a

2. B. alvinipulli

No

Humans

Symbols: +, positive; −, negative; nr, not reported; nd, not detected.

Demonstrated pathogenicity (animal)c Hemolysis type Growth rated Cell size (mm) Cell end shape Flagella per cell DNA G+C content (mol%) Metabolic endproducts (mmol/ml): e Acetate Butyrate CO2 H2 Ethanol 16S rRNA signaturef 16S rRNA sequence similarity:g B. aalborgi 513AT B. hyodysenteriae B78T Biochemical reaction profile: d-Ribose fermentationh Indole production Hippurate hydrolysis a-Galactosidase activity b-Glucosidase activity

Host

Characteristic

1. B. aalborgi

Table 130.  Brachyspira species characteristics a,b

Genus I. Brachyspira 533

534

Family II. Brachyspiraceae

Compound

1. B. aalborgi

2. B. alvinipulli

3. B. hyodysenteriae

4. B. innocens

5. B. intermedia

6. B. murdochii

7. B. pilosicoli

Table 131.  Brachyspira growth/fermentation substratesa,b

d-Glucose d-Fructose Sucrose d-Trehalose d-Galactose d-Mannose N-Acetyl-d-glucosamine d-Glucosamine d-Maltose d-Cellobiose l-Fucose d-Ribosec d-Xylose Lactose Pyruvate

+ + nr + + + nr nr + nr nr nr nr + nr

+ + − − − + + + + nr − − − − −

+ + + + + + + + + − − − − + +

+ + + + + + + + + + + − − d +

+ + + + + − + − + − + − − − +

+ + + + − + + − + + + − − + +

+ d + + d d + + d + + + d nr +

a Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive); −, 0–15% positive; nr, not reported.

Fermentation substrates for Brachyspira aalborgi were determined by phenol red measurements of acid production (Ochiai et al., 1997). Growth substrates for the other six Brachyspira species were determined by monitoring culture growth in HS broth to which the various substrates were added. Different substrates support different growth yields of cells. Data are for Brachyspira aalborgi NCTC 11492 (Ochiai et al., 1997), Brachyspira hyodysenteriae strains B78T, B204, and B169 (Stanton and Lebo, 1988), Brachyspira innocens B256T (Trott et al., 1996a), Brachyspira pilosicoli strains P43/6/78T, 3295, 1648, Hrm7, Kar, WesB (Trott et al., 1996a, b), Brachyspira intermedia PWS/AT (Stanton et al., 1997), Brachyspira murdochii 56-150T (Stanton et al., 1997), and Brachyspira alvinipulli C1T (Stanton et al., 1998). In the original studies, additional compounds were tested and were not substrates for any species. b

Figure 97.  Electron micrograph of one end of Brachyspira alvinipulli

C1T cell negatively stained with 2% phosphotungstic acid (pH 7.0). Disrupted outer sheath enables insertion sites of 15 periplasmic flagella to be seen (white arrowheads). Marker bar = 0.25 mm. (Reproduced with permission from T.B. Stanton et  al., 1998. Int. J. Syst. Bacteriol. 48: 669–676.)

­ urrows, 1980; Plaza et  al., 1997; Stanton, 1987; Stanton and B Cornell, 1987). The cellular fatty acids of Brachyspira hyodysenteriae are distinct from those of Borrelia and Leptospira species (Livesley et  al., 1993). The cellular phospholipids and glycolipids of Brachyspira hyodysenteriae and Brachyspira innocens contain acyl and alkenyl side chains and the spirochetes apparently have some capacity for fatty acid and lipid biosynthesis (Matthews and Kinyon, 1984; Matthews et al., 1980a, b). Glucose and pyruvate metabolism.  Brachyspira hyodysenteriae uses the Embden–Meyerhof–Parnas (EMP) pathway for converting glucose to pyruvate. Pyruvate is catabolized by a clostridialtype clastic reaction to acetyl-CoA, H2, and CO2. Acetyl-CoA is converted to either acetate or butyrate via a branched fermentation pathway. The ATP-yielding mechanisms are substratelevel phosphorylation reactions mediated by phosphoglycerate kinase and pyruvate kinase in the EMP pathway and by acetate kinase in the conversion of acetyl phosphate to acetate ­(Stanton, 1989, 1997). In Brachyspira hyodysenteriae cells, NADH-H+ produced ­during glycolysis can be recycled or oxidized to NAD+ by three pathways: (a) 3-hydroxybutyryl-CoA dehydrogenase and butyrylCoA dehydrogenase (butyrate pathway); (b) NADH-ferredoxin ­oxidoreductase plus hydrogenase; and (c) NADH oxidase (Stanton, 1997). The Brachyspira hyodysenteriae NADH oxidase is a water-forming, FAD-linked enzyme (Stanton and Jensen, 1993) and the nox gene has been cloned (GenBank no. U19610) (Stanton and Sellwood, 1999). Brachyspira hyodysenteriae noxdefective mutant strains are sensitive to oxygen and are avirulent (Stanton et al., 1999).

In a study of swine Brachyspira isolates, d-ribose fermentation was found be a unique trait of Brachyspira pilosicoli strains (Fossi and Skrzypczak, 2006).

c

NADH oxidase is universally present in Brachyspira species (Atyeo et al., 1999; Stanton et al., 1995). Brachyspira species produce higher amounts of H2 than CO2, indicative of the NADHferredoxin oxidoreductase reaction. Ethanol is produced in cultures of Brachyspira pilosicoli, Brachyspira alvinipulli, Brachyspira murdochii, Brachyspira intermedia, and Brachyspira innocens (Stanton, 1989; Stanton et  al., 1997, 1998; Stanton and Lebo, 1988; Trott et al., 1996b, c). The major endproducts of glucose metabolism by growing cells of various Brachyspira species are the same as those of Brachyspira hyodysenteriae (Table 130). For this reason, the species are likely to have similar catabolic routes. Iron metabolism.  Several observations suggest that specific iron uptake mechanisms are present and are important for brachyspire growth in animal hosts. Brachyspira hyodysenteriae cells grow in broth containing an iron chelator, 2,2¢-dipyridyl, and increase the expression of three unidentified high molecular mass proteins, >200, 134, and 109 kDa (Li et  al., 1995). A Brachyspira hyodysenteriae genome locus, designated bit (“Brachyspira iron transport”), encodes six proteins that are likely to form an iron ATP-binding transport system (Dugourd et al., 1999). Genome properties.  The Brachyspira hyodysenteriae B78T ­chromosome is circular and 3.2 Mbp (megabase pairs) in size

Genus I. Brachyspira

(Zuerner and Stanton, 1994). Brachyspira pilosicoli P43/6/78T has a circular chromosome approximately 2.4 Mb (Zuerner et al., 2004). The Brachyspira hyodysenteriae and Brachyspira pilosicoli genomes are currently being sequenced under proprietary circumstances by private corporations. The genomes of the spirochetes Brachyspira hyodysenteriae, Leptospira interrogans, Borrelia spp., Treponema pallidum and Treponema denticola differ from one another in terms of chromosomal conformation (linear and circular), chromosomal numbers (two for Leptospira interrogans), size (0.95–4.9 Mb), and number and arrangement of rRNA genes (Zuerner, 1997). Plasmids.  Uncharacterized plasmids and extrachromosomal DNAs have been reported for various Brachyspira species (Cattani et al., 1998; Combs et al., 1989; Turner and Sellwood, 1997). Some of the extrachromosomal DNAs (Combs et  al., 1989; Turner and Sellwood, 1997) are likely to be DNA from VSH-1-like bacteriophage particles spontaneously produced in brachyspire cultures. Plasmid DNA was not detected for Brachyspira hyodysenteriae B78T (Zuerner and Stanton, 1994). Prophage-like gene transfer agent (VSH-1).  When treated with mitomycin C, both Brachyspira hyodysenteriae and Brachyspira innocens cells lyse and release bacteriophage-like particles (Humphrey et  al., 1995). One of these prophages was purified from Brachyspira hyodysenteriae strain B204 cultures and was named VSH-1 (Humphrey et al., 1997). VSH-1 virions package random, 7.5 kb linear fragments of bacterial DNA. The purified virions are noninfectious, that is, they do not lyse bacteria or form plaques, and behave like generalized transducing phages (Humphrey et  al., 1997; Stanton et  al., 2001). The VSH-1 genome (GenBank no. AY971355) encodes head, tail, and lytic proteins (Matson et al., 2005) and is 16.5 kb (substantially larger than the virion packaged DNA). Based on MLEE analysis of Brachyspira hyodysenteriae strains, Trott et al. (1997c) concluded that substantial genetic recombination, likely mediated by VSH-1 gene transfer, has shaped the overall population structure of this species. VSH-1 is the first natural gene transfer mechanism to be discovered for a spirochete. Bacteriophages have also been detected by electron microscopy in both mitomycin C-treated and untreated cultures of weakly hemolytic human intestinal spirochetes (Calderaro et al., 1998a, b). These bacteriophages resemble VSH-1 in size and morphology. Genetic techniques – mutagenesis.  Brachyspira hyodysenteriae strains can be mutated by allelic exchange at specific loci (Li et  al., 2000; Rosey et  al., 1995, 1996; Stanton et  al., 1999; ter Huurne et al., 1992). By this method, Brachyspira hyodysenteriae genes cloned in Escherichia coli are inactivated in vitro by inserting an antimicrobial (kanamycin, chloramphenicol) resistance marker into the gene. The constructs are then introduced as plasmids into Brachyspira hyodysenteriae cells by electroporation. The allelic exchange technique yields mutant strains that are isogenic, except for single genetic loci, to their progenitor strains. Such strains are invaluable in determining bacterial virulence traits. UV mutagenesis has been used to generate coumermycin A1-resistant Brachyspira hyodysenteriae strains with mutations in gyrB (DNA gyrase) genes (Stanton et al., 2001). Coumermycin resistance is an antibiotic selection marker for genetic manipulations of Brachyspira. Another useful selection marker is tylosin resistance. Some Brachyspira hyodysenteriae strains are naturally

535

resistant to tylosin, owing to a nucleotide base change in the 23S rRNA gene (Karlsson et al., 1999). Serotype analysis.  Brachyspira hyodysenteriae strains have been subdivided into serotypes based on the immunological reactivities of lipo-oligosaccharides (LOS) in hot water-­phenol extracts of whole cells (Baum and Joens, 1979; Li et al., 1991). A more elaborate system of immunological identification assigns Brachyspira hyodysenteriae strains to serogroups and subdivides the groups into serovars (Hampson et al., 1989, 1997; Lau and Hampson, 1992). This latter system has not been widely adopted because serogroups are not entirely consistent with genetic groupings based on MLEE analysis (Lee et  al., 1993; Trott et al., 1997a). Enzyme-linked immunosorbent assay (ELISA) methods based on antibodies to LOS have been used to detect animal herd exposure to Brachyspira hyodysenteriae strains (Joens et al., 1982; Wright et al., 1989). Antibiotic sensitivity.  Brachyspira hyodysenteriae and other Brachyspira species are naturally insensitive to the antibiotics rifampin, spectinomycin, polymyxin B, and colistin at concentrations which limit growth of other intestinal bacteria (Buller and Hampson, 1994; Messier et  al., 1990; Taylor and Trott, 1997; Trott et al., 1996c). Consequently, individual antibiotics or combinations of these antibiotics are useful for selectively culturing Brachyspira hyodysenteriae and other species (Jenkinson and Wingar, 1981; Kunkle and Kinyon, 1988). There is evidence that Brachyspira hyodysenteriae and Brachyspira pilosicoli strains are becoming resistant to antimicrobials used to treat swine dysentery and porcine intestinal spirochetosis. These antimicrobials belong to the macrolide (i.e., tylosin, erythromycin), lincosamide (i.e., lincomycin, clindamycin), and pleuromutilin (i.e., tiamulin) classes of antibiotics (Duhamel et  al., 1998a; Karlsson et  al., 2003; Lobova et  al., 2004; Pringle et  al., 2006). Simultaneous resistance to both macrolide and lincosamide antimicrobials results from single nucleotide base changes in the 23S rRNA sequences of these spirochetes (Karlsson et al., 1999, 2004). Tiamulin resistance in a clinical isolate of Brachyspira hyodysenteriae has been associated with an amino acid change in ribosomal protein L3 (Pringle et al., 2004). Brachyspira diseases and virulence-associated traits.  Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira intermedia, and Brachyspira alvinipulli cause intestinal disease when inoculated into their healthy host animals (Table 130). Most disease-related research has focused on Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. Several publications have described and reviewed brachyspire diseases from the viewpoints of host manifestations, clinical detection methods, therapies, and experimental models (Barrett, 1997; Galvin et al., 1997; Hampson et al., 1997; Hampson and Trott, 1999; Harris et al., 1999; Swayne, 2003; Swayne and McLaren, 1997; Taylor and Trott, 1997). Swine dysentery (Brachyspira hyodysenteriae).  Swine dys­ entery (bloody scours or black scours) is a severe intestinal ­disease that affects piglets, primarily in the postweaning stage of growth (8–14 weeks after birth). The disease has been reported worldwide in every major pig producing country. A typical sign of the disease is profuse bleeding into the large bowel lumen through lesions induced by Brachyspira hyodysenteriae cells. Afflicted animals pass loose stools containing blood and mucus and spirochetes readily seen by microscopy. These are presumptive signs of the disease. Culturing and identifying Brachyspira

536

Family II. Brachyspiraceae

hyodysenteriae cells, along with histopathological observations, provide conclusive evidence of swine dysentery. Up to 90–100% of a herd can become infected, and without effective treatment, 20–30% of infected animals may die. Economic losses result from death, poor weight gain/feed efficiency, and medication expenses. Swine management strategies, including ­segregation by age and prophylactic administration of antibiotics (such as tylosin, lincomycin, and carbadox), are credited with reducing swine dysentery in the United States. Swine ­dysentery can be experimentally produced by feeding or intragastrically inoculating normal swine with Brachyspira hyodysenteriae cultures (Kennedy et al., 1988; Kinyon et al., 1977). However, the type strain B78T is weakly virulent and not useful for experimental infections (Jensen and Stanton, 1993). ­Various approaches have been used to develop whole cell or cell subunit-based vaccines for swine dysentery (Lee et  al., 2000). A commercial vaccine for swine dysentery is based on pepsin-digested Brachyspira hyodysenteriae cells. The immunological properties of the vaccine are now being examined (Waters et al., 2000). Swine are the common, but not the exclusive, animal hosts for Brachyspira hyodysenteriae. Strains of the spirochete also have been isolated from juvenile rheas with a severe necrotizing typhlitis (Buckles et al., 1997; Jensen et al., 1996; Sagartz et al., 1992). For experimental infections, mice ( Joens and Glock, 1979; Nibbelink and Wannemuehler, 1992; Rosey et al., 1996; ter Huurne et al., 1992) and 1-day-old chicks (Sueyoshi et al., 1987) have been used. Nevertheless, nuances or inconsistencies are associated with the use of these surrogate animal models (Achacha et al., 1996; Jensen and Stanton, 1993). For this reason, conclusions regarding Brachyspira hyodysenteriae pathogenesis based on alternative animal models should be confirmed through the use of swine infections. Several Brachyspira hyodysenteriae properties are putative or demonstrated virulence-associated traits. They include lipo-­ oligosaccharide (Greer and Wannemuehler, 1989; Nibbelink and Wannemuehler, 1991; Nuessen et al., 1982, 1983), hemolysin/hemolytic activity (Hsu et  al., 2001; Hutto and Wannemuehler, 1999; Kent et  al., 1988; Lysons et  al., 1991; Saheb et al., 1980, 1981; ter Huurne et al., 1992, 1994), chemotaxis/motility (Glock et al., 1974; Kennedy et al., 1988; Kennedy and Yancey, 1996; Milner and Sellwood, 1994), oxygen metabolism/NADH oxidase (Stanton, 1997; Stanton and Jensen, 1993), and variable surface proteins (Gabe et  al., 1998). The swine host immune response also likely contributes to the pathology of swine dysentery (Hontecillas et al., 2005). The ability to create strains with specific gene mutations has enabled direct evidence of a link between virulence and NADH oxidase activity, motility/flagella, and hemolytic activity. Mutant strains with specific deletions of nox (Stanton et al., 1999), flaA or flaB (Kennedy et al., 1997), or tlyA (Hyatt et al., 1994; Joens, 1997; ter Huurne et al., 1992) are avirulent compared to their isogenic wild-type counterparts. Although tlyA, tlyB, and tlyC were originally identified as hemolysin genes (Muir et al., 1992; ter Huurne et al., 1992, 1994), a subsequent study raises questions about that identification (Hsu et  al., 2001). Spirochetal colitis and spirochetal diarrhea (Brachyspira pilosicoli).  Spirochetal colitis caused by Brachyspira pilosicoli is a mild to moderate diarrheal disease of swine, birds, and possibly humans (Hampson and Trott, 1999; Swayne and McLaren, 1997; Taylor and Trott, 1997). Spirochetal colitis of swine

resembles a mild case or early stage of swine dysentery. Watery, mucoid diarrhea and a reduction of growth rate of affected animals are common clinical signs. The disease has been experimentally produced by inoculating pure cultures of Brachyspira pilosicoli into healthy swine (Taylor and Alexander, 1971; Thomson et al., 1997; Trott et al., 1996a). Evidence that Brachyspira pilosicoli is a pathogen of humans is circumstantial but multifarious. Brachyspira pilosicoli strains have been isolated from humans, including some (homosexual males and persons living in developing countries) with intestinal disorders and who are immunocompromised (Barrett, 1997; Trott et  al., 1997a, b). Human strains are virulent for healthy piglets (Trott et al., 1996a). A human volunteer became colonized after drinking cultures of Brachyspira pilosicoli strain Wes B (Oxberry et  al., 1998). Finally, Brachyspira pilosicoli has been isolated from the blood of human patients (Trott et al., 1997a). The significance and the capacity of Brachyspira pilosicoli cells to leave the intestinal tract and circulate throughout the host’s body have not been sufficiently investigated. Virulence factors of Brachyspira pilosicoli are unknown. By virtue of their location, outer-membrane proteins undoubtedly mediate interactions between spirochete cells and their environment and are logically involved in host colonization and virulence (Trott et  al., 2001, 2004). Cell motility, chemotaxis, and spirochete end-on attachment to host tissues are likely to be associated with colonization of the intestinal tract and therefore important for pathogenesis. In the absence of gross lesions, extensive colonization of intestinal tissues by Brachyspira pilosicoli cells with associated damage to microvilli could interfere with intestinal absorptive processes and lead to diarrhea (Gad et al., 1977; Taylor and Trott, 1997). Avian intestinal spirochetosis.  Many studies of avian intestinal infections associated with spirochetes were made before Brachyspira species had been characterized and taxonomically defined (Davelaar et al., 1986; Dwars et al., 1989; Griffiths et al., 1987). Avian diarrheal diseases can be caused by different Brachyspira species (Swayne, 2003; Swayne and McLaren, 1997). In addition to Brachyspira pilosicoli and Brachyspira hyodysenteriae, Brachyspira alvinipulli C1T, isolated from a diarrheic chicken, is a chicken enteropathogen. The C1T cells colonize the ceca of 1-day-old chicks and 14-month-old hens and produce mild typhlitis with discolored and watery cecal contents (Swayne et  al., 1995). Brachyspira alvinipulli has been isolated only from poultry and resembles an uncharacterized enteropathogenic spirochete (Davelaar et al., 1986). Brachyspira intermedia appears to be another common avian enteropathogen, inasmuch as spirochetes of that species have been isolated from birds with moderate intestinal colitis ­(Stephens and Hampson, 2001; Swayne, 2003; Swayne and McLaren, 1997). One-day-old chicks inoculated with pure cultures of an intestinal spirochete strain 1380, later identified by MLEE analysis as a strain of Brachyspira intermedia (Swayne and McLaren, 1997), shed watery feces containing spirochetes and had body weight reduction compared to control birds (Dwars et al., 1992). Avian Brachyspira intermedia strain HB60 isolated from a hen with ­diarrhea causes reduced egg production and watery feces when inoculated into healthy hens (Hampson and McLaren, 1999). A Brachyspira intermedia strain, freshly isolated from a swine herd with diarrhea, however, was not pathogenic for swine ( Jensen et al., 2000). Virulence traits of avian enteropathogenic Brachyspira species have yet to be determined.

Genus I. Brachyspira

Enrichment and isolation procedures.  Brachyspira hyodysenteriae and other Brachyspira species can be isolated by inoculating intestinal contents or tissues onto solid agar culture media containing antibiotics selective for the growth of those spirochetes (Calderaro et al., 2001; Hovind-Hougen et al., 1982; Jenkinson and Wingar, 1981; Kunkle and Kinyon, 1988). Achacha and Messier (1992) compared various selective media and found BJ medium containing spiramycin, rifampin, vancomycin, colistin, and spectinomycin provided the highest rate of isolation of Brachyspira hyodysenteriae from feces of experimentally infected swine. Physical methods (filtration, agar migration) can be used to isolate Brachyspira species from intestinal contents and tissues (Harris et al., 1972b; Olson, 1996; Taylor and Alexander, 1971). These methods take advantage of the small diameter of brachyspire cells and their active motility in agar-containing media.

Maintenance procedures Broth cultures of most Brachyspira species remain viable when stored at 5°C for 1–2 weeks and can be used as “working” stock cultures to inoculate fresh cultures for experiments. The refrigerated cultures should be in exponential growth phase (approx. 2 × 108 cells/ml, direct microscope counts). Oxygen exposure must be prevented for example by sealing stoppered culture tubes with plastic tape (3M Scotch #471) before storage. Longterm in vitro passage of strains (with possible loss of virulence) should be avoided by starting fresh working stock cultures from long-term stock cultures every several months. Agar plate cultures with colonies of Brachyspira hyodysenteriae can be stored at room temperature in an anaerobic atmosphere for at least a week. The plates should be sealed (Parafilm) to prevent desiccation. A slowly growing spirochete identified as Brachyspira aalborgi remained viable when kept at room temperature in an anaerobic jar for over 3 months (Kraaz et al., 2000). For long-term storage, brachyspire broth cultures in the exponential phase of growth are harvested by centrifugation (15 min, 5000 × g). The pelleted bacteria are resuspended at 50–100 times their original concentration in fresh sterile broth medium containing dimethylsulfoxide [DMSO; 10% (v/v), final concentration]. The cell suspension is dispensed into Nunc cryovials (0.5–1.0 ml/ vial). The sealed vials are placed upright in a beaker containing enough 95% ethanol to equal the fluid level of the suspensions (and well below the tops of the cryovials). The ethanol bath provides a more uniform rate of freezing of the cells and, with the DMSO as a cryoprotectant, prevents ice crystals from damaging the bacteria. The beaker is placed in an ultra-cold freezer (−75°C). After 24 h, the frozen stock cultures are transferred to storage boxes in the freezer. After 7–10 d, a cryovial should be examined to insure recovery of contaminant-free brachyspire cells. Brachyspire cells have remained viable in frozen stocks prepared in this way for over 20 years (T.B. Stanton, unpublished observations). Completely thawed stocks should not be refrozen. However, it is often possible to subculture without thawing the frozen stocks, by scraping the surface of a frozen cell suspension with a sterile inoculation loop and inoculating fresh media with the ice scrapings. Brachyspira hyodysenteriae cultures also have been preserved by lyophilization (Stanton and Lebo, 1988).

Taxonomic comments In the past 25 years, there have been several taxonomic changes for spirochetes now assigned to the genus Brachyspira. Initially, the designation Treponema hyodysenteriae was applied to both patho-

537

genic (strongly hemolytic) and nonpathogenic (weakly hemolytic) strains of intestinal spirochetes from swine. Miao and colleagues (1978) found that these pathogenic and nonpathogenic strains share only 28% DNA homology, and the nonpathogenic strains were reclassified as a new species, Treponema innocens (Kinyon and Harris, 1979). In 1991, Stanton et al. proposed a reclassification of Treponema hyodysenteriae and Treponema innocens to a new genus “Serpula” on the basis primarily of 16S rRNA sequence and DNA homology analyses. The genus name Serpula was subsequently changed to Serpulina after it was determined that Serpula had prior use as a name for a genus of fungi (Stanton, 1992). More recently, Ochiai et al. (1997) proposed the unification of the genera Serpulina and Brachyspira. The genus name Brachyspira was first given to a human intestinal isolate, Brachyspira aalborgi, in 1982 (Hovind-Hougen et al., 1982). Due to the use of the genus name Brachyspira prior to the genus designation Serpulina, this proposed change is consistent with International Taxonomic rules governing bacterial nomenclature. This action raised Brachyspira aalborgi, currently considered a commensal member of the human intestinal microbiota, to the status of type species of the genus Brachyspira. Brachyspira aalborgi has been characterized to a limited extent (Hovind-Hougen et al., 1982; Ochiai et al., 1997) and additional biochemical, physiological, and genetic investigations of this Brachyspira type species are needed. Ochiai et al. (1997) proposed the reassignment of Serpulina hyodysenteriae (the Serpulina type species), Serpulina innocens, and Serpulina pilosicoli to the genus Brachyspira. Unfortunately, prior to the publication of this proposal, the species Serpulina intermedia and Serpulina murdochii were described and validly named as two new species within the genus Serpulina based on their similarities to Serpulina hyodysenteriae (Stanton et al., 1997). For taxonomic and phylogenetic reasons, Serpulina intermedia and Serpulina murdochii should be reassigned to the genus Brachyspira and a proposal has been submitted for this reassignment (T.B. Stanton, unpublished). Thus, both species are included in this section describing the genus Brachyspira. 16S rRNA gene sequence comparisons indicate that members of the genus Brachyspira belong within the family Spirochetaceae and are clearly distinct from the other genera of spirochetes (Figure 98). Members of the genus Brachyspira cluster closely together, with several species at greater than 99% similarity. However, a phylogenetic tree with similar topology is also obtained for the phylogenetic analysis of the sequences of NADH (nox) genes (Mikosza et  al., 2004). Other methods described below, such as MLEE, have been used to better differentiate closely related members of the genus.

Differentiation of the species of the genus Brachyspira For taxonomic purposes, Brachyspira species can be differentiated by MLEE (multilocus enzyme electrophoresis) and DNA homology methods. In the clinical laboratory, the cultivation of strongly (b-) hemolytic spirochete colonies from the feces of dysenteric animals has been used for reliable identification of Brachyspira hyodysenteriae when this hemolytic type is linked to histopathology and animal disease signs. It is more difficult to differentiate “non-hyodysenteriae” Brachyspira species, all of which form weakly hemolytic (nonhemolytic) colonies for several ­reasons. Nonpathogenic and pathogenic species which are both nonhemolytic can be present in the same clinical samples. ­Diseases caused by the weakly hemolytic species are often ­economic diseases associated with poor animal performance

538

Family II. Brachyspiraceae 2%

Brachyspira aalborgi “Brachyspira ibaraki ”; AB079583 Brachyspira strain Trb03; AF228819 Brachyspira pilosicoli Brachyspira intermedia Brachyspira hyodysenteriae “Brachyspira suanatina ”; DQ473577 Brachyspira alvinipulli “Brachyspira pulli ”; EF164986 “Brachyspira canis ”; AY349936 Brachyspira innocens Brachyspira murdochii Figure 98.  Dendrogram illustrating the phylogenetic positions of

members of the genus Brachyspira. Species that have provisional names are indicated by quotation marks.

rather than diseases with high mortality and severe clinical signs. A further complicating factor for any clinical diagnostic test is that additional, uncharacterized Brachyspira species are likely to be in the intestinal tracts of humans and animals ­( Jansson et al., 2001; Pettersson et al., 2000). Species differentiation based on biochemical tests ­(hippurate hydrolysis, indole production, and a-galactosidase and b-­ glucosidase) (Table 130) and 16S rRNA based RFLP-PCR ­methods have been proposed for differentiating swine brachyspires (Fellstrom et al., 1995, 1997). There is growing ­evidence, however, that Brachyspira species from swine at ­different geographical locations

and from different animal ­species give inconsistent results in these biochemical tests ­(Fellstrom et al., 1999; Fossi et al., 2004; Stephens et  al., 2005). Both the 16S rRNA and 23S rRNA sequences of Brachyspira ­species are highly conserved, although a 16S rRNA signature region was identified for Brachyspira pilosicoli and has been used effectively in a PCR assay for that species (Park et al., 1995). Various PCR techniques based on the nox gene are currently being tested to differentiate Brachyspira species in poultry and swine (La et al., 2003; Rohde et al., 2002; Townsend et al., 2005).

Further reading Hampson, D.J. and T.B. Stanton. 1997. Intestinal Spirochetes in Domestic Animals and Humans. CAB International, Wallingford. Hampson, D.J. and J.R. Thomson. 2004. Brachyspira research special issue on colonic spirochetes of medical and veterinary significance. J. Med. Microbiol. 53: 263–265. Hampson, D.J. and D.J. Trott. 1999. Spirochetal diarrhea/porcine intestinal spirochetosis. In Diseases of Swine, 8th edn (edited by Straw, D’Allaire, Mengeling and Taylor). Iowa State University Press, Ames, pp. 553–562. Harris, D.L., D.J. Hampson and R.D. Glock. 1999. Swine Dysentery. In Diseases of Swine, 8th edn (edited by Straw, D’Allaire, ­Mengeling and Taylor). Iowa State University Press, Ames, IA, pp. 579–600. Stanton, T.B. 2006. Genus Brachyspira. In The Prokaryotes: A Handbook on the Biology of Bacteria, 3rd edn, vol. 7, Proteobacteria: Delta and Epsilon Subclasses. Deeply Rooting Bacteria (edited by ­Dworkin, Falkow, Rosenberg, Schleifer and Stackebrandt). Springer, New York, pp. 712–740. Swayne, D.E. 2003. Avian intestinal spirochetosis. In Diseases of ­Poultry, 11th edn (edited by Saif, Barnes, Fadly, Glisson, Mcdougald and Swayne). Iowa State University Press, Ames, IA, pp. 826–836.

List of species of the genus Brachyspira 1. Brachyspira aalborgi Hovind-Hougen, Birch-Andersen, Henrik-Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1983, 896VP (Effective publication: Hovind-Hougen, BirchAndersen, Henrik-Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1982, 1135VP.) a.al.bor¢gi. N.L. gen. n. aalborgi of Aalborg, named for the Danish town Aalborg in which the rectal biopsies containing the spirochete were taken from human diarrheic patients. Exhibit characteristics common to the genus Brachyspira. Additional characteristics of the species are given in Tables 130 and 131. Electron microphotographs of Brachyspira aalborgi cells have been published (Hovind-Hougen et  al., 1982). ­Differentiated from other Brachyspira species by DNA–DNA relative reassociation. Brachyspira aalborgi 513A exhibits 17–22% DNA–DNA relative reassociation with Brachyspira ­hyodysenteriae B78T, Brachyspira innocens B256T, and Brachyspira pilosicoli P43/6/78T (filter hybridization method) (Ochiai et al., 1997). Weakly hemolytic colonies on trypticase soy blood agar. Negative for indole production and positive for esculin hydrolysis. Ferments soluble carbohydrates (Table 131). Products of glucose metabolism have not been determined. Possess b-galactosidase and esterase, acid phosphatase, and phosphoamidase activities. Catalase-negative. Brachyspira aalborgi is not considered a pathogen.

Source: human intestinal tissues and contents. DNA G+C content (mol%): 27.1 (Tm) for strain 513A. Type strain: 513A, ATCC 43994, CIP 104603, NCTC 11492. Sequence accession no. (16S rRNA gene): Z22781 (NCTC 11492). 2. Brachyspira alvinipulli Stanton, Postic and Jensen 1998, 675VP al.vi.ni.pul¢li. L. adj. alvinus -a -um suffering from diarrhea; L. n. pullus a young fowl, a chicken; N.L. gen. n. alvinipulli of a diarrhaeic chicken, referring to the host animal from which spirochete was isolated. Exhibit characteristics common to the genus Brachyspira. A phase-contrast microphotograph and an electron micrograph of Brachyspira alvinipulli C1 cells have been published (Stanton et al., 1998). Additional characteristics of the species are given in Tables 130 and 131. Differentiated from other Brachyspira species by DNA–DNA relative reassociation and MLEE analysis. Exhibits 24–39% DNA–DNA relative reassociation with Brachyspira hyodysenteriae B78T, Brachyspira innocens B256T, Brachyspira pilosicoli P43/6/78T, Brachyspira intermedia PWS/AT, and Brachyspira murdochii 56-150T based on the S1 nuclease method (Stanton et al., 1998). Weakly hemolytic colonies on trypticase soy blood agar. Grows optimally (3 × 108 to 109 cells/ml, direct cell counts) at 37–39°C in BHIS broth or HS broth containing carbohydrates (Table 131). Products of glucose metabolism include

Genus I. Brachyspira

acetate, butyrate, CO2, H2, and ethanol. Positive in tests for hippurate hydrolysis, b-glucosidase, b-galactosidase, alkaline phosphatase, indoxyl acetate hydrolysis, arginine aminopeptidase, alanine aminopeptidase, and glycine aminopeptidase. Negative for indole production and catalase activity. Shares high 16S rRNA gene sequence similarity ­(96–99%) with other Brachyspira species. Intestinal pathogen of ­chickens. DNA G+C content (mol%): 24.6 (Tm) (strain C1). Type strain: C1 (91–1207/C1), ATCC 51933, CIP 105681. Sequence accession no. (16S rRNA gene): U23030 (strain C1). 3. Brachyspira hyodysenteriae (Harris, Glock, Christensen and Kinyon 1972a) emend. Ochiai, Adachi and Mori 1998, 327VP (Treponema hyodysenteriae Harris, Glock, Christensen and Kinyon 1972a, 64AL). Other synonyms: Serpula hyodysenteriae (Harris, Glock, Christensen and Kinyon 1972a) Stanton, Jensen, Casey, Tordoff, Dewhirst and Paster 1991, 56; ­Serpulina hyodysenteriae (Harris, Glock, Christensen and Kinyon 1972a) Stanton 1992, 189 hyo.dy.sen.te.ri¢ae. Gr. n. hyos hog, pig; L. n. dysenteria a flux, dysentery; N.L. gen. n. hyodysenteriae of hog dysentery. In ­recognition of the species as the etiologic agent of swine ­dysentery. Exhibits characteristic common to the genus Brachyspira. Phase-contrast photomicrographs and electron photomicrographs of cells have been published (Kinyon et al., 1977; Trott et al., 1996b). Additional characteristics of the species are given in Tables 130 and 131. Differentiated from other Brachyspira species by DNA–DNA relative reassociation, MLEE analysis. Strain B78T exhibits 22% DNA–DNA relative reassociation with Brachyspira aalborgi NCTC 11492T, based on the filter hybridization method (Ochiai et al., 1997). Strain B78T exhibits 27–57% DNA–DNA relative reassociation with Brachyspira innocens B256T, Brachyspira pilosicoli P43/6/78T, Brachyspira intermedia PWS/AT, Brachyspira murdochii 56-150T, and Brachyspira alvinipulli C1T, based on the S1 nuclease method (Stanton et  al., 1998; Trott et  al., 1996b). Unlike other Brachyspira species, Brachyspira hyodysenteriae cells form b-hemolytic (strongly hemolytic) colonies on trypticase soy blood agar and are positive for indole production. Strains have been differentiated according to serotypes, based on soluble antigens in the water phase of cells extracted with hot phenol-water. Grows at 37–39°C in a variety of anaerobically prepared culture broth media, attaining population densities ³109 cells/ml (direct cell counts), under optimum conditions. Cholesterol in nutrient amounts is required for growth, presumably for cell membrane biosynthesis. Uses carbohydrates as growth substrates (Table 131). Products of glucose metabolism include acetate, butyrate, CO2, and H2. Although considered an anaerobic species, growing cells under certain conditions require low (1%) oxygen tensions and consume substrate amounts of oxygen. Cells possess NADH oxidase, superoxide dismutase, NADH peroxidase, and inducible catalase activities. Hydrogen is produced via a clostridial-type phosphoroclastic mechanism and via NADH-ferredoxin oxidoreductase. Exhibits alkaline phosphatase, C4 esterase, C8 esterase, lipase, phosphatase acid, b-galactosidase, a-glucosidase, and b-glucosidase activities. Positive for esculin hydrolysis and most strains positive for indole production.

539

Isolated from intestinal contents and feces of swine, rheas, and other mammals (dogs) in contact with dysenteric feces. Brachyspira hyodysenteriae is an intestinal pathogen of swine and rheas. Brachyspira hyodysenteriae cells colonize within and between intestinal epithelial cells and the overlying intestinal mucus without cell attachment. The type strain B78 has become nonpathogenic for swine likely due to longterm passage in culture. Strain B204 (ATCC 31212) has commonly been used in the United States for experimental swine infections. DNA G+C content (mol%): 25.7–25.9% (Tm) for strains B204 and A1. Type strain: B78, ATCC 27164, CCUG 46668, NCTC 13041. Sequence accession no. (16S rRNA gene): M57743, U14930 (B78). 4. Brachyspira innocens (Kinyon and Harris 1979, 108) emend. Ochiai, Adachi and Mori 1998, 327VP (Treponema innocens Kinyon and Harris 1979, 108AL). Other synonyms: Serpula innocens (Kinyon and Harris 1979) Stanton, Jensen, Casey, Tordoff, Dewhirst and Paster 1991, 56; Serpulina innocens (Kinyon and Harris 1979) Stanton 1992, 189 in¢no.cens. L. fem. adj. innocens harmless, inoffensive (referring to non-pathogenic nature of the species for swine). Exhibit characteristics common to the genus Brachyspira. Additional characteristics of the species are given in Tables 130 and 131. Differentiated from other Brachyspira species by DNA–DNA relative reassociation and MLEE analysis. Strain B256 exhibits 19% DNA–DNA relative-reassociation with Brachyspira aalborgi NCTC 11492, based on the filter hybridization method (Ochiai et  al., 1997). Strain B256 exhibits 26–66% relative reassociation with Brachyspira hyodysenteriae B78T, Brachyspira pilosicoli P43/6/78T, Brachyspira intermedia PWS/AT, Brachyspira murdochii 56-150T, and Brachyspira alvinipulli C1T based on the S1 nuclease method (Stanton et al., 1998; Trott et al., 1996b). Weakly hemolytic colonies on trypticase soy blood agar. Uses carbohydrates as growth substrates (Table 131). Grows optimally (9 × 108 to 1.5 × 109 cells/ml, direct counts) at 37–39°C in BHIS broth or HS broth containing carbohydrates (Table 131). Products of glucose metabolism include acetate, butyrate, CO2, H2, and ethanol. Population doubling times are 3–5 h. Does not produce indole, hydrolyzes esculin. Brachyspira innocens is not considered a pathogen. Source: intestinal contents of healthy pigs, dogs, chickens. DNA G+C content (mol%): 25.6–25.8% (Tm) (strain B256). Type strain: B256, ATCC 29796, CCUG 17081. Sequence accession no. (16S rRNA gene): M57744 (strain B256). 5. Brachyspira intermedia (Stanton, Fournié-Amazouz, Postic, Trott, Grimont, Baranton, Hampson and Saint Girons 1997) emend. Hampson and La 2006, 1011VP (Serpulina intermedia Stanton, Fournié-Amazouz, Postic, Trott, Grimont, Baranton, Hampson and Saint Girons 1997, 1011) in.ter.me¢di.a. L. fem. adj. intermedia which is in the middle, referring to the fact that biochemical traits are intermediate between those possessed only by Brachyspira hyodysenteriae or by Brachyspira innocens. Exhibit characteristics common to the genus Brachyspira. Additional characteristics of the species are given in Tables

540

Family II. Brachyspiraceae

130 and 131. Differentiated from other Brachyspira species by DNA–DNA relative reassociation and MLEE analysis. Also called “Serpulina intermedius”. Some intestinal spirochetes referred to as “Treponema hyodysenteriae biotype 2” or “intermediate type” may be Brachyspira intermedia strains. Strain PWS/A exhibits 26–68% DNA–DNA relative reassociation with Brachyspira hyodysenteriae B78T, Brachyspira innocens B256T, Brachyspira pilosicoli P43/6/78T, Brachyspira murdochii 56-150T, and Brachyspira alvinipulli C1T, based on the S1 nuclease method (Stanton et al., 1997, 1998). Weakly to “intermediate” hemolytic colonies on trypticase soy blood agar. Uses carbohydrates as growth substrates (Table 131). Grows optimally (1.1 × 109 to 1.6 × 109 cells/ml, direct counts) at 37–39°C in BHIS broth or HS broth containing carbohydrates (Table 131). Products of glucose metabolism include acetate, butyrate, CO2, H2, and ethanol. Hydrolyzes esculin. Does not hydrolyze hippurate. Lacks a-galactosidase and possesses a-glucosidase and b-glucosidase activities. Brachyspira intermedia strains have been isolated from swine, including swine with diarrhea and from commercial poultry flocks exhibiting diarrhea. Avian intestinal infections by Brachyspira intermedia are aptly described as “economic disease” due to production losses in flocks. Birds colonized by Brachyspira intermedia typically do not die and may even appear healthy. There is some thought that disease severity is either strain related or influenced by the animal diet or other environmental factors. DNA G+C content (mol%): 25 (Tm) (strain PWS/AT). Type strain: PWS/AT, ATCC 51140, CIP 105833. Sequence accession no. (16S rRNA gene): U23033 (strain PWS/A). 6. Brachyspira murdochii (Stanton, Fournié-Amazouz, Postic, Trott, Grimont, Baranton, Hampson and Saint Girons 1997) emend. Hampson and La 2006, 1011VP (Serpulina intermedia Stanton, Fournié-Amazouz, Postic, Trott, Grimont, Baranton, Hampson and Saint Girons 1997, 1011) mur.do¢chi.i. N.L. masc. gen. n. murdochii of Murdoch, named in recognition of work conducted at the Murdoch University in Western Australia where the type strain was identified. Exhibit characteristics common to the genus Brachyspira. Additional characteristics of the species are given in Tables 130 and 131. Differentiated from other Brachyspira species by DNA–DNA relative reassociation and MLEE analysis. Strain 56-150T exhibits 27–66% DNA–DNA relative reassociation with Brachyspira hyodysenteriae B78T, Brachyspira innocens B256T, Brachyspira pilosicoli P43/6/78T, Brachyspira intermedia PWS/ AT, and Brachyspira alvinipulli C1T, based on the S1 nuclease method (Stanton et al., 1997, 1998). Weakly hemolytic colonies on trypticase soy blood agar. Grows optimally (9 × 108 to 1.5 × 109 cells/ml, direct counts) at 37–39°C in BHIS broth or HS broth containing carbohydrates (Table 131). Products of glucose metabolism include acetate, butyrate, CO2, H2, and ethanol. Does not produce indole. Does not hydrolyze hippurate. Cells lack a-galactosidase, a-glucosidase, and possess b-glucosidase activities.

Not considered a pathogen. Source: Intestinal contents of healthy swine and rats. DNA G+C content (mol%): 27 (Tm) (strain 56-150). Type strain: 56-150, ATCC 51254, CIP 105832, DSM 12563. Sequence accession no. (16S rRNA gene): AY312492 (strain 56-150). 7. Brachyspira pilosicoli (Trott, Stanton, Jensen, Duhamel, Johnson and Hampson 1996b) emend. Ochiai, Adachi and Mori 1998, 327VP (Serpulina pilosicoli Trott, Stanton, Jensen, Duhamel, Johnson and Hampson 1996b, 213) pi.lo.si¢co.li. L. adj. pilosus -a -um hairy, napped, shaggy; L. n. colon or colum the colon; N.L. gen. n. pilosicoli, of a hairy colon (referring to the fact that infection and attachment by this intestinal spirochete can result in the histological appearance of a hairy covering, false brush border, on the surface of the colon). Exhibit characteristics common to the genus Brachyspira. Additional characteristics of the species are given in Tables 130 and 131. Phase-contrast photomicrographs and electron microphotographs of Brachyspira pilosicoli cells have been published (Trott et  al., 1996b). Differentiated from other Brachyspira species by DNA–DNA relative reassociation, MLEE analysis, and by a signature nucleotide sequence (5¢-AGUUUUUUCGCUUCA-3¢) in the 16S rRNA. The 16S rRNA signature sequence has been useful in clinical identification of Brachyspira pilosicoli. Alternative designations are Anguillina coli and Serpulina pilosicoli. Strain P43/6/78 exhibits 17% DNA–DNA relative reassociation with Brachyspira ­aalborgi NCTC 11492 based on the filter hybridization method (Ochiai et al., 1997). Strains P43/6/78 and WES-B exhibit 21–28% DNA–DNA relative reassociation with Brachyspira hyodysenteriae B78T, Brachyspira innocens B256T, Brachyspira intermedia PWS/AT, Brachyspira murdochii 56-150T, and Brachyspira alvinipulli C1T, based on the S1 nuclease method (Stanton et al., 1997, 1998). Weakly hemolytic colonies on trypticase soy blood agar. Grows optimally (1.5 × 109 to >2.0 × 109 cells/ml, direct counts) at 37–39°C in BHIS broth or HS broth containing carbohydrates (Table 131). Products of glucose metabolism include acetate, butyrate, CO2, H2, and ethanol. Strains lack b-glucosidase activity. Brachyspira pilosicoli is considered an intestinal pathogen, the etiological agent of intestinal spirochetosis. Isolated from swine with watery, mucoid diarrhea. Swine intestinal spirochetosis clinically resembles early, mild stages of swine dysentery. Typical of the disease is the attachment of Brachyspira pilosicoli cells by one end to colonic epithelial cells. Brachyspira pilosicoli has been found in intestinal contents of birds, dogs, humans, and non-human primates. Brachyspira pilosicoli is being investigated as a possible enteropathogen of humans, dogs, and poultry. DNA G+C content (mol%): 25 ± 1 (Tm) (strain P43/6/78). Type strain: P43/6/78, ATCC 51139. Sequence accession no. (16S rRNA gene): U23032 (strain P43/6/78).

Other organisms There are reports of intestinal spirochetes that are likely to represent new Brachyspira species based on their MLEE profiles and comparative analyses of 16S rRNA and NADH oxi-

dase genes (Mikosza et al., 2004). The strains have only been partially characterized and some have been given provisional designations. For example, there are dog-associated spirochetes

Genus I. Brachyspira

“Brachyspira canis” (Duhamel et al., 1998b), chicken-associated spirochetes “Brachyspira pulli” also known as group “d” spirochetes (McLaren et al., 1997; Stephens and Hampson, 1999),

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Hampson, D.J., J.R. Mhoma, B. Combs and J.R. Buddle. 1989. Proposed revisions to the serological typing system for Treponema hyodysenteriae. Epidemiol. Infect. 102: 75–84. Hampson, D.J., R.F. Atyeo and B.G. Combs. 1997. Swine dysentery. In Intestinal Spirochaetes in Domestic Animals and Humans (edited by Hampson and Stanton). CAB International, Wallingford, pp. 175–209. Harris, D.L., R.D. Glock, C.R. Christensen and J.M. Kinyon. 1972a. Swine dysentery. I. Inoculation of pigs with the Treponema hyodysenteriae (new species) and reproduction of the disease. Vet. Med. 67: 61–64. Harris, D.L., J.M. Kinyon, M.T. Mullin and R.D. Glock. 1972b. Isolation and propagation of spirochetes from the colon of swine dysentery affected pigs. Can. J. Comp. Med. 36: 74–76. Harris, D.L., D.J. Hampson and R.D. Glock. 1999. Swine dysentery. In Diseases of Swine, 8th edn (edited by Straw, D’Allaire, Mengeling and Taylor). Iowa State University Press, Ames, IA, pp. 579–600. Hontecillas, R., J. Bassaganya-Riera, J. Wilson, D.L. Hutto and M.J. Wannemuehler. 2005. CD4+ T-cell responses and distribution at the colonic mucosa during Brachyspira hyodysenteriae-induced colitis in pigs. Immunology 115: 127–135. Hovind-Hougen, K., A. Birch-Andersen, R. Henrik-Nielsen, M. Orholm, J.O. Pedersen, P.S. Teglbjaerg and E.H. Thaysen. 1982. Intestinal spirochetosis: morphological characterization and cultivation of the spirochete Brachyspira aalborgi gen. nov., sp. nov. J. Clin. Microbiol. 16: 1127–1136. Hovind-Hougen, K., A. Birch-Andersen, R. Henrik-Nielsen, M. Orholm, J.O. Pedersen, P.S. Teglbjaerg and E.H. Thaysen. 1983. In Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 12. Int. J. Syst. Bacteriol. 33: 896–897. Hsu, T., D.L. Hutto, F.C. Minion, R.L. Zuerner and M.J. Wannemuehler. 2001. Cloning of a beta-hemolysin gene of Brachyspira (Serpulina) hyodysenteriae and its expression in Escherichia coli. Infect. Immun. 69: 706–711. Humphrey, S.B., T.B. Stanton and N.S. Jensen. 1995. Mitomycin C induction of bacteriophages from Serpulina hyodysenteriae and Serpulina innocens. FEMS Microbiol. Lett. 134: 97–101. Humphrey, S.B., T.B. Stanton, N.S. Jensen and R.L. Zuerner. 1997. Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae. J. Bacteriol. 179: 323–329. Hutto, D.L. and M.J. Wannemuehler. 1999. A comparison of the morphologic effects of Serpulina hyodysenteriae or its beta-hemolysin on the murine cecal mucosa. Vet. Pathol. 36: 412–422. Hyatt, D.R., A.A. ter Huurne, B.A. van der Zeijst and L.A. Joens. 1994. Reduced virulence of Serpulina hyodysenteriae hemolysin-negative mutants in pigs and their potential to protect pigs against challenge with a virulent strain. Infect. Immun. 62: 2244–2248. Jansson, D.S., C. Brojer, D. Gavier-Widen, A. Gunnarsson and C. Fellstrom. 2001. Brachyspira spp. (Serpulina spp.) in birds: a review and results from a study of Swedish game birds. Anim. Health Res. Rev. 2: 93–100. Jansson, D.S., K.E. Johansson, T. Olofsson, T. Rasback, I. Vagsholm, B. Pettersson, A. Gunnarsson and C. Fellstrom. 2004. Brachyspira hyodysenteriae and other strongly beta-haemolytic and indole-positive spirochaetes isolated from mallards (Anas platyrhynchos). J. Med. Microbiol. 53: 293–300. Jenkinson, S.R. and C.R. Wingar. 1981. Selective medium for the isolation of Treponema hyodysenteriae. Vet. Rec. 109: 384–385. Jensen, N.S. and T.B. Stanton. 1993. Comparison of Serpulina hyodysenteriae B78, the type strain of the species, with other S. hyodysenteriae strains using enteropathogenicity studies and restriction fragment length polymorphism analysis. Vet. Microbiol. 36: 221–231. Jensen, N.S. and T.B. Stanton. 1994. Production of an inducible sucrase Activity by Serpulina hyodysenteriae. Appl. Environ. Microbiol. 60: 3429–3432. Jensen, N.S., T.B. Stanton and D.E. Swayne. 1996. Identification of the swine pathogen Serpulina hyodysenteriae in rheas (Rhea americana). Vet. Microbiol. 52: 259–269. Jensen, T.K., K. Moller, M. Boye, T.D. Leser and S.E. Jorsal. 2000. Scanning electron microscopy and fluorescent in situ hybridization of

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Family II. Brachyspiraceae

Stanton, T.B. 1997. Physiology of ruminal and intestinal spirochaetes. In Intestinal Spirochaetes in Domestic Animals and Humans (edited by Hampson and Stanton). CAB International, Wallingford, pp. 7–45. Stanton, T.B. and C.P. Cornell. 1987. Erythrocytes as a source of essential lipids for Treponema hyodysenteriae. Infect. Immun. 55: 304–308. Stanton, T.B. and N.S. Jensen. 1993. Purification and characterization of NADH oxidase from Serpulina (Treponema) hyodysenteriae. J. Bacteriol. 175: 2980–2987. Stanton, T.B. and D.F. Lebo. 1988. Treponema hyodysenteriae growth under various culture conditions. Vet. Microbiol. 18: 177–190. Stanton, T.B. and R. Sellwood. 1999. Cloning and characteristics of a gene encoding NADH oxidase, a major mechanism for oxygen metabolism by the anaerobic spirochete, Brachyspira (Serpulina) ­hyodysenteriae. Anaerobe 5: 539–546. Stanton, T.B., N.S. Jensen, T.A. Casey, L.A. Tordoff, F.E. Dewhirst and B.J. Paster. 1991. Reclassification of Treponema hyodysenteriae and Treponema innocens in a new genus, Serpula gen. nov., as Serpula ­hyodysenteriae comb. nov. and Serpula innocens comb. nov. Int. J. Syst. Bacteriol. 41: 50–58. Stanton, T.B., B.L. Hanzelka and N.S. Jensen. 1995. Survey of intestinal spirochaetes for NADH oxidase by gene probe and by enzyme assay. Microb. Ecol. Health Dis. 8: 93–100. Stanton, T.B., D.J. Trott, J.I. Lee, A.J. McLaren, D.J. Hampson, B.J. Paster and N.S. Jensen. 1996. Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons. FEMS Microbiol. Lett. 136: 181–186. Stanton, T.B., E. Fournié-Amazouz, D. Postic, D.J. Trott, P.A. Grimont, G. Baranton, D.J. Hampson and I. Saint Girons. 1997. Recognition of two new species of intestinal spirochetes: Serpulina intermedia sp. nov. and Serpulina murdochii sp. nov.. Int. J. Syst. Bacteriol. 47: 1007–1012. Stanton, T.B., D. Postic and N.S. Jensen. 1998. Serpulina alvinipulli sp. nov., a new Serpulina species that is enteropathogenic for chickens. Int. J. Syst. Bacteriol. 48: 669–676. Stanton, T.B., E.L. Rosey, M.J. Kennedy, N.S. Jensen and B.T. Bosworth. 1999. Isolation, oxygen sensitivity, and virulence of NADH oxidase mutants of the anaerobic spirochete Brachyspira (Serpulina) hyodysenteriae, etiologic agent of swine dysentery. Appl. Environ. Microbiol. 65: 5028–5034. Stanton, T.B., E.G. Matson and S.B. Humphrey. 2001. Brachyspira ­(Serpulina) hyodysenteriae gyrB mutants and interstrain transfer of coumermycin A(1) resistance. Appl. Environ. Microbiol. 67: 2037–2043. Stephens, C.P. and D.J. Hampson. 1999. Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia. Avian Pathol. 28: 447–454. Stephens, C.P. and D.J. Hampson. 2001. Intestinal spirochete infections of chickens: a review of disease associations, epidemiology and control. Anim. Health Res. Rev. 2: 83–91. Stephens, C.P., S.L. Oxberry, N.D. Phillips, T. La and D.J. Hampson. 2005. The use of multilocus enzyme electrophoresis to characterise intestinal spirochaetes (Brachyspira spp.) colonising hens in commercial flocks. Vet. Microbiol. 107: 149–157. Sueyoshi, M., Y. Adachi and S. Shoya. 1987. Enteropathogenicity of Treponema hyodysenteriae in young chicks. Zentralbl. Bakteriol. Mikrobiol. Hyg. [A]. 266: 469–477. Swayne, D.E. 2003. Avian intestinal spirochetosis. In Diseases of ­Poultry, 11th edn (edited by Saif, Barnes, Fadly, Glisson, Mcdougald and Swayne). Iowa State University Press, Ames, IA, pp. 826–836. Swayne, D.E. and A.J. McLaren. 1997. Avian intestinal spirochaetes and avian intestinal spirochaetosis. In Intestinal Spirochaetes in Domestic Animals and Humans (edited by Hampson and Stanton). CAB International, Wallingford, pp. 267–300. Swayne, D.E., K.A. Eaton, J. Stoutenburg, D.J. Trott, D.J. Hampson and N.S. Jensen. 1995. Identification of a new intestinal spirochete with pathogenicity for chickens. Infect. Immun. 63: 430–436. Taylor, D.J. and T.J. Alexander. 1971. The production of dysentery in swine by feeding cultures containing a spirochaete. Br. Vet. J. 127: 58–61. Taylor, D.J. and D.J. Trott. 1997. Porcine intestinal spirochaetosis and spirochaetal colitis. In Intestinal Spirochaetes in Domestic Animals

and Humans (edited by Hampson and Stanton). CAB International, Wallingford, pp. 211–241. ter Huurne, A.A., M. van Houten, S. Muir, J.G. Kusters, B.A. van der Zeijst and W. Gaastra. 1992. Inactivation of a Serpula (Treponema) hyodysenteriae hemolysin gene by homologous recombination: importance of this hemolysin in pathogenesis in mice. FEMS Microbiol. Lett. 92: 109–114. ter Huurne, A.A., S. Muir, M. van Houten, B.A. van der Zeijst, W. Gaastra and J.G. Kusters. 1994. Characterization of three putative Serpulina hyodysenteriae hemolysins. Microb. Pathog. 16: 269–282. Thomson, J.R., W.J. Smith, B.P. Murray and S. McOrist. 1997. Pathogenicity of three strains of Serpulina pilosicoli in pigs with a naturally acquired intestinal flora. Infect. Immun. 65: 3693–3700. Tompkins, D.S., S.J. Foulkes, P.G. Godwin and A.P. West. 1986. Isolation and characterisation of intestinal spirochaetes. J. Clin. Pathol. 39: 535–541. Townsend, K.M., V.N. Giang, C. Stephens, P.T. Scott and D.J. Trott. 2005. Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia. J. Vet. Diagn. Invest. 17: 103– 109. Trott, D.J., C.R. Huxtable and D.J. Hampson. 1996a. Experimental infection of newly weaned pigs with human and porcine strains of Serpulina pilosicoli. Infect. Immun. 64: 4648–4654. Trott, D.J., T.B. Stanton, N.S. Jensen, G.E. Duhamel, J.L. Johnson and D.J. Hampson. 1996b. Serpulina pilosicoli sp. nov.: the agent of porcine intestinal spirochetosis. Int. J. Syst. Bacteriol. 46: 206–215. Trott, D.J., T.B. Stanton, N.S. Jensen and D.J. Hampson. 1996c. Phenotypic characteristics of Serpulina pilosicoli the agent of intestinal spirochaetosis. FEMS Microbiol. Lett. 142: 209–214. Trott, D.J., B.G. Combs, A.S. Mikosza, S.L. Oxberry, I.D. Robertson, M. Passey, J. Taime, R. Sehuko, M.P. Alpers and D.J. Hampson. 1997a. The prevalence of Serpulina pilosicoli in humans and domestic animals in the Eastern Highlands of Papua New Guinea. Epidemiol. Infect. 119: 369–379. Trott, D.J., N.S. Jensen, I. Saint Girons, S.L. Oxberry, T.B. Stanton, D. Lindquist and D.J. Hampson. 1997b. Identification and characterization of Serpulina pilosicoli isolates recovered from the blood of critically ill patients. J. Clin. Microbiol. 35: 482–485. Trott, D.J., S.L. Oxberry and D.J. Hampson. 1997c. Evidence for Serpulina hyodysenteriae being recombinant, with an epidemic population structure. Microbiology 143: 3357–3365. Trott, D.J., D.P. Alt, R.L. Zuerner, M.J. Wannemuehler and T.B. Stanton. 2001. The search for Brachyspira outer membrane proteins that interact with the host. Anim. Health Res. Rev. 2: 19–30. Trott, D.J., D.P. Alt, R.L. Zuerner, D.M. Bulach, M.J. Wannemuehler, J. Stasko, K.M. Townsend and T.B. Stanton. 2004. Identification and cloning of the gene encoding BmpC: an outer-membrane ­lipoprotein associated with Brachyspira pilosicoli membrane vesicles. Microbiology 150: 1041–1053. Turner, A.K. and R. Sellwood. 1997. Extracellular DNA from Serpulina hyodysenteriae consists of 6.5 kbp random fragments of chromosomal DNA. FEMS Microbiol. Lett. 150: 75–80. Waters, W.R., B.A. Pesch, R. Hontecillas, R.E. Sacco, F.A. Zuckermann and M.J. Wannemuehler. 2000. Cellular immune responses of pigs induced by vaccination with either a whole-cell sonciate or pepsin-digested Brachyspira (Serpulina) hyodysenteriae bacterin. Vaccine 18: 711–719. Wright, J.C., G.R. Wilt, R.B. Reed and T.A. Powe. 1989. Use of an enzymelinked immunosorbent assay for detection of Treponema ­hyodysenteriae infection in swine. J. Clin. Microbiol. 27: 411–416. Zuerner, R.L. 1997. Genetic organization in spirochaetes. In Intestinal Spirochaetes in Domestic Animals and Humans (edited by Hampson and Stanton). CAB International, Wallingford, pp. 63–89. Zuerner, R.L. and T.B. Stanton. 1994. Physical and genetic map of the ­Serpulina hyodysenteriae B78T chromosome. J. Bacteriol. 176: 1087–1092. Zuerner, R.L., T.B. Stanton, F.C. Minion, C. Li, N.W. Charon, D.J. Trott and D.J. Hampson. 2004. Genetic variation in Brachyspira: chromosomal rearrangements and sequence drift distinguish B. pilosicoli from B. hyodysenteriae. Anaerobe 10: 229–237.

Genus I. Brevinema

545

Family III. Brevinemataceae fam. nov. Bruce J. Paster Bre.vi.ne.ma.ta.ce’ae. N.L. fem. n. Brevinema -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Brevinemataceae the Brevinema family. The family Brevinemataceae was circumscribed for this volume on the basis of phylogenetic analysis of 16S rRNA gene sequences. The family contains only one genus, Brevinema.

Description is the same as for the genus Brevinema. Type genus: Brevinema Defosse, Johnson, Paster, Dewhirst and Fraser 1995, 83VP.

Genus I. Brevinema Defosse, Johnson, Paster, Dewhirst and Fraser 1995, 83VP Bruce J. Paster Bre.vi. ne¢ma. L. adj. brevis short; Gr. neut. n. nema thread; N.L. neut. n. Brevinema a short thread.

Helical cells are 0.2–0.3 mm in diameter by 4–5 mm in length, displaying one to two helical turns. Irregular wavelengths of the cells range from 2 to 3 mm. Sheathed periplasmic flagella are in a 1:2:1 arrangement. No cytoplasmic tubules have been observed. Cells are motile by flexing, rotation, and translation. Microaerophilic, host-associated, isolated from blood and other tissues of short-tailed shrews (Blarina brevicauda) and whitefooted mice (Peromyscus leucopus). Infectious for laboratory mice and Syrian hamsters. DNA G+C content (mol%): 34–36 (Tm). Type species: Brevinema andersonii Defosse, Johnson, Paster, Dewhirst and Fraser 1995, 83VP.

Further descriptive information Strains of Brevinema andersonii are homogeneous as based on enzymic, protein profile, and immunoblot data. Furthermore, there are no significant differences in fatty acid composition among the strains analyzed (Defosse et  al., 1995). The major fatty acid components of Brevinema cells are myristic acid (C14:0), palmitic acid (C16:0), and oleic acid (C18:1) with smaller amounts of stearic acid (C18:0) and linoleic acid (C18:2). Several fatty acids are present at low levels (less than 1%). Restriction enzyme analysis and SDS–PAGE patterns also demonstrate little or no differences among strains. Consequently, it was suggested that Brevinema andersonii represents a genetically homologous group, despite the diverse hosts and different geographic origins (Defosse et al., 1995).

Enrichment and isolation procedures Brevinema andersoni has been isolated from blood and other tissues of the short-tailed shrew and the white-footed mouse using Shrew-Mouse Spirochete medium under microaerophilic conditions (Defosse et al., 1995). Fetal bovine serum, reducing agents, and peptones are required for growth. Neither supplemental bovine serum albumin, N-acetylglucosamine, nor pyruvate is required for growth. Optimal growth is at 30–34°C at pH 7.4 with a generation time of 11–14 h.

Differentiation of the genus Brevinema from other genera Brevinema andersonii is serologically distinct from other spirochetes (Anderson et al., 1987; Defosse et al., 1995). There is little or no DNA–DNA hybridization between Brevinema andersonii and members of other spirochetal genera using Southern blot analysis (LeFebvre and Perng, 1989; LeFebvre et al., 1989). At the species level, Brevinema andersonii is differentiated using restriction enzyme analysis, SDS-PAGE, or fatty acid composition.

Taxonomic comments Brevinema andersoni is the only named species for the genus. Based on 16S rRNA gene sequence comparisons, Brevinema belongs within the family Spirochetaceae and is clearly distinct from the other genera of spirochetes (Paster and Dewhirst, 2000). 16S rRNA sequences of Brevinema andersonii do not possess a 20- to 30-base extension at the 5¢ end, which is typical of 16S rRNA sequences of species of Treponema, Spirocheta, Leptospira, and Leptonema (Defosse et al., 1995; Paster et al., 1991).

List of species of the genus Brevinema 1. Brevinema andersonii Defosse, Johnson, Paster, Dewhirst and Fraser 1995, 83VP an.der.so¢ni.i. N.L. masc. gen. n. andersonii of Anderson, named for John F. Anderson, who first described the organism. The characteristics are as described for the genus. Chemoorganotrophic. Microaerophilic and catalase-negative. Growth occurs in modified BSK medium at an optimal growth temperature of 30–34°C and an optimal pH of 7.4; under these conditions the generation time is 11–14 h. Does not grow at

25°C. Exhibits the following enzymic activities: C4, C5, C6, C8, C9, and C10 esterases, C4 esterase lipase, alkaline phosphatase, acid phosphatase, and b-glucuronidase. Source: tissues of a short-tailed shrew (Blarina brevicauda) captured in West Haven, Connecticut, USA (Anderson et al., 1987). DNA G+C content (mol%): 34–36 (Tm). Type strain: ATCC 43811, CT11616. Sequence accession no. (16S rRNA gene): M59179.

546

Family IV. Leptospiraceae

References Anderson, J.F., R.C. Johnson, L.A. Magnarelli, F.W. Hyde and T.G. Andreadis. 1987. New infectious spirochete isolated from shorttailed shrews and white-footed mice. J. Clin. Microbiol. 25: 1490– 1494. Defosse, D.L., R.C. Johnson, B.J. Paster, F.E. Dewhirst and G.J. Fraser. 1995. Brevinema andersonii gen. nov., sp. nov., and infectious spirochete isolated from the short-tailed shrew (Blarina brevicauda) and the white-footed mouse (Peromyscus leucopus). Int. J. Syst. Bacteriol. 45: 78–84.

LeFebvre, R.B. and G.C. Perng. 1989. Genetic and antigenic characterization of Borrelia coriaceae, putative agent of epizootic bovine abortion. J. Clin. Microbiol. 27: 389–393. LeFebvre, R.B., G.C. Perng and R.C. Johnson. 1989. Characterization of Borrelia burgdorferi isolates by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 27: 636–639. Paster, B. and F. Dewhirst. 2000. Phylogenetic foundation of spirochetes. J. Mol. Microbiol. Biotechnol. 2: 341–344. Paster, B.J., F.E. Dewhirst, W.G. Weisburg, L.A. Tordoff, G.J. Fraser, R.B. Hespell, T.B. Stanton, L. Zablen, L. Mandelco and C.R. Woese. 1991. Phylogenetic analysis of the spirochetes. J. Bacteriol. 173: 6101–6109.

Family IV. Leptospiraceae Hovind-Hougen 1979, 245AL emend. Levett, Morey, Galloway, Steigerwalt and Ellis 2005, 1499 Richard L. Zuerner Lep.to.spi.ra.ce¢ae. N.L. fem. n. Leptospira type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Leptospiraceae the Leptospira family. Helical cells, 0.1–0.3 mm in diameter and 3.5–20 mm in length. Cells have right-handed helical conformation. Cells at rest and those that are fixed have hooked ends. Actively motile cells have a spiral anterior end and a hook at the posterior end of the cell. One periplasmic flagellum (historically also referred to as axial filaments, endoflagella, or flagella), is inserted subterminally at each end of cell, but flagella rarely overlap in the center of the cell. Periplasmic flagella lie along the helix axis. The diamino acid in peptidoglycan is a,e-diaminopimelic acid. Obligate aerobes or microaerophilic. Chemoorganotrophic. Utilize long-chain fatty acids and fatty alcohols as carbon and energy sources. Do not use carbohydrates/amino acids as carbon or energy sources. Free-living or in association with animal and human hosts. Some species are pathogenic. Species examined by 16S rRNA sequence analysis are distinct from members of Spirochetaceae. DNA G+C content (mol%): 33–53. Type genus: Leptospira Noguchi 1917, 755AL.

Key to the genera of the family Leptospiraceae 1. DNA G+C content (mol%) is 33–43% (Tm and genomic sequence analysis). Cells are 0.1 mm in diameter and 6–20 mm in length. Aerobe or microaerophilic. Long-chain fatty acids and long-chain fatty alcohols serve as carbon and energy

sources. Free-living in aquatic environments, including mud, sediments, and water of ponds, lakes, and streams. May be found in fresh water and marine environments. Some species are found in association with animals. Some species are pathogenic.   →Genus I. Leptospira 2. DNA G+C content (mol%) is 54% (Tm and Bd).Cells are 0.1–0.2 mm in diameter and 13–15 mm in length, with wavelength of 0.7 mm. Aerobe. Long-chain fatty acids and longchain fatty alcohols serve as carbon and energy sources. Can grow on trypticase broth. Free-living in aquatic environments, including mud, sediments, and water of ponds, lakes, and streams. May be found in fresh water and marine environments. Type strain was found in association with animals (cattle). Nonpathogenic for hamsters.   →Genus II. Leptonema 3. DNA G+C content (mol%) is 48% (Tm and Bd). Cells are 0.3 mm in diameter and 3.5–7.5 mm in length, with wavelength of 0.3–0.5 mm. Obligate aerobe. One periplasmic flagellum. Long-chain fatty acids and long-chain fatty alcohols serve as carbon and energy sources. Oxidase-positive. Found in tap water and in association with animals. Not pathogenic for hamsters.   →Genus III. Turneriella

Genus I. Leptospira Noguchi 1917, 755AL emend. Faine and Stallman 1982, 461 Richard L. Zuerner Lep.to.spi¢ra. Gr. adj. leptos thin, narrow, fine; L. fem. n. spira a coil, helix; N.L. fem. n. Leptospira a thin helix or coil, referring to the morphology of the bacterium.

Leptospira are long, thin, flexible rods, 0.1 mm in diameter and 6–12 mm in length, with a regular right-handed helical coiling pattern (Carleton et al., 1979). These bacteria are unicellular but may be observed as dividing pairs or short chains of three or more cells in actively growing cultures. Resting stages are not known, but long term survival in water, with the appearance of aggregates has been described (Trueba et al., 2004). Spherically

shaped cells form under unfavorable growth conditions. Bacteria stain as Gram-negative. Due to the small diameter of these bacteria, unstained cells are not visible by bright-field microscopy. Dark-field or phase-contrast microscopy is required for visualization of unstained cells. These are highly motile aerobic or microaerophilic bacteria. Optimum growth temperature is 28–30°C, with a generation time of 6–16 h, although many

Genus I. Leptospira

primary pathogenic isolates may grow slower. Chemoorganotrophic bacteria that consume long-chain fatty acids and alcohols as primary carbon and energy sources, and carry out respiration with oxygen as the terminal electron acceptor. Optimal growth occurs in semi-solid (0.1–0.2%) agar media. Growth on 1–2% solid agar results in the formation of clear to turbid surface or subsurface colonies. Colony formation is enhanced by the addition of pyruvate. Oxidase, catalase, and/or peroxidase-positive. Some strains are b-hemolytic. Some strains are pathogenic for humans and animals, while other strains are saprophytic and found in freshwater and marine environments. The genus Leptospira forms a deep unique branch of spirochetes, separate from other genera based on comparison of 16S rRNA gene sequences. Species are differentiated by DNA–DNA relative reassociation analysis and by unique sequence polymorphisms in 16S rRNA. DNA G+C content (mol%): 35–43. Type species: Leptospira interrogans (Stimson 1907) Wenyon 1926, 1281 emend. Faine and Stallman 1982, 462 (Spirochaeta interrogans Stimson 1907, 541).

Further descriptive information Taxonomic history.  The taxonomy of Leptospira has undergone substantial revisions in the past 20 years with the use of 16S rRNA sequence comparison and DNA–DNA reassociation studies. DNA–DNA reassociation studies helped differentiate pathogenic and saprophytic species (Haapala et  al., 1969). Subsequent studies by Brendle et  al. (1974), Ramadass et  al. (1992), and Yasuda et  al. (1987) helped to revise and better define Leptospira species. The key paper by Brenner et  al. (1999) compared 303 strains of Leptospira, leading to a refined definition of 12 different species and identification of five new genomospecies, one of which was given the designation Leptospira alexanderi. One of the species included in the Brenner et al. (1999) study was Leptospira parva, originally described by Hovind-Hougen et al. (1981). Due to extensive DNA sequence and 16S rRNA differences from other Leptospira species and to the difference from Leptonema illini, it was placed in a new genus of Leptospiraceae, Turneriella parva (Levett et  al., 2005). Three new Leptospira species have been described, Leptospira broomii (Levett et al., 2006), Leptospira licerasiae (Matthias et al., 2008), and Leptospira wolffii (Slack et al., 2008). Thus, at the current time, the genus Leptospira contains 15 named species and the Subcommittee on the taxonomy of Leptospiraceae has recommended names for four genomospecies identified by Brenner et al., (1999) (Levett and Smythe, 2008). Following the initial use of 16S rRNA sequence comparisons to define taxonomic relationships among spirochete genera (Paster et al., 1984, 1991); Hookey et al. (1993) expanded these analyses to help differentiate Leptospira species and differentiate

547

the genera Leptonema and Leptospira. Comparison of 16S rRNA sequences and DNA–DNA reassociation studies suggest the presence of three groups of pathogenic species; Group I contains Leptospira interrogans and Leptospira kirschneri, Group II contains Leptospira weilii, Leptospira borgpetersenii, and Leptospira santarosai, and Group III contains Leptospira noguchi and Leptospira meyeri (Hookey, 1993). There are five species having intermediate pathogenicity: Leptospira fainei, Leptospira inadai, Leptospira broomi, Leptospira licerasiae, and Leptospira wolffii. The latter three species were recently recognized by the Subcommittee on the taxonomy of Leptospiraceae (Levett and Smythe, 2008). Saprophytic species include Leptospira biflexa and Leptospira wolbachii (Yasuda et al., 1987). The status of Leptospira meyeri is unclear because strains fit into both saprophytic (Yasuda et al., 1987) and pathogenic groups (Hookey et al., 1993; Victoria et al., 2008). Many of the diverse free-living strains of Leptospira described by Ramadass et  al. (1992) and halophiles described by Cinco et al. (1975) were not included in the study by Brenner et al. (1999), so the taxonomic status of these strains is unknown. Brenner et al. (1999) discovered and described four Leptospira genomospecies tentatively named Leptospira alstonii (genomospecies  1), Leptospira vanthielii (genomospecies  3), Leptospira terpstrae (genomospecies 4), and Leptospira yanagawae (genomospecies 5) (Levett and Smythe, 2008). Cell morphology, motility, and ultrastructure.  Leptospira cells are typically visualized by dark-field light microscopy due to their long slender cell dimensions. The basic cell morphology of Leptospira resembles other members of the order Spirochetales; Leptospira species typically appear as long helical cells with loose coils (Figure 99). One flagellum is inserted subterminally at each end of the cell and lies along the helix axis in the periplasmic space (Goldstein et  al., 1996). Scanning electron microscopic analysis shows these cells form right-handed helices, and helical handedness may affect cell motility (Carleton et al., 1979). During translational movement, the trailing end of the cell bends to form a hook thought to function as a propeller, with the leading end maintaining a spiral twist, guiding the forward movement of the bacterium (Charon et al., 1984; Charon et  al., 1981). Translational movement increases with the viscosity of the medium (Greenberg and Canale-Parola, 1977a, b), a finding that suggests these bacteria are viscotaxic (Petrino and Doetsch, 1978). Leptospira have a typical spirochete ultrastructure, having an outer membrane surrounding a protoplasmic cylinder. The cell wall contains peptidoglycan containing the diamino acid alpha, epsilon diaminopimelic acid (Azuma et al., 1975). Two flagella (also referred to as axial filaments) are inserted subterminally at each end of the cell, and wrap around the protoplasmic cylinder in the periplasmic space. The ends of the flagella rarely overlap near the midpoint of the cell. The outer membrane is

Figure 99.  Transmission electron micrograph of Leptospira biflexa serovar Patoc. Bar = 500 nm.

548

Family IV. Leptospiraceae

loosely attached to the cell and easily removed by detergents (Haake et al., 1991; Zuerner et al., 1991). Rapid displacement of antibody-coated latex beads adhered to Leptospira cells suggests the outer membrane is quite fluid (Charon et al., 1981). Outer membrane composition.  Protein composition of the Leptospira outer membrane varies depending on virulence and growth conditions. For example, few proteins have been detected on the surface of virulent Leptospira kirschneri, while outer-membrane protein density is substantially higher in avirulent strains (Haake et  al., 1991). LipL36 is expressed under normal in vitro growth conditions, but down-regulated during mammalian infection (Haake et al., 1998). Changes in osmolarity also alter outer-membrane protein expression. For example, expression of LigA and LigB, two bacterial immunoglobulinlike proteins increase as osmolarity rises (Choy et al., 2007; Matsunaga et al., 2007a, b; Matsunaga et al., 2005). Under normal in vitro growth, the most predominant proteins on the surface of pathogenic Leptospira are LipL32, LipL21, and LipL41 (Cullen et al., 2002, 2003, 2005). LipL32 is the major outer-membrane protein and is unique to pathogenic Leptospira species. LipL32 is heat labile and, in the absence of calcium ions, is degraded by an endogenous protease to two smaller peptides (Haake et al., 2000; Zuerner et al., 1991). Although the original report describing LipL21 identified this protein as unique to pathogenic Leptospira species (Cullen et al., 2003), genome analysis of the Leptospira biflexa genome revealed the presence of a gene encoding a protein with extensive similarity to LipL21 (Picardeau et  al., 2008). Protein OmpLI is an integral transmembrane porin (Haake et al., 1993). Access to genomic sequences for three species of Leptospira, including two pathogenic species, has resulted in cloning and analysis of many previously unknown outer-membrane proteins. Of particular interest are proteins from pathogenic strains that interact with the host. LigA and Lsa24 (also referred to as LfhA) bind extracellular matrix proteins (Barbosa et al., 2006; Choy et  al., 2007), and Lsa24 binds complement factor H (Verma et al., 2006). The leptospiral LPS is thought to be the primary antigen associated with determining serovar, and variation in LPS biosynthetic genes may occur via lateral transfer (de la PenaMoctezuma et  al., 1999) or mutation (Zuerner and Trueba, 2005), resulting in expression of antigenically distinct LPS. Leptospiral LPS is not as complex in structure as often found in typical Gram-stain-negative bacteria, and does not resolve as a ladder of variable sized products during polyacrylamide gel electrophoresis. Purified leptospiral LPS activates mouse cells via TLR2 and TLR4 (Nahori et  al., 2005; Werts et  al., 2001). Purified Leptospira interrogans Lipid A, the membrane anchor for LPS, does not induce Limulus amebocyte lysates to gel (Nahori et al., 2005; Que-Gewirth et al., 2004). The Leptospira interrogans lipid A has unusual features including a methylated 1-phosphate group (Nahori et al., 2005). Nutrition and growth conditions.  Leptospira are obligate aerobes but vary greatly in their ability to grow in vitro. Although many Leptospira can grow in a defined medium of relatively simple composition referred to as EMJH (Ellinghausen and McCullough, 1965; Johnson and Harris, 1967a), addition of rabbit serum is often necessary for growth of many pathogenic strains, and early attempts to grow Leptospira utilized media rich in rabbit serum (Fletcher, 1928; Stuart, 1946). Growth yields in

EMJH media vary from 3 to 5 × 108 to ~1010 cells/ml, with generation times of 6–16 h, depending on the strain. Leptospira consume long chain fatty acids or alcohols as primary carbon and energy sources, which are metabolized by beta-oxidation (Baseman and Cox, 1969; Henneberry and Cox, 1970). Fatty acids are commonly added to Leptospira media preparations through the addition of polysorbate (e.g., Tweens), nonionic detergents derived from long chain fatty acids. Tween 80, predominantly containing oleic acid, is used most commonly, but addition of Tween 40 (primarily palmitic acid) and/or Tween 60 (primarily steric acid) can be useful to support growth of fastidious strains of Leptospira (e.g., Leptospira borgpetersenii serovar Hardjo) (Ellinghausen, 1983). Leptospiral media often contains purified bovine serum albumin to bind free fatty acids and reduce their toxicity. Protein-free media for growth of some strains of Leptospira have been reported (Bey and Johnson, 1978; Shenberg, 1967), but these media do not support prolonged propagation of many pathogenic species. Vitamins B1 and B12 are typically included in the growth medium for Leptospira. However, Leptospira synthesize heme (Guegan et  al., 2003), consistent with possessing vitamin B12 biosynthesis genes. Furthermore, de novo synthesis of B12 may be required as genomic analysis suggests Leptospira may be deficient in the ability to transport exogenous B12 (Rodionov et al., 2003). Ammonium is provided by addition of ammonium salts to the media as amino acids do not appear to be a significant source of ammonia for Leptospira (Johnson and Rogers, 1964c). Leptospira appear to synthesize all amino acids except isoleucine by standard pathways (Charon et  al., 1974). Isoleucine is synthesized either by the standard pathway, being derived from threonine, or by a pathway involving condensation of pyruvate with acetyl-S-coenzyme A (Charon et al., 1974). Leptospira synthesize isoleucine either by a combination of threonine and pyruvate pathways or by exclusive use of the threonine or pyruvate pathways (Westfall et al., 1983). Exogenous purines are incorporated by Leptospira (Johnson and Rogers, 1964a). However, Leptospira do not incorporate exogenous pyrimidines, thus incorporation of pyrimidine analogs such as 5-fluorouracil is a useful method for suppressing the growth of other bacteria and providing an enrichment method for isolating Leptospira (Johnson and Rogers, 1964b). Iron is required for growth of Leptospira (Faine, 1959) and is usually supplied as iron sulfate in the media. Recent analysis of iron transport in Leptospira revealed that iron sulfate-free media supports growth of Leptospira interrogans, presumably by supplying trace iron, but Leptospira biflexa requires addition of exogenous iron to the media for growth (Louvel et al., 2006). Leptospira are rarely grown on agar plate media due to their slow growth. Colony formation can be observed in about 10 days for saprophytic species, but it may take 6–8 weeks for pathogenic species to form colonies. Colonies are diffuse, nonpigmented, clear to turbid, and may form below the surface. Growth, especially on solid agar media, is enhanced by the addition of pyruvate (Johnson et al., 1973). Optimal growth conditions include pH 7.2–7.4, 30°C, and media containing low salt (17–60 mM), although halophilic strains require 0.22–0.44 M for growth (Cinco and Cociancich, 1975). Genome properties.  Physical mapping of Leptospira interrogans serovars Pomona and Icterohemorrhagiae using pulsedfield gel electrophoresis (PFGE) and DNA hybridization revealed the presence of two chromosomal replicons in this

Genus I. Leptospira

species (Baril et  al., 1992; Zuerner, 1991). Comparative mapping revealed extensive rearrangements that alter genetic organization within the same species (Zuerner et  al., 1993). The genomes of four pathogenic Leptospira strains have been published: Leptospira interrogans serovar Copenhageni (Nascimento et  al., 2004) and Leptospira interrogans serovar Lai (Ren et  al., 2003), and two strains of Leptospira borgpetersenii serovar Hardjo (Bulach et al., 2006). Genome size ranges from 3.9 to 4.7 Mbp, consisting of one large and one small chromosome. Genome sequencing of the free-living Leptospira biflexa serovar Patoc shows the presence of three stable replicons, corresponding to the large and small chromosomes of pathogenic Leptospira, and a third replicon of unknown function (Picardeau et  al., 2008). Comparative analysis of Leptospira interrogans serovar Lai and Copenhageni confirm previous mapping studies that suggest recombination between transposable elements contribute to changes in genetic organization by identifying an inversion flanked at both ends by insertion sequences that differentiates genetic organization of these two serovars (Nascimento et al., 2004). Extensive rearrangements differentiate the organization of Leptospira interrogans from Leptospira borgpetersenii, and strains of Leptospira borgpetersenii are likewise differentiated by the presence of chromosomal rearrangements. Approximately 7% of the Leptospira borgpetersenii serovar Hardjo genome is comprised of transposable elements, and nearly 17% of pseudogenes in Leptospira borgpetersenii are associated with insertion sequences (IS), leading to the conclusion that the genome of this species is undergoing IS-mediated genome erosion (Bulach et al., 2006). A primary consequence of genome erosion in Leptospira borgpetersenii is the apparent loss of viability in water (Bulach et al., 2006), thereby limiting dissemination of viable organisms via the environment, a common means of disease transmission for Leptospira interrogans. Additional variability in Leptospira interrogans may result from lateral transfer of genomic islands (Bourhy et al., 2007). An unusual feature of Leptospira genomes is the organization of rRNA genes (Fukunaga and Mifuchi, 1989a, b). Individual rRNA genes are not linked closely to each other in Leptospira, but are dispersed around the large chromosome (Zuerner et  al., 1993). There are two copies each of the 16S and 23S rRNA genes in Leptospira; there is one copy of the 5S rRNA gene in pathogenic Leptospira, but two copies of the 5S rRNA gene in free-living Leptospira (Fukunaga and Mifuchi, 1989b). Phage, plasmid, and transposable elements and genetic ­ anipulation in Leptospira.  Although naturally occurring m mechanisms for genetic exchange are not yet defined for Leptospira, several recent discoveries and advances in genetic manipulation of members of this genus suggest lateral transfer of DNA contributes to genetic variation. Bacteriophages have been detected by electron microscopy of Leptospira cultures. Saint Girons et al. (Saint Girons et al., 1990) described the first successful isolation and propagation of bacteriophage that infect Leptospira biflexa. The replication origin of bacteriophage LE1 can direct autonomous replication of a plasmid in Leptospira biflexa, and this discovery led to development of an Escherichia coli- Leptospira biflexa shuttle vector facilitating genetic manipulation of Leptospira (Girons et  al., 2000). In a similar manner, discovery of genetic islands in Leptospira interrogans led to development of a limited-host range shuttle vector (Bourhy et al., 2007). Autonomous replication of these genetic islands

549

may contribute to lateral transfer of DNA in nature and affect pathogenicity by spreading virulence determinants (Bourhy et al., 2007). Recent demonstration of conjugative transfer of a shuttle vector from Escherichia coli to Leptospira is consistent with evidence for lateral transfer of genetic material into Leptospira (Picardeau, 2008). Intervening sequences are inserted into the 23S rRNA gene in some Leptospira species (Ralph and McClelland, 1993). During post-transcriptional processing, the intervening sequences are excised without ligation, therefore bacterial strains containing these elements lack an intact 23S rRNA and instead have 14S and 17S rRNA species (Hsu et al., 1990). As noted above, genetic rearrangements occur frequently in Leptospira strains, and these rearrangements are often associated with transposable elements including IS-elements. There are several different IS-like elements found in Leptospira (BoursauxEude et al., 1995; Bulach et al., 2006; Nascimento et al., 2004; Ralph and McClelland, 1993; Zuerner, 1994; Zuerner and Huang, 2002; Zuerner and Trueba, 2005) that share substantial sequence similarity to elements found in diverse bacterial genera, yet lateral transfer of these elements has not been demonstrated. The presence of high numbers (~90 copies) of IS1533 in Leptospira borgpetersenii serovar Hardjo is hypothesized to have a prominent role in genome degradation by generating nonfunctional pseudogenes that restrict serovar Hardjo to a host to host transmission cycle (Bulach et al., 2006). Transposition of the genetic elements derived from the eukaryotic transposon himar1 provides a means to generate selectable random mutants in Leptospira species (Bourhy et al., 2005), opening up new possibilities in genetic manipulation and analysis in this genus. Serotype analysis.  Leptospira are antigenically diverse and are clustered by serological analysis into serogroups. Serogroups are further subdivided into serovars, which are considered the primary serotype taxon. Serovar determination is achieved by agglutination reactions using a microscopic agglutination test (Cole et  al., 1973; Ryu, 1970). Serovars are differentiated by cross-absorption agglutination reactions using high titer sera. Two strains are designated different serovars if, after sufficient cross-absorption with each sera using the heterologous strain, at least one of the sera retains 10% or more of the initial homologous agglutination titer (Stallman, 1984). Use of monoclonal antibodies can also help differentiate strains belonging to different serovars (Terpstra et al., 1985; World Health Organization, 2003). Serovar designation has historical relevance and may be essential in understanding the relationships between these bacteria and their normal maintenance hosts; maintenance hosts appear to have a stable relationship with bacteria in the same serovar but may undergo acute infection with a different serovar (Faine et al., 1999). Likewise, the same strain in its normal maintenance host rarely displays signs of acute infection, but accidental infection by that strain of a non-maintenance host can result in severe acute infection (Faine et al., 1999). Complicating the importance and relevance of serovar designation is the discovery of several serovars represented by strains belonging to different Leptospira species (Levett, 2001). Antibiotic sensitivity.  Leptospira exhibit in vitro sensitivity to a large number of antibiotics, including beta-lactams (penicillin, ampicillin, and amoxycillin), rifampin, tetracycline and doxycycline, cephalosporins (cefotaxime and ceftizoxime),

550

Family IV. Leptospiraceae

aminoglycosides including streptomycin (Oie et  al., 1983), macrolides (erythromycin and azithromycin), and fluoroquinolones including ciprofloxacin (Hospenthal and Murray, 2003). Immediate treatment of suspected infections is recommended. Traditionally, leptospirosis has been treated with intravenous penicillin for severe cases, or oral antibiotics including amoxycillin, ampicillin, doxycycline, and erythromycin (Terpstra et  al., 1985; World Health Organization, 2003). There is limited clinical data on effective use of newer antibiotics to treat leptospirosis. Environmental range.  Pathogenic Leptospira species cause leptospirosis, one of the most widespread zoonosis known. Wild and domesticated animals are reservoirs of infection, and animal to human transmission may occur either through direct exposure to blood or urine from an infected animal, or indirectly from urine-contaminated water. After infection, the bacteria disseminate via the blood and concentrate in the kidney. Leptospira are voided in the urine (Figure 100), facilitating infection of new hosts (Faine et al., 1999). Leptospirosis ranges in severity from a mild influenza-like infection to a severe acute infection often resulting in death from organ failure. Chronically infected animal maintenance hosts often do not exhibit clinical signs of infection. Several Leptospira species exhibit an intermediate pathogenicity in humans and animal hosts, and may be opportunistic pathogens. The normal environment for saprophytic Leptospira species is water or moist soil (Henry and Johnson, 1978). Cinco et al. (1975) have reported isolation of halophilic Leptospira, but these have not been subjected to taxonomic characterization. Recent surveys using 16S rRNA sequence analysis to assess the diversity of microbes in different environments suggest Leptospira

and Leptonema are widely distributed, including in petroleum contaminated soil (Kasai et al., 2005), marine sediments (GenBank accession EU386041), and geothermal regions (GenBank accession EF205465). Leptospirosis and virulence-associated traits.  Virulence determinants of pathogenic Leptospira remain poorly characterized. This is due, in part, to the limited tools available for genetic manipulation; until quite recently, genetic manipulation of pathogenic Leptospira species was not possible. Thus, to date, only one protein has been clearly shown to have an affect on virulence. Mutation of the gene encoding Loa22, a protein localized in the outer membrane, resulted in attenuation of Leptospira interrogans (Ristow et al., 2007). Genetic complementation of a Loa22− mutant partially restored virulence near the level seen with the wild-type parental strain (Ristow et al., 2007). Hemolysins, including phospholipase C, sphingomylinase-like proteins, and pore-forming proteins, are potential virulence factors (Artiushin et al., 2004; Bernheimer and Bey, 1986; del Real et al., 1989; Lee et al., 2000, 2002; Matsunaga et al., 2007b; Segers et  al., 1990, 1992). Several proteins, including bacterial immunoglobulin-like proteins LigA and LigB (Choy et al., 2007), a group of endostatin-like proteins (Barbosa et al., 2006; Stevenson et al., 2007), and the major outer-membrane protein LipL32 (Hoke et al., 2008), facilitate attachment of pathogenic Leptospira to mammalian extracellular matrix proteins and probably have important roles during infection. Lsa24 may also help the pathogenic Leptospira evade the antibacterial effects of complement by binding factor H (Verma et al., 2006), consistent with earlier findings that virulent Leptospira are resistant to serum (Johnson and Harris, 1967b). The unusual ability of Leptospira to undergo translational movement in highly viscous media (Greenberg and Canale-Parola, 1977a) may be an important factor in tissue penetration.

Enrichment and isolation procedures

Figure 100.  Leptospira in urine. A sample of urine was obtained from

a dog exhibiting clinical signs of leptospirosis and stained with FITCconjugated anti-Leptospira rabbit sera, counterstained with Flazo-orange, then visualized using a 60× objective on an Olympus fluorescent microscope (final magnification 600×). Under these conditions, the bacteria appear green. Individual cells and cell masses are visible.

Pathogenic Leptospira can be isolated from blood and, occasionally, spinal fluid during the early stages of infection (1–2 weeks) by growth in semi-solid (0.1–0.2% agar) or liquid growth media. Blood treated with heparin to prevent coagulation or untreated blood added directly to transport media (1% bovine serum albumin in phosphate buffer) can be used to inoculate growth media. One to two weeks after infection, and for several weeks to months following infection, Leptospira can be isolated from urine. Leptospira reside primarily in kidney and liver, therefore inoculation of media with tissue homogenates prepared from these organs collected during necropsy often results in successful isolation of bacteria. Addition of 5¢-fluorouracil (100 mg/ ml) to the isolation medium is useful for primary isolation of pathogenic Leptospira, especially from urine, by helping to suppress growth of contaminating bacteria. Primary isolation of pathogenic strains may require incubation of cultures up to 6 months with periodic microscopic evaluation. Direct intraperitoneal inoculation of weanling Golden Syrian hamsters using tissue, soil, or water samples is an alternative method to enrich for pathogenic Leptospira (Brendle and Alexander, 1974; Glosser et al., 1974). Saprophytic Leptospira reside in streams, lakes, and moist soil (Henry and Johnson, 1978). To prepare environmental ­samples for culture, suspend soil in sterile distilled water, or use surface water directly, and filter through 0.45 or 0.22 mm filters before inoculating culture media. Incorporation of 5¢-­fluorouracil

Genus I. Leptospira

­ uring initial isolation of environmental samples is important d for restricting growth of non-Leptospira bacteria. Isolation of Leptospira from contaminated cultures can be achieved either by filtration through 0.22 mm filters or by spotting a few microliters of culture on solid culture medium. Leptospira will tend to migrate away from the spot, forming a halo of growth from which the bacteria can be isolated. Leptospira growth in semi-solid media typically results in formation of a dense zone of growth (referred to as a Dinger disk) a few millimeters below the surface of the media.

Maintenance procedures Leptospira cultures are maintained in semi-solid (0.1–0.2% agar) media at 30°C. The frequency of subculture varies greatly, dependent upon strain specific growth characteristics; some fastidious strains may take 6 months of incubation before visible growth, while more vigorous strains can be subcultured weekly. Long term cultivation of pathogenic Leptospira may result in loss of virulence, and therefore pathogenic strains should be frozen and stored in liquid nitrogen after initial isolation or within 1–2 in  vitro subcultures. To prepare cultures for cryopreservation, mix a fresh Dinger disk of Leptospira growth in semi-solid media with an equal volume transport media and freeze at −70°C before placing in liquid nitrogen. Free-living Leptospira may be frozen or kept in semi-solid media for long term storage. Storage of Leptospira cultures by lyophilization is not recommended.

Differentiation of the genus Leptospira from other closely related taxa Leptospira are obligate aerobes that consume fatty acids or alcohols as primary carbon and energy sources, distinguishing this genus from other spirochetes. Although morphologically similar to other spirochetes, Leptospira form tighter coils, often with at least one hooked end. These bacteria are resistant to 5¢-fluorouracil, whereas other spirochetes are sensitive to this compound. The cell wall contains diaminopimelic acid vs ornithine in other spirochetes.

Further reading Charon, N.W. and S.F. Goldstein. 2002. Genetics of motility and chemotaxis of a fascinating group of bacteria: the spirochetes. Annu. Rev. Genet. 36: 47–73. Faine, S. 1994. Leptospira and Leptospirosis. CRC Press, Boca Raton, FL.

Differentiation of species of the genus Leptospira Characteristics that differentiate species of Leptospira are shown in Table 132. Leptospira species are defined by DNA–DNA ­reassociation analysis. In addition, 16S rRNA sequence comparison (Hookey, 1993), mapped-restriction site ­polymorphisms of amplified rRNA sequences (Ralph et  al., 1993), and secY sequence comparison (Ahmed et al., 2006; ­Victoria et al., 2008)

551

are also useful for differentiating ­Leptospira ­species. Several methods have proven helpful for strain and serovar differentiation, including restriction endonuclease analysis (Marshall et al., 1981; Robinson et al., 1982) and ­macrorestriction ­endonuclease analysis by pulsed-field gel ­electrophoresis (Galloway and Levett, 2008; Herrmann et al., 1991, 1992). The results of strain comparison using restriction endonuclease DNA digestion ­patterns usually correlate with serological ­analysis that separates strains based on agglutination reactions using specific sera (Ellis et al., 1991, 1988; Robinson et  al., 1982, 1986; ­Thiermann et  al., 1985). There are several examples of serovars found in more than one species (Levett, 2001), indicating the need to characterize strains by both serological and DNA-based techniques. Extensive phenotypic diversity precludes use of differential growth characteristics for differentiation of Leptospira species.

Taxonomic comments The previous edition of the Manual described three species of Leptospira, namely Leptospira interrogans, representing pathogens, Leptospira biflexa, representing free-living leptospires, and ­Leptospira illini species incertae sedis. DNA–DNA renaturation ­studies have revealed substantial diversity within the previous classifications of Leptospira interrogans and Leptospira biflexa, and have supported the establishment of two new genera, ­Leptonema, in which Leptonema illini species incertae sedis now resides, and Turneriella parva. The taxonomic status of halophilic Leptospira has not been determined. Environmental surveys using 16S rRNA sequence analysis to monitor bacterial populations ­suggests the distribution of Leptospira and Leptonema species is diverse, and additional species are likely to be discovered. The serovar is the primary Leptospira taxon, but this classification scheme is distinct from molecular taxonomy based on DNA– DNA reassociation kinetics or 16S rRNA sequence analysis. Strains representing the same serovar occur in two or more species. Likewise, serogroups also occur in different species (Table 133). Differentiation of Leptospira species is difficult because these bacteria are morphologically similar and there is substantial variation in biochemical and growth characteristics within each species. Generally, free-living Leptospira and Leptonema illini share the ability to grow at 13°C. Leptonema is the only genus in Leptospiraceae that can grow on trypticase media and possesses cytoplasmic tubules. Pathogenic Leptospira are sensitive to 8-azaguanine, but nonpathogenic and strains with inter­ mediate pathogenic status can vary in their capacity to grow in the presence of this compound. Likewise, 2,6-diaminopurine or copper sulfate inhibits growth of most pathogenic strains, but nonpathogenic strains and strains with intermediate pathogenic status vary in their ability to grow in the presence of these compounds. Therefore, accurate differentiation of Leptospira species must rely on molecular characterization comparing 16S rRNA sequences or DNA–DNA reassociation analysis (Levett and Smythe, 2006).

List of species of the genus Leptospira 1. Leptospira interrogans (Stimson 1907) Wenyon 1926, 1281AL emend. Faine and Stallman 1982, 462 (Spirocheta ­interrogans Stimson 1907, 541) in.ter¢ro.gans. L. part. adj. interrogans asking, inquiring, interrogating, here meaning shaped like a question mark.

Exhibits morphological and cultural features common to the genus Leptospira (Figure 101). Grows at 30°C, but does not grow at 13°C. Growth is inhibited by 8-azaguanine (225 mg/ml) or CuSO4 (100 p.p.m.). Growth of some strains is inhibited by 2,6-diaminopurine (10 mg/ml). Production

2 − d + d + − + 38

2

− d

+ + d − + 35

L. interrogans nr

L. alexanderi

+

L. biflexa d d d + + 38.9

− +

2

−

L. borgpetersenii + + − − + 39.8

− −

2

+

L. broomi nr nr nr nr + 42

− nr

2

+

L. fainei p nr nr + + nr

− nr

2

+

L. inadai d d d v + 42.6

− d

2

i

+ d d − + nr

− d

2

+

+ nr nr nr + 43.9

− nr

2

i

L. meyeri − − − − + 33.5

− +

2

v

L. noguchi + + + − + 36.5

− d

2

+

L. santarosai + + d − + 40.7

− −

2

+

L. weilii + + + − + 40.5

− −

2

+

L. wolbachii − − + − + 37.2

− +

2

−

L. wolffii − nr nr − + 41.8

− nr

2

v

“L. alstonii” + + + − + 39.8

− −

2

nr

− − + − + 38.9

− −

2

nr

“L. terpstrae”

a

Symbols: +, >85% positive; −, 0–15% positived, some strains; v, variation among strains; p, partial growth inhibition; i, infectious, pathogenicity not demonstrated; nr, not reported.

Pathogenic in humans No. periplasmic flagella Cytoplasmic tubules Lipase activity Growth inhibited by: 8-Azaguanine CuSO4 2,6-Diaminopurine Growth at 11−13°C Growth at 30°C DNA G+C content (mol%)

Characteristic

L. kirschneri

Table 132.  Differentiation of the species of the genera Leptospira, Leptonema, and Turneriella a

L. licerasiae

“L. vanthielii” + + + − + 43.4

− −

2

nr

“L. yanagawae” − − + − + 37.9

− −

2

nr

Leptonema illini − + + − + 54.2

+ +

2

−

Turneriella parva − − + − + 47–48

− +

2

−

552 Family IV. Leptospiraceae

Genus I. Leptospira

553

Table 133.  Distribution of serogroups in different Leptospira species

Species L. interrogans

L. alexanderi L. biflexa L. borgpetersenii

L. broomi L. fainei L. inadai L. kirschneri

L. licerasiae L. meyeri L. noguchi

L. santarosai

L. weilii

Serogroup Australis, Autumnalis, Bataviae, Canicola,  Djasiman, Grippotyphosa, Hebdomadis, Icterohemorrhagiae, Louisiana, Mini, Pomona, Pyrogenes, Ranarum, Sarmin, Sehgali, Sejroe Hebdomadis, Javanica, Manhoa, Mini   Semaranga Australis, Autumnalis, Ballum, Bataviae,  Celledoni Hebdomadis, Javanica, Mini, Pyrogenes, Sejroe, Tarassovi Undesignated Hurstbridge Canicola, Icterohemorrhagiae, Javanica,   Lyme, Manhoa, Panama, Shermani, Tarassovi Australis, Autumnalis, Bataviae, Canicola,  Cynopteri, Djasiman, Grippotyphosa, Hebdomadis, Icterohemorrhagiae, Pomona Iquitos Javanica, Mini, Ranarum, Sejroe, Semaranga Australis, Autumnalis, Bataviae, Djasiman,  Louisiana, Panama, Pomona, Pyrogenes, Shermani, Tarassovi Autumnalis, Bataviae, Cynopteri,  Grippotyphosa, Hebdomadis, Javanica, Mini, Pomona, Pyrogenes, Sarmin, Sejroe, Shermani, Tarassovi Celledoni, Hebdomadis, Icterohemorrhagiae,  Javanica, Manhoa, Mini, Pyrogenes, Sarmin, Sejroe, Tarassovi Codice Undesignated Ranarum

L. wolbachii L. wolffii “L. alstonii ” (genomospecies 1) “L. vanthielii ” Holland (genomospecies 3) “L. terpstrae ” Icterohemorrhagiae (genomospecies 4) “L. yanagawae ” Semaranga (genomospecies 5)

in situ. Sections of hamster kidney were isolated after infection with ­Leptospira interrogans serovar Pomona, fixed with formalin, then processed for histology. The tissue was stained with a modified PAS Steiner process. Bacteria appear black due to the precipitation of silver on the bacterial surface. A final magnification of 400× is shown. Bar = 25 mm.

Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but does not grow at 11 or 37°C. Growth is inhibited by 8-azaguanine (225 mg/ml) or 2,6-diaminopurine (10 mg/ml). Strains vary in the ability to grow in the presence of CuSO4 (100 p.p.m.), and production of lipase activity is variable. Pathogenicity for animals not reported. Source: isolated in China from an unknown source. DNA G+C content (mol%): 38 (Tm). Type strain: L 60 serovar Manhao 3, ATCC 700520. Sequence accession no. (16S rRNA gene): AY631880. 3. Leptospira biflexa (Wolbach and Binger 1914) Noguchi 1918, 585AL emend. Faine and Stallman 1982, 462 (Spirocheta biflexa Wolbach and Binger 1914, 23)

of lipase varies among strains of this species. Pathogenic in mammals with disease manifestations ranging from mild influenza-like symptoms to acute, lethal infection. The genome sequences for two strains of Leptospira interrogans have been determined: serovar Copenhageni strain Fiocruz L1–130 (GenBank accession nos AE016823 and AE016824) and serovar Lai strain 56601 (GenBank accession nos AE010300 and AE010301). Source: bacteria localize in the kidneys of mammals, facilitating shedding via urine. DNA G+C content (mol%): 35 (Tm). Type strain: Leptospira interrogans serogroup Icterohaemorrhagiae, serovar Icterohaemorrhagiae, strain RGA, ATCC 23581, ATCC 43642, CCUG 5117, KCTC 2880. Sequence accession no. (16S rRNA gene): AY631894, FJ154549. 2. Leptospira alexanderi Brenner, Kaufmann, ­Steigerwalt, Rogers and Weyant 1999, 856VP

Figure 101.  Silver-stained Leptospira interrogans serovar Pomona

Sulzer,

a.le.xan.de¢ri. N.L. masc. gen. n. alexanderi of Alexander, named to honor Aaron D. Alexander, an American microbiologist.

bi.fle¢xa. L. adv. num. bis twice; L. part. adj. flexus -a -um (from. L. v. flecto) bent, winding; N.L. fem. adj. biflexa bent twice. Exhibits morphological and cultural features common to the genus Leptospira. Grows at 13°C and at 30°C. Strains often grow in the presence of 8-azaguanine (225 mg/ml), 2,6-diaminopurine (10 mg/ml), and CuSO4 (100 p.p.m.). All strains produce lipase. Strains are typically found in flowing or still freshwater sources, tap water, and occasionally animals. Pathogenicity not demonstrated, thought to be nonpathogenic for mammals. The genome sequence for the type strain and one of its derivatives has been determined (GenBank accession nos CP00777–CP00779 and CP007786–CP007788). Source: a stream in Italy. DNA G+C content (mol%): 38.9 (genomic sequencing). Type strain: Leptospira biflexa serovar Patoc strain Patoc I, ATCC 23582. Sequence accession no. (16S rRNA gene): AY631876, Z12821. 4. Leptospira borgpetersenii Yasuda, Steigerwalt, Sulzer, Kaufmann, Rogers and Brenner 1987, 414VP

554

Family IV. Leptospiraceae

borg.pe¢ter.sen¢i.i. N.L. masc. gen. n. borgpetersenii of BorgPetersen, named for C. Borg-Petersen, the Danish physician who made significant early contributions to the epidemiology and microbiology of leptospirosis in Europe. Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but does not grow at 11°C. Growth is inhibited by 8-azaguanine (225 mg/ ml) or CuSO4 (100 p.p.m.), but not by 2,6-diaminopurine (10 mg/ml). Does not produce lipase. The type strain was isolated from a Java house rat in Indonesia. Pathogenic in mammals with disease manifestations ranging from mild influenza-like symptoms to acute, lethal infection. Bacteria localize in the kidneys of their host, facilitating shedding via urine. The genome sequences for two strains of Leptospira borgpetersenii serovar Hardjo (L550 and JB197) have been determined (GenBank accession nos. CP000348-CP000351). Source: a Java house rat in Indonesia. DNA G+C content (mol%): 39.8 (Tm). Type strain: Veldrat Bataviae 46 serovar Javanica, ATCC 43292. Sequence accession no. (16S rRNA gene): AY461862, AY887899, AM050572, DQ991483, FJ154600, Z21630. 5. Leptospira broomii Levett, Morey, Galloway and Steigerwalt 2006, 673VP bro.o¢mi.i. N.L. masc. gen. n. broomii of Broom, named for John Constable Broom (1902–1960), a Scottish bacteriologist who made substantial contributions to the study of leptospirosis. Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but growth at other temperatures not reported. Pathogenic in mammals with disease manifestations ranging from mild influenza-like symptoms to acute, lethal infection. Bacteria localize in the kidneys of their host, facilitating shedding via urine. However, clusters with other species (Leptospira fainei, Leptospira inadai, and Leptospira licerasiae) considered intermediate in pathogenicity based on 16S rRNA sequence comparison. Source: blood of human patient with leptospirosis; additional isolates from blood, cerebrospinal fluid, and urine of human patients with leptospirosis. DNA G+C content (mol%): 42 (Tm). Type strain: 5399, ATCC BAA-1107, KIT 5399. Sequence accession no. (16S rRNA gene): AY796065. 6. Leptospira fainei Perolat, Chappel, Adler, Baranton, Bulach, Billinghurst, Letocart, Merien and Serrano 1998, 857VP fai¢ne.i. N.L. masc. gen. n. fainei of Faine, named for Solomon Faine, an Australian medical microbiologist who made definitive contributions to the knowledge of the physiopathology and epidemiology of leptospirosis. Exhibits morphological and cultural features common to the genus Leptospira. Grows at 13°C and 30°C. Growth is partially inhibited by 8-azaguanine (225 mg/ml, 30°C). May induce disease manifestations ranging from mild influenzalike symptoms to acute, potentially lethal infection. Bacteria localize in the kidneys of their host, facilitating shedding via urine. Clusters with other species (Leptospira broomii,

Leptospira inadai, and Leptospira licerasiae) ­considered i­ntermediate in pathogenicity based on 16S rRNA sequence comparison. Species contains a single serovar, Hurstbridge. Source: the uterus of a sow in Australia; strains isolated from chronic human infections also reported. DNA G+C content (mol%): not reported. Type strain: BUT 6 serovar Hurstbridge. Sequence accession no. (16S rRNA gene): AY631885, FJ154578, U60594. 7. Leptospira inadai Yasuda, Steigerwalt, Sulzer, Kaufmann, Rogers and Brenner 1987, 414VP i.na¢da.i. N.L. masc. gen. n. inadai of Inada, named for ­Ryokichi Inada, the Japanese microbiologist who is regarded by some to have first isolated leptospires from human patients. Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but no growth at 11°C. Growth of some strains is inhibited by 8-azaguanine (225 mg/ml), CuSO4 (100 p.p.m.), or 2,6-diaminopurine (10 mg/ml). Some strains produce lipase. Strains of this species are considered to have intermediate pathogenic status, but clinical manifestations and pathogenicity have not been clearly demonstrated. Clusters with other species (Leptospira broomii, Leptospira fainei, and Leptospira licerasiae) considered intermediate in pathogenicity based on 16S rRNA sequence comparison. Source: the skin of a patient with a concurrent, unrelated Lyme disease infection; strains have been isolated from a variety of rodents. DNA G+C content (mol%): 42.6 (Tm). Type strain: 10 serovar Lyme, ATCC 43289. Sequence accession no. (16S rRNA gene): AY631896, Z21634. 8. Leptospira kirschneri Ramadass, Jarvis, Corner, Penny and Marshall 1992, 219VP kirsch¢ne.ri. N.L. masc. gen. n. kirschneri of Kirschner, named for Leopold Kirschner, a Dutch medical microbiologist who worked on leptospirosis research in Indonesia before coming to New Zealand to work at the Otago Medical School in Dunedin and whose pioneering work on leptospirosis helped focus attention on the human and animal health problem that existed at the time. Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but no growth at 13°C. Growth is inhibited by 8-azaguanine (225 mg/ml). Most strains are inhibited by CuSO4 (100 p.p.m.) or 2,6-diaminopurine (10 mg/ml). Lipase is not produced. Strains of this species are pathogenic for mammals with disease manifestations ranging from mild influenza-like symptoms to acute, lethal infection. Bacteria localize in the kidneys of their host, facilitating shedding via urine. Source: Indonesia,from a short-headed fruit bat. DNA G+C content (mol%): not reported. Type strain: 3522 C serovar Cynopteri, ATCC 49945. Sequence accession no. (16S rRNA gene): AY631895, DQ991475, FJ154546, Z21628. 9. Leptospira licerasiae Matthias, Ricaldi, Cespedes, Diaz, ­Galloway, Saito, Steigerwalt, Patra, Ore, Gotuzzo, Gilman, Levett and Vinetz 2009, 1VP (Effective publication: Matthias,

Genus I. Leptospira

Ricaldi, Cespedes, Diaz, Galloway, Saito, Steigerwalt, Patra, Ore, Gotuzzo, Gilman, Levett and Vinetz 2008, 11.) li.ce.ra.si¢ae. N.L. fem. gen. n. licerasiae of Liceras, named to honor Julia Liceras de Hidalgo, who obtained the first leptospiral isolates in Peru. Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C. Growth at other temperatures not reported. Growth is inhibited by 8-azaguanine (225 mg/ml). However, clusters with other species (Leptospira broomi, Leptospira fainei, and Leptospira inadai) considered intermediate in pathogenicity based on 16S rRNA sequence comparison. Although these strains are associated with human infections, experimental inoculation of hamsters and guinea pigs did not induce clinical signs of leptospirosis. Source: blood obtained from human patients with undifferentiated fever. DNA G+C content (mol%): not reported. Type strain: VAR010 serovar Varillal, ATCC BAA-1110, KIT VAR 010, WPR VAR 010. Sequence accession no. (16S rRNA gene): EF612284. 10. Leptospira meyeri Yasuda, Steigerwalt, Sulzer, Kaufmann, Rogers and Brenner 1987, 414VP me.ye¢ri. N.L. masc. gen. n. meyeri of Meyer, named to honor Karl F. Meyer, the veterinarian who established veterinary public health in the United States through his broad interests in the zoonoses, including leptospirosis.

555

san.ta.ro¢sa.i. N.L. masc. gen. n. santarosai of Santa Rosa, named to honor Carlos A. Santa Rosa, the Brazilian veterinary microbiologist who was a pioneer in the study of leptospirosis as a human and animal health problem in Brazil. Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but does not grow at 11°C. Growth is inhibited by 8-azaguanine (225 mg/ml) or CuSO4 (100 p.p.m.). Growth of some strains is inhibited by 2,6-diaminopurine (10 mg/ml). Lipase is not produced. Pathogenic in mammals with disease manifestations ranging from mild influenza-like symptoms to acute, lethal infection. Bacteria localize in the kidneys of their host, facilitating shedding via urine. Source: the Panama Canal Zone, from a spiney rat. DNA G+C content (mol%): 40.7 (Tm). Type strain: LT 821 serovar Shermani, ATCC 43286. Sequence accession no. (16S rRNA gene): AY461889, AY631883. 13. Leptospira weilii Yasuda, Steigerwalt, Sulzer, Kaufmann, Rogers and Brenner 1987, 413VP weil¢i.i. N.L. masc. gen. n. weilii of Weil, named to honor Adolph Weil, a German physician who was among the first to clinically differentiate leptospirosis (Weil¢s disease) from other types of infectious jaundice.

Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but not at 11°C. Grows in the presence 8-azaguanine (225 mg/ml), 2,6-diaminopurine (10 mg/ml), or CuSO4 (100 p.p.m.). Produces lipase. Virulence has not been demonstrated, but some strains share genetic and antigenic similarity to pathogens. Source: United States, from a leopard frog. DNA G+C content (mol%): 33.5 (Tm). Type strain: Iowa City Frog serovar Ranarum, ATCC 43782. Sequence accession no. (16S rRNA gene): AY631878.

Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but does not grow at 11°C. Growth is inhibited by 8-azaguanine (225 mg/ml), 2,6-diaminopurine (10 mg/ml), or CuSO4 (100 p.p.m.). Lipase is not produced. Pathogenic in mammals with disease manifestations ranging from mild influenza-like symptoms to acute, lethal infection. Bacteria localize in the kidneys of their host, facilitating shedding via urine. Source: Australia, from blood of a patient with ­leptospirosis. DNA G+C content (mol%): 40.5 (Tm). Type strain: Celledoni serovar Celledoni, ATCC 43285. Sequence accession nos (16S rRNA gene): AY631877, DQ991486, FJ154580, U12676, Z21637.

11. Leptospira noguchii Yasuda, Steigerwalt, Sulzer, Kaufmann, Rogers and Brenner 1987, 413VP

14. Leptospira wolbachii Yasuda, Steigerwalt, Sulzer, Kaufmann, Rogers and Brenner 1987, 414VP

no.gu¢chi.i. N.L. masc. gen. n. noguchii of Noguchi, named to honor Hideyo Noguchi, the Japanese microbiologist who named the genus Leptospira.

wol.ba¢chi.i. N.L. masc. gen. n. wolbachii of Wolbach, named to honor Simeon Burt Wolbach (1880–1954), the American microbiologist who first identified Leptospira [Spirochaeta] biflexa.

Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but no growth at 11°C. Growth is inhibited by 8-azaguanine (225 mg/ml), 2,6-diaminopurine (10 mg/ml), or CuSO4 (100 p.p.m.). Lipase is usually produced. Thought to be pathogenic with disease manifestations ranging from mild influenza-like symptoms to acute, lethal infection. Bacteria localize in the kidneys of their host, facilitating shedding via urine. Source: parasitic strains isolated from mammals. DNA G+C content (mol%): 36.5 (Tm). Type strain: CZ 214 serovar Panama, ATCC 43288. Sequence accession no. (16S rRNA gene): AY631886, DQ991500, Z21635. 12. Leptospira santarosai Yasuda, Steigerwalt, Sulzer, Kaufmann, Rogers and Brenner 1987, 413VP

Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but does not grow at 11°C. Grows in the presence of 8-azaguanine (225 mg/ml) or 2,6-diaminopurine (10 mg/ml), but does not grow in the presence of CuSO4 (100 p.p.m.). All strains produce lipase. Pathogenicity not demonstrated; thought to be nonpathogenic for mammals. Source: water in the United States. DNA G+C content (mol%): 37.2 (Tm). Type strain: CDC serovar Codice, ATCC 43284. Sequence accession no. (16S rRNA gene): AY631879. 15. Leptospira wolffii Slack, Kalambaheti, Symonds, Dohnt, Galloway, Steigerwalt, Chaicumpa, Bunyaraksyotin, Craig, Harrower and Smythe 2008, 2307VP

556

Family IV. Leptospiraceae

wolf¢fi.i. N.L. masc. gen. n. wolffii of Wolff, named to honor Jan Wolff, a Dutch bacteriologist. Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C and 37°C, but does not grow at 13°C. Grows in the presence of 8-azaguanine (225 mg/ml). Pathogenicity not demonstrated, but thought to be of intermediate pathogenicity based on 16S rRNA

and DNA:DNA reassociation similarity to Leptospira broomii, ­Leptospira fainei, and Leptospira inadai. Source: urine of a human patient with symptoms consistent with leptospirosis. DNA G+C content (mol%): 41.8 (Tm). Type strain: Khorat-H2, KIT Khorat-H2, WHO LT1686. Sequence accession no. (16S rRNA gene): EF025496.

Other organisms 1. “Leptospira genomospecies 1” Brenner, Kaufmann, Sulzer, Steigerwalt, Rogers and Weyant 1999, 857 Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but does not grow at 11°C. Growth is inhibited by 8-azaguanine (225 mg/ml), 2,6-diaminopurine (10 mg/ml), or CuSO4 (100 p.p.m.). Does not produce lipase. Pathogenicity not reported. Source: a frog in China. DNA G+C content (mol%): 39.8 (Tm). Type strain: 79601 serovar Sichuan, ATCC 700521. Note: ATCC does not list this strain. Sequence accession no. (16S rRNA gene): AY631881. Taxonomic note: the Subcommittee on the taxonomy of Leptospiraceae proposed this species be named Leptospira alstonii.

3. “Leptospira genomospecies 4” Brenner, Kaufmann, Sulzer, Steigerwalt, Rogers and Weyant 1999, 857 Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but does not grow at 11°C. Growth is inhibited by 2,6-diaminopurine (10 mg/ml), but growth is not inhibited by 8-azaguanine (225 mg/ml) or CuSO4 (100 p.p.m.). Does not produce lipase. Pathogenicity not reported. Source: China from an unknown source. DNA G+C content (mol%): 38.9 (Tm). Type strain: H 2 serovar Hualin, ATCC 700522. Note ATCC lists this strain as genomospecies 3. Sequence accession no. (16S rRNA gene): AY631888. Taxonomic note: the Subcommittee on the taxonomy of Leptospiraceae proposed this species be named Leptospira terpstrae.

2. “Leptospira genomospecies 3” Brenner, Kaufmann, Sulzer, Steigerwalt, Rogers and Weyant 1999, 857 Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but does not grow at 11°C. Growth is inhibited by 8-azaguanine (225 mg/ml), 2,6-diaminopurine (10 mg/ml), or CuSO4 (100 p.p.m.). Does not produce lipase. Pathogenicity not reported. Source: water in the Netherlands. DNA G+C content (mol%): 43.4 (Tm). Type strain: WaZ Holland serovar Holland, ATCC 700522. Sequence accession no. (16S rRNA gene): AY631897. Taxonomic note: the Subcommittee on the taxonomy of Leptospiraceae proposed this species be named Leptospira ­vanthielii.

4. “Leptospira genomospecies 5” Brenner, Kaufmann, Sulzer, Steigerwalt, Rogers and Weyant 1999, 857 Exhibits morphological and cultural features common to the genus Leptospira. Grows at 30°C, but does not grow at 11°C. Growth is inhibited by 2,6-diaminopurine (10 mg/ml), but growth is not inhibited by 8-azaguanine (225 mg/ml) or CuSO4 (100 p.p.m.). Does not produce lipase. Pathogenicity not reported. Source: China from an unknown source. DNA G+C content (mol%): 37.9 (Tm). Type strain: Sao Paulo serovar Saopaulo, ATCC 700523. Sequence accession no. (16S rRNA gene): AY631882. Taxonomic note: the Subcommittee on the taxonomy of Leptospiraceae proposed this species be named Leptospira yanagawae.

Genus II. Leptonema Hovind-Hougen 1983, 439VP (Effective publication: Hovind-Hougen 1979, 250.) Richard L. Zuerner Lep.to.ne¢ma. Gr. adj. leptos thin, narrow, fine; Gr. neut. n. nema a filament or thread; N.L. neut. n. Leptonema a thin filament or thread, describing a bacterium that resembles a thin filament or thread.

Long, thin, flexible rods, 0.1–0.2 mm in diameter and 13–21 mm in length, with a regular helical coiling pattern having a wavelength of 0.6–0.7 mm. Unicellular but may be observed as dividing pairs or short chains in actively growing cultures. ­Resting stages are not known. Gram-stain-negative. Due to small ­diameter, unstained cells are not visible by bright-field microscopy. Dark-field or phase-contrast microscopy is required for visualization of unstained cells. Highly motile. Obligate ­aerobes. Growth temperatures range between 13 and 30°C, with optimal growth at 28–30°C. Chemoorganotrophic; consume long-chain

fatty acids and fatty alcohols as primary carbon and energy sources. Can grow on trypticase media. Uses respiration with oxygen as the terminal electron acceptor. Optimal growth occurs in semi-solid (0.2%) agar media. Growth on 1–2% solid agar results in the formation of clear to turbid subsurface colonies. Oxidase-positive. Lipase-positive. Nonpathogenic for cattle, gerbils, guinea pigs, hamsters, and mice. Free-living in aquatic environments and soil. Some strains found in association with animals. Does not share significant levels of sequence similarity with other Leptospiraceae as ­determined by DNA

Genus II. Leptonema

hybridization analysis. The 16S rRNA sequence is distinct from other Leptospiraceae. DNA G+C content (mol%): 51–54% Type species: Leptonema illini (Hanson, Tripathy, Evans and Alexander 1974) Hovind-Hougen 1983, 439 (Effective publi­ cation: Hovind-Hougen 1979, 251.) (Leptospira illini Hanson, Tripathy, Evans and Alexander 1974, 355).

Further descriptive information Taxonomic history.  The previous taxonomic designation for Leptospira illini as Leptospira interrogans serovar Illini and Leptospira illini has led to considerable confusion in the literature. In the previous edition of this manual, the taxonomic status of the genus Leptonema was uncertain, and the species illini was designated a species incertae sedis. The International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Leptospira approved the genus designation at its meeting in 1986 (Stallman, 1987). Leptonema illini strain 3055, the type strain for this genus and species, was originally isolated from the urine of a healthy bull in Illinois (Hanson et  al., 1974). An antigenically similar strain, A177, was isolated from a turtle in the same geographical region in the year following isolation of strain 3055T. Early DNA hybridization studies led to the discovery that strain 3055T lacked significant homology to either the “pathogenic” complex (Leptospira interrogans sensu lato), or the “saprophytic” complex (Leptospira biflexa sensu lato) (Brendle et  al., 1974). Furthermore, the DNA G+C content (51–54 mol%) of Leptonema illini is substantially higher than known Leptospira strains (Brendle et al., 1974). Subsequent DNA hybridization studies by Ramadass et  al. (1990) and Brenner et  al. (1999), and analyzes of Leptospiraceae 16S rRNA sequences (Hookey et al., 1993; Morey et al., 2006; Paster et al., 1991) support a familial relationship between Leptospira and Leptonema, while supporting formation of a distinct genus Leptonema with a single known species, Leptonema illini. Cell morphology, ultrastructure, and motility.  Analysis of Leptonema illini has played a critical role in understanding cell structure and motility of the Leptospiraceae. Leptonema illini cells have a typical spirochete ultrastructure including a long slender helical morphology (Figure 102), forming right-handed helices (Carleton et  al., 1979). Cells undergoing translational movement have a spiral anterior end and a hooked posterior end (Goldstein and Charon, 1990). The ability to form hooked ends is essential for translational movement and is governed by

557

the shape of the two periplasmic flagella inserted subterminally at each end of the cell (Bromley and Charon, 1979). Nonhelical mutants are nonmotile and have periplasmic flagella with no defined shape, whereas wild-type cells and motile revertants have hooked ends and highly coiled flagella (Bromley and Charon, 1979), indicating these structures are rigid. Leptonema cells retain a gentle helical morphology regardless of whether the periplasmic flagella are straight or coiled (Bromley and Charon, 1979), and this is consistent with a model suggesting that the periplasmic flagella lie along the helical axis of the cell (Goldstein et al., 1996). Rotation of the periplasmic flagella is thought to cause the cytoplasmic cylinder to rotate around the axis of the flagella, and, depending on the direction of flagellar rotation, induce either a spiral or hook shape (Berg et al., 1978; Charon et  al., 1984; Goldstein and Charon, 1990). A coordinated change in the cell ends from spiral to hook or from hook to spiral, enables the bacteria to rapidly change direction during translational movement (Charon et al., 1984). A feature unique to Leptonema among the Leptospiraceae is the presence of cytoplasmic tubules that start near the insertion of the periplasmic flagella, and are about the same length as the flagella (Hovind-Hougen, 1979). These structures are also seen in species of Treponema and Spirocheta, but have not been observed in other members of Leptospiraceae. Nutrition and growth conditions.  Although Leptonema is traditionally grown on bovine serum albumin, Tween-based media used to cultivate Leptospira, e.g., EMJH (Ellinghausen and McCullough, 1965; Johnson and Harris, 1967a), members of this genus are distinguished by the ability to grow on trypticase media (Hanson et  al., 1974). Large (8–10 mm) colonies appear on solid EMJH agar media within 7–10 d. Little information is known about the specific nutritional requirements of Leptonema illini. However, physiological profiles of Leptospiraceae using aminopeptidase substrates clustered the Leptonema strains together in a distinct pattern separate from either Leptospira or Turneriella (Neill et al., 1987). Leptonema illini and Leptospira biflexa are more resistant to killing by UV or mitomycin C treatments than pathogenic Leptospira (Stamm and Charon, 1988). rRNA sequence analysis.  As noted above, 16S rRNA sequence analysis is useful for distinguishing Leptonema from Leptospira or Turneriella (Hookey et al., 1993; Morey et al., 2006; Paster et  al., 1991). Unlike Leptospira species, which have one gene encoding 5S rRNA (rrn) and two genes each encoding 16S (rrs) and 23S (rrl) rRNA species, Leptonema has two genes

Figure 102.  Transmission electron micrograph of Leptonema illini strain 3055T. Bar = 2 mm.

558

Family IV. Leptospiraceae

encoding each rRNA species (Fukunaga et al., 1991). In addition, the two ­Leptonema rrn genes are closely linked to each other (Fukunaga and Mifuchi, 1989b), and the rrs and rrl genes are separated by a 435 bp intergenic spacer (Woo et al., 1996). In contrast, the rRNA genes in Leptospira species are dispersed throughout the large chromosome. Bacteriophage.  Early electron microscopy studies of Leptonema illini strain 3055T cultures revealed the presence of bacteriophage-like particles with 45–50 nm heads and 60–65 × 15–20 nm tails (Hanson et al., 1974; Ritchie and Ellinghausen, 1969). Intracellular forms of these phage-like particles were also detected. Subsequent studies to characterize these particles have not been reported. Ecology.  The initial isolations of Leptonema illini were from cattle and turtles. Subsequent isolations of Leptonema illini having distinctly different antigenic profiles from the original isolates have been reported from fresh water (Bazovska et  al., 1983) and a lymphocyte culture from a HIV-I infected human patient (Rocha et  al., 1993). Several spirochete isolates from animal and water sources by Neill et al. (1987) have

­aminopeptidase substrate profiles similar to Leptonema illini, but these have not been characterized further by either DNA hybridization or 16S rRNA sequence analysis. Recent studies using 16S rRNA sequence analysis to profile microbial communities suggest Leptonema illini is broadly distributed in nature; 16S rRNA sequences matching Leptonema illini were detected in petroleum-contaminated soil in Japan (Kasai et  al., 2005) and diseased coral communities near the Bahamas (Sekar et al., 2006). Pathogenesis and antigenicity.  Although serological surveys of cattle and swine in Illinois showed >60% positive reaction with strain 3055T (Tripathy and Hanson, 1973a), this strain is not pathogenic for a wide variety of animal species including cattle, gerbils, guinea pigs, hamsters, and mice (Tripathy and Hanson, 1973b). Serological testing of strains 3055T and A177 showed these strains comprised a novel antigenic group, distinct from known Leptospira serovars (Hanson et al., 1974). The discovery of additional serovars of Leptonema illini suggests this genus may also be antigenically diverse (Rocha et al., 1993).

List of species of the genus Leptonema 1. Leptonema illini (Hanson, Tripathy, Evans and Alexander 1974) Hovind-Hougen 1983, 439VP (Effective publication: Hovind-Hougen 1979, 251.) (Leptospira illini Hanson, Tripathy, Evans and Alexander 1974, 355) il.li¢ni. N.L. gen. n. illini of Illinois, named after the state of Illinois, USA, where the first isolate was obtained. Morphologically similar to Leptospira. Cytoplasmic tubules extend from near the insertion of periplasmic flagella and are about the same length. Aerobe. Long-chain fatty acids and long-chain fatty alcohols serve as carbon and energy sources. Can grow on trypticase media, unlike Leptospira or Turneriella.

Three serovars described. Lacks significant homology to Leptospira or Turneriella as measured by DNA hybridization analysis. Has a unique 16S rRNA sequence profile. Not pathogenic for cattle, gerbils, guinea pigs, hamsters, and mice. Source: urine of a clinically normal bull in Illinois, USA, in 1965 (Hanson et al., 1974); free living in soil or aquatic environments; some strains are found in association with animals. DNA G+C content (mol%): 51–54% (Tm and Bd). Type strain: 3055, NCTC 11301. Sequence accession no. (16S rRNA gene): AY714984, Z21632.

Genus III. Turneriella Levett, Morey, Galloway, Steigerwalt and Ellis 2005, 1499VP Richard L. Zuerner Tur.ne.ri¢el.la. N.L. fem. dim. n. Turneriella named after Leslie Turner, an English microbiologist who made definitive contributions to the knowledge of leptospirosis.

Flexible helical rods, 0.3 × 3.5–7.5 mm with a wavelength of 0.3– 0.5 mm. Gram-stain-negative. Unicellular. Resting stages are not known. Dark-field or phase-contrast microscopy is required for visualization of unstained cells. Obligate aerobes. Grows slowly at 13, 30, and 37°C, with temperature optimum of 28–30°C. Chemoorganotrophic bacteria that consume long-chain fatty acids and fatty alcohols as primary carbon and energy sources. Optimal growth occurs in semi-solid (0.2%) agar media. Oxidase-positive. Lipase-positive. Isolated from contaminated culture medium, tap water, and uterus of a sow. DNA hybridization and 16S rRNA sequence analyzes show this genus is distinct from other Leptospiraceae. DNA G+C content (mol%): 47–48. Type species: Turneriella parva (Hovind-Hougen, Ellis and Birch-Andersen 1981) Levett, Morey, Galloway, Steigerwalt and

Ellis 2005, 1499VP (Leptospira parva Hovind-Hougen, Ellis and Birch-Andersen 1981, 352).

Further descriptive information Taxonomic history.  The type strain was isolated from contaminated bovine serum albumin culture media and provisionally named Leptospira parva (Hovind-Hougen et al., 1981). Analysis of 16S rRNA sequence data indicated that this strain was different from other Leptospira and Leptonema species, and distinct from Spirochetaceae (Hookey et  al., 1993). Additionally, this strain has an unusual aminopeptidase profile as compared to other Leptospiraceae (Neill et al., 1983, 1987). These data were consistent with DNA hybridization studies that showed no significant sequence similarity to other Leptospiraceae (Brenner et al., 1999). Furthermore, the G+C content of genomic DNA,

Genus III. Turneriella

at 47–48 mol% (Hovind-Hougen et al., 1981; Neill et al., 1987), is inconsistent with known Leptospira and Leptonema species. The unique position of the type strain among Leptospiraceae led to a proposal for a new genus, Turneria that was approved by the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Leptospiraceae in 1990 (Marshall, 1992). However, the genus name Turneria was found to be illegitimate due to its use as genus names for plants and animals. A description of this bacterial genus was not published in a timely manner, leading to further confusion in the literature, with references to “Turneria” and Leptospira parva incertae sedis. The accepted name of Turneriella was approved by the taxonomic subcommittee in 2005 (Levett and Smythe, 2006) and a description published by Levett et al. (2005).

Enrichment and isolation procedures The type strain and original isolate was obtained from contaminated bovine serum albumin (BSA) based media used for the cultivation of Leptospira (Hovind-Hougen et al., 1981).

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Presumably, the metabolic capabilities are similar to other Leptospiraceae. Cells grow slowly at 13, 30, and 37°C in liquid, solid, and semi-solid BSA-Tween based media (EMJH) capable of supporting the growth of Leptospira.

Maintenance procedures These bacteria are maintained in liquid nitrogen by placing fresh growth in semi-solid BSA-Tween media, e.g., EMJH (Ellinghausen and McCullough, 1965; Johnson and Harris, 1967a), in sterile cryogenic vials. Cultures are frozen slowly to −70°C before long-term storage in liquid nitrogen.

Differentiation of Turneriella from other genera The G+C content of genomic DNA is 47–48 mol%, which is distinct from Leptospira (33–43%) and Leptonema (54%). Phylogenetic analysis of 16S rRNA sequences distinguish Turneriella from other Leptospiraceae (Levett et al., 2005). Morphologically, Turneriella cells are shorter and have a shorter wavelength than other Leptospiraceae.

List of species of the genus Turneriella 1. Turneriella parva (Hovind-Hougen, Ellis and Birch­Andersen 1981) Levett, Morey, Galloway, Steigerwalt and Ellis 2005, 1499VP (Leptospira parva Hovind-Hougen, Ellis and Birch-Andersen 1981, 352) par¢va. L. fem. adj. parva small. In addition to the description of the genus, this ­species  has the following characteristics. Growth is

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i­nhibited by 200 mg 8-azaguanine/ml and 10 mg 2,6­diaminopurine/ml. Source: contaminated bovine serum albumin (BSA)-based media. DNA G+C content (mol%): 47–48 (Tm). Type strain: H, ATCC BAA-1111, NCTC 11395. Sequence accession no. (16S rRNA gene): AY293856, Z21636.

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Neill, S.D., L.R. Reid and W.A. Ellis. 1983. Aminopeptidase activity of Leptospira strains. J. Gen. Microbiol. 129: 395–400. Neill, S.D., R.L. Reid, S.T. Weatherup and W.A. Ellis. 1987. The use of aminopeptidase substrate specificity profiles to identify leptospires. Zentralbl. Bakteriol. Mikrobiol. Hyg. [A] 264: 137–144. Noguchi, H. 1917. Spirochaeta icterohaemorrhagiae in American wild rats and its relation to the Japanese and European strains. J. Exp. Med. 25: 755–763. Noguchi, H. 1918. Morphological characteristics and nomenclature of Leptospira (Spirochaeta) icterohaemorrhagiae (Inada and Ido). J. Exp. Med. 27: 575–592. Oie, S., K. Hironaga, A. Koshiro, H. Konishi and Z. Yoshii. 1983. In vitro susceptibilities of five Leptospira strains to 16 antimicrobial agents. Antimicrob. Agents Chemother. 24: 905–908. Paster, B.J., E. Stackebrandt, R.B. Hespell, C.M. Hahn and C.R. Woese. 1984. The phylogeny of the spirochetes. Syst. Appl. Microbiol. 5: 337–351. Paster, B.J., F.E. Dewhirst, W.G. Weisburg, L.A. Tordoff, G.J. Fraser, R.B. Hespell, T.B. Stanton, L. Zablen, L. Mandelco and C.R. Woese. 1991. Phylogenetic analysis of the spirochetes. J. Bacteriol. 173: 6101–6109. Perolat, P., R.J. Chappel, B. Adler, G. Baranton, D.M. Bulach, M.L. Billinghurst, M. Letocart, F. Merien and M.S. Serrano. 1998. Leptospira fainei sp. nov., isolated from pigs in Australia. Int. J. Syst. Bacteriol. 48: 851–858. Petrino, M.G. and R.N. Doetsch. 1978. ‘Viscotaxis’, a new behavioural response of Leptospira interrogans (biflexa) strain B16. J. Gen. Microbiol. 109: 113–117. Picardeau, M. 2008. Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool. Appl. Environ. Microbiol. 74: 319–322. Picardeau, M., D.M. Bulach, C. Bouchier, R.L. Zuerner, N. Zidane, P.J. Wilson, S. Creno, E.S. Kuczek, S. Bommezzadri, J.C. Davis, A. McGrath, M.J. Johnson, C. Boursaux-Eude, T. Seemann, Z. Rouy, R.L. Coppel, J.I. Rood, A. Lajus, J.K. Davies, C. Medigue and B. Adler. 2008. Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis. PLoS ONE 3: e1607. Que-Gewirth, N.L., A.A. Ribeiro, S.R. Kalb, R.J. Cotter, D.M. Bulach, B. Adler, I.S. Girons, C. Werts and C.R. Raetz. 2004. A methylated phosphate group and four amide-linked acyl chains in Leptospira interrogans lipid A. The membrane anchor of an unusual lipopolysaccharide that activates TLR2. J. Biol. Chem. 279: 25420–25429. Ralph, D. and M. McClelland. 1993. Intervening sequence with conserved open reading frame in eubacterial 23S rRNA genes. Proc. Natl. Acad. Sci. U. S. A. 90: 6864–6868. Ralph, D., M. McClelland, J. Welsh, G. Baranton and P. Perolat. 1993. Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCRamplified rRNA genes. J. Bacteriol. 175: 973–981. Ramadass, P., B.D.W. Jarvis, R.J. Corner, M. Cinco and R.B. Marshall. 1990. DNA Relatedness among Strains of Leptospira biflexa. Int. J. Syst. Bacteriol. 40: 231–235. Ramadass, P., B.D. Jarvis, R.J. Corner, D. Penny and R.B. Marshall. 1992. Genetic characterization of pathogenic Leptospira species by DNA hybridization. Int. J. Syst. Bacteriol. 42: 215–219. Ren, S.X., G. Fu, X.G. Jiang, R. Zeng, Y.G. Miao, H. Xu, Y.X. Zhang, H. Xiong, G. Lu, L.F. Lu, H.Q. Jiang, J. Jia, Y.F. Tu, J.X. Jiang, W.Y. Gu, Y.Q. Zhang, Z. Cai, H.H. Sheng, H.F. Yin, Y. Zhang, G.F. Zhu, M. Wan, H.L. Huang, Z. Qian, S.Y. Wang, W. Ma, Z.J. Yao, Y. Shen, B.Q. Qiang, Q.C. Xia, X.K. Guo, A. Danchin, I. Saint Girons, R.L. Somerville, Y.M. Wen, M.H. Shi, Z. Chen, J.G. Xu and G.P. Zhao. 2003. Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. Nature 422: 888–893. Ristow, P., P. Bourhy, F.W. da Cruz McBride, C.P. Figueira, M. Huerre, P. Ave, I.S. Girons, A.I. Ko and M. Picardeau. 2007. The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathog. 3: e97.

Ritchie, A.E. and H.C. Ellinghausen. 1969. Bacteriophage-like entities associated with a Leptospire. Proceedings of the Electron Microscope Society of America, Baton Rouge, pp. 228–229. Robinson, A.J., P. Ramadass, A. Lee and R.B. Marshall. 1982. Differentiation of subtypes within Leptospira interrogans serovars Hardjo, Balcanica and Tarassovi, by bacterial restriction-endonuclease DNA analysis (BRENDA). J. Med. Microbiol. 15: 331–338. Rocha, T., E.A. Cardoso, A.M. Terrinha, J.F. Nunes, K. Hovind-Hougen and M. Cinco. 1993. Isolation of a new serovar of the genus Leptonema in the family Leptospiraceae. Zentralbl. Bakteriol. 279: 167–172. Rodionov, D.A., A.G. Vitreschak, A.A. Mironov and M.S. Gelfand. 2003. Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes. J. Biol. Chem. 278: 41148–41159. Ryu, E. 1970. Rapid microscopic agglutination test for Leptospira without non-specific reaction. Bull. Off. Int. Epizoot. 73: 49–58. Saint Girons, I., D. Margarita, P. Amouriaux and G. Baranton. 1990. First isolation of bacteriophages for a spirochaete: potential genetic tools for Leptospira. Res. Microbiol. 141: 1131–1138. Segers, R.P., A. van der Drift, A. de Nijs, P. Corcione, B.A. van der Zeijst and W. Gaastra. 1990. Molecular analysis of a sphingomyelinase C gene from Leptospira interrogans serovar hardjo. Infect. Immun. 58: 2177–2185. Segers, R.P., J.A. van Gestel, G.J. van Eys, B.A. van der Zeijst and W. Gaastra. 1992. Presence of putative sphingomyelinase genes among members of the family Leptospiraceae. Infect. Immun. 60: 1707–1710. Sekar, R., D.K. Mills, E.R. Remily, J.D. Voss and L.L. Richardson. 2006. Microbial communities in the surface mucopolysaccharide layer and the black band microbial mat of black band-diseased Siderastrea siderea. Appl. Environ. Microbiol. 72: 5963–5973. Shenberg, E. 1967. Growth of pathogenic Leptospira in chemically defined media. J. Bacteriol. 93: 1598–1606. Slack, A.T., T. Kalambaheti, M.L. Symonds, M.F. Dohnt, R.L. Galloway, A.G. Steigerwalt, W. Chaicumpa, G. Bunyaraksyotin, S. Craig, B.J. Harrower and L.D. Smythe. 2008. Leptospira wolffii sp. nov., isolated from a human with suspected leptospirosis in Thailand. Int. J. Syst. Evol. Microbiol. 58: 2305–2308. Stallman, N.D. 1984. International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Leptospira: Minutes of the Meeting, 6 to 10 August 1982, Boston, Massachusetts. Int. J. Syst. Bacteriol. 34: 258–259. Stallman, N.D. 1987. International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Leptospira: Minutes of the Meeting, 5 and 6 September 1986, Manchester, England. Int. J. Syst. Bacteriol. 37: 472–473. Stamm, L.V. and N.W. Charon. 1988. Sensitivity of pathogenic and freeliving Leptospira spp. to UV radiation and mitomycin C. Appl. Environ. Microbiol. 54: 728–733. Stevenson, B., H.A. Choy, M. Pinne, M.L. Rotondi, M.C. Miller, E. Demoll, P. Kraiczy, A.E. Cooley, T.P. Creamer, M.A. Suchard, C.A. Brissette, A. Verma and D.A. Haake. 2007. Leptospira interrogans endostatin-like outer membrane proteins bind host fibronectin, laminin and regulators of complement. PLoS ONE 2: e1188. Stimson, A.M. 1907. Note on an organism found in yellow-fever tissue. Public Health Rep. 22: 541. Stuart, R.D. 1946. The preparation and use of a simple culture medium for leptospirae. J. Pathol. Bacteriol. 58: 343–349. Terpstra, W.J., H. Korver, J. van Leeuwen, P.R. Klatser and A.H. Kolk. 1985. The classification of Sejroe group serovars of Leptospira interrogans with monoclonal antibodies. Zentralbl. Bakteriol. Mikrobiol. Hyg. A 259: 498–506. Thiermann, A.B., A.L. Handsaker, S.L. Moseley and B. Kingscote. 1985. New method for classification of leptospiral isolates belonging to serogroup pomona by restriction endonuclease analysis: serovar kennewicki. J. Clin. Microbiol. 21: 585–587. Thiermann, A.B., A.L. Handsaker, J.W. Foley, F.H. White and B.F. Kingscote. 1986. Reclassification of North American leptospiral isolates

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Hindgut spirochetes of termites and Cryptocercus punctulatus Bruce J. Paster and John A. Breznak Spirochetes are commonly observed in the hindguts of termites (Table 134) and the wood-eating cockroach Cryptocercus punctulatus (Grimstone, 1963). Early workers referred to these motile bacteria as spirilla (Leidy, 1877) or vibrios (Leidy, 1881), assigned them to currently recognized spirochete genera (see Taxonomic comments), or “spirochetes” (Damon, 1926). Later studies using electron microscopy confirmed that these bacteria were indeed true spirochetes in that they possessed ultrastructural characteristics typical of spirochetes. (Bermudes et  al., 1988). Hindgut spirochetes occur free in the gut fluid as well as attached to the surfaces of hindgut protozoa. Recently, two species of spirochetes from termite hindguts have been grown in pure culture and, on the basis of 16S rRNA sequence comparisons, were determined to be species of Treponema, namely Treponema azotonutricium and Treponema primitia (Graber et al., 2004; see chapter on Treponema, in this volume). However, most of spirochetes from the termite and cockroach hindguts have not been isolated and grown in pure or mixed culture. The size of free hindgut spirochetes ranges from about 0.2 mm in diameter × 3 mm long (Breznak and Pankratz, 1977) to as large as 1.0 mm in diameter × 100 mm long (Hollande and Gharagozlou, 1967). Likewise, the number of periplasmic flagella ranges from a few per cell to as many as 100 or more in the larger forms (Bermudes et al., 1988; To et al., 1978; Wier et al., 2000). However, the multiple periplasmic flagella in most of the large forms do not generally occur in a tight bundle as in Cristispira. Bermudes et al. (1988) presented taxonomic considerations of the large uncultivable hindgut spirochetes as based on size,

number of flagella, amplitude and wavelength of coils, and other ultrastructural traits. These distinctive features include the following: (a) a crenulated outer sheath (Hollande and Gharagozlou, 1967) (Figure 103); (b) a helicoidal groove or “sillon”, an invagination of the outer membrane that appears to be in contact to the inner membrane (Gharagozlou, 1968; Hollande and Gharagozlou, 1967) (Figure 104); (c) the thickness of the outer and inner coat of the outer membrane (Bermudes et al., 1988); (d) a polar organelle (Bermudes et al., 1988); and (e) cytoplasmic tubules (Bermudes et al., 1988). Some hindgut spirochetes attach by one end to the surface of certain flagellate protozoa found only in the lower termites (i.e., families Mastotermitidae, Kalotermitidae, Hodotermitidae, Serritermitidae, and Rhinotermitidae) and Cryptocercus punctulatus. These spirochetes may be uniformly distributed over the surface or localized to specific regions (Ball, 1969; Kirby, 1941). Some spirochetes have a structural modification of one end of the cell in their attachment to the surfaces of Pyrsonympha (from Reticulitermes flavipes and Reticulitermes tibialis) and Barbulanympha (from Cryptocercus punctulatus) (Bloodgood and Fitzharris, 1976; Bloodgood et al., 1974). In contrast, some protozoa have structural modifications to facilitate spirochetal attachment. For example, in Mixotrichia paradoxa in the termite Mastotermes darwiniensis, bracket-like elements in the plasma membranes serve as attachment points (Cleveland and Grimstone, 1964), whereas in polymastigotes from Reticulitermes flavipes the attachment points are screw-like structures (Smith et al., 1975a, b). In other termite species, both the protozoan plasma

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Hindgut spirochetes

Table 134.  Distribution of spirochetes in the hindgut of termites and wood-eating cockroaches Host genus Cockroach: Cryptocercus punctulatus Termite: Bifiditermes condonesis Calcaritermes (Kalotermes) nigriceps Ceratokalotermes spoliator Coptotermes acinaciformis Coptotermes formosanus Coptotermes lacteus Cryptotermes brevis Cryptotermes cavifrons Cryptotermes gearyi Glyptotermes neotuberculatus Glyptotermes (Kalotermes) iridipennis Heterotermes aureus Incisitermes rnilleri Kalotermes (Incisitermes) minor Kalotermes (Incisitermes) schwarzi Kalotermes (Neotermes) jouteli Kalotermes approximatus Kalotermes banksiae Kalotermes flavicollis Kalotermes snyderi Leucopitermes lucifugus Leucopitermes tenuis Marginitermes (Kalotermes) hubbardi Mastotermes darwiniensis Nasutitermes costalis Nasutitermes exitiosus Nasutitermes morio Neotermes castaneus Neotermes insularis Paraneotermes simplicicornis Porotermes adamsoni Postelectrotertnes (Kalotermes) praecox Postelectrotertnes militaris Pterotermes occidentis Reticulitermes flavipes Reticulitermes hageni Reticulitermes hesperus Reticulitermes lucifugus Reticulitermes tibialis Reticulitermes virginicus Zootermopsis angusticollis Zootermopsis nevadensis

Source location USA Australia British Guinea Australia Australia Hawaii Australia USA USA Australia Australia Australia USA USA USA USA USA USA Australia France, Spain USA Bastia, Corsica British Guinea USA Australia Puerto Rico Australia Puerto Rico USA Australia USA Australia Portugal Mexico, USA USA USA USA Japan, Italy USA USA USA USA

membrane and the spirochetal poles are modified to form an attachment complex (Smith and Arnott, 1974). Cleveland and Grimstone (1964) demonstrated that adherent spirochetes serve a locomotory function for Mixotrichia paradoxa, although not likely for propulsion. Based on these observations, Margulis et  al. (1979) proposed that eukaryotic flagella and cilia evolved from ectosymbiotic spirochetes. However, antibiotic treatment of Mixotrichia paradoxa reduces the number of ectobionts and leads to a disintegration of the attachment systems rendering the protozoan immotile (Radek and Nitsch, 2007). Furthermore, the antibiotic treatment causes attached spirochetes to lose their helical shape and form round bodies (Radek and Nitsch, 2007). From the data on the two cultivable hindgut spirochetes (Graber et al., 2004), Treponema azotonutricium ferments carbo-

References Hollande and Gharagozlou (1967) To et al. (1978) Damon (1926) To et al. (1978) To et al. (1978) To et al. (1978) Eutick et al. (1978) Damon (1926); To et al. (1978) To et al. (1978) To et al. (1978) To et al. (1978) To et al. (1978) To et al. (1978) To et al. (1978) To et al. (1978) Damon (1926); To et al. (1978) Margulis et al. (1981) Margulis et al. (1981) Margulis et al. (1981) Gharagozlou (1968); To et al. (1978) Bermudes et al. (1988) Hollande (1922) Damon (1926) To et al. (1978) Cleveland and Grimstone (1964); To et al. (1978) To et al. (1978) Eutick et al. (1978) Damon (1926) To et al. (1978) To et al. (1978) To et al. (1978) To et al. (1978) Hollande and Gharagozlou (1967) Dobell (1910, 1912) To et al. (1978) Breznak (1984); Damon (1926); Leidy (1877, 1881) Damon (1926) Margulis et al. (1981) Ghidini and Archetti (1939); von Prowazek (1910) Bloodgood and Fitzharris (1976) Damon (1926) Damon (1926); To et al. (1978) Damon (1926); To et al. (1978)

hydrates to acetate, ethanol, CO2, and H2 as major products and is noteworthy in that this species does have nitrogenase activity. On the other hand, Treponema primitia ferments carbohydrates only to acetate, with little or no nitrogenase activity. Treponema azotonutricium and Treponema primitia are obligate anaerobes, and it is likely that other hindgut spirochetes are anaerobic as they become nonmotile and begin to disintegrate when exposed to air. Hindgut spirochetes do not invade the hindgut epithelium, and the insects harboring them appear vigorous and healthy. It has been suggested that hindgut spirochetes may benefit the host. Eutick et al. (1978) observed that Nasutitermes exitiosus termites had a reduced life span when spirochetes were eliminated from the hindgut. Motile spirochetes have been observed within the cytoplasm of hindgut protozoa (Kirby, 1941; Margulis et al., 1979;

Hindgut spirochetes

FIGURE 103.  Transmission electron micrograph of transverse section of large spirochete from the hindgut of Reticulitermes flavipes, showing the proposed genus “Pillotina” with a crenulated outer sheath (OS), sillon or groove (G) and periplasmic flagella (PF). Scale bar = 0.2 µm.

To et al., 1978). However, it is not clear whether these intracellular spirochetes are truly symbiotic or were endocytosed into food vacuoles.

Taxonomic comments Early workers classified spirochete-like organisms from termite hindguts as belonging to known spirochete genera such as “Spirochaeta termitis” (Dobell, 1910), “Spirochaeta minei” (von Prowazek, 1910), “Spirochaeta leucotermitis” (Hollande, 1922), and “Spirochaeta staphylina” (Ghidini and Archetti, 1939); “Treponema termitis” and “Treponema minei” (Dobell, 1912); and “Cristispira termitis” (Hollande, 1922). Electron microscopy was later used to confirm that these organisms possessed ultrastructural features characteristic of spirochetes such as a protoplasmic cylinder, periplasmic flagella (internal organelles), and an outer sheath (Margulis and Hinkle, 1992). Based on morphology and electron microscopic studies, new generic and specific epithets for true spirochetes of termite hindguts have been proposed. The framework for the morphometric analysis of large uncultivable spirochetes was formally proposed to revive four earlier-proposed species, namely Pillotina calotermitidis, Diplocalyx calotermitidis, Hollandina pterotermitidis, and ­Clevelandina

References Ball, G.H. 1969. Organisms lining on and in protozoa. Res. Protozool. 3: 567–718. Bermudes, D., D. Chase and L. Margulis. 1988. Morphology as a basis for taxonomy of large spirochetes symbiotic in wood-eating cockroaches and termites: Pillotina gen. nov., nom. rev., Pillotina calotermitidis sp. nov., nom. rev., Diplocalyx gen. nov., nom. rev., Diplocalyx calotermitidis sp. nov., nom. rev., Hollandina gen. nov., nom. rev., Hollandina pterotermitidis sp. nov., nom. rev., and Clevelandina reticulitermitidis gen. nov., sp. nov. Int. J. Syst. Bacteriol. 38: 291–302.

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FIGURE 104.  Transmission electron micrograph of transverse section of large spirochete from the hindgut of Reticulitermes flavipes, showing the proposed genus “Clevelandina” with sillon or groove (G), outer sheath (OS), and periplasmic flagella (PF). Scale bar = 0.2 µm.

r­ eticulitermitidis (Bermudes et  al., 1988; Margulis and Hinkle, 1992). More recently, also on the basis of morphometric analysis, two new, large pillotinaceous spirochetes, “Canaleparolina darwiniensis” and “Diplocalyx cryptotermitidis”, have been proposed (Wier et al., 2000). “Canaleparolina darwiniensis”, the large spirochete attached to the protozoan Mixotrichia paradoxa, is 0.5 mm × 25 mm in length and has multiple flagella in a 16:32:16 flagellar arrangement (Wier et  al., 2000). “Diplocalyx cryptotermitidis”, observed in the hindguts of the dry wood-eating termite Cryptotermes cavifrons, is smaller in diameter and has fewer flagella than the other large hindgut spirochetes. Spirochetal sequences have been obtained from 16S rRNA clonal analysis of enrichments of Mixtotricha paradoxa and are deposited in GenBank under the accession numbers AJ458944, AJ458945, AJ458946, and AJ458947. These sequences represent four separate phylotypes that cluster deeply with the genus Treponema at about 90% similarity to treponemal phylotypes from termite hindguts. However, in situ hybridization experiments have not yet been performed to verify that the sequences obtained were derived from the spirochetes attached to Mixotrichia paradoxa. Consequently, these large spirochetes from the hindguts of termites and Cryptocercus punctulatus will be included in the family Spirochaetaceae until pure cultures are obtained and the phylogenetic placement among the spirochetal genera has been verified. Bloodgood, R.A., K.R. Miller, T. Fitzharris and J.R. McIntosh. 1974. The ultrastructure of Pyrsonympha and its associated microorganisms. J. Morphol. 143: 77–105. Bloodgood, R.A. and T.P. Fitzharris. 1976. Specific associations of prokaryotes with symbiotic flagellate protozoa from hindgut of termite Reticulitermes and wood-eating roach Cryptocercus. Cytobios 17: 103–122. Breznak, J.A. and H.S. Pankratz. 1977. In situ morphology of the gut microbiota of wood-eating termites (Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki). Appl. Environ. Microbiol. 33: 406–426.

566

Hindgut spirochetes

Breznak, J.A. 1984. Hindgut spirochetes of termites and Cryptocercus puntulatus. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 67–70. Cleveland, L.R. and A.V. Grimstone. 1964. The fine structure of the flagellate Mixotrichia paradoxa and its associated micro-organisms. Proc. R. Soc. Lond. Ser. B Biol. Sci. 159: 668–686. Damon, S.R. 1926. A note on the spirochaetes of termites. J. Bacteriol. 11: 31–36. Dobell, C.C. 1910. On some parasitic protozoa from Ceylon. Spolia Zeylanica 7: 65–87. Dobell, C.C. 1912. Researches on the spirochaetes and related organisms. Arch. Protistenkd. 26: 117–240. Eutick, H.L., P. Veivers, R.W. O’Brien and M. Slaytor. 1978. Dependence of higher termite, Nasutitermes exitiosus and lower termite, Coptotermes lacteus on their gut flora. J. Insect Physiol. 24: 363–368. Gharagozlou, I.D. 1968. Aspect infrastructural de Diplocalyx calotermitidis nov. gen., nov. sp., spirochaetale de l’intestin de Calotermes flavicollis. C. R. Acad. Sci. Ser. D 266: 494–496. Ghidini, G.M. and I. Archetti. 1939. Studi sulle termit; 2 - Le spirochete presenti in Reticulitermes lucifugus. Rossi. Riv. Biol. Coloniale 2: 125– 140. Graber, J.R., J.R. Leadbetter and J.A. Breznak. 2004. Description of Treponema azotonutricium sp. nov. and Treponema primitia sp. nov., the first spirochetes isolated from termite guts. Appl. Environ. Microbiol. 70: 1315–1320. Grimstone, A.V. 1963. A note on the fine structure of a spirochaete. Q. J. Microsc. Sci. 104: 145–153. Hollande, A.C. 1922. Les spirochètes de termites; processus de division: formation du schizoplaste. Arch. Zool. Espt. Gén. Notes Rev. 61: 23–28. Hollande, A.C. and I.D. Gharagozlou. 1967. Morphologie infrastructurale de Pillotina calotermitidis nov. gen., nov. sp. spirochaetale de l’intestin de Calotermes praecox. C. R. Acad. Sci. 265: 1309–1312. Kirby, H., Jr. 1941. Organisms living on and in Protozoa. In Protozoa in Biological Research. Columbia University Press, New York, pp. 1009–1113.

Leidy, J. 1877. On intestinal parasites of Termes flavipes. Proc. Acad. Nat. Sci. 29: 146–149. Leidy, J. 1881. The parasites of the termites. J. Acad. Nat. Sci. 8: 425–447. Margulis, L., D. Chase and L.P. To. 1979. Possible evolutionary significance of spirochaetes. Proc. R. Soc. Lond. B 204: 189–198. Margulis, L., L.P. To and D. Chase. 1981. The genera Pillotina, Hollandina and Diplocalyx. In The Prokaryotes: a Handbook on Habitats, Isolation, and Identification of Bacteria (edited by Starr, Stolp, Trüper, Balows and Schlegel). Springer, New York, pp. 548–554. Margulis, L. and G. Hinkle. 1992. Large symbiotic spirochetes: Clevelandina, Cristispira, Diplocalyx, Hollandina, and Pillotina. In The Prokaryotes: a Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, 2nd edn (edited by Balows, Trüper, Dworkin, Harder and Schleifer). Springer, New York, pp. 3965–3978. Radek, R. and G. Nitsch. 2007. Ectobiotic spirochetes of flagellates from the termite Mastotermes darwiniensis: attachment and cyst formation. Eur. J. Protistol. 43: 281–294. Smith, H.E. and H.J. Arnott. 1974. Epibiotic and endobiotic bacteria associated with Pyrsonympha vertens-symbiotic protozoan of the termite Reticulitermes flavipes. Trans. Am. Microsc. Soc. 93: 180–194. Smith, H.E., H.E. Buhse and S.J. Stamler. 1975a. Possible formation and development of spirochaete attachment sites found on the surface of symbiotic polymastigote flagellates of the termite Reticulitermes flavipes. BioSystems 7: 374–379. Smith, H.E., S.J. Stamler and H.E. Buhse. 1975b. A scanning electron microscope survey of the surface features of polymastigote flagellates from Reticulitermes flavipes. Trans. Am. Microsc. Soc. 94: 401–410. To, L.P., L. Margulis and A.T.W. Cheung. 1978. Pillotinas and hollandinas: distribution and behavior of large spirochetes symbiotic in termites. Microbios 22: 103–133. von Prowazek, S. 1910. Parasitische Protozoen aus Japan, gesammelt von Herrn Dr. Mine in Fukuoka. Arch. Schiffs-Trop. Hyg. 14: 297–302. Wier, A., J. Ashen and L. Margulis. 2000. Canaleparolina darwiniensis, gen. nov., sp. nov., and other pillotinaceous spirochetes from insects. Int. Microbiol. 3: 213–223.

Phylum XVI. Tenericutes Murray 1984a, 356VP (Effective publication: Murray 1984b, 33.) Daniel R. Brown Ten.er¢i.cutes. L. adj. tener tender; L. fem. n. cutis skin; N.L. fem. n. Tenericutes prokaryotes of a soft pliable nature indicative of a lack of a rigid cell wall.

Members of the Tenericutes are wall-less bacteria that do not synthesize precursors of peptidoglycan.

Further descriptive information The nomenclatural type by monotypy (Murray, 1984a) is the class Mollicutes, which consists of very small prokaryotes that are devoid of cell walls. Electron microscopic evidence for the absence of a cell wall was mandatory for describing novel species of mollicutes until very recently. Genes encoding the pathways for peptidoglycan biosynthesis are absent from the genomes of more than 15 species that have been annotated to date. Some species do possess an extracellular glycocalyx. The absence of a cell wall confers such mechanical plasticity that most mollicutes are readily filterable through 450 nm pores and many species have some cells in their populations that are able to pass through 220 nm or even 100 nm filters. However, they may vary in shape from coccoid to flask-shaped cells or helical filaments that reflect flexible cytoskeletal elements.

Taxonomic comments To provide greater definition and formal nomenclature for vernacular names used in the 8th edition of Bergey’s Manual of Determinative Bacteriology (Bergey VIII; Buchanan and Gibbons, 1974), Gibbons and Murray (1978) proposed that the higher taxa of prokaryotes be subdivided primarily according to the presence and character, or absence, of a rigid or semirigid cell wall as reflected in the determinative Gram reaction. Similar to the non-hierarchical groupings of Bergey VIII, which were based on a few readily determined criteria, the “wall-deficient” organisms grouped together in the first edition of The Prokaryotes included the mollicutes (Starr et al., 1981). While acknowledging the emerging 16S rRNA-based evidence that indicated a phylogenetic relationship between mollicutes and certain Gram-stain-positive bacteria in the division Firmicutes, Murray (1984b) proposed the separate division Tenericutes for the stable and distinctive group of wall-less species that are not simply an obvious subset of the Firmicutes. The approved divisional rank of Tenericutes and the assignment of class Mollicutes as its nomenclatural type (Murray, 1984a) were adopted by the International Committee on Systematic Bacteriology’s Subcommittee on the Taxonomy of Mollicutes (Tully, 1988) and subsequent valid taxonomic descriptions assigned novel species of mollicutes to the Tenericutes. However, the second (1992) and third (2007) editions of The Prokaryotes described the mollicutes instead as Firmicutes with low G+C DNA. The Subcommittee considered this to be an unfortunate ­grouping: “While

the ­organisms are evolutionarily related to certain clostridia, the absence of a cell wall cannot be equated with Gram reaction positivity or with other members of the Firmicutes. It is unfortunate that workers involved in determinative bacteriology have a reference in which wall-free prokaryotes are described as Gram-positive bacteria” (Tully, 1993a). Despite numerous valid assignments of novel species of mollicutes to the Tenericutes during the intervening years, the class Mollicutes was still included in the phylum Firmicutes in the most recent revision of the Taxonomic Outline of Bacteria and Archaea (TOBA), which is based solely on the phylogeny of 16S rRNA genes (Garrity et al., 2007). The taxon Tenericutes is not recognized in the TOBA, although paradoxically it is the phylum consisting of the Mollicutes in the most current release of the Ribosomal Database Project (Cole et al., 2009). Mollicutes are specifically excluded from the most recently emended description of the Firmicutes in Bergey’s Manual of ­Systematic Bacteriology (2nd edition, volume 3; De Vos et  al., 2009) on the grounds of their lack of rigid cell walls plus analyses of strongly supported alternative universal phylogenetic markers, including RNA polymerase subunit B, the chaperonin GroEL, several different aminoacyl tRNA synthetases, and subunits of F0F1-ATPase (Ludwig et  al., 2009; Ludwig and Schleifer, 2005). The taxonomic dignity of Tenericutes bestowed by its original formal validation, and upheld by a quarter of a century of valid descriptions of novel species of mollicutes, has therefore been respected in this volume of Bergey’s Manual. Type order: Mycoplasmatales Freundt 1955, 71AL emend. Tully, Bové, Laigret and Whitcomb 1993b, 382.

References Buchanan, R.E. and N.E. Gibbons (editors). 1974. Bergey’s Manual of Determinative Bacteriology, 8th edn. Williams & Wilkins, Baltimore. Cole, J.R., Q. Wang, E. Cardenas, J. Fish, B. Chai, R.J. Farris, A.S. KulamSyed-Mohideen, D.M. McGarrell, T. Marsh, G.M. Garrity and J.M. Tiedje. 2009. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 37: (Database issue): D141–D145. De Vos, P., G. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.H. Schleifer and W.B. Whitman. 2009. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3. Springer, New York. Freundt, E.A. 1955. The classification of the pleuropneumoniae group of organisms (Borrelomycetales). Int. Bull. Bacteriol. Nomencl. Taxon. 5: 67–78. Garrity, G.M., T.G. Lilburn, J.R. Cole, S.H. Harrison, J. Euzéby and B.J. Tindall. 2007. The Taxonomic Outline of the Bacteria and Archaea, Release 7.7, Part 11 – The Bacteria: Phyla Planctomycetes, Chlamydiae, Spirochaetes, Fibrobacteres, Acidobacteria, Bacteroidetes, Fusobacteria,

567

568

Phylum XVI. Tenericutes

­ errucomicrobia, Dictyoglomi, Gemmatomonadetes, and Lentisphaerae. pp. V 540–595. (http://www.taxonomicoutline.org/). Gibbons, N.E. and R.G.E. Murray. 1978. Proposals concerning the higher taxa of bacteria. Int. J. Syst. Bacteriol. 28: 1–6. Ludwig, W. and K.H. Schleifer. 2005. Molecular phylogeny of bacteria based on comparative sequence analysis of conserved genes. In Microbial Phylogeny and Evolution, Concepts and Controversies, (edited by Sapp). Oxford University Press, New York, pp. 70–98. Ludwig, W., K.H. Schleifer and W.B. Whitman. 2009. Revised road map to the phylum Firmicutes. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3, The Firmicutes (edited by De Vos, Garrity, Jones, Krieg, Ludwig, Rainey, Schleifer and Whitman). Springer, New York, pp. 1–13. Murray, R.G.E. 1984a. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 15. Int. J. Syst. Bacteriol. 34: 355–357. Murray, R.G.E. 1984b. The higher taxa, or, a place for everything…? In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 31–34.

Starr, M.P., H. Stolp, H.G. Trüper, A. Balows and H.G. Schlegel ­(editors). 1981. The Prokaryotes. Springer, Berlin. Tully, J.G. 1988. International Committee on Systematic Bacteriology, Subcommittee on the Taxonomy of Mollicutes, Minutes of the Interim Meeting, 25 and 28 August 1986, Birmingham, Alabama. Int. J. Syst. Bacteriol. 38: 226–230. Tully, J.G. 1993a. International Committee on Systematic Bacteriology, Subcommittee on the Taxonomy of Mollicutes, Minutes of the Interim Meetings, 1 and 2 August, 1992, Ames, Iowa. Int. J. Syst. ­Bacteriol. 43: 394–397. Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993b. Revised ­taxonomy of the class Mollicutes – proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. ­Bacteriol. 43: 378–385.

Class I. Mollicutes Edward and Freundt 1967, 267AL Daniel R. Brown, Meghan May, Janet M. Bradbury and Karl-Erik Johansson Mol¢li.cutes or Mol.li.cu¢tes. L. adj. mollis soft, pliable; L. fem. n. cutis skin; N.L. fem. pl. n. Mollicutes class with pliable cell boundary. Very small prokaryotes totally devoid of cell walls. Bounded by a plasma membrane only. Incapable of synthesis of peptidoglycan or its precursors. Consequently resistant to penicillin and its derivatives and sensitive to lysis by osmotic shock, detergents, alcohols, and specific antibody plus complement. Gramstain-negative due to lack of cell wall, but constitute a distinct phylogenetic lineage within the Gram-stain-positive bacteria (Woese et  al., 1980). Pleomorphic, varying from spherical or flask-shaped structures to branched or helical filaments. The coccoid and flask-shaped cells usually range from 200–500 nm in diameter, although cells as large as 2000 nm have been seen. Replicate by binary fission, but genome replication may precede cytoplasmic division, leading to the formation of multinucleated filaments. Colonies on solid media are very small, usually much less than 1 mm in diameter. The organisms tend to penetrate and grow inside the solid medium. Under suitable conditions, almost all species form colonies that have a characteristic fried-egg appearance. Usually nonmotile, but some species show gliding motility. Species that occur as helical filaments show rotary, flexional, and translational motility. No resting stages are known. The species recognized so far can be grown on artificial cellfree media of varying complexity, although certain strains may be more readily isolated by cell-culture procedures. Many “Candidatus” species have been proposed and characterized at the molecular level, but not yet cultivated axenically. Most cultivable species require sterols and fatty acids for growth. However, members of some genera can grow well in either serum-free media or serum-free media supplemented with polyoxyethylene sorbitan. Most species are facultative anaerobes, but some are obligate anaerobes that are killed by exposure to minute quantities of oxygen. No tricarboxylic acid cycle enzymes, quinones, or cytochromes have been found. All mollicutes are commensals or parasites, occurring in a wide range of vertebrate, insect, and plant hosts. Many are significant­

pathogens of humans, animals, insects, or plants. Genome sizes range from 580 to 2200 kbp, among the smallest recorded in prokaryotes. The genomes of more than 20 species have been completely sequenced and annotated to date (Table 135). The G+C content of the DNA is usually low, ~23–34 mol%, but in some species is as high as ~40 mol% (Bd, Tm). Can be distinguished from other bacteria in having only one or two rRNA operons (one species of Mesoplasma has three) and an RNA polymerase that is resistant to rifampin. The 5S rRNA contains fewer nucleotides than that of other bacteria and there are fewer tRNA genes. In some genera, instead of a stop, the UGA codon encodes tryptophan. Plasmids and viruses (phage) occur in some species. Type order: Mycoplasmatales Freundt 1955, 71AL emend. Tully, Bové, Laigret and Whitcomb 1993, 382.

Further descriptive information Table 136 summarizes the present classification of the Mollicutes into families and genera and provides the major distinguishing characteristics of these taxa. The trivial term mycoplasma has been used to denote any species included in the class Mollicutes, but the term mollicute(s) is now considered most appropriate as the trivial name for all members of the class, so that the trivial name mycoplasma can be retained only for members of the genus Mycoplasma. Hemotropic mycoplasmas are referred to by the trivial name hemoplasmas. The trivial names ureaplasma, entomoplasma, mesoplasma, spiroplasma, acholeplasma, anaeroplasma, and asteroleplasma are commonly used when reference is made to members of the corresponding genus. Their 16S rRNA gene sequences usefully place the mollicutes into phylogenetic groups ( Johansson, 2002; Weisburg et  al., 1989) and an analysis of 16S rRNA gene sequences is now mandatory for characterization of novel species (Brown et  al., 2007). 16S rRNA gene sequences have also shown that certain ­hemotropic bacteria, previously considered to be ­members of the Rickettsia, belong to the order Mycoplasmatales.

0.82 30

631

nd

80

Dybvig et al. (2008)

PG2T

0.88 29

742

50

87

SirandPugnet et al. (2007)

Strain

Size (mb) DNA G+C content (mol%) Open reading frames Hypothetical genes (%) Coding density (%) References

Mycoplasma arthritidis

Mycoplasma capricolum subsp. capricolum J. Glass and others, unpublished.

88

nd

812

ATCC 27343T 1.0 23

Mycoplasma conjunctivae

91

37

726

0.99 31

R

Calderon- Papazisi Copete et al. et al. (2003) (2009)

90

45

727

0.85 28

HRC/581T

Mycoplasma genitalium Fraser et al. (1995)

90

21

475

0.58 31

G-37T

Mycoplasma hominis

Mycoplasma hyopneumoniae b 87

nd

657

0.90 28

JT

Mycoplasma mobile 90

27

633

0.78 25

163KT

Mycoplasma mycoides subsp. mycoides SC 80

41

1016

1.21 24

PG1T

88

41

1037

1.36 26

HF-2

Mycoplasma penetrans

Pereyre Vasconcelos Jaffe Westberg Sasaki et al. et al. et al. et al. et al. (2009) (2005) (2004) (2004) (2002)

90

36

537

0.67 27

PG21T

Mycoplasma pneumoniae Himmelreich et al. (1996)

87

38

689

0.82 40

M129

b

a

91

33

659

0.80 28

53

Mycoplasma synoviae Cham­baud Vasconcelos et al. (2001) et al. (2005)

90

38

782

0.96 26

UAB CTIP

Mycoplasma pulmonis

nd, Not determined; na, not available. Mycoplasma hyopneumoniae strains 232 and 7448 were sequenced by Minion et al. (2004) and Vasconcelos et al. (2005). c Data for Ureaplasma parvum and Ureaplasma urealyticum refer to serovars 3 and 10, respectively; serovars 1–14 were sequenced and deposited directly into GenBank. d AYWB, Aster yellows witches’ broom; the onion yellows strain was sequenced by Oshima et al. (2004).

158L3-1

Mycoplasma agalactiae

Species

Mycoplasma gallisepticum

Table 135.  Characteristics of sequenced mollicute genomesa

Mesoplasma florum Knight et al. (2004)

92

nd

687

0.79 27

L1T

Ureaplasma parvumc Glass et al. (2000)

91

33

653

ATCC 700970 0.75 25

Ureaplasma urealyticumc na

89

nd

692

ATCC 33699 0.87 25

Acholeplasma laidlawii na

90

nd

1433

1.5 31

PG8T

Bai et al. (2006)

73

nd

671

0.71 26

AYWB

“Candidatus Phytoplasma asteris”d

Mycoplasmataceae Mycoplasmataceae Incertae sedis Incertae sedis Entomoplasmataceae Entomoplasmataceae Spiroplasmataceae Acholeplasmataceae Incertae sedis Anaeroplasmataceae Anaeroplasmataceae

Family

Numbers of species: valid, Candidatus, incertae sedis, invalid.

Mycoplasma Ureaplasma Eperythrozoon Haemobartonella Entomoplasma Mesoplasma Spiroplasma Acholeplasma “Candidatus Phytoplasma” Anaeroplasma Asteroleplasma

Genus

c

H, human; A, vertebrate animal; N, invertebrate animal; P, plant. d Affiliation of the constituent genera within the Mycoplasmatales has not been formalized.

b

a

nd, Not determined; PES, polyoxyethylene sorbitan.

I. Mycoplasmatales I. Mycoplasmatales I. Mycoplasmatalesd I. Mycoplasmatalesd II. Entomoplasmatales II. Entomoplasmatales II. Entomoplasmatales III. Acholeplasmatales III. Acholeplasmatales IV. Anaeroplasmatales IV. Anaeroplasmatales

Order

Table 136.  Description of the class Mollicutes a

116, 9, 1, 4 7, 0, 0, 0 4, 0, 0, 0 1, 0, 0, 0 6, 0, 0, 0 11, 0, 0, 0 37, 0, 0, 0 18, 0, 0, 0 0, 27, 0, 0 4, 0, 0, 0 1, 0, 0, 0

Speciesb 580–1,350 760–1,140 Nd Nd 870–900 825–930 780–2,220 1,500–1,650 530–1,350 1,500–1,600 1,500

Genome size range (kbp) + + nd nd + − + − nd + −

Cholesterol requirement

H, A H, A A A N, P N, P N, P A, N, P N, P A A

Habitatc

Not yet cultured in vitro Strictly anaerobic Strictly anaerobic

Growth with PES Helical morphology

Urea hydrolysis Hemotropic Hemotropic

Defining features

570 Phylum XVI. Tenericutes

Phylum XVI. Tenericutes

The ­phytoplasmas, a large group of uncultivated mollicutes occurring as agents that can cycle between plant and invertebrate hosts, have been given a provisional “Candidatus Phytoplasma” genus designation. The 16S rRNA gene sequences from at least ten unique phylotypes, recently discovered among the human microbial flora through global 16S rRNA gene PCR (Eckburg et al., 2005), cluster distinctly enough to suggest the existence of a yet-uncircumscribed order within the class (May et al., 2009). Non-helical mollicutes isolated from insects and plants have been placed in the order Entomoplasmatales, the two genera of which are distinguished by their requirement for cholesterol (Tully et al., 1993). Members of the genus Entomoplasma require cholesterol; those of the genus Mesoplasma do not. However, sterol requirements do not correlate well with phylogenetic analyses in other groups. At least four species of spiroplasmas do not require sterol for growth, but they do not form a phylogenetic group. Within the order including the obligately anaerobic mollicutes Anaeroplasmatales, members of the genus Anaeroplasma require sterols for growth, whereas members of the genus Asteroleplasma do not (Robinson et al., 1975; Robinson and Freundt, 1987). Thus, sterol requirement is a useful phylogenetic marker only in the Acholeplasmatales and Anaeroplasmatales. In the past, there was some risk of confusing mollicutes with wall-less “L (Lister)-phase” variants of certain other bacteria, but simple PCR-based analyses of 16S rRNA or other gene sequences now obviate that concern. Wall-less members of the genus Thermoplasma, previously assigned to the Mollicutes, are Archaea and differ from all other members of this class in their 16S rRNA nucleotide sequences plus a number of important features relating to their mode of life and metabolism. Thus, they are quite unrelated to this class (Fox et al., 1980; Razin and Freundt, 1984; Woese et al., 1980). Members of the Erysipelothrix line of descent, also formerly assigned to the Mollicutes, are now assigned to the class Erysipelotrichi in the phylum Firmicutes (Stackebrandt, 2009; Verbarg et al., 2004).

Taxonomic comments The origin of mollicutes and their relationships to other prokaryotes was controversial for many years, especially since their small genomes and comparative phenotypic simplicity suggested that they might have descended from a primitive organism. The first comparative phylogenetic analysis of the origin of mollicutes was carried out by oligonucleotide mapping of 16S rRNA gene sequences by Woese et al. (1980). The organisms then assigned to the genera Mycoplasma, Spiroplasma, and Acholeplasma seemed to have arisen by reductive evolution as a deep branch of the clostridial lineage leading to the genera Bacillus and Lactobacillus. This relationship had been proposed earlier (Neimark, 1979) because the low G+C mollicutes, streptococci, and lactic acid bacteria share characteristic enzymes. In particular, acholeplasma and streptococcus aldolases show high amino acid sequence similarity. These findings were generally confirmed by studies of 5S rRNA gene sequences (Rogers et al., 1985), which included a number of acholeplasmas, anaeroplasmas, mycoplasmas, ureaplasmas, and Clostridium innocuum. Dendrograms constructed from evolutionary distance matrices indicated that the mollicutes form a coherent phylogenetic group that developed as

571

a branch of the Firmicutes. The initial event in this evolution was proposed to be the formation of the Acholeplasma branch, although the position of the Anaeroplasma species (Anaeroplasma bactoclasticum and Anaeroplasma abactoclasticum) was not definitely established within these dendrograms. Formation of the acholeplasmas may have coincided with a reduction in genome size to about 1500–1700 kb and loss of the cell wall. With a genome size similar to the acholeplasmas, the spiroplasmas may have formed from the acholeplasmas. Later independent genome reductions to 500–1000 kb may have led to the origins of the sterol-requiring mycoplasma and ureaplasma lineages. The more extensive phylogenetic analysis of Weisburg et al. (1989) examined the 16S rRNA gene sequences of about 50 species of mollicutes and confirmed a number of these observations and provided additional insights into mollicute evolution. These results also indicated that the acholeplasmas formed upon the initial divergence of mollicutes from clostridial ancestors. Further divergence of this stem led to the sterolrequiring, anaerobic Anaeroplasma and the non-sterol-requiring Asteroleplasma branches. The Spiroplasma branch also appeared to originate from within the acholeplasmas, with further evolution leading to a series of repeated and independent genome reductions from nearly 2000 kb to 600–1200 kb to yield the Mycoplasma and Ureaplasma lineages. Based on the phylogeny of 16S rRNA genes, the class ­Mollicutes was included in the phylum Firmicutes in the most recent revision of the Taxonomic Outline of Bacteria and Archaea ­(Garrity et  al., 2007). However, the Mollicutes are excluded from the most recently emended description of the Firmicutes (De Vos et al., 2009) based on alternative phylogenetic markers, including RNA polymerase subunit B, the chaperonin GroEL, several different aminoacyl tRNA synthetases, and subunits of F0F1-ATPase (Ludwig and Schleifer, 2005). The Weisburg et  al. (1989) study also proposed five additional phylogenetic groupings within the mollicutes, including the anaeroplasma, asteroleplasma, spiroplasma, pneumoniae, and hominis groups (Figure 105). Phytoplasmas are similar to acholeplasmas in their 16S rRNA gene sequences and UGA codon usage (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004). They probably diverged from acholeplasmas at about the same time as the split of spiroplasmas into helical and non-helical lineages (Maniloff, 2002). The modern species concept for mollicutes is justified principally by DNA–DNA hybridization, serology, and 16S rRNA gene sequence similarity (Brown et al., 2007). A large number of individual species have been assigned to phylogenetic groups, clusters, and subclusters that also share other characteristics, although the cluster boundaries are sometimes subjective (Harasawa and Cassell, 1996; Johansson, 2002; Pettersson et al., 2000, 2001). Lastly, the type order Mycoplasmatales is assigned to the class as this clearly appeared to be the intention of Edward and Freundt (1967) in their paper entitled “Proposal for Mollicutes as name of the class established for the order Mycoplasmatales”.

Acknowledgements The lifetime achievements in mycoplasmology and major contributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged. Daniel R. Brown and Meghan May were supported by NIH grant 5R01GM076584.

572

Phylum XVI. Tenericutes Mycoplasma hominis Ureaplasma urealyticum Mycoplasma pneumoniae

*

Mycoplasma coccoides Spiroplasma apis Mycoplasma mycoides subsp. mycoides Entomoplasma ellychniae Mesoplasma florum Spiroplasma citri Spiroplasma ixodetis Acholeplasma laidlawii ‘Candidatus Phytoplasma’ strain OY-M Anaeroplasma abactoclasticum Asteroleplasma anaerobium Clostridium innocuum

Scale:

0.1 substitutions / site

Figure 105.  Phylogenetic grouping of the class Mollicutes. The phylogram was based on a Jukes–Cantor corrected distance matrix and weighted neighbor-joining analysis of the 16S rRNA gene sequences of the type genera, plus representatives of other major clusters within the Mycoplasmatales and Entomoplasmatales and a phytoplasma. Clostridium innocuum was the outgroup. All bootstrap values (100 replicates) are >50% except where indicated (asterisk).

Further reading Barile, M.F., S. Razin, J.G. Tully and R.F. Whitcomb (Editors). 1979, 1985, 1989. The Mycoplasmas (five volumes). Academic Press, New York. Maniloff, J., R.N. McElhaney, L.R. Finch and J.B. Baseman (editors). 1992. Mycoplasmas: Molecular Biology and Pathogenesis. American Society for Microbiology, Washington, D.C. Murray, R.G.E. 1984. The higher taxa, or, a place for everything…?. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 31–34. Razin, S. and J.G.E. Tully. 1995. Molecular and Diagnostic Procedures in Mycoplasmology, vol. 1. Academic Press, San Diego.

References Bai, X., J. Zhang, A. Ewing, S.A. Miller, A. Jancso Radek, D.V. Shevchenko, K. Tsukerman, T. Walunas, A. Lapidus, J.W. Campbell and S.A. Hogenhout. 2006. Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts. J. Bacteriol. 188: 3682–3696. Brown, D., R. Whitcomb and J. Bradbury. 2007. Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. Calderon-Copete, S.P., G. Wigger, C. Wunderlin, T. Schmidheini, J. Frey, M.A. Quail and L. Falquet. 2009. The Mycoplasma conjunctivae genome sequencing, annotation and analysis. BMC Bioinformatics 10 Suppl 6: S7. Chambaud, I., R. Heilig, S. Ferris, V. Barbe, D. Samson, F. Galisson,  I. Moszer, K. Dybvig, H. Wroblewski, A. Viari, E.P. Rocha and A. Blanchard. 2001. The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis. Nucleic Acids Res. 29: 2145–2153.

Taylor-Robinson, D. and J. Bradbury. 1998. Mycoplasma diseases. In Topley and Wilson’s Principles and Practice of Microbiology, vol. 3 (edited by Hausler and Sussman). Edward Arnold, London, pp. 1013–1037. Taylor-Robinson, D. and J.G. Tully. 1998. Mycoplasmas, ureaplasmas, spiroplasmas, and related organisms. In Topley and Wilson, Principles and Practice of Microbiology, 9th edn, vol. 2 (edited by Balows and Duerden). Arnold Publishers, London, pp. 799–827. Tully, J.G. and S. Razin (editors). 1996. Molecular and ­Diagnostic ­Procedures in Mycoplasmology, vol. 2. Academic Press, San Diego, CA.

De Vos, P., G. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K. H. Schleifer and W.B. Whitman. 2009. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3. Springer, New York. Dybvig, K., C. Zuhua, P. Lao, D.S. Jordan, C.T. French, A.H. Tu and A.E. Loraine. 2008. Genome of Mycoplasma arthritidis. Infect. Immun. 76: 4000–4008. Eckburg, P., E. Bik, C. Bernstein, E. Purdom, L. Dethlefsen, M. Sargent, S. Gill, K. Nelson and D. Relman. 2005. Diversity of the human intestinal microbial flora. Science 308: 1635–1638. Edward, D.G.ff. and E.A. Freundt. 1967. Proposal for Mollicutes as name of the class established for the order Mycoplasmatales. Int. J. Syst. ­Bacteriol. 17: 267–268. Fox, G.E., E. Stackebrandt, R.B. Hespell, J. Gibson, J. Maniloff, T.A. Dyer, R.S. Wolfe, W.E. Balch, R.S. Tanner, L.J. Magrum, L.B. Zablen, R. Blakemore, R. Gupta, L. Bonen, B.J. Lewis, D.A. Stahl, K.R. Luehrsen, K.N. Chen and C.R. Woese. 1980. The phylogeny of prokaryotes. Science 209: 457–463. Fraser, C.M., J.D. Gocayne, O. White, M.D. Adams, R.A. Clayton, R.D. Fleischmann, C.J. Bult, A.R. Kerlavage, G. Sutton, J.M. Kelley and

Phylum XVI. Tenericutes et al. 1995. The minimal gene complement of Mycoplasma genitalium. Science 270 : 397–403. Freundt, E.A. 1955. The classification of the pleuropneumoniae group of organisms (Borrelomycetales). Int. Bull. Bacteriol. Nomencl. Taxon. 5: 67–78. Garrity, G.M., T.G. Lilburn, J.R. Cole, S.H. Harrison, J. Euzéby and B.J. Tindall. 2007. The Taxonomic Outline of the Bacteria and Archaea, Release 7.7, Part 11 – The Bacteria: phyla Planctomycetes, Chlamydiae, Spirochaetes, Fibrobacteres, Acidobacteria, Bacteroidetes, ­Fusobacteria, ­Verrucomicrobia, Dictyoglomi, Gemmatomonadetes, and ­Lentisphaerae. pp. 540–595. (http://www.taxonomicoutline.org/). Glass, J.I., E.J. Lefkowitz, J.S. Glass, C.R. Heiner, E.Y. Chen and G.H. Cassell. 2000. The complete sequence of the mucosal pathogen Ureaplasma urealyticum. Nature 407: 757–762. Harasawa, R. and G.H. Cassell. 1996. Phylogenetic analysis of genes coding for 16S rRNA in mammalian ureaplasmas. Int. J. Syst. Bacteriol. 46: 827–829. Himmelreich, R., H. Hilbert, H. Plagens, E. Pirkl, B.C. Li and R. Herrmann. 1996. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res. 24: 4420–4449. IRPCM Phytoplasma/Spiroplasma Working Team - Phytoplasma Taxonomy Group. 2004. Description of the genus ‘Candidatus Phytoplasma’, a taxon for the wall-less non-helical prokaryotes that colonize plant phloem and insects. Int. J. Syst. Evol. Microbiol. 54: 1243–1255. Jaffe, J.D., N. Stange-Thomann, C. Smith, D. DeCaprio, S. Fisher, J. Butler, S. Calvo, T. Elkins, M.G. Fitzgerald, N. Hafez, C.D. Kodira, J. Major, S. Wang, J. Wilkinson, R. Nicol, C. Nusbaum, B. Birren, H.C. Berg and G.M. Church. 2004. The complete genome and proteome of Mycoplasma mobile. Genome Res. 14: 1447–1461. Johansson, K.-E. 2002. Taxonomy of Mollicutes. In Molecular Biology and Pathogenicity of Mycoplasmas (edited by Razin and Herrmann). Kluwer Academic/Plenum Publishers, New York, pp. 1–29. Knight, T.F., Jr. 2004. Reclassification of Mesoplasma pleciae as Acholeplasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene sequence data. Int. J. Syst. Evol. Microbiol. 54: 1951–1952. Ludwig, W. and K.H. Schleifer. 2005. Molecular phylogeny of bacteria based on comparative sequence analysis of conserved genes. In Microbial Phylogeny and Evolution, Concepts and Controversies (edited by Sapp). Oxford University Press, New York, pp. 70–98. Maniloff, J. 2002. Phylogeny and Evolution. In Molecular Biology and Pathogenicity of Mycoplasmas. Kluwer Academic/Plenum Publishers, pp. 31–43. May, M., R.F. Whitcomb and D.R. Brown. 2009. Mycoplasma and related organisms. In CRC Practical Handbook of Microbiology, 2nd edn. (edited by Goldman and Green). Taylor & Francis, pp. 456–479. Minion, F.C., E.J. Lefkowitz, M.L. Madsen, B.J. Cleary, S.M. Swartzell and G.G. Mahairas. 2004. The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J. Bacteriol. 186: 7123–7133. Neimark, H.C. 1979. Phylogenetic relationships between mycoplasmas and other prokaryotes. In The Mycoplasmas, vol. 1 (edited by Barile and Razin). Academic Press, New York, pp. 43–61. Oshima, K., S. Kakizawa, H. Nishigawa, H.Y. Jung, W. Wei, S. Suzuki, R. Arashida, D. Nakata, S. Miyata, M. Ugaki and S. Namba. 2004. Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nat. Genet. 36: 27–29. Papazisi, L., T. Gorton, G. Kutish, P. Markham, G. Browning, D. Nguyen, S. Swartzell, A. Madan, G. Mahairas and S. Geary. 2003. The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain R(low). Microbiology 149 : 2307–2316. Pereyre, S., P. Sirand-Pugnet, L. Beven, A. Charron, H. Renaudin, A. Barre, P. Avenaud, D. Jacob, A. Couloux, V. Barbe, A. de Daruvar, A. Blanchard and C. Bebear. 2009. Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas. PLoS. Genet. 5: e1000677.

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Pettersson, B., J.G. Tully, G. Bolske and K.E. Johansson. 2000. Updated phylogenetic description of the Mycoplasma hominis cluster (Weisburg et al. 1989) based on 16S rDNA sequences. Int. J. Syst. Evol. Microbiol. 50: 291–301. Pettersson, B., J.G. Tully, G. Bolske and K.E. Johansson. 2001. Re-evaluation of the classical Mycoplasma lipophilum cluster ­(Weisburg et al. 1989) and description of two new clusters in the hominis group based on 16S rDNA sequences. Int. J. Syst. Evol. Microbiol. 51: 633–643. Razin, S. and E.A. Freundt. 1984. The Mollicutes, Mycoplasmatales, and Mycoplasmataceae. In Bergey’s Manual of Systematic Bacteriology, vol.  1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 740–742. Robinson, I.M., M.J. Allison and P.A. Hartman. 1975. Anaeroplasma abactoclasticum gen. nov., sp. nov., obligately anaerobic mycoplasma from rumen. Int. J. Syst. Bacteriol. 25: 173–181. Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended classification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81. Rogers, M.J., J. Simmons, R.T. Walker, W.G. Weisburg, C.R. Woese, R.S. Tanner, I.M. Robinson, D.A. Stahl, G. Olsen, R.H. Leach and J. Maniloff. 1985. Construction of the mycoplasma evolutionary tree from 5S rRNA sequence data. Proc. Natl. Acad. Sci. U.S.A. 82: 1160–1164. Sasaki, Y., J. Ishikawa, A. Yamashita, K. Oshima, T. Kenri, K. Furuya, C. Yoshino, A. Horino, T. Shiba, T. Sasaki and M. Hattori. 2002. The complete genomic sequence of Mycoplasma penetrans, an intracellular bacterial pathogen in humans. Nucleic Acids Res. 30 : 5293–5300. Sirand-Pugnet, P., C. Lartigue, M. Marenda, D. Jacob, A. Barre, V. Barbe, C. Schenowitz, S. Mangenot, A. Couloux, B. Segurens, A. de Daruvar, A. Blanchard and C. Citti. 2007. Being pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome. PLoS. Genet. 3: e75. Stackebrandt, E. 2009. Class III. Erysipelotrichia. In Bergey’s Manual of Systematic Bacteriology, vol. 3 (edited by de Vos, Garrity, Jones, Krieg, Ludwig, Rainey, Schleifer and Whitman). Springer, New York, p. 1298. Tully, J.G., J.M. Bove, F. Laigret and R.F. Whitcomb. 1993. Revised taxonomy of the class Mollicutes - proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank ­(Entomoplasmatales ord. nov.), with provision for familial rank to ­separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378–385. Vasconcelos, A.T. and a. coauthors. 2005. Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae. J. Bacteriol. 187: 5568–5577. Verbarg, S., H. Rheims, S. Emus, A. Fruhling, R.M. Kroppenstedt, E. Stackebrandt and P. Schumann. 2004. Erysipelothrix inopinata sp. nov., isolated in the course of sterile filtration of vegetable peptone broth, and description of Erysipelotrichaceae fam. nov. Int. J. Syst. Evol. Microbiol. 54: 221–225. Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. Mandelco, J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic analysis of the mycoplasmas: basis for their classification. J. Bacteriol. 171: 6455–6467. Westberg, J., A. Persson, A. Holmberg, A. Goesmann, J. Lundeberg, K.E. Johansson, B. Pettersson and M. Uhlen. 2004. The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res. 14: 221–227. Woese, C.R., J. Maniloff and L.B. Zablen. 1980. Phylogenetic analysis of the mycoplasmas. Proc. Natl. Acad. Sci. U. S. A. 77: 494–498.

574

Phylum XVI. Tenericutes

Order I. Mycoplasmatales Freundt 1955, 71AL emend. Tully, Bové, Laigret and Whitcomb 1993, 382 Daniel R. Brown, Meghan May, Janet M. Bradbury, Karl-Erik Johansson and Harold Neimark My.co.plas.ma.ta¢les. N.L. neut. n. Mycoplasma, -atos type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Mycoplasmatales the Mycoplasma order. The first order in the class Mollicutes is assigned to a group of sterol-requiring, wall-less prokaryotes that occur as commensals or pathogens in a wide range of vertebrate hosts. The description of the order is essentially the same as for the class. A single family Mycoplasmataceae with two genera, Mycoplasma and Urea­ plasma, recognizes the prominent and distinct characteristics of the assigned organisms, based on their sterol requirements for growth, the capacity of some to hydrolyze urea, and conserved 16S rRNA gene sequences. Type genus: Mycoplasma Nowak 1929, 1349 nom. cons. Jud. Comm. Opin. 22, 1958, 166.

Further descriptive information The entire class Mollicutes was encompassed initially by a single order. The elevation of acholeplasmas to ordinal rank (Achole­ plasmatales Freundt, Whitcomb, Barile, Razin and Tully 1984) recognized their major distinctions in nutritional, biochemical, physiological, and genetic characteristics from other members of the class Mollicutes. Subsequently, additional orders were proposed to recognize the anaerobic mollicutes and the wallless prokaryotes from plants and insects which were phylogenetically related to the remaining Mycoplasmatales. Thus, the Anaeroplasmatales (Robinson and Freundt, 1987) recognized the strictly anaerobic, wall-less prokaryotes first isolated from the bovine and ovine rumen, and Entomoplasmatales (Tully et al., 1993) provided a classification for a number of the mollicutes regularly associated with plant and insect hosts. On the basis of 16S rRNA gene sequence similarities (Johansson and

References Edward, D.G. 1971. Determination of sterol requirement for Mycoplas­ matales. J. Gen. Microbiol. 69 : 205–210. Freundt, E.A. 1955. The classification of the pleuropneumoniae group of organisms (Borrelomycetales). Int. Bull. Bacteriol. Nomencl. Taxon. 5: 67–78. Freundt, E.A., R.F. Whitcomb, M.F. Barile, S. Razin and J.G. Tully. 1984. Proposal for elevation of the family Acholeplasmataceae to ordinal rank: Acholeplasmatales. Int. J. Syst. Bacteriol. 34: 346–349. Johansson, K.E., Pettersson B. 2002. Taxonomy of Mollicutes. In Molecular biology and pathogenicity of mycoplasmas (edited by Razin and Herrmann). Kluwer Academic, New York, pp. 1–30. Judicial Commission. 1958. Opinion 22. Status of the generic name Asterococcus and conservation of the generic name Mycoplasma. Int. Bull. Bacteriol. Nomencl. Taxon. 8: 166–168. Nowak, J. 1929. Morphologie, nature et cycle évolutif du microbe de la péripneumonie des bovidés. Ann. Inst. Pasteur (Paris) 43: 1330–1352.

Pettersson, 2002), the Mycoplasmatales and Entomoplasmatales represent a clade deeply split from the Acholeplasmatales and Anaeroplasmatales. A growth requirement for cholesterol or serum is shared by the organisms assigned to the order Mycoplasmatales, as well as most other organisms within the class Mollicutes. Therefore, tests for cholesterol requirements are essential to classification. Earlier assessments of the growth requirements for cholesterol were based upon the capacity of organisms to grow in a number of serum-free broth preparations to which various concentrations of cholesterol were added (Edward, 1971; Razin and Tully, 1970). In this test, species that do not require exogenous sterol usually show no significant growth response to increasing cholesterol concentrations. Polyoxyethylene sorbitan (Tween 80) and palmitic acid should be included in the base medium because acholeplasmas such as Acholeplasma axanthum and Acholeplasma morum require additional fatty acids for adequate growth. A modified method utilizing serial passage in selective medium has been applied successfully to a large number of mollicutes (Rose et al., 1993; Tully, 1995). The Acholeplasmatales grow through end-point dilutions in serum-containing medium and in serum-free preparations, or occasionally in serum-free medium supplemented with Tween 80. Mesoplasmas from the order Entomoplasmatales grow in serum-containing medium and in serum-free medium supplemented only with Tween 80. Most spiroplasmas, also from the Entomoplasmatales, and all members of the order Mycoplasmatales grow only in serum-containing medium.

Razin, S. and J.G. Tully. 1970. Cholesterol requirement of mycoplasmas. J. Bacteriol. 102: 306–310. Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended classification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81. Rose, D.L., J.G. Tully, J.M. Bove and R.F. Whitcomb. 1993. A test for measuring growth responses of Mollicutes to serum and polyoxyethylene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532. Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised taxonomy of the class Mollicutes - proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Ento­ moplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378–385. Tully, J.G. 1995. Determination of cholesterol and polyoxyethylene sorbitan growth requirements of mollicutes. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, San Diego, pp. 381–389.

Genus I. Mycoplasma

575

Family I. Mycoplasmataceae Freundt 1955, 71AL emend. Tully, Bové, Laigret and Whitcomb 1993, 382 Daniel R. Brown, Meghan May, Janet M. Bradbury, Karl-Erik Johansson and Harold Neimark My.co.plas.ma.ta.ce′ae. N.L. neut. n. Mycoplasma, -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Mycoplasmataceae the Mycoplasma family. Pleomorphic usually coccoid cells, 300–800 nm in diameter, to slender branched filaments of uniform diameter. Some cells have a terminal bleb or tip structure that mediates adhesion to certain surfaces. Cells lack a cell wall and are bounded only by a plasma membrane. Gram-stain-negative due to the absence of a cell wall. Usually nonmotile. Facultatively anaerobic in most instances, possessing a truncated flavin-terminated electron transport chain devoid of quinones and cytochromes. Colonies of Mycoplasma are usually less than l mm in diameter and colonies of Ureaplasma are much smaller than that. The typical colony has a fried-egg or “cauliflower head” appearance. Usually catalase-negative. Chemo-organotrophic, usually using either sugars or arginine, but sometimes both, or having an obligate requirement for urea as the major energy source. Require cholesterol or related sterols for growth. Commensals or pathogens of a wide range of vertebrate hosts. The genome size ranges from about 580 to 1350 kbp, as measured by pulsed field gel electrophoresis (PFGE) or complete DNA sequencing. DNA G+C content (mol%): about 23–40 (Bd, Tm).

Type genus: Mycoplasma Nowak 1929, 1349 nom. cons. Jud. Comm. Opin. 22, 1958, 166.

Further descriptive information This family and its type genus Mycoplasma are polyphyletic. Two genera, Mycoplasma and Ureaplasma, are currently accepted within the family. The genus Mycoplasma is further divisible into phylogenetic groups on the basis of 16S rRNA gene sequence similarities (Johansson and Pettersson, 2002), including an ecologically, phenotypically, and genetically cohesive group called the mycoides cluster, which includes the type species Mycoplasma mycoides and other major pathogens of ruminant animals. The taxonomic position of the mycoides cluster is an important anomaly because molecular markers based upon rRNA and other gene sequences indicate that it is closely related to other genera usually associated with plant and insect hosts and currently classified within the order Entomoplasmatales. Members of the genus Ureaplasma are distinguished by their tiny colony size and ­capacity to hydrolyze urea.

Genus I. Mycoplasma Nowak 1929, 1349 nom. cons. Jud. Comm. Opin. 22, 1958, 166AL Daniel R. Brown, Meghan May, Janet M. Bradbury, Mitchell F. Balish, Michael J. Calcutt, John I. Glass, Séverine Tasker, Joanne B. Messick, Karl-Erik Johansson and Harold Neimark My.co.plas¢ma. Gr. masc. n. myces a fungus; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Mycoplasma fungus form.

Pleomorphic cells, 300–800 nm in diameter, varying in shape from spherical, ovoid or flask-shaped, or twisted rods, to slender branched filaments ranging in length from 50 to 500 nm. Cells lack a cell wall and are bounded by a single plasma membrane. Gram-stain-negative due to the absence of a cell wall. Some have a complex internal cytoskeleton. Some have a specific tip structure that mediates attachment to host cells or other surfaces. Usually nonmotile, but gliding motility has been demonstrated in some species. Aerobic or facultatively anaerobic. Optimum growth at 37°C is common, but permissive growth temperatures range from 20 to 45°C. Chemo-organotrophic, usually using either sugars or arginine as the major energy source. Require cholesterol or related sterols for growth. Colonies are usually less than l mm in diameter. The typical colony has a fried-egg appearance. The genome size of species examined ranges from 580 kbp to about 1350 kbp. The codon UGA encodes tryptophan in all species examined. Commensals or pathogens in a wide range of vertebrate hosts. DNA G+C content (mol%): 23–40. Type species: Mycoplasma mycoides (Borrel, Dujardin­Beaumetz, Jeantet and Jouan 1910) Freundt 1955, 73 (Asterococcus mycoides Borrel, Dujardin-Beaumetz, Jeantet and Jouan 1910, 179).

Further descriptive information The shape of these organisms (trivial name, mycoplasmas) can depend on the osmotic pressure, nutritional quality of the

culture medium, and the growth phase. Some mycoplasmas are filamentous in their early and exponential growth phases or when attached to surfaces or other cells. This form can be transitory, and the filaments may branch or fragment into chains of cocci or individual vegetative cells. Many species are typically coccoid and never develop a filamentous phase. Some species develop specialized attachment tip structures involved in colonization and virulence (Figure 106). In Romanowskytype stained blood smears, hemotropic species (trivial name, hemoplasmas) appear as round to oval cells on the surface of erythrocytes (Figure 107). They may be found individually or, during periods of high parasitemia, in pairs or chains giving the appearance of pleomorphism. Their small size and the absence of cell wall components provide considerable plasticity to the organisms, so that cells of most species are readily filterable through 450 nm pores, and many species have some cells in the population that are able to pass through 220 nm or even 100 nm filters (Tully, 1983). Descriptions of the morphology, ultrastructure, and motility of mycoplasmas should be based on correlation of the appearance of young exponential-phase broth cultures under phase-contrast or dark-field microscopy with their appearance using negative-staining or electron microscopy (Biberfeld and Biberfeld, 1970; Boatman, 1979; Carson et al., 1992; Cole, 1983). Special attention to the osmolarity of the fixatives and buffers is required since these may alter the size and shape of the organisms. The classical isolated colony is umbonate with a fried-egg appearance, but others may have

576

Family I. Mycoplasmataceae

Figure 106.  Diverse cellular morphology in the genus Mycoplasma. Scanning electron micrographs of cells of (a) Mycoplasma penetrans, (b) Mycoplasma pneumoniae, (c) “Mycoplasma insons”, and (d) Mycoplasma genitalium. Bar = 1 mm. Images provided by Dominika Jurkovic, Jennifer Hatchel, Ryan Relich and Mitchell Balish.

Figure 107.  Hemotropic mycoplasmas. (a) Scanning electron micrograph of Mycoplasma ovis cells colonizing

the surface of an erythrocyte (Neimark et al., 2004); bar = 500 nm. (b) Transmission electron micrograph showing fibrils bridging the space between a “Candidatus Mycoplasma kahaneii” cell and a depression in the surface of a colonized erythrocyte (Neimark et al., 2002a); bar = 250 nm. Images used with permission.

either cauliflower-like or smooth colony surfaces (Figure 108), with smooth, irregular or scalloped margins, depending on the species, agar concentration, and other growth conditions. A significant minority of species exhibit cell polarization. This depends on Triton X-100-insoluble cytoskeletal structures

involved in morphogenesis, motility, cytadherence, and cell division (Balish and Krause, 2006). In the distantly related species Mycoplasma pneumoniae and Mycoplasma mobile, the cytoskeleton underlies a terminal organelle. This prominent extension of the cytoplasm and cell membrane is the principal focus of adherence

Genus I. Mycoplasma

577

Figure 108.  Diverse colonial morphology in the genus Mycoplasma. (a) Mycoplasma mycoides PG1T (diameter 0.50–0.75 mm), (b) Mycoplasma hyopneumoniae NCTC 10110T (diameter 0.15–0.20 mm), (c) Mycoplasma pneumoniae NCTC 10119T (diameter 0.05–0.10 mm), and (d) Mycoplasma hyorhinis ATCC 29052 (diameter 0.25–0.30 mm) after 3, 7, 5, and 6 d growth, respectively, on Mycoplasma Experience Solid Medium at 36°C in 95% nitrogen/5% carbon dioxide. Original magnification 25×. Images provided by Helena Windsor and David Windsor.

and is the leading end of cells engaged in gliding motility. In Mycoplasma pneumoniae, adhesin proteins are located either all over the surface of the organelle or at its distal tip; an unrelated adhesin is concentrated at the base of the terminal organelle in Mycoplasma mobile (Balish, 2006). Both the formation of this attachment organelle of Mycoplasma pneumoniae and the localization of the adhesins depend upon cytoskeletal proteins that form an electron-dense core within its cytoplasm, which is surrounded by an electron-lucent space (Krause and Balish, 2004). The overall appearance of this core is that of two parallel, flat rods of differing thickness, with a bend near the cell-proximal end (Henderson and Jensen, 2006; Seybert et  al., 2006). A bilobed button constitutes its distal end and its proximal base terminates in a bowl-like structure. Overall, both the core and the attachment organelle are 270–300 nm in length (Hatchel and Balish, 2008). Around the onset of DNA replication, a second attachment organelle is constructed (Seto et  al., 2001). The motile force provided by the first organelle reorganizes the cell such that the new organelle is moved to the opposite cell pole before cell division (Hasselbring et  al., 2006). These observations suggest that complex coordination exists between attachment organelle biogenesis, motor activity, the DNA replication machinery, and the cytokinetic machinery. Similar structures are present in other species of the Mycoplasma pneumoniae cluster, but in most cases the attachment organelle is shorter, resulting in much of the core protruding into the cell body (Hatchel and Balish, 2008). In Mycoplasma mobile, the terminal organelle is completely dissimilar, consisting of a cell-distal sphere with numerous tentacle-like strands extending into the cytoplasm (Nakane and Miyata, 2007). It is comprised of proteins unrelated to those

found in Mycoplasma pneumoniae, suggesting that it has evolved independently. Further distinct cytoskeletal structures appear in Mycoplasma penetrans (Jurkovic and Balish, unpublished), “Mycoplasma insons” (Relich et  al., 2009), and several species of the mycoides cluster (Peterson et al., 1973). Attachment to eukaryotic host cells is important for the natural survival and transmission of mycoplasmas. The prominent attachment organelle of species in the Mycoplasma pneumoniae cluster is the most extensively characterized determinant of cytadherence. In other species, multiple adhesin proteins are involved in cytadherence. When one adhesin is blocked, cytadherence is reduced, but not completely lost. For this reason, the adhesins appear to be functionally redundant rather than synergistic in action. Numerous species possess multigene families of antigenically variable proteins, some of which have been implicated in host cell attachment or hemagglutination. While this attachment may serve as a supplemental binding mechanism in species such as Mycoplasma gallisepticum and Mycoplasma hominis, variable surface proteins are currently the only known mechanism for cytadherence and hemagglutination of Mycoplasma synoviae and Mycoplasma pulmonis. The avidity of adherence may differ among variants in Mycoplasma pulmonis and Mycoplasma hominis. Though one or more attachment mechanisms have been described for numerous species, there remains a greater number of species with no documented system for cytadherence. Strains that lose the capacity to cytadhere are almost invariably unable to survive in their hosts, but highly invasive species such as Mycoplasma ­alligatoris may not require host cell attachment for infection. The mycoplasmas possess a typical prokaryotic plasma ­membrane composed of amphipathic lipids and proteins

578

Family I. Mycoplasmataceae

(­ McElhaney, 1992a, b, c; Smith, 1992; Wieslander et al., 1992). At one time, demonstration of a single unit membrane was mandatory for defining all novel species of mollicutes (Tully, 1995a). Now, when the 16S rRNA gene sequence of a novel species is determined and the candidate is placed in one of the phylogenetic clusters of mollicutes, in the majority of cases it can be safely inferred that the organism lacks a cell wall, because the majority of others in that cluster will have been shown to be solely membrane-bound (Brown et  al., 2007). The lack of a cell wall explains the resistance of the organisms to lysis by lysozyme and their susceptibility to lysis by osmotic shock and various agents causing the lysis of bacterial protoplasts (Razin, 1979, 1983). In certain species, the extracellular surface is textured with capsular material or a nap, which can be stained with ruthenium red in some cases (Rosenbusch and Minion, 1992). These organisms represent some of the most nutritionally fastidious prokaryotes, as expected from their greatly reduced or minimalist genomes, close association with vertebrate hosts as commensals and pathogens, and total dependence upon the host to meet all nutritional requirements. They have very limited capacity for intermediary metabolism, which restricts the utility of conventional biochemical tests for identification. Detailed information on carbohydrate (Pollack, 1992, 1997, 2002; Pollack et al., 1996), lipid (McElhaney, 1992a), and amino acid (Fischer et al., 1992) metabolism is available. All species examined have truncated respiratory systems, lack a complete tricarboxylic acid cycle, and lack quinones or cytochromes, which precludes their capacity to carry out oxidative phosphorylation. Instead only low levels of ATP may be generated through glycolysis or the arginine dihydrolase pathway (Miles, 1992a, b). Fermentative species catabolize glucose or other carbohydrates to produce ATP and acid and, consequently, lower the pH of the medium. Non-fermentative species hydrolyze arginine to yield ammonia, some ATP, and carbon dioxide, and consequently raise the pH of the medium. Species such as Mycoplasma fermentans have both pathways. Species such as Mycoplasma bovis evidently lack both pathways, but are capable oxidizing pyruvate or lactate to yield ATP (Miles, 1992a; Taylor et al., 1994). Some species cause a pronounced “film and spots” reaction on media incorporating heat-inactivated horse serum or egg yolk: a wrinkled film composed of cholesterol and phospholipids forms on the surface of the medium and dark spots containing salts of fatty acids appear around the colonies. Most mycoplasmas are aerobes or facultative anaerobes, but some species such as Mycoplasma muris prefer an anaerobic environment. The optimum growth of species isolated from homeothermic hosts is commonly at 37°C and the permissive temperature range of species from poikilothermic fish and reptiles is always above 20–25°C. Thus, growth of the mycoplasmas is restricted to mesophilic temperatures. Growth in liquid cultures usually produces at most light turbidity and few sedimented cells, except for the heavy turbidity and sediments usually observed with members of the Mycoplasma mycoides cluster. Tully (1995b) described in detail the most commonly used culture media formulations. Although colonies are occasionally first detected on blood agar, complex undefined media such as American Type Culture Collection (ATCC) medium 988 (SP-4) are usually required for primary isolation and maintenance. Cell-wall-targeting antibiotics are included to discourage growth of other bacteria. Phenol red facilitates detection of species that excrete acidic or alkaline metabolites. Growth of

a­ rginine-hydrolyzing species can be enhanced by supplementing media with arginine. Commonly used alternatives such as Frey’s, Hayflick’s and Friis’ media differ from SP-4 mainly in the proportions of inorganic salts, amino acids, serum sources, and types of antibiotics. For species that utilize both sugars and arginine as carbon sources, the pH of the medium may initially decrease before rising later during the course of growth (Razin et  al., 1998). Defined mycoplasma culture media have been described in detail (Rodwell, 1983), but provision of lipids and amino acids in the appropriate ratios is difficult technically (Miles, 1992b). Many mobile genetic elements occur in the genus. Four plasmids have been identified in members of the mycoides cluster (Bergemann and Finch, 1988; Djordjevic et al., 2001; King and Dybvig, 1994). Each plasmid is apparently cryptic, with no discernible determinants for virulence or antibiotic resistance. DNA viruses have been isolated from Mycoplasma bovirhinis (Howard et  al., 1980), Mycoplasma hyorhinis (Gourlay et  al., 1983) Mycoplasma pulmonis (Tu et  al., 2001), and Mycoplasma arthritidis (Voelker and Dybvig, 1999). The Mycoplasma pulmonis P1 virus and the lysogenic bacteriophage MAV1 of Mycoplasma arthritidis do not share sequence similarity (Tu et  al., 2001; Voelker and Dybvig, 1999), whereas the Mycoplasma fermentans MFV1 prophage is strikingly similar in genetic organization to MAV1 (Röske et al., 2004). No role in pathobiology has been demonstrated for any virus or prophage. The most abundant mobile DNAs in Mycoplasma are insertion sequence (IS) elements. The first identified units (IS1138 of Mycoplasma pulmonis, IS1221 of Mycoplasma hyorhinis, IS1296 of Mycoplasma mycoides subsp. mycoides and ISMi1 of Mycoplasma fermentans) are members of the IS3 family (Bhugra and Dybvig, 1993; Ferrell et al., 1989; Frey et al., 1995; Hu et al., 1990). More recently, multiple IS elements of divergent subgroups have been identified. Members of the IS4 family include IS1634 and ISMmy1 of Mycoplasma mycoides subsp. mycoides (Vilei et al., 1999; Westberg et  al., 2002), ISMhp1 of Mycoplasma hyopneumoniae, ISMhp1-like unit of Mycoplasma synoviae, and four distinct elements of Mycoplasma bovis (Lysnyansky et al., 2009). Among the IS30 family members identified are IS1630 of Mycoplasma fermentans, ISMhom1 from Mycoplasma hominis, ISMag1 of Mycoplasma agalactiae (Pilo et al., 2003), and two IS units of Mycoplasma bovis. IS-like elements have also been identified in Mycoplasma leachii, Mycoplasma penetrans (belonging to four different families), Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma orale. Transposases that reside within IS units are also discernable in the genome of Mycoplasma gallisepticum (Papazisi et  al., 2003). In select instances, almost identical IS units have been found in species from different phylogenetic clades, which strongly suggests lateral gene transfer between species. Despite their widespread distribution, IS elements are not ubiquitous in the genus. Although the type strains of Mycoplasma bovis (54 IS units of seven different types) and Mycoplasma mycoides subsp. mycoides (97 elements of three different types) possess large numbers of elements, the sequenced genomes of Mycoplasma arthritidis, Mycoplasma genitalium, Mycoplasma pneumoniae, and Mycoplasma mobile lack detectable IS units. Although IS units only encode genes related to transposition, large integrating elements have also been identified in diverse Mycoplasma species. The Integrative Conjugal Elements (ICE) of Mycoplasma fermentans strain PG18T comprise >8% of the genome and related units have been identified in Mycoplasma

Genus I. Mycoplasma

agalactiae, Mycoplasma bovis, Mycoplasma capricolum, Mycoplasma hyopneumoniae, and Mycoplasma mycoides subsp. mycoides. In general, such units encode 18–30 genes, can be detected in extrachromosomal forms, and are strain-variable in distribution and chromosomal insertion site. Two additional large mobile DNAs, designated Tra Islands, were identified in Mycoplasma capricolum California kidT. The presence of putative conjugation genes and the variability in genomic location of Tra Islands and ICE suggest that these are agents of lateral gene transfer. The best-studied mycoplasmas are primary pathogens of humans or domesticated animals (Baseman and Tully, 1997). About half of the listed species occur in the absence of disease, but are occasional opportunistic or secondary pathogens. The principal human pathogens are Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium, with Mycoplasma penetrans added to this list due to its association with HIV infections (Blanchard, 1997; Blanchard et al., 1997; Tully, 1993; Waites and Talkington, 2005). Mycoplasma pneumoniae is one of the main agents of community-acquired pneumonia, bronchitis, and other respiratory complications (Atkinson et  al., 2008). Mycoplasma pneumoniae infections can also involve extra-pulmonary complications including central nervous system, cardiovascular, and dermatological manifestations. Outbreaks cause considerable morbidity and require rapid and effective therapeutic intervention (Hyde et al., 2001; Meyer and Clough, 1993). Mycoplasma hominis occurs more frequently in the urogenital tract of women than men and is often found in the genital tract of women with vaginitis, bacterial vaginosis, or localized intrauterine infections (Keane et  al., 2000). The organism can gain access to a fetus from uterine sites and it is associated with perinatal morbidity and mortality (Gonçalves et  al., 2002; Waites et  al., 1988). It is also clearly associated with septicemias and respiratory infections and with transplant or joint infections in immunosuppressed persons (Brunner et  al., 2000; Busch et  al., 2000; Fernandez Guerrero et  al., 1999; Garcia-Porrua et  al., 1997; Gass et  al., 1996; Hopkins et al., 2002; Mattila et al., 1999; Tully, 1993; Zheng et al., 1997). Mycoplasma genitalium has been associated with nongonococcal urethritis in men (Gambini et al., 2000; Jensen, 2004; Jensen et al., 2004; Taylor-Robinson et al., 2004; Taylor-Robinson and Horner, 2001; Totten et al., 2001) and urogenital disease in women (Baseman et al., 2004; Blaylock et al., 2004). Mycoplasma genitalium occurs more frequently in the vagina than in the cervix or urethra, but it may be involved in cervicitis (Casin et al., 2002; Manhart et  al., 2001). Mycoplasmas are common agents of chronic joint inflammation in a wide variety of hosts (Cole et al., 1985). Species associated with arthritis in humans include Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma salivarium, and possibly Mycoplasma pneumoniae in juvenile arthritis (Waites and Talkington, 2005). Humans are also susceptible to opportunistic zoonotic mycoplasmosis; immunosuppressed persons are highly susceptible. The recent molecular confirmation of a Mycoplasma haemofelis-like infection in an HIVpositive patient (dos Santos et al., 2008) highlights the zoonotic potential of the hemoplasmas. Mycoplasmas colonize fish, reptiles, birds, and terrestrial and aquatic mammals. Some cause significant diseases of cattle and other ruminants, swine, poultry, or wildlife, and others are opportunistic or secondary veterinary pathogens (Simecka et  al., 1992; Tully and Whitcomb, 1979). The principal bovine pathogens include the serovars historically called “Small Colony”

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types of Mycoplasma mycoides subsp. mycoides, and Mycoplasma bovis. Mycoplasma mycoides subsp. mycoides has caused major losses of livestock globally in the twentieth century due to contagious bovine pleuropneumonia and currently remains a problem in Asia and Africa (Lesnoff et al., 2004). Mycoplasma bovis is a widespread agent of otitis media, pneumonia, mastitis, polyarthritis, and urogenital disease in cattle and buffaloes. Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum, and Mycoplasma agalactiae are important causes of arthritis, mastitis, and agalactia in goats and sheep. Mycoplasma mycoides subsp. capri (type strain PG3T) properly includes all of the serovars historically called “Large Colony” types of subspecies mycoides (MansoSilván et al., 2009; Shahram et al., 2010). Mycoplasma capricolum subsp. capripneumoniae (type strain F38T) causes severe contagious pleuropneumonia in goats (Leach et al., 1993; McMartin et al., 1980). Contagious bovine and caprine pleuropneumonia, and mycoplasmal agalactia of sheep or goats are subject to control through listing in the Terrestrial Animal Health Code of the Office International des Epizooties (http://oie.int) as well as strict notification and export regulations by individual countries. Mycoplasma hyopneumoniae, one of the most difficult species to cultivate, causes primary enzootic pneumonia in pigs and exacerbates other porcine respiratory diseases leading to substantial economic burdens. Mycoplasma hyosynoviae is carried in the upper respiratory tract, but causes nonsuppurative polyarthritis, usually without other serositis, especially in growing pigs. The most important poultry pathogens are Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis, but more than 20 other species have been isolated from birds as diverse as ostriches, raptors, and penguins (Bradbury and ­Morrow, 2008). Mycoplasma gallisepticum can be transmitted vertically, venereally, by other direct contact, or by aerosol to cause respiratory disease and its sequelae in chickens, turkeys, and other birds. It also causes decreased egg production and egg quality in chickens. Mycoplasma synoviae can cause a syndrome of synovitis, tendonitis, and bursitis in addition to respiratory disease in chickens and turkeys, whereas the developmental abnormalities and airsacculitis associated with congenital or acquired Mycoplasma meleagridis infection seem restricted to turkeys. Mycoplasma gallisepticum and Mycoplasma synoviae are also listed in the OIE’s Terrestrial Animal Health Code. Pathogenicity of specific mycoplasmas has also been reported for companion animals (Chalker, 2005; Lemcke, 1979; ­Messick, 2003) and a number of wild animal hosts (Brown et al., 2005). The respiratory, reproductive, and joint diseases caused in rodents by Mycoplasma pulmonis and Mycoplasma arthritidis (Schoeb, 2000) are important models of infection and immunity in humans and other animals. Hemoplasmas infect a variety of wild and domesticated ­animals and are relatively host-specific, although cross-­infection of related hosts has been reported. Transmission can be achieved by ingestion of infected blood or by percutaneous inoculation. Arthropod vector transmission of some species is also supported by experimentation and by the ­clustered ­geographic distribution of hemoplasmosis in some studies (Sykes et al., 2007; Willi et al., 2006a). The pathogenicity of different hemoplasma species is variable and strain virulence also likely plays a key role in the development of disease. For example, Mycoplasma haemofelis can induce acute clinical disease in non-splenectomized, immunocompetent cats, whereas Mycoplasma haemocanis appears able

580

Family I. Mycoplasmataceae

to induce disease only in immunosuppressed or splenectomized dogs. Clinical syndromes range from acute fatal hemolytic anemia to chronic insidious anemia and ill-thrift. Signs may include anemia, pyrexia, anorexia, dehydration, weight loss, and infertility. The presence of erythrocyte-bound antibodies (including cold agglutinins), indicated by positive Coombs’ testing, has been demonstrated in some hemoplasma-infected animals and may contribute to anemia. Animals can remain chronic asymptomatic carriers of hemoplasmas after acute infection. PCR is the diagnostic test of choice for hemoplasma infection. Contamination of eukaryotic cell cultures with mollicutes is still a common and important yet often unrecognized problem (Tully and Razin, 1996). More than 20 species have been isolated from contaminated cell lines, but more than 90% of the contamination is thought to be caused by just five species of mycoplasma: Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma hyorhinis, and Mycoplasma orale, plus Acholeplasma laidlawii. Mycoplasma pirum and Mycoplasma salivarium account for most of the remainder (Drexler and Uphoff, 2002). Culture medium components of animal origin, passage of contaminated cultures, and laboratory personnel are likely to be the most significant sources of cell culture contaminants. PCR-based approaches to detection achieve sensitivity and specificity far superior to fluorescent staining methods (Masover and Becker, 1996). Another method of detection is based on mycoplasma-specific ATP synthesis activity present in contaminated culture medium (Robertson and Stemke, 1995; MycoAlert, Lonza Group). Eradication through treatment of contaminated cultures with antibiotics (Del Giudice and Gardella, 1996) is rarely successful. Strategies for prevention and control of mycoplasmal contamination of cell cultures have been described in detail (Smith and Mowles, 1996). Several categories of potential virulence determinants are encoded in the metagenome of pathogenic mycoplasmas. Some species possess multiple types of virulence factors. Determinants such as adhesins and accessory proteins, extracellular polysaccharide structures, and pro-inflammatory or pro-­apoptotic membrane lipoproteins are produced by multiple species. Several species excrete potentially toxic by-products of intermediary metabolism, including hydrogen peroxide, superoxide radicals, or ammonia. Other determinants such as extracellular endopeptidases, nucleases, and glycosidases seem irregularly distributed in the genus, whereas the ADP-ribosylating and vacuolating cytotoxin (pertussis exotoxin S1 subunit analog) of Mycoplasma pneumoniae and the T-lymphocyte mitogen (superantigen) of Mycoplasma arthritidis are evidently unique to those species. Reports of a putative exotoxin elaborated by Mycoplasma neurolyticum have not been substantiated by later work (Tryon and Baseman, 1992). Candidate virulence mechanisms, such as motility, biofilm formation, or facultative intracellular invasion, are expressed by a range of pathogenic species. Several species possess systems of variable surface antigens that are thought to be important in evasion of the hosts’ adaptive immune responses. In addition, a large number of species can suppress or inappropriately stimulate host immune cells and their receptors and cytokines through diverse, poorly characterized mycoplasmal components. Although candidate virulence factor discovery has accelerated significantly in recent years through whole genome annotation, the molecular basis for pathogenicity and causal relationships with disease still remain to be definitively established for most of these factors (Razin and Herrmann, 2002; Razin et al., 1998).

Because they lack lipopolysaccharide and a cell wall, and do not synthesize their own nucleotides, mycoplasmas are intrinsically resistant to polymixins, b-lactams, vancomycin, fosfomycin, sulfonamides, and trimethoprim. They are also resistant to rifampin because their RNA polymerase is not affected by that antibiotic (Bébéar and Kempf, 2005). Individual species exhibit an even broader spectrum of antibiotic resistance, such as the resistance to erythromycin and azithromycin exhibited by several species, which is apparently mediated by mutation in the 23S rRNA (Pereyre et al., 2002). Treatment of mycoplasmosis often involves the use of antibiotics that inhibit protein synthesis or DNA replication. Certain macrolides or ketolides are used when tetracyclines or fluoroquinolones are inappropriate. Fluoroquinolones, aminoglycosides, pleuromutilins, and phenicols are not widely used to treat human mycoplasmosis at present, with the exception of chloramphenicol for neonates with mycoplasmosis of the central nervous system unresponsive to other antibiotics (Waites et al., 1992), but their use in veterinary medicine is more common. The long-term antimicrobial therapy often required may be due to mycoplasmal sequestration in privileged sites, potentially including inside host cells. Mycoplasmosis in immunodeficient patients is very difficult to control with antibiotic drugs (Baseman and Tully, 1997).

Enrichment and isolation procedures Techniques for isolation of mycoplasmas from humans, various species of animals, and from cell cultures have been described (Neimark et al., 2001; Tully and Razin, 1983). Typical steps in the isolation of mycoplasmas were outlined in the recently revised minimal standards for descriptions of new species (Brown et al., 2007). Initial isolates may contain a mixture of species, so cloning by repeated filtration through membrane filters with a pore size of 450 or 220 nm is essential. The initial filtrate and dilutions of it are cultured on solid medium and an isolated colony is subsequently picked from a plate on which only a few colonies have developed. This colony is used to found a new cultural line, which is then expanded, filtered, plated, and picked two additional times. Hemoplasmas have not yet been successfully grown in continuous culture in vitro, although recent work (Li et al., 2008) suggests that in vitro maintenance of Mycoplasma suis may be possible.

Maintenance procedures Cultures of mycoplasma can be preserved by lyophilization or cryogenic storage (Leach, 1983). The serum in the culture medium provides effective cryoprotection, but addition of sucrose may enhance survival following lyophilization. Hemoplasmas can be frozen in heparin- or EDTA-anticoagulated blood cryopreserved with dimethylsulfoxide. Most species can be recovered with little loss of viability even after storage for many years.

Taxonomic comments This polyphyletic genus is divisible on the basis of 16S rRNA and other gene sequence similarities into a large paraphyletic clade of over 100 species in two groups called hominis and pneumoniae (Johansson and Pettersson, 2002; Figure 109), plus the ecologically, phenotypically, and genetically cohesive “mycoides cluster” of five species including the type species Mycoplasma mycoides (Cottew et al., 1987; Manso-Silván et al., 2009; ­Shahram et al., 2010). The priority of Mycoplasma mycoides as the type species of the genus Mycoplasma and, hence, the family Mycoplasmataceae and the order Mycoplasmatales is, in retrospect, unfortunate.

Genus I. Mycoplasma

The phylogenetic position of the mycoides cluster is eccentrically situated to the remaining species of the order Mycoplasmatales, amidst genera that are properly classified in the order Entomoplasmatales. When the order Entomoplasmatales was established, a century after the discovery of Mycoplasma mycoides, it was explicitly accepted that the taxonomic anomaly created by the phylogenetic position of the mycoides cluster will remain impractical to resolve (Tully et al., 1993). The few species in the mycoides cluster cannot simply be renamed, because confusion and peril would result, especially regarding “Small Colony” PG1T-like strains of Mycoplasma mycoides subsp. mycoides and F38T-like strains of Mycoplasma capricolum subsp. capripneumoniae, which are highly virulent pathogens and subject to strict international regulations. Another controversy involves the nomenclature of uncultivated hemotropic bacteria originally assigned to the genera Eperythrozoon or Haemobartonella. It is now undisputed that, on the bases of their lack of a cell wall, small cell size, low G+C content, use of the codon UGA to encode tryptophan, regular association with vertebrate hosts, and 16S rRNA gene sequences that are most similar (80–84%) to species in the pneumoniae group of Mycoplasma, these organisms are properly affiliated with the Mycoplasmatales. However, the proposed transfers of Eperythrozoon and Haemobartonella species to the genus Mycoplasma (Neimark et al., 2001, 2005) were opposed on the grounds that the degree of 16S rRNA gene sequence similarity is insufficient (Uilenberg et al., 2004, 2006). The principal objection to establishing the hemoplasmas in a third genus in the ­Mycoplasmataceae ­(Uilenberg et al., 2006) is that this would compound the polyphyly within the pneumoniae group solely on the basis of a capacity to adhere to erythrocytes in vivo. In addition, the transfer of the type species Eperythrozoon coccoides to the genus Mycoplasma is complicated by priority because Eperythrozoon predates Mycoplasma. The alternative, to transfer all mycoplasmas to the genus Eperythrozoon, would be completely impractical and perilous in part because the epithet Eperythrozoon does not indicate an affiliation with Mycoplasmatales. The Judicial Commission of the International Committee on Systematics of Prokaryotes (ICSP) declined to rule on a request for an opinion in this matter (Neimark et al., 2005) during their 2008 meeting, but a provisional placement in the genus Mycoplasma has otherwise been embraced by specialists in the molecular biology and clinical pathogenicity of these and similar hemotropic organisms. At present, the designation “Candidatus” must still be used for new types. Mycoplasma feliminutum was first described during a time when the only named genus of mollicutes was Mycoplasma. Its publication coincided with the first proposal of the genus Acholeplasma (Edward and Freundt, 1969, 1970), with which Mycoplasma feliminutum is properly affiliated through established phenotypic (Heyward et al., 1969) and 16S rRNA gene sequence (Brown et al., 1995) similarities. This explains the apparent inconsistencies with its assignment to the genus Mycoplasma. The name Mycoplasma feliminutum should therefore be revised to Acholeplasma feliminutum comb. nov. The type strain is BenT (=ATCC 25749T; Heyward et al., 1969). Recent work at the J. Craig Venter Institute, including complete chemical synthesis and cloning of an intact Mycoplasma genitalium chromosome (Gibson et  al., 2008) and other work with Mycoplasma mycoides “Large Colony” (Lartigue et al., 2009), suggests that de novo synthesis of two species of mollicute is imminent. Transplantation of isolated deproteinized tetR-selectable chromosomes from donor Mycoplasma mycoides “Large Colony” into recipient Mycoplasma capricolum cells displaced the recipient genome and conferred the genotype and phenotype of the donor

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(Lartigue et  al., 2007). Cloning in yeast and subsequent resurrection of Mycoplasma mycoides “Large Colony” genomes as living bacteria demonstrate that it is possible to enliven a prokaryotic genome constructed in a eukaryotic cell (Lartigue et al., 2009). The ICSP subcommittee on the taxonomy of Mollicutes may be the first to accommodate a system of nomenclature and classification for species of novel prokaryotes that originate by entirely artificial speciation events (Brown and Bradbury., 2008).

Differentiation of the genus Mycoplasma from other genera Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et  al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. Aerobic or facultatively anaerobic growth in artificial medium and a growth requirement for sterols exclude assignment to the genera Anaeroplasma, Asteroleplasma, Acholeplasma, or “Candidatus Phytoplasma”. Non-spiral cellular morphology and regular association with a vertebrate host or fluids of vertebrate origin support exclusion from the genera Spiroplasma, Entomoplasma, or Mesoplasma. The inability to hydrolyze urea excludes assignment to the genus Ureaplasma.

Acknowledgements The lifetime achievements in mycoplasmology and substantial contributions to the preparation of this material by Joseph G. Tully are gratefully acknowledged. Daniel R. Brown and Meghan May were supported by NIH grant 5R01GM076584. Séverine Tasker was supported by Wellcome Trust grant WT077718.

Further reading Blanchard, A. and G. Browning (editors). 2005. Mycoplasmas: Molecular Biology, Pathogenicity, and Strategies for Control. Horizon Press, Norwich, UK. Maniloff, J., R.N. McElhaney, L.R. Finch and J.B. Baseman (editors). 1992. Mycoplasmas: Molecular Biology and Pathogenesis. American Society for Microbiology, Washington, D.C. Razin, S. and J.G. Tully (editors). 1995. Molecular and Diagnostic ­Procedures in Mycoplasmology, vol. 1, Molecular Characterization. Academic Press, San Diego. Tully, J.G. and S. Razin (editors). 1996. Molecular and Diagnostic Procedures in Mycoplasmology, vol. 2, Diagnostic Procedures. Academic Press, San Diego.

Differentiation of the species of the genus Mycoplasma Glucose fermentation and arginine hydrolysis are discriminating phenotypic markers (Table 137), but the pleomorphism and metabolic simplicity of mycoplasmas has led to a current reliance principally on the combination of 16S rRNA gene sequencing and reciprocal serology for species differentiation. Failure to crossreact with antisera against previously recognized species provides substantial evidence for species novelty. For this reason, deposition of antiserum against a novel type strain into a recognized collection is still mandatory for novel species descriptions (Brown et al., 2007). Preliminary differentiation can be by PCR and DNA sequencing using primers specific for bacterial 16S rRNA genes or the 16S–23S intergenic region. A similarity matrix relating the candidate strain to its closest neighbors, usually species with >94% 16S rRNA gene sequence similarity, will suggest related species that should be examined for serological cross-reactivities.

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Family I. Mycoplasmataceae

hominis group

*

*

*

*

* *

Scale: Figure 109.  (Continued)

Mycoplasma equigenitalium Mycoplasma elephantis Mycoplasma bovis Mycoplasma agalactiae * Mycoplasma primatum Mycoplasma opalescens Mycoplasma spermatophilum Mycoplasma fermentans * Mycoplasma caviae Mycoplasma adleri ** Mycoplasma felifaucium * Mycoplasma leopharyngis Mycoplasma maculosum Mycoplasma lipofaciens Mycoplasma bovigenitalium Mycoplasma californicum ** Mycoplasma simbae Mycoplasma phocirhinis Mycoplasma meleagridis * Mycoplasma gallinarum Mycoplasma iners Mycoplasma columbinasale * Mycoplasma columbinum * Mycoplasma lipophilum Mycoplasma hyopharyngis Mycoplasma sphenisci Mycoplasma synoviae Mycoplasma verecundum Mycoplasma gallinaceum Mycoplasma corogypsi Mycoplasma glycophilum Mycoplasma gallopavonis Mycoplasma buteonis * Mycoplasma felis * Mycoplasma mustelae * Mycoplasma leonicaptivi Mycoplasma bovirhinis Mycoplasma cynos * Mycoplasma edwardii Mycoplasma canis Mycoplasma columborale Mycoplasma oxoniensis Mycoplasma citelli Mycoplasma sturni Mycoplasma pullorum Mycoplasma anatis Mycoplasma crocodyli Mycoplasma alligatoris Mycoplasma hominis Mycoplasma equirhinis Mycoplasma phocidae * Mycoplasma falconis Mycoplasma spumans Mycoplasma arthritidis * Mycoplasma phocicerebrale * Mycoplasma auris * Mycoplasma alkalescens * * Mycoplasma canadense Mycoplasma gateae Mycoplasma arginini Mycoplasma cloacale Mycoplasma anseris Mycoplasma buccale Mycoplasma hyosynoviae Mycoplasma orale * Mycoplasma indiense Mycoplasma faucium Mycoplasma subdolum Mycoplasma gypis Mycoplasma pulmonis strain UAB CTIP Mycoplasma agassizii Mycoplasma testudineum Mycoplasma sualvi Mycoplasma moatsii Mycoplasma mobile Mycoplasma neurolyticum Mycoplasma cricetuli Mycoplasma collis Mycoplasma molare Mycoplasma lagogenitalium Mycoplasma iguanae Mycoplasma hyopneumoniae * Mycoplasma flocculare Mycoplasma ovipneumoniae Mycoplasma dispar Mycoplasma bovoculi Mycoplasma conjunctivae Mycoplasma hyorhinis Mycoplasma vulturis

0.1 substitutions/site

equigenitalium cluster

bovis cluster

lipophilum cluster

synoviae cluster

hominis cluster

pulmonis cluster sualvi cluster

neurolyticum cluster

583

Genus I. Mycoplasma Mycoplasma insons Mycoplasma cavipharyngis Mycoplasma fastidiosum

pneumoniae group

*

hemotropic cluster

Mycoplasma pneumoniae Mycoplasma genitalium Mycoplasma amphoriforme Mycoplasma testudinis Mycoplasma alvi Mycoplasma pirum Mycoplasma gallisepticum strain R Mycoplasma imitans Ureaplasma urealyticum Ureaplasma parvum serovar 3 Ureaplasma gallorale Ureaplasma diversum Ureaplasma felinum Ureaplasma canigenitalium Mycoplasma penetrans strain HF-2 Mycoplasma iowae Mycoplasma muris Mycoplasma microti

*

hemotropic cluster

*

Scale:

fastidiosum cluster

pneumoniae cluster

Ureaplasma cluster

muris cluster

Mycoplasma coccoides Mycoplasma haemofelis Mycoplasma haemocanis ‘Candidatus Mycoplasma haemobos’ Mycoplasma haemomuris Mycoplasma suis Mycoplasma wenyonii Mycoplasma ovis

0.1 substitutions /site

Figure 109.  Phylogenetic relationships in the Mycoplasma hominis and Mycoplasma pneumoniae groups of the order Mycoplasmatales. The phylogram was based on a Jukes–Cantor corrected distance matrix and weighted neighbor-joining analysis of the 16S rRNA gene sequences of the type strains, except where noted. Acholeplasma (formerly Mycoplasma) feliminutum was the outgroup. The major groups and clusters are defined in terms of positions in 16S rRNA showing characteristic base composition and signature positions, plus higher-order structural synapomorphies (Johansson and ­Pettersson, 2002; Weisburg et al., 1989). Bootstrap values (100 replicates) 99%) genomes: serovar 2 strain ATCC 27814, NZ_ABFL00000000; serovar 4 strain ATCC 27816, NZ_AAYO00000000; serovar 5 strain ATCC 27817, NZ_AAZR00000000; serovar 7 strain ATCC 27819, NZ_AAYP00000000; serovar 8 strain ATCC 27618, NZ_AAYN00000000; serovar 9 strain ATCC 33175, NZ_ AAYQ00000000; serovar 10 strain ATCC 33699, NC_011374; serovar 11 strain ATCC 33695, NZ_AAZS00000000; serovar 12 strain ATCC 33696, NZ_AAZT00000000; serovar 13 strain ATCC 33698, NZ_ABEV00000000. 2. Ureaplasma canigenitalium Harasawa, Imada, Kotani, Koshimizu and Barile 1993, 644VP ca.ni.ge.ni.ta¢li.um. L. n. canis dog; L. pl. n. genitalia the genitals; N.L. pl. gen. n. canigenitalium of canine genitals. Cells are coccoid and about 500 nm in diameter; coccobacillary forms are seen. Colonies are £20–140 mm diameter with fried-egg morphology. Serogroup I strains represented by D6P-CT are serologically distinct from all other established species in the genus and from the other three serogroups of

ureaplasmas isolated from dogs (represented by the strains DIM-C, D29M, and D11N-A). The species designation refers only to serogroup I strains, although strain D11N-A shows a one-way, serological cross-reaction with D6P-CT. It produces an IgA protease which specifically cleaves canine myeloma IgA, but not human or murine IgA. Genome size is 860 kbp (PFGE). DNA reassociation values: between D6P-CT and the other three canine strains (DIM-C, D29M, and D11N-A) are 41–63% versus 33% with Ureaplasma urealyticum (strain T960T). Source: habitat is the prepuce, vagina, and oral and nasal cavities of canines. DNA G+C content (mol%): 29.4 (HPLC). Type strain: D6P-C, ATCC 51252, CIP 106087. Sequence accession no. (16S rRNA gene): D78648 (type strain). 3. Ureaplasma cati Harasawa, Imada, Ito, Koshimizu, Cassell and Barile 1990a, 50VP ca¢ti. L. gen. n. cati of a cat. Cells are coccoid and ³675 nm diameter, exceeding the 450–550 nm range of most named Ureaplasma species. Coccobacillary forms are seen and occasionally filaments. Colonies are £15–140 mm in diameter with diffuse, granular appearance; some fried-egg colonies may appear after passaging. Distinct from other established species in the genus, including Ureaplasma felinum, antigenically and in PAGE (Harasawa et al., 1990a) and RFLP patterns (Harasawa et al., 1984). Genome size has not been determined. DNA reassociation values: 83–100% within feline serogroup SII strains (Ureaplasma cati) versus 85% positive; −, 0–15% positive; nd, not determined.

a

*Deceased 21 December 2007.

v­ iability after 7–10 d. Arginine hydrolysis and “film and spot” lipase reactions are rare among species described to date. Entomoplasmas were shown to lack some key metabolic ­activities found in other mollicutes, especially PPi-dependent phosphofructokinase and dUTPase, and to possess uracil DNA glycosylase activity. Although the latter pyrimidine enzymic activity distinguished Entomoplasma from Mesoplasma species, only two Entomoplasma species and three Mesoplasma species have been tested so far for these activities (Pollack et al., 1996). Antisera to whole cell antigens of entomoplasmas have been used extensively to provide specific identification to the species level with a variety of serologic techniques, including growth inhibition, metabolism inhibition, and agar plate immunofluorescence (Tully et al., 1989, 1990, 1998). There is no evidence for the pathogenicity of entomoplasmas to either plant or insect hosts. Like other mollicutes, the entomoplasmas are resistant to 500 U/ml penicillin G.

Enrichment and isolation procedures Flowers and other plant material should be cut in the field and placed in plastic bags without touching by hand. In the laboratory, plant materials are rinsed briefly in either SP-4 or M1D media (May et  al., 2008). In both of these media, fetal bovine serum is a critical component for successful growth of these organisms (Hackett and Whitcomb, 1995; Tully, 1995). The rinse medium is immediately decanted and passed through a sterile membrane filter, usually of 450 nm porosity. The filtrate is then passed through at least several tenfold dilutions in the selected culture medium. The retentate may be frozen at −70°C for later use or for retesting. The cultures are incubated at 27–30°C and monitored by dark-field microscopy and/or by observing acidification of the medium. It is important to note that several nonsterol-requiring Acholeplasma species have also been isolated from plant and insect material (Tully et al., 1994b). Insect material, primarily from gut contents or hemolymph obtained by dissection or by fine-pointed glass pipettes, should be added to small volumes of SP-4 or M1D medium and filtered through a 450 nm membrane filter. Serial tenfold dilutions of the filtrate should be incubated at 27–30°C and observed for a decrease in pH of the medium. After two to three serial passages, the organisms should be purified by conventional filtercloning techniques (Tully, 1983) and stocks of various clones and early passage isolates frozen for further identification procedures (Whitcomb and Hackett, 1996).

Maintenance procedures Stock cultures of entomoplasmas can be maintained well in SP-4 and/or M1D broth medium containing about 17% fetal bovine serum. Most strains in the group can be adapted to grow in a broth medium containing bovine serum. Stock cultures in broth medium can be stored at −70°C for indefinite periods. For optimum preservation, the organisms should be lyophilized as broth cultures in the early exponential phase of growth and the dried cultures should be sealed under vacuum and stored at 4°C.

Genus I. Entomoplasma

Differentiation of the genus Entomoplasma from other genera Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et  al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. Aerobic or facultative anaerobic growth in artificial media and the necessity for sterols for growth exclude assignment to the genera Anaeroplasma, Asteroleplasma, Acholeplasma, Mesoplasma, or “Candidatus Phytoplasma”. Non-helical cellular morphology and regular association with arthropod or plant hosts support exclusion from the genera Spiroplasma or Mycoplasma. The inability to hydrolyze urea excludes assignment to the genus Ureaplasma. However, the difficulty in assigning novel species to this genus is well demonstrated by the earlier difficulties in establishing accurately the taxonomic status of these organisms (Tully et al., 1993). The availability of 16S rRNA gene sequence analyses was critical to the differentiation of these organisms from other mollicutes. Although isolates from vertebrates are very unlikely to be entomoplasmas, two bona fide Mycoplasma species, Mycoplasma iowae and Mycoplasma equigenitalium, have been isolated from plants [Grau et al., 1991; J.C. Vignault, J.M. Bové and J.G. Tully, unpublished (see ATCC 49192)].

Taxonomic comments The landmark studies of Weisburg et al. (1989), using 16S rRNA gene sequences of about 50 species of mollicutes, were critical in the resolution of certain taxonomic conflicts regarding the species that became Entomoplasma. The first entomoplasmas to be recognized were serologically related isolates from the flowers of Melaleuca and Grevillea trees (McCoy et al., 1979). Others, found in a wide range of insect species (Tully et  al., 1987), included strain ELCN-1T from the hemolymph of the firefly beetle Ellychnia corrusca (Tully et al., 1989) and three serologically distinct strains isolated from gut contents of Pyractomena and Photinus beetles (Williamson et al., 1990). Although these nonhelical, sterol-requiring mollicutes were initially placed in the genus Mycoplasma, 16S rRNA gene sequence analysis clearly indicated that strain M1T, previously designated Mycoplasma melaleucae, and strain ELCN-1T, previously designated Mycoplasma ellychniae, were most closely affiliated with the Spiroplasma lineage of helical organisms isolated primarily from arthropods. These findings prompted a proposal to reclassify the nonhelical mollicutes from arthropods and plants in a new order, Entomoplasmatales, and new family, Entomoplasmataceae, with the genus Entomoplasma reserved for sterol-requiring ­species (Tully et  al., 1993). Strains M1T and ELCN-1T were renamed as Entomoplasma melaleucae and Entomoplasma ellychniae, respectively. Subsequent phylogenetic analysis of Mycoplasma freundtii, later renamed Entomoplasma freundtii, confirmed the placement (Tully et al., 1998).

647

The paraphyletic relationship between the genera Entomoplasma and Mesoplasma is currently an unresolved problem in the systematics of this genus. It is possible that these genera, separated by the single criterion of sterol requirement, should be combined into the single genus Entomoplasma. However, Knight (2004) showed that Mesoplasma pleciae (Tully et al., 1994b) should belong to the genus Acholeplasma based on 16S rRNA gene sequence similarity and the preferred use of UGG rather than UGA as the codon for tryptophan. Therefore, transfer of the currently remaining members of genus Mesoplasma to other genera cannot be endorsed until similar analyses have been completed for all of those species (D.V. Volokhov, unpublished).

Acknowledgements We thank Karl-Erik Johansson for helpful comments and suggestions and Gail E. Gasparich for her landmark contributions regarding the phylogenetics of the Entomoplasmatales. The major contributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged.

Further reading Tully, J.G. 1989. Class Mollicutes: new perspectives from plant and arthropod studies. In The Mycoplasmas, vol. 5 (edited by Whitcomb and Tully). Academic Press, San Diego, pp. 1–31. Tully, J.G. 1996. Mollicute–host interrelationships: current concepts and diagnostic implications. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, San Diego, pp. 1–21.

Differentiation of the species of the genus Entomoplasma The primary technique for differentiation of Entomoplasma species is 16S rRNA gene sequence comparisons, confirmed by serology (Brown et al., 2007). Nonhelical mollicutes that belong to a known species isolated from arthropods or plants can be readily identified serologically provided that a battery of potent antisera for classified species is available. Growth inhibition tests, performed by placing paper discs saturated with type-specific antisera on agar plates inoculated with the organism, are perhaps the most convenient and rapid serological technique to differentiate species (Clyde, 1983). The agar plate immunofluorescence test is also a convenient and rapid means of mollicute species identification. In the absence of specific conjugated antiserum, an indirect immunofluorescence test can be performed with type-specific antiserum and a fluorescein-conjugated secondary antibody. The metabolism inhibition test (Taylor-Robinson, 1983) has also been applied to differentiation of Entomoplasma species (Tully et al., 1998).

List of species of the genus Entomoplasma 1. Entomoplasma ellychniae (Tully, Rose, Hackett, Whitcomb, Carle, Bové, Colflesh and Williamson 1989) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma ellychniae Tully, Rose, Hackett, Whitcomb, Carle, Bové, Colflesh and Williamson 1989, 288)

el.lych.ni¢ae. N.L. n. Ellychnia a genus of firefly beetles; N.L. gen. n. ellychniae of Ellychnia, from which the organism was first isolated. This is the type species of the genus Entomoplasma. Cells are nonhelical, pleomorphic filaments, with some ­branching;

648

Family I. Entomoplasmataceae

small coccoid forms, ranging in diameter from 200 to 300 nm, also occur. Passage of broth cultures through 450 and 300 nm porosity membrane filters does not reduce viable cell ­numbers, whereas passage through 220 nm porosity reduces cell populations by about 10%. Grows well in SP-4 medium with fetal bovine serum supplements. Does not grow well in horse serumsupplemented broth or agar media. Optimum temperature for broth growth is 30°C; can grow at 18–32°C. Colonies incubated at 30°C under anaerobic conditions have a fried-egg appearance. Does not hemadsorb guinea pig erythrocytes. No evidence for pathogenicity for insects. Source: isolated from the hemolymph of the firefly beetle Ellychniae corrusca. DNA G+C content (mol%): 27.5 (Bd). Type strain: ELCN-1, ATCC 43707, NCTC 11714. Sequence accession no. (16S rRNA gene): M24292. 2. Entomoplasma freundtii Tully, Whitcomb, Hackett, Williamson, Laigret, Carle, Bové, Henegar, Ellis, Dodge and Adams 1998, 1203VP freund¢ti.i. N.L. masc. gen. n. freundtii of Freundt, named after Eyvind Freundt, a Danish pioneer in the taxonomy and classification of mollicutes. Cells are predominantly coccoid in shape, ranging from 300 to 1200 nm in diameter. Organisms are readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm; more than 90% of viable cells in broth culture are able to pass 220 nm porosity membranes. The temperature range for growth is 10–32°C, with an optimum at 30°C. Colonies under anaerobic conditions are granular and frequently exhibit multiple satellite forms although the organism is considered nonmotile. The organism grows well in SP-4 broth medium or other media containing horse serum supplements. No evidence for pathogenicity for insects. Source: isolated from the gut contents of a green tiger beetle (Coleoptera: Cicindelidae). DNA G+C content (mol%): 34.1 (Bd). Type strain: BARC 318, ATCC 51999. Sequence accession no. (16S rRNA gene): AF036954. 3. Entomoplasma lucivorax (Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma lucivorax Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990, 164) lu.ci.vo¢rax. L. fem. n. lux lucis light; L. neut. adj. vorax gluttonous, devouring; N.L. neut. adj. lucivorax light devouring, referring to the predacious habit of the host insect, which preys on other luminescent firefly species. Cells are either pleomorphic coccoidal or subcoccoidal, with a diameter of 200–300 nm, or are short, branched or unbranched filaments. Cells are readily filterable through membrane filters with mean pore diameters of 450, 300, and 220 nm, but do not pass 100 nm porosity membranes. Optimum temperature for growth is 30°C; can grow at 10–32°C. Nonmotile. Colonies under anaerobic conditions usually have a fried-egg appearance. Grows well in SP-4 broth medium or other media containing horse serum supplements. Colonies do not hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for insects or plants. Source: first isolated from the gut of a firefly beetle (Photinus pyralis); also isolated from a flower (Spirea ulmaria; C. Chastel, unpublished). DNA G+C content (mol%): 27.4 (Bd). Type strain: PIPN-2, ATCC 49196, NCTC 11716. Sequence accession no. (16S rRNA gene): AF547212. 4. Entomoplasma luminosum (Williamson, Tully, Rose, ­Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma luminosum Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990, 163) lu.mi.no¢sum. L. neut. adj. luminosum luminous, emitting light, referring to the luminescence of the adult host from which the organism was isolated. Cells are pleomorphic and coccoidal or subcoccoidal with a diameter of 200–300 nm. Cells also occur as short, branched or unbranched filaments. The organisms are readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm, but do not pass 100 nm porosity membranes. The temperature range for growth is 10–32°C, with an optimum at 32°C. Nonmotile. Colonies under anaerobic conditions have a fried-egg appearance. The organism grows well in SP-4 broth medium or other media containing horse serum supplements. Colonies hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for insects. Source: isolated from the gut of the firefly beetle (Photinus marginata). DNA G+C content (mol%): 28.8 (Bd). Type strain: PIMN-1, ATCC 49195, NCTC 11717. Sequence accession no. (16S rRNA gene): AY155670. 5. Entomoplasma melaleucae (Tully, Rose, McCoy, Carle, Bové, Whitcomb and Weisburg 1990) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma melaleucae Tully, Rose, McCoy, Carle, Bové, Whitcomb and Weisburg 1990, 146) me la.leu¢cae. N.L. n. Melaleuca a genus of tropical trees having white flowers with sweet fragrance; N.L. gen. n. melaleucae of Melaleuca, the plant from which the type strain was isolated. Cells are pleomorphic and coccoidal or subcoccoidal, with few filamentous forms. Coccoidal forms have mean diameters of 250–300 nm. Cells are readily filterable through 450 and 300 nm porosity membrane filters, with few cells passing 220 nm porosity membranes. The temperature range for growth is 10–30°C, with an optimum at about 23°C. Nonmotile. Colonies under anaerobic conditions at 23–30°C display a friedegg appearance. Grows well in SP-4 broth or in modified Edward medium containing fetal bovine serum. The organism does not grow well in horse serum-based broth medium. Agar colonies do not adsorb guinea pig erythrocytes. No evidence of pathogenicity for insects or plants. Source: isolated from flower surfaces of a subtropical plant, Melaleuca quinquenervia, in south Florida. Related strains have been isolated from flowers of other subtropical trees in Florida, Melaleuca decora and Grevillea robusta (silk oak), and from an anthophorine bee (Xylocopa micans) in the same geographic area.

Genus II. Mesoplasma

DNA G+C content (mol%): 27.0 (Bd). Type strain: M1, ATCC 49191, NCTC 11715. Sequence accession nos (16S rRNA gene): M24478, AY345990. Further comment: the 16S rRNA gene sequence is more similar to that of members of genus Mesoplasma than to others in the genus Entomoplasma. 6. Entomoplasma somnilux (Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma ­somnilux Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990, 163) som.ni¢lux. L. masc. n. somnus sleep; L. fem. n. lux light; N.L. n. somnilux intended to mean sleeping light, referring to the quiescent pupal stage of the host from which the organism was isolated, which precedes the luminescent adult stage.

649

Cells are pleomorphic and coccoidal or subcoccoidal, with a diameter of 200–300 nm; also occur as short, branched or unbranched filaments. Readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm. The temperature range for growth is 10–32°C, with ­optimum growth at 30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C have a fried-egg appearance. The organism grows well in SP-4 broth medium or other media containing horse serum supplements. Colonies do not adsorb guinea pig erythrocytes. No evidence of pathogenicity for insects. Source: isolated from a pupal gut of the firefly beetle (Pyractomena angulata). DNA G+C content (mol%): 27.4 (Bd). Type strain: PYAN-1, ATCC 49194, NCTC 11719. Sequence accession no. (16S rRNA gene): AY157871.

Genus II. Mesoplasma Tully, Bové, Laigret and Whitcomb 1993, 380VP Daniel R. Brown, Janet M. Bradbury and Robert F. Whitcomb* Me.so.plas¢ma. Gr. adj. mesos middle; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Mesoplasma middle form, name intended to denote a middle position with respect to sterol or cholesterol requirement.

Cells are nonhelical and nonmotile, generally coccoid or short filamentous forms. Coccoid cells are usually 220–300 nm in dia­ meter, but some cells in some species can be as large as 400–500 nm. Most strains ferment glucose and most, but not all, lack the ability to hydrolyze arginine. Species possess the phosphoenolpyruvatedependent sugar-phosphotransferase system. Neither serum nor cholesterol is required for growth, but strains show sustained growth in a serum-free or cholesterol-free medium when the medium is supplemented with 0.04% PES. The optimum temperature for growth is usually near 28–32°C, with some strains able to grow well at temperatures as low as 23°C or as high as 37°C. Genome sizes range from 825 to 930 kbp (PFGE). DNA G+C content (mol%): 26–32. Type species: Mesoplasma florum (McCoy, Basham, Tully, Rose, Carle and Bové 1984) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Acholeplasma florum McCoy, Basham, Tully, Rose, Carle and Bové 1984, 14).

Further descriptive information Cells are predominantly coccoid in the exponential phase of growth when examined by dark-field microscopy. Cells from broth cultures examined by transmission electron microscopy are also coccoid, with individual cells usually 220–500 nm in diameter and clearly defined by a single cytoplasmic membrane. Colony growth is best obtained on SP-4 agar medium. Plates incubated under anaerobic conditions at about 30°C usually display characteristic fried-egg type colonies after 5–7 d incubation. Several mesoplasmas lack certain key metabolic activities found in other mollicutes, especially PPi-dependent phosphofructokinase, dUTPase, and uracil DNA glycosylase activity (Pollack et al., 1996). Most mesoplasmas were isolated in M1D medium containing 15% fetal bovine serum (Whitcomb, 1983), but adapt well to growth in SP-4 broth containing 15–17% fetal *Deceased 21 December 2007.

bovine serum, or in broth medium containing a 1% bovine serum fraction supplement (Tully, 1984; Tully et al., 1994a). All species show strong fermentation of glucose with acid production (Table 141), with a rapid decline in pH of the medium and loss of viability. Arginine hydrolysis has been observed only with the type strain (PUPA-2T) of Mesoplasma photuris. Antisera directed against whole-cell antigens of filter-cloned mesoplasmas have been used extensively to establish species and to provide species identifications. There is no evidence of pathogenicity of any currently established species in the genus for either an insect or plant host. Mesoplasmas are resistant to 500 U/ml penicillin.

Enrichment, isolation, and maintenance procedures The culture media and procedures for isolation and maintenance of entomoplasmas from plant and insect sources can also be effectively applied for mesoplasmas.

Differentiation of the genus Mesoplasma from other genera Properties that fulfill criteria for assignment to this genus are the same as those for the genus Entomoplasma, with the exception that the genus Mesoplasma is currently reserved for species that are able to grow in serum-free medium supplemented with PES (Tully et al., 1993).

Taxonomic comments The existence of a flora of nonhelical, wall-less prokaryotes associated with arthropod or plant hosts was first documented by T.B. Clark, S. Eden-Green, and R.E. McCoy and colleagues. Some of the plant isolates were clearly related to previously described Acholeplasma species, such as Acholeplasma oculi (EdenGreen and Tully, 1979), whereas others were established as novel Acholeplasma species, able to grow well in broth media without any cholesterol, serum, or fatty acid supplements. However, a significant group of other similarly derived strains were able to

650

Family I. Entomoplasmataceae

M. coleopterae

M. corruscae

M. entomophilum

M. grammopterae

M. lactucae

M. photuris

M. seiffertii

M. syrphidae

M. tabanidae

Glucose fermentation Arginine hydrolysis Hemadsorption of guinea pig red blood cells DNA G+C content (mol%)

M. chauliocola

Characteristic

M. florum

Table 141.  Differential characteristics of species of the genus Mesoplasma a

+ − − 27.3

+ − + 28.3

+ − − 27.7

+ − + 26.4

+ − + 30

+ − − 29.1

+ − + 30

+ + − 28.8

+ − + 30

+ − + 27.6

+ − − 28.3

Symbols: +, >85% positive; −, 0–15% positive.

a

grow in serum-free or cholesterol-free media only when small amounts of PES were added to the medium. Because these strains grew in the absence of cholesterol or serum, several of them were initially described as Acholeplasma species, including Acholeplasma florum (McCoy et al., 1984), Acholeplasma entomophilum (Tully et al., 1988), and Acholeplasma seiffertii (Bonnet et al., 1991). Although the growth response to PES in serum-free or cholesterol-free media suggested that there were fundamental differences between such mollicutes and classic acholeplasmas, conclusive taxonomic evidence was lacking. The subsequent analysis of 16S rRNA gene sequences by Weisburg et al. (1989) showed that the PES-requiring organisms were closely related to the spiroplasma group of mollicutes and were phylogenetically distant from acholeplasmas. On the basis of these findings and additional phylogenetic data, a proposal was made that the plant- and insect-derived mollicutes with growth responses to PES in serum-free or cholesterol-free media would be assigned to a new family, Entomoplasmataceae, and a new genus, Mesoplasma (Tully et al., 1993). Three of the plant-derived strains previously described as Acholeplasma species (Acholeplasma f­lorum, Acholeplasma entomophilum, and Acholeplasma seiffertii) were transferred to the genus Mesoplasma, with retention of their species epithets. A single plant-derived strain that had previously been described as Mycoplasma lactucae, and later found to grow in serum-free or cholesterol-free media supplemented with PES, was renamed Mesoplasma lactucae. Later, eight novel Mesoplasma species were described (Tully et al., 1994a). The paraphyletic relationship between the genera Entomoplasma and Mesoplasma is a currently unresolved problem in the systematics of this genus. It is possible that these genera,

s­ eparated by the single criterion of sterol requirement, should be combined into the single genus Entomoplasma. However, Knight (2004) showed that Mesoplasma pleciae (Tully et  al., 1994a) should belong to the genus Acholeplasma based on 16S rRNA gene sequence similarity and the preferred use of UGG rather than UGA as the codon for tryptophan. Therefore, transfer of the currently remaining members of the genus Mesoplasma to other genera cannot be endorsed until similar analyses have been completed for all of those species (D.V. Volokhov, unpublished).

Acknowledgements We thank Karl-Erik Johansson for helpful comments and suggestions and Gail E. Gasparich for her landmark contributions regarding the phylogenetics of the Entomoplasmatales. The major contributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged.

Further reading Tully, J.G. 1989. Class Mollicutes: new perspectives from plant and arthropod studies. In The Mycoplasmas, vol. 5 (edited by Whitcomb and Tully). Academic Press, San Diego, pp. 1–31. Tully, J.G. 1996. Mollicute-host interrelationships: current concepts and diagnostic implications. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, San Diego, pp. 1–21.

Differentiation of the species of the genus Mesoplasma The techniques for differentiation of Mesoplasma species are the same as those for genus Entomoplasma.

List of species of the genus Mesoplasma 1. Mesoplasma florum (McCoy, Basham, Tully, Rose, Carle and Bové 1984) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Acholeplasma florum McCoy, Basham, Tully, Rose, Carle and Bové 1984, 14) flo¢rum. L. gen. pl. n. florum of flowers, indicating the recovery site of the organism. This is the type species of the genus. Cells are oval or coccoid. The organism is readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm, but does not pass a membrane with 100 nm porosity. Temperature range for growth is 18–37°C, with an optimum at 28–30°C. Colonies on agar medium containing horse serum supplements have a typical fried-egg appearance after anaerobic

incubation at 37°C. Colonies on agar do not hemadsorb guinea pig erythrocytes. The 16S rRNA gene sequence is identical to that of Mesoplasma entomophilum (GenBank accession no. AF305693), but antiserum against Mesoplasma florum did not inhibit growth of Mesoplasma entomophilum or label the surfaces of Mesoplasma entomophilum colonies on agar (Tully et al., 1988). There are additional phenotypic distinctions between the two species. No evidence of pathogenicity for plants or insects. Source: first isolated from surface of flowers on a lemon  tree (Citrus limon) in Florida, with subsequent ­isolations from floral surfaces of grapefruit (Citrus

Genus II. Mesoplasma

651

p­ aradisi) and ­powderpuff trees (Albizia julibrissin) in Florida (McCoy et al., 1979). Also isolated from a variety of plants and from the gut tissues of numerous species of insects (Clark et  al., 1986; Tully et  al., 1990; Whitcomb et al., 1982). DNA G+C content (mol%): 27.3 (Bd, whole genome sequence). Type strain: L1, ATCC 33453, NCTC 11704. Sequence accession nos: AF300327 (16S rRNA gene), NC_006055 (strain L1T genome sequence).

­ emperature range for growth is 10–32°C, with an optimum T of 30°C. Nonmotile. Colonies incubated anaerobically at 30°C usually have a fried-egg appearance. Colonies hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for plants or insects. Source: original isolation was from the gut of an adult ­firefly (Ellychnia corrusca). DNA G+C content (mol%): 26.4 (Bd, Tm, HPLC). Type strain: ELCA-2, ATCC 49579. Sequence accession no. (16S rRNA gene): AY168929.

2. Mesoplasma chauliocola Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP

5. Mesoplasma entomophilum (Tully, Rose, Carle, Bové, Hackett and Whitcomb 1988) Tully, Bové, Laigret and ­Whitcomb 1993, 380VP (Acholeplasma entomophilum Tully, Rose, Carle, Bové, Hackett and Whitcomb 1988, 166)

chau.li.o¢co.la. N.L. n. chaulio first part of the genus name of goldenrod beetle (Chauliognathus); L. suff. -cola (from L. masc. or fem. n. incola) inhabitant; N.L. masc. n. chauliocola inhabitant of the goldenrod beetle. Cells are primarily coccoid, ranging in size from 300 to 500 nm in diameter. Cells are readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm, with a small number of cells able to pass through 100 nm porosity filters. Temperature range for growth is 10–37°C, with an optimum of 32–37°C. Nonmotile. Colonies incubated anaerobically at 32–37°C show fried-egg morphology. Colonies hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for plants or insects. Source: originally isolated from gut fluid of an adult goldenrod soldier beetle (Chauliognathus pennsylvanicus). DNA G+C content (mol%): 28.3 (Bd, Tm, HPLC). Type strain: CHPA-2, ATCC 49578. Sequence accession no. (16S rRNA gene): AY166704. 3. Mesoplasma coleopterae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 692VP co.le.op.te¢rae. N.L. fem. gen. n. coleopterae of Coleoptera, referring to the order of insects (Coleoptera) from which the organism was first isolated. Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Organisms are readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm. Temperature range for growth is 10–37°C, with an optimum of 30–37°C. Nonmotile. Colonies incubated anaerobically at 30°C usually have a fried-egg appearance. Agar colonies do not hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for plants or insects. Source: original isolation was from the gut of an adult ­soldier beetle (Chauliognathus sp.). DNA G+C content (mol%): 27.7 (Bd, Tm, HPLC). Type strain: BARC 779, ATCC 49583. Sequence accession no. (16S rRNA gene): DQ514605 (partial sequence). 4. Mesoplasma corruscae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP cor.rus¢cae. N.L. fem. gen. n. corruscae of corrusca, referring to the species of firefly beetle (Ellychnia corrusca) from which the organism was first isolated. Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Cells are readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm.

en.to.mo.phi¢lum. Gr. n. entomon insect; N.L. neut. adj. ­philum (from Gr. neut. adj. philon) friend, loving; N.L. neut. adj. entomophilum insect-loving. Cells are pleomorphic, but primarily coccoid, ranging from 300 to 500 nm in diameter. Cells are readily filterable through 220 nm porosity membrane filters. The temperature range for growth is 23–32°C, with an optimum at 30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C usually have a fried-egg appearance. Colonies hemadsorb guinea pig erythrocytes. The 16S rRNA gene sequence is identical to that of ­Mesoplasma florum (GenBank accession no. AF300327), but antiserum against Mesoplasma florum did not inhibit growth of Mesoplasma entomophilum or label the surfaces of Mesoplasma entomophilum colonies on agar (Tully et  al., 1988). There are additional phenotypic distinctions between the two species. No evidence of pathogenicity for plants or insects. Source: original isolation was from the gut contents of a tabanid fly (Tabanus catenatus). Also isolated from a variety of other species of insects. DNA G+C content (mol%): 30 (Bd). Type strain: TAC, ATCC 43706, NCTC 11713. Sequence accession no. (16S rRNA gene): AF305693. 6. Mesoplasma grammopterae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP gram.mop.te¢rae. N.L. fem. gen. n. grammopterae of Grammoptera, referring to the genus of beetle (Grammoptera) from which the organism was first isolated. Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Cells are readily filterable through membrane filters with mean pore diameters of 450, 300, and 220 nm. Temperature range for growth is 10–37°C, with an optimum at 30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C have a fried-egg appearance. Colonies do not hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for plants or insects. Source: original isolation was from the gut contents of an adult long-horned beetle (Grammoptera sp.). Other isolations were made from adult soldier beetle (Cantharidae sp.) and from an adult mining bee (Andrena sp.). DNA G+C content (mol%): 29.1 (Bd, Tm, HPLC). Type strain: GRUA-1, ATCC 49580. Sequence accession no. (16S rRNA gene): AY174170.

652

Family I. Entomoplasmataceae

7. Mesoplasma lactucae (Rose, Kocka, Somerson, Tully, ­Whitcomb, Carle, Bové, Colflesh and Williamson 1990) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma lactucae Rose, Kocka, Somerson, Tully, Whitcomb, Carle, Bové, Colflesh and Williamson 1990, 141) lac.tu¢cae. L. fem. n. lactuca lettuce; L. gen. n. lactucae of lettuce, referring to the plant from which the organism was first isolated. Cells are primarily coccoid, ranging in size from 300 to 500 nm in diameter, with only occasional short, nonhelical, pleomorphic filaments. Cells are readily filterable through membrane filters with mean pore diameters of 450, 300, and 220 nm, and a few cells are able to pass 100 nm porosity membranes. Temperature range for growth is 18–37°C, with optimal growth at 30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C have a fried-egg appearance. Colonies hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for plants or insects. Source: original isolation was from lettuce (Lactuca sativa). DNA G+C content (mol%): 30 (Bd). Type strain: 831-C4, ATCC 49193, NCTC 11718. Sequence accession no. (16S rRNA gene): AF303132. Has been reported to possess three rRNA operons (Grau, 1991). 8. Mesoplasma photuris Tully, Whitcomb, Hackett, Rose, ­Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP pho.tu¢ris. N.L. gen. n. photuris of Photuris, referring to the genus of firefly beetle (Photuris sp.) from which the organism was first isolated. Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Readily filterable through membrane filters with mean pore diameters of 450, 300, and 220 nm. Temperature range for growth is 10–32°C, with optimum at 30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C have a fried-egg appearance. Colonies do not hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for plants or insects. Source: original isolation was from gut fluids of larval and adult fireflies (Photuris lucicrescens and other Photuris spp.). One isolate (BARC 1976) was obtained by F.E. French from the gut of a horse fly (Tabanus americanus). DNA G+C content (mol%): 28.8 (Bd, Tm, HPLC). Type strain: PUPA-2, ATCC 49581. Sequence accession no. (16S rRNA gene): AY177627. 9. Mesoplasma seiffertii (Bonnet, Saillard, Vignault, Garnier, Carle, Bové, Rose, Tully and Whitcomb 1991) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Acholeplasma seiffertii Bonnet, Saillard, Vignault, Garnier, Carle, Bové, Rose, Tully and Whitcomb 1991, 48) seif.fer¢ti.i. N.L. masc. gen. n. seiffertii of Seiffert, in honor of Gustav Seiffert, a German microbiologist who performed pioneering studies on mollicutes that occur in soil and compost and do not require sterols for growth. Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Cells are readily filterable through membranes with mean pore diameters of 450, 300, and

220  nm. Temperature range for growth is 20–35°C, with optimum at about 28–30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C have a fried-egg appearance. Colonies hemadsorb guinea pig erythrocytes. Three insect isolates of Mesoplasma seiffertii, two from ­mosquitoes and one from a horse fly, were compared to strain F7T of plant origin. High relatedness values of 78–98% DNA–DNA reassociation under high stringency conditions were obtained (Gros et al., 1996). No evidence of pathogenicity for plants or insects. Source: first isolated from floral surfaces of a sweet orange tree (Citrus sinensis) and from wild angelica (Angelica sylvestris). Also isolated from insects. DNA G+C content (mol%): 30 (Bd). Type strain: F7, ATCC 49495. Sequence accession no. (16S rRNA gene): L12056. 10. Mesoplasma syrphidae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP syr.phi¢dae. N.L. fem. gen. n. syrphidae of a syrphid, referring to the syrphid fly family (Syrphidae), from which the organism was first isolated. Cells are primarily coccoid, ranging in size from 300 to 500 nm in diameter. Cells readily pass membrane filters with mean pore diameters of 450, 300, and 220 nm. Temperature range for growth is 10–32°C, with optimum at 23–25°C. Nonmotile. Colonies incubated under anaerobic conditions at 23–25°C have a fried-egg appearance. Colonies hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for insects. Source: original isolation was from the gut of an adult syrphid fly (Diptera: Syrphidae). Similar strains have been isolated from a bumblebee (Bombus sp.) and a skipper ­(Lepidoptera: Hesperiidae). DNA G+C content (mol%): 27.6 (Bd, Tm, HPLC). Type strain: YJS, ATCC 51578. Sequence accession no. (16S rRNA gene): AY231458. 11. Mesoplasma tabanidae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 692VP ta.ba.ni.dae. N.L. fem. gen. n. tabanidae of a tabanid, referring to the horse fly family (Tabanidae), the host from which the organism was first isolated. Cells are primarily coccoid, ranging in size from 300 to 500 nm in diameter. Cells readily pass membrane filters with mean pore diameters of 450, 300, and 220 nm. Temperature range for growth is 10–37°C, with optimum at 37°C. Nonmotile. Colonies incubated under anaerobic conditions at 37°C display a fried-egg appearance. Colonies do not hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for insects. Source: original isolation was from the gut of an adult horse fly (Tabanus abactor). DNA G+C content (mol%): 28.3 (Bd, Tm, HPLC). Type strain: BARC 857, ATCC 49584. Sequence accession no. (16S rRNA gene): AY187288.

Genus II. Mesoplasma

References Bonnet, F., C. Saillard, J.C. Vignault, M. Garnier, P. Carle, J.M. Bové, D.L. Rose, J.G. Tully and R.F. Whitcomb. 1991. Acholeplasma seiffertii sp. nov., a mollicute from plant surfaces. Int. J. Syst. Bacteriol. 41: 45–49. Brown, D.R., R.F. Whitcomb and J.M. Bradbury. 2007. Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. Clark, T.B., J.G. Tully, D.L. Rose, R. Henegar and R.F. Whitcomb. 1986. Acholeplasmas and similar nonsterol-requiring mollicutes from insects: missing link in microbial ecology. Curr. Microbiol. 13: 11–16. Clyde, W.A., Jr. 1983. Growth inhibition tests. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 405–410. Eden-Green, S.J. and J.G. Tully. 1979. Isolation of Acholeplasma spp. from coconut palms affected by lethal yellowing disease in Jamaica. Curr. Microbiol. 2: 311–316. Grau, O. 1991. Analyse des gènes ribosomiques des mollicutes, application à l’identification d’un mollicute non classé et conséquences taxonomiques [thesis]. Bordeaux, France. Grau, O., F. Laigret, P. Carle, J.G. Tully, D.L. Rose and J.M. Bové. 1991. Identification of a plant-derived mollicute as a strain of an avian pathogen, Mycoplasma iowae, and its implications for mollicute taxonomy. Int. J. Syst. Bacteriol. 41: 473–478. Gros, O., C. Saillard, C. Helias, F. LeGoff, M. Marjolet, J.M. Bové and C. Chastel. 1996. Serological and molecular characterization of Mesoplasma seiffertii strains isolated from hematophagous dipterans in France. Int. J. Syst. Bacteriol. 46 : 112–115. Hackett, K.J. and R.F. Whitcomb. 1995. Cultivation of spiroplasmas in undefined and defined media. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). ­Academic Press, San Diego, pp. 41–53. Knight, T.F., Jr. 2004. Reclassification of Mesoplasma pleciae as Acholeplasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene sequence data. Int. J. Syst. Evol. Microbiol. 54: 1951–1952. May, M., R.F. Whitcomb and D.R. Brown. 2008. Mycoplasma and related organisms. In Practical Handbook of Microbiology (edited by Goldman and Green). CRC Press, Boca Raton, pp. 467–491. McCoy, R.E., D.S. Williams and D.L. Thomas. 1979. Isolation of mycoplasmas from flowers. Proceedings of the Republic of China-United States Cooperative Science Seminar, Symposium series 1, National Science Council, Taipei, Taiwan, pp. 75–81. McCoy, R.E., H.G. Basham, J.G. Tully, D.L. Rose, P. Carle and J.M. Bové. 1984. Acholeplasma florum, a new species isolated from plants. Int. J. Syst. Bacteriol. 34: 11–15. Pollack, J.D., M.V. Williams, J. Banzon, M.A. Jones, L. Harvey and J.G. Tully. 1996. Comparative metabolism of Mesoplasma, Entomoplasma, Mycoplasma, and Acholeplasma. Int. J. Syst. Bacteriol. 46: 885–890. Rose, D.L., J.P. Kocka, N.L. Somerson, J.G. Tully, R.F. Whitcomb, P. Carle, J.M. Bové, D.E. Colflesh and D.L. Williamson. 1990. Mycoplasma lactucae sp. nov., a sterol-requiring mollicute from a plant surface. Int. J. Syst. Bacteriol. 40: 138–142. Rose, D.L., J.G. Tully, J.M. Bove and R.F. Whitcomb. 1993. A test for measuring growth responses of Mollicutes to serum and polyoxyethylene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532. Taylor-Robinson, D. 1983. Metabolism inhibition tests. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 411–421. Tully, J.G. 1983. Cloning and filtration techniques for mycoplasmas. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 173–177. Tully, J.G. 1984. Genus Acholeplasma. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 775–781.

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Tully, J.G. 1995. Determination of cholesterol and polyoxyethylene ­sorbitan growth requirements of mollicutes. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, San Diego, pp. 381–389. Tully, J.G., D.L. Rose, R.F. Whitcomb, K.J. Hackett, T.B. Clark, R.B. Henegar, E. Clark, P. Carle and J.M. Bové. 1987. Characterization of some new insect-derived acholeplasmas. Isr. J. Med. Sci. 23: 699–703. Tully, J.G., D.L. Rose, P. Carle, J.M. Bové, K.J. Hackett and R.F. Whitcomb. 1988. Acholeplasma entomophilum sp. nov. from gut contents of a wide-range of host insects. Int. J. Syst. Bacteriol. 38: 164–167. Tully, J.G., D.L. Rose, K.J. Hackett, R.F. Whitcomb, P. Carle, J.M. Bové, D.E. Colflesh and D.L. Williamson. 1989. Mycoplasma ellychniae sp. nov., a sterol-requiring mollicute from the firefly beetle Ellychnia corrusca. Int. J. Syst. Bacteriol. 39: 284–289. Tully, J.G., D.L. Rose, R.E. McCoy, P. Carle, J.M. Bové, R.F. Whitcomb and W.G. Weisburg. 1990. Mycoplasma melaleucae sp. nov., a sterolrequiring mollicute from flowers of several tropical plants. Int. J. Syst. Bacteriol. 40: 143–147. Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised taxonomy of the class Mollicutes - proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378–385. Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Rose, R.B. Henegar, J.M. Bové, P. Carle, D.L. Williamson and T.B. Clark. 1994a. Taxonomic descriptions of eight new non-sterol-requiring Mollicutes assigned to the genus Mesoplasma. Int. J. Syst. Bacteriol. 44: 685–693. Tully, J.G., R.F. Whitcomb, D.L. Rose, J.M. Bové, P. Carle, N.L. Somerson, D.L. Williamson and S. Edengreen. 1994b. Acholeplasma brassicae sp. nov. and Acholeplasma palmae sp. nov., two ­non-sterol-requiring mollicutes from plant surfaces. Int. J. Syst. Bacteriol. 44: 680–684. Tully, J.G., D.L. Rose, C.E. Yunker, P. Carle, J.M. Bové, D.L. Williamson and R.F. Whitcomb. 1995. Spiroplasma ixodetis sp. nov., a new species from Ixodes pacificus ticks collected in Oregon. Int. J. Syst. Bacteriol. 45: 23–28. Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Williamson, F. Laigret, P. Carle, J.M. Bové, R.B. Henegar, N.M. Ellis, D.E. Dodge and J. Adams. 1998. Entomoplasma freundtii sp. nov., a new species from a green tiger beetle (Coleoptera: Cicindelidae). Int. J. Syst. Bacteriol. 48: 1197–1204. Weisburg, W.G., J.G. Tully, D.L. Rose, J.P. Petzel, H. Oyaizu, D. Yang, L. Mandelco, J. Sechrest, T.G. Lawrence, J. Van Etten, J. Maniloff and C.R. Woese. 1989. A phylogenetic analysis of the mycoplasmas: basis for their classification. J. Bacteriol. 171: 6455–6467. Whitcomb, R.F. 1983. Culture media for spiroplasmas. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 147–158. Whitcomb, R.F. and K.J. Hackett. 1996. Identification of mollicutes from insects. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, San Diego, pp. 313–322. Whitcomb, R.F., J.G. Tully, D.L. Rose, E.B. Stephens, A. Smith, R.E. McCoy and M.F. Barile. 1982. Wall-less prokaryotes from fall flowers in central United States and Maryland. Curr. Microbiol. 7: 285–290. Williamson, D.L., J.G. Tully, D.L. Rose, K.J. Hackett, R. Henegar, P. Carle, J.M. Bové, D.E. Colflesh and R.F. Whitcomb. 1990. Mycoplasma somnilux sp. nov., Mycoplasma luminosum sp. nov., and Mycoplasma lucivorax sp. nov., new sterol-requiring mollicutes from firefly beetles (Coleoptera, Lampyridae). Int. J. Syst. Bacteriol. 40 : 160–164.

654

Family II. Spiroplasmataceae

Family II. Spiroplasmataceae Skripal 1983, 408VP David L. Williamson, Gail E. Gasparich, Laura B. Regassa, Collette Saillard, Joël Renaudin, Joseph M. Bové and Robert F. Whitcomb* Spi.ro.plas.ma.ta.ce′ae. N.L. neut. n. Spiroplasma, -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Spiroplasmataceae the Spiroplasma family. Cells are helical during exponential growth, with rotatory, flexional, and translational motility. Genome size is variable: 780–2220 kbp. Variable sterol requirement for growth. Procedures for determining sterol requirement are as described for Family I (Entomoplasmataceae). Possess a phosphoenolpyruvate phosphotransferase system for glucose uptake. Reduced

nicotinamide adenine dinucleotide (NADH) oxidase activity is located only in the cytoplasm. Unable to synthesize fatty acids from acetate. Other characteristics are as described for the class and order. Type genus: Spiroplasma Saglio, L’Hospital, Laflèche, Dupont, Bové, Tully and Freundt 1973, 201AL.

Genus I. Spiroplasma Saglio, L’Hospital, Laflèche, Dupont, Bové, Tully and Freundt 1973, 201AL David L. Williamson, Gail E. Gasparich, Laura B. Regassa, Collette Saillard, Joël Renaudin, Joseph M. Bové and Robert F. Whitcomb* Spi.ro.plas¢ma. Gr. n. speira (L. transliteration spira) a coil, spiral; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Spiroplasma spiral form.

Cells are pleomorphic, varying in size and shape from helical and branched nonhelical filaments to spherical or ovoid. The helical forms, usually 100–200 nm in diameter and 3–5 mm in length, generally occur during the exponential phase of growth and in some species persist during stationary phase. The cells of some species are short (1–2 mm). In certain cases, helical cells may be very tightly coiled, or the coils may show continuous variation in amplitude. Spherical cells ~300 nm in diameter and nonhelical filaments are frequently seen in the stationary phase, where they may not be viable, and in all growth phases in suboptimal growth media, where they may or may not be viable. In some species during certain phases, spherical forms may be the replicating form. Helical filaments are motile, with flexional and twitching movements, and often show an apparent rotatory motility. Fibrils are associated with the membrane, but flagellae, periplasmic fibrils, or other organelles of locomotion are absent. Fimbriae and pili observed on the cell surface of insect- and plant-pathogenic spiroplasmas are believed to be involved in host-cell attachment and conjugation (Ammar et  al., 2004; Özbek et  al., 2003), but not in locomotion. Cells divide by binary fission, with doubling times of 0.7–37 h. Facultatively anaerobic. The temperature growth range varies among species, from 5 to 41°C. Colonies on solid media are frequently diffuse, with irregular shapes and borders, a condition that reflects the motility of the cells during active growth (Figure 111). Colony type is strongly dependent on the agar concentration. Colony sizes vary from 0.1 to 4.0 mm in diameter. Colonies formed by nonmotile variants or mutants, or by cultures growing on inadequate media are typically umbonate with diameters of 200 mm or less. Some species, such as Spiroplasma platyhelix, have barely visible helicity along most of their length and display little rotatory or flexing motility. Colonies of motile, fast-growing spiroplasmas are diffuse, often with satellite colonies developing from foci adjacent to the initial site of colony development. Light turbidity may be produced in liquid cultures. Chemo-organotrophic. Acid is produced from glucose. Hydrolysis of arginine is variable. Urea, arbutin, and *Deceased 21 December 2007.

FIGURE 111.  Colonial morphology of Spiroplasma lampyridicola strain

PUP-lT grown on SP-4 agar under anaerobic conditions for 4 d at 30°C. The diffuse appearance and indistinct margins reflect the motility of spiroplasmas during active growth. Bar = 50 mm. (Reprinted with permission from Stevens et al., 1997. Int. J. Syst. Bacteriol. 47: 709–712.)

esculin are not hydrolyzed. Sterol requirements are variable. An optimum osmolality, usually in the range of 300–800 mOsm, has been demonstrated for some spiroplasmas. Media containing mycoplasma broth base, serum, and other supplements are required for primary growth, but after adaptation, growth often occurs in less complex media. Defined or semi-defined media are available for some species. Resistant to 10,000 U/ml penicillin. Insensitive to rifampicin, sensitive to erythromycin and tetracycline. Isolated from the surfaces of flowers and other plant parts, from the guts and hemolymph of various insects and crustaceans, and from tick triturates. Also isolated from vascular plant fluids (phloem sap) and insects that feed on the fluids. Specific host associations are common. The type species, Spiroplasma citri, is pathogenic for citrus (e.g., orange and

Genus I. Spiroplasma

grapefruit), producing “stubborn” disease. Experimental or natural infections also occur in horseradish, periwinkle, radish, broad bean, carrot, and other plant species. Spiroplasma kunkelii is a maize pathogen. Some species are pathogenic for insects. Certain species are pathogenic, under experimental conditions, for a variety of suckling rodents (rats, mice, hamsters and rabbits) and/or chicken embryos. Genome sizes vary from 780 to 2220 kbp (PFGE). DNA G+C content (mol%): 24–31 (Tm, Bd). Type species: Spiroplasma citri Saglio, L’Hospital, Laflèche, Dupont, Bové, Tully and Freundt 1973, 202AL.

Further descriptive information Morphology.  The morphology of spiroplasmas is most easily observed in suspensions with the light microscope under dark-field illumination (Williamson and Poulson, 1979). In the exponential phase in liquid media, most spiroplasma cells are helical filaments 90–250 nm in diameter and of variable length (Figure 112). Fixed and negatively stained cells usually show a blunt and a tapered end (Williamson, 1969; Williamson and Whitcomb, 1974). The tapered ends of the cells are a consequence of the constriction process preceding division (Garnier et  al., 1981, 1984). However, they are adapted as attachment sites in some species (Ammar et al., 2004). Motility.  Helical spiroplasma cells exhibit flexing, twitching, and apparent rotation about the longitudinal axis (Cole et al., 1973; Davis and Worley, 1973). Spiroplasmas exhibit temperature-dependent chemotactic movement toward higher concentrations of nutrients, such as carbohydrates and amino acids

FIGURE 112.  Electron micrograph of Drosophila willistoni strain B3SR sex-ratio spiroplasmas. Hemolymph suspension in phosphate buffered saline, glutaraldehyde vapor-fixed, and negatively stained with 1% phosphotungstic acid, pH 7.2. (Reprinted with permission from Whitcomb et al., 2007. Biodiversity and Conservation 16: 3877–3894.)

655

(Daniels and Longland, 1984, 1980); but motility is random in the absence of attractants (Daniels and Longland, 1984). Both natural (Townsend et  al., 1980b, 1977) and engineered (Cohen et al., 1989; Duret et al., 1999; Jacob et al., 1997) motility mutants have been described. These mutants form perfectly umbonate colonies on solid medium. Mutational analysis has highlighted the involvement of the smc1 gene in motility. Jacob et al. (1997) demonstrated that a Tn4001 insertion mutant with reduced flexional motility and no rotational motility could be complemented with the wild-type scm1 gene. The scm1 gene encodes a 409 amino acid polypeptide having ten transmembrane domains but no significant homology with known proteins. In another study, the scm1 gene was inactivated through homologous recombination, abolishing motility (Duret et  al., 1999). The disrupted scm1− mutant was injected into the leafhopper vector (Circulifer haematoceps); it multiplied actively in the insect vector and was then transmitted to periwinkle plants. The mutant induced symptoms that were indistinguishable from those caused by the motile wild-type strain showing that spiroplasma motility is not essential for phytopathogenicity and transmission to the plant host (Duret et al., 1999). Fibrils and motility.  Microfibrils 3.6 nm in width have been envisioned in the membranes of some spiroplasmas. These structures have repeat intervals of 9 nm along their lengths (Williamson, 1974) and form a ribbon that extends the entire length of the helix (Charbonneau and Ghiorse, 1984; Williamson et al., 1984). The sequence of the fibril protein gene has been determined (Williamson et al., 1991) and the calculated mass of the fibril protein is 59 kDa. The flat, monolayered, membrane-bound ribbon composed of several well-ordered fibrils represents the internal spiroplasmal cytoskeleton. The spiroplasmal cytoskeletal ribbon follows the shortest helical line on the cellular coil. Recent studies have focused on the detailed cellular and molecular organization of the cytoskeleton in Spiroplasma melliferum and Spiroplasma citri (Gilad et al., 2003; Trachtenberg, 2004; Trachtenberg et  al., 2003a, b; Trachtenberg and Gilad, 2001). Each cytoskeletal ribbon contains seven fibril pairs (or 14 fibrils) and the functional unit is a pair of aligned fibrils (Trachtenberg et al., 2003a). Paired fibrils can be viewed as chains of tetramers composed of 59 kDa monomers. Cryo-electron tomography has been used to elucidate the native state, cytoskeletal structure of Spiroplasma melliferum and suggested the presence of three parallel ribbons under the membrane: two appear to be composed of the fibril protein and the third is composed of the actin-like MreB protein (Kürner et al., 2005). Subsequent studies suggest the presence of a single ribbon structure (Trachtenberg et  al., 2008). The subunits in the fibrils undergo conformational changes from circular to elliptical, which results in shortening of the fibrils and helix contraction, or from elliptical to circular, leading to a length increase of the fibrils and cell helix. The cytoskeleton, which is bound to the spiroplasmal membrane over its entire length, acts as a scaffold and controls the helical shape of the cell. The cell shape is therefore dynamic. Movement appears to be driven by the propagation of a pair of kinks that travel down the length of the cell along the fibril ribbons (Shaevitz et al., 2005; Wada and Netz, 2007; Wolgemuth and Charon, 2005). The contractile cytoskeleton can thus be seen as a “linear motor” in contrast to the common “rotary motor” that is part of the flagellar apparatus in bacteria (Trachtenberg, 2006).

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Family II. Spiroplasmataceae

There are several adherent proteins that copurify with the cytoskeleton, ranging in size from 26 to 170 kDa (Townsend et  al., 1980a; Trachtenberg, 2006; Trachtenberg and Gilad, 2001). These proteins are apparently membrane-associated and may function as anchor proteins (Trachtenberg and Gilad, 2001). The structural organization of the cytoskeleton-associated proteins of Spiroplasma melliferum is beginning to be elucidated (Trachtenberg et  al., 2008). The 59 kDa polypeptide is the cytoskeletal fibril protein. The 26 kDa polypeptide is probably spiralin, the major spiroplasmal membrane protein. However, the involvement of spiralin in helicity and motility is unlikely (see “Spiralin” section below), especially since spiralin is anchored on the outside surface of the cell (Bévén et al., 1996; Bové, 1993; Brenner et al., 1995; Foissac et al., 1996) and spiralin-deficient mutants maintain helicity and motility (Duret et al., 2003). The 45 kDa protein may correspond to the product of the scm1 gene, shown to be essential for motility (Jacob et al., 1997), and the 34 kDa protein may be the product of the mreB1 gene (W. Maccheroni and J. Renaudin, unpublished). MreB is the bacterial homolog to eukaryotic actin (Jones et al., 2001; Van den Ent et  al., 2001). Early work provided evidence for the presence of actin-like proteins in spiroplasmas. Antisera prepared against SDS-denatured invertebrate actin coupled to horseradish peroxidase specifically stained cells of Spiroplasma citri (Williamson et al., 1979a). Also, a protein with a molecular mass similar to that of actin (protein P25) was isolated from Spiroplasma citri and reacted with IgG directed against rabbit actin (Mouches et al., 1982b, c, 1983b). Monospecific antibodies raised against the P25 protein recognized not only P25 of Spiroplasma citri, but also a homologous protein from Mycoplasma mycoides PG50 and Ureaplasma urealyticum serotype V (Mouches et al., 1983b). More recent work has focused on the molecular organization of the genes. mreB genes are present in rod-shaped, filamentous, and helical bacteria, but not in coccoid, spherical bacteria, regardless of whether or not they are Gram-stain-positive or Gramstain-negative. mreB genes are also absent from the pleomorphic mycoplasmas. However, Spiroplasma citri contains five homologs of Bacillus subtilis mreB genes (Maccheroni et al., 2002). Four of these (mreB2, 3, 4, and 5) form a cluster on the genome and are transcribed in two separate operons. Gene mreB1 is transcribed as a monocistronic operon and at a much higher level. Growth characteristics.  Spiroplasma cells increase in length and divide by constriction. Pulse labeling of the membrane with tritiated amino acids revealed a polar growth of the helix. Polarity was also observed by tellurium-labeling of oxido-reduction sites (Garnier et al., 1984). In the stationary or death phase, the cells are usually distorted, often forming either subovoid bodies or nonhelical filaments. Within cultured insect cells, all the spiroplasma cells were subovoid, but presumably viable (Wayadande and Fletcher, 1998). Thus, the ability of cells to grow and divide is not linked inextricably to helicity. Growth rate.  Enumerated microscopically (Rodwell and Whitcomb, 1983), spiroplasmas reach titers of 108–1011 cells/ ml in medium containing horse or fetal bovine serum. Growth rates of related strains tend to be similar. Konai et al. (1996a) calculated doubling times from the time required for medium acidification. In general, spiroplasmas adapted to complex cycles or single hosts had slower growth rates than spiroplasmas known or suspected to be transmitted on plant surfaces.

Temperature.­­  Konai et al. (1996a) determined temperature ranges and optima for a large number of spiroplasma strains. The ranges of some strains (e.g., Spiroplasma apis) were very wide (5–41°C), but some group I strains from leafhoppers and plants grew only at 25° and 30°C. Although some spiroplasmas grew well at 41°C, none grew at 43°C. Biochemical reactions.  All tested spiroplasmas ferment glucose with concomitant acid production, although the utilization rates may vary. Some strains of group I (e.g., members of subgroups I-4 and I-6) and all strains of Spiroplasma mirum ferment glucose slowly. With Spiroplasma citri, all strains tested grew actively on fructose and strain GII3 grew on fructose, glucose, or trehalose. The ability of spiroplasmas to utilize arginine varies (Hackett et al., 1996a). Arginine hydrolysis by some spiroplasmas can be observed only if glucose is also present in the medium. In other cases, aggressive glucose metabolism interferes with detection of arginine hydrolysis (Hackett et al., 1996a). Regulation of the fructose and trehalose operons of Spiroplasma citri.  The fructose operon of Spiroplasma citri (Gaurivaud et al., 2000a) became of special interest when fructose utilization was implicated in Spiroplasma citri phytopathogenicity (see “Mechanism of Spiroplasma citri phytopathogenicity” below). In particular, the role of the first gene of the operon, fruR, was investigated. In vivo transcription of the operon is greatly enhanced by the presence of fructose in the growth medium, whereas glucose has no effect. When fruR is not expressed (fruR− mutants), transcription of the operon is not stimulated by fructose and the rate of fructose fermentation is decreased, indicating that FruR is an activator of the fructose operon (Gaurivaud et al., 2001). Trehalose is the major sugar in leafhoppers and other insects. The trehalose operon of Spiroplasma citri has a gene organization very similar to that of the fructose operon and the first gene of the trehalose operon, treR, also encodes a transcriptional activator of the operon (André et al., 2003). Sterol utilization.  It was originally thought that all spiroplasmas require sterol for growth. Subsequent screening by Rose et al. (1993) showed that a minority of the spiroplasmas tested were able to sustain growth in mycoplasma broth base medium without sterols. The discovery that the sterol requirement in Mollicutes is polyphyletic greatly diminished the significance of sterol requirements in mollicute taxonomy (Tully et al., 1993). Metabolic pathways and enzymes.  The intermediary meta­ bolism of Mollicutes has been reviewed (Miles, 1992; Pollack, 2002a, b; Pollack et al., 1997). Like all mollicutes, Spiroplasma species apparently lack both cytochromes and, except for malate dehydrogenase, the enzymes of the tricarboxylic acid cycle. They do not have an electron-transport system and their respiration is characterized as being flavin-terminated. McElwain et al. (1988) studied Spiroplasma citri and Pollack et al. (1989) screened ten spiroplasma species for 67 enzyme activities. All spiroplasmas were fermentative; their 6-phosphofructokinases (6-PFKs) required ATP for substrate phosphorylation during glycolysis. This enzymic requirement is common to all mollicutes except Acholeplasma and Anaeroplasma spp. The 6-PFKs of the species in these genera require pyrophosphate and cannot use ATP. Additionally, except for Spiroplasma floricola, all Spiroplasma species have dUTPase activity. Pollack et al. (1989) also

Genus I. Spiroplasma

reported that all spiroplasmas except Spiroplasma floricola have deoxyguanosine kinase activity. They found that deoxyguanosine, but no other nucleoside, could be phosphorylated to GMP with ATP. Spiroplasmal proteins with multiple functions have been described. The CpG-specific methylase from Spiroplasma monobiae appears to also have topoisomerase activity (Matsuo et al., 1994). Protein P46 of Spiroplasma citri is a bifunctional protein in which the N-terminal domain represents ribosomal protein L29, whereas the C-terminal domain is capable of binding a specific inverted repeat sequence. It could be involved in regulation (Le Dantec et al., 1998). Such protein multifunctionality may reflect genomic economy in the small mollicute genome (Pollack, 2002b). However, functional redundancy has also been reported; Spiroplasma citri apparently has two distinct membrane ATPases (Simoneau and Labarère, 1991). Genome size, genomic maps, and chromosomal rearrangements.  PFGE revealed that the genome size range for spiroplasmas varied continuously (Pyle and Finch, 1988) from 780 kbp for Spiroplasma platyhelix to 2220 kbp for Spiroplasma ixodetis (Carle et al., 1995, 1990). There is a general trend for genomic simplification in Spiroplasma lineages. This trend culminated in loss of helicity and motility in the Entomoplasmataceae and eventually to the host transfer events forming the mycoides group of mycoplasmas (Gasparich et al., 2004). The genome size of Spiroplasma citri varies among strains from 1650 to 1910 kbp (Ye et al., 1995). It was found that the relative positions of mapped loci were conserved in most of the strains, but that differences in the sizes of certain fragments permitted genome size variation. Genome size can fluctuate rapidly in spiroplasma cultures after a relatively short number of in vitro passages (Melcher and Fletcher, 1999; Ye et al., 1996). The genome of Spiroplasma melliferum is 360 kbp shorter than that of Spiroplasma citri strain R8-A2T, but DNA hybridization has shown that the two spiroplasmas share extensive DNA hybridization (65%). Comparison of their genomic maps revealed that the genome region, which is shorter in Spiroplasma melliferum, corresponds to a variable region in the genomes of Spiroplasma citri strains and that a large region of the Spiroplasma melliferum genome is inverted in comparison with Spiroplasma citri. Therefore, chromosomal rearrangements and deletions were probably major events during evolution of the genomes of Spiroplasma citri and Spiroplasma melliferum. In addition, a large amount of noncoding DNA is present as repeat sequences (McIntosh et al., 1992; Nur et al., 1986, 1987) and integrated viral DNA (Bébéar et al., 1996) may also account for differences in genome sizes of closely related species. Base composition.  The DNA G+C content for most spiroplasma groups and subgroups has been determined (Carle et al., 1995, 1990; Williamson et al., 1998). Most group I spiroplasmas and Spiroplasma poulsonii have a G+C content of 25–27 mol%. However, the G+C content of subgroup I-6 Spiroplasma insolitum is significantly higher, indicating that the base composition of spiroplasmal DNA may shift over relatively short evolutionary periods. The range of G+C content of 25–27 mol% is modal for Spiroplasma and is also common in the Apis clade. However, Spiroplasma mirum (group V), strains of Spiroplasma apis (group IV), and group VIII strains have a G+C content of about 29–31 mol%. Restriction sites containing only G and C

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nucleotides are not uniformly distributed over the genome (Ye et al., 1992). Methylated bases.  Methylated bases have been detected in spiroplasmal DNA (Nur et al., 1985). The gene encoding the CpG methylase in Spiroplasma monobiae has been cloned (Renbaum et  al., 1990) and its mode of action studied (Renbaum and Razin, 1992). DNA restriction patterns.  Restriction patterns of spiroplasmal DNA, as determined by polyacrylamide gel electrophoresis, may be highly similar among strains of a given species (Bové et al., 1989). Variations in restriction fragment length patterns among strains of Spiroplasma corruscae correlated imperfectly with serological variation, so their significance was uncertain (Gasparich et al., 1998). RNA genes.  Some spiroplasmas, such as Spiroplasma citri, have only one rRNA operon, whereas others, such as Spiroplasma apis, have two (Amikam et al., 1984, 1982; Bové, 1993; Grau et  al., 1988; Razin, 1985). The three rRNA genes are linked in the classical order found in bacteria: 5¢-16S–23S-5S-3¢. The sequence of the 16S rRNA gene (rDNA) of most spiroplasma species has been determined for phylogenetic studies (Gasparich et al., 2004; Weisburg et al., 1989). A gene cluster of ten tRNAs (Cys, Arg, Pro, Ala, Met, Ile, Ser, fMet, Asp, Phe) was identified in Spiroplasma melliferum (Rogers et al., 1987). Similar tRNA gene clusters have been cloned and sequenced from Spiroplasma citri (Citti et al., 1992). Codon usage.  In spiroplasmas, UGA is not a stop codon but encodes tryptophan. The universal tryptophan codon, UGG, is also used (Citti et al., 1992; Renaudin et al., 1986). Codon usage also reflects the A+T richness of spiroplasmal DNA (usually about 74 mol% A+T). For example, in Spiroplasma citri, UGA is used to code for tryptophan eight times more frequently than the universal tryptophan codon UGG (Bové, 1993; Citti et al., 1992; Navas-Castillo et al., 1992). Also, synonymous codons with U or A at the 5¢ or 3¢ ends are preferentially used over those with a C or G in that position. RNA polymerase and spiroplasmal insensitivity to rifampicin.  Spiroplasmas are insensitive to rifampicin. DNAdependent RNA polymerases from Spiroplasma melliferum and Spiroplasma apis were at least 1000 times less sensitive to rifampicin than the corresponding Escherichia coli enzyme (Gadeau et  al., 1986). Rifampicin insensitivity of Spiroplasma citri and all other mollicutes tested was found to be associated with the presence of an asparagine residue at position 526 in RpoB. The importance of the asparagine residue was confirmed by site-directed mutagenesis of the histidine codon (CAC) to an asparagine codon (AAC) at position 526 of Escherichia coli RpoB, resulting in a rifampicin-resistant mutant (Gaurivaud et  al., 1996). The genetic organization surrounding the rpoB gene in spiroplasmas is also atypical. In many bacteria, rpoB is part of the b operon in which the four genes rplK, rplA, rplJ, and rplL, encoding ribosomal proteins L11, L1, L10, and L12, respectively, are located immediately upstream of rpoB; rpoC is immediately downstream of rpoB. In Spiroplasma citri, the gene organization is different in that the hsdS gene, encoding a component of a type I restriction-modification system, is upstream of rpoB. Sequences showing similarities with insertion elements are found between hsdS and rpoB (Laigret et al., 1996).

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Family II. Spiroplasmataceae

DNA polymerases and other proteins involved in DNA replication and repair.  From genomic studies, it appears that Mycoplasma species carry the essential, multimeric enzyme for genomic DNA replication, DNA polymerase III. The subunit responsible for actual DNA biosynthesis is subunit a, encoded by polC (dnaE). The polC gene has been identified in all sequenced mollicute genomes, including Spiroplasma citri. The genes encoding the other subunits, dnaN (subunit b) and dnaX (subunits t and g), are also shared by the Spiroplasma and Mycoplasma species studied to date. So, it seems that spiroplasmas, like other mollicutes, possess DNA polymerase III and that it is probably the major DNA replication enzyme. However, there is also evidence for two additional DNA polymerases. A second gene for a DNA polymerase (enzyme B) was found in the Spiroplasma citri genome and there is evidence that the Spiroplasma kunkelii polA gene may encode a full-length DNA polymerase I protein (Bai and Hogenhout, 2002). DNA polymerase I is a single polypeptide that has, in addition to DNA synthesis activity, two exonuclease activities: exo-3¢ to 5¢ as well as exo-5¢ to 3¢. At this stage, it is not possible to determine the equivalence between the three spiroplasmal DNA polymerases identified by sequencing (Pol III, enzyme B, and Pol I) and those originally detected biochemically (ScA, ScB, ScC) (Charron et al., 1979, 1982). As the Spiroplasma citri genome sequencing project has progressed, the following Spiroplasma citri genes involved in DNA replication have been detected: dnaA, dnaB, polA, dnaE, polC, dnaN, dnaX, holB, dinB (truncated), dnaJ, dnaK, gyrA, gyrB, parC, parE, topA, rnhB, rnhC, rnpA, rnR, rnc, yrrc, xseA, xseB, and ssb (Carle et al., 2010; accession numbers AM285301–AM285339). Genes encoding DNA replication proteins have also been identified in Spiroplasma kunkelii (Bai and Hogenhout, 2002). Spiroplasma citri is highly sensitive to UV irradiation (Labarère and Barroso, 1989) and the organism has no functional recA gene, since a significant portion of the C-terminal part of the gene is lacking (Marais et al., 1996).

region and an amino acid sequence repetition, including a VTKXE consensus sequence, are present in all spiralins analyzed (Foissac et al., 1997a). Spiralin confers a significant amount of the antigenic activity in group I spiroplasmas (Whitcomb et al., 1983) and has a high degree of species specificity, although minor cross-reactions have been detected (Zaaria et al., 1990). The spiralin genes of Spiroplasma citri and Spiroplasma melliferum species, which have about 65% overall DNA–DNA hybridization, shared 89% nucleotide sequence identity and 75% deduced amino acid sequence similarity (Bové et al., 1993). Spiralin mutants were constructed through homologous recombination in Spiroplasma citri to examine the role of spiralin in  vivo (Duret et  al., 2003). Phenotypic characterization of mutant 9a2 showed that, in spite of a total lack of spiralin, it maintained helicity and motility similar to the wild-type strain GII3 (Duret et  al., 2003). When injected into the leafhopper vector, Circulifer haematoceps, the mutant multiplied to a high titer, but transmission efficiency to periwinkle plants was very low compared to the wild-type strain. In the infected plants, however, the spiralin-deficient mutant multiplied well and produced the typical symptoms of the disease. In addition, preliminary results indicated that the mutant could not be acquired by insects feeding on 9a2-infected plants, suggesting that spiralin may mediate spiroplasma invasion of insect tissues (Duret et al., 2003). In order to test this possibility, Circulifer haematoceps leafhopper proteins were screened as putative Spiroplasma citri-binding molecules using Far-Western analysis (Killiny et al., 2005).These experiments showed that spiralin is a lectin capable of binding to insect 50 and 60 kDa mannose glycoproteins. Hence, spiralin could play a key role in insect transmission of Spiroplasma citri by mediating spiroplasma adherence to epithelial cells of the insect vector gut or salivary gland (Killiny et al., 2005). This would also explain why the spiralin-negative mutant 9a2 is poorly transmitted by the vector and is not acquired by insects feeding on 9a2-infected plants.

Origin of DNA replication.  Even before the Spiroplasma citri genome project was initiated, some fragments with multiple open reading frames had been completely sequenced. For example, Ye et al. (1994b) sequenced a 5.6 kbp fragment containing genes for the replication initiation protein (dnaA), the beta subunit of DNA polymerase III (dnaN), and the DNA gyrase subunits A and B (gyrA and gyrB). Several dnaA-box consensus sequences were found upstream and downstream of the dnaA gene. From these data, it was established that the dnaA region was the origin of replication in Spiroplasma citri (Ye et al., 1994b). Zhao et al. (2004a) cloned a cell division gene cluster from Spiroplasma kunkelii and functionally characterized the key division gene, ftsZsk, and showed that it encodes a cell division protein similar to FtsZ proteins from other bacteria.

Viruses.  Four different virus types have been found in Spiroplasma, SpV1-SpV4. Use of SpV1 viruses for recombinant DNA studies in Spiroplasma citri is described later in the section on “Tools for molecular genetics of Spiroplasma citri ”. Cells of many spiroplasma species contain filamentous/rodshaped viruses (SVC1 = SpV1) that are associated with nonlytic infections (Bové et  al., 1989; Ranhand et  al., 1980; Renaudin and Bové, 1994). They belong to the Plectrovirus group within the Inoviridae. SpV1 viruses have circular, single-stranded DNA genomes (7.5 to 8.5 kbp), some of which have been sequenced (Renaudin and Bové, 1994). SpV1 sequences also occur as prophages in the genome of the majority of Spiroplasma citri strains studied (Renaudin and Bové, 1994). These insertions take place at numerous sites in the chromosomes of Spiroplasma citri (Ye et al., 1992) and Spiroplasma melliferum (Ye et al., 1994a). The SpV1-ORF3 and the repeat sequences could be part of an IS-like element of chromosomal origin. Resistance of spiroplasmas to virus infection may be associated with integration of viral DNA sequences in the chromosome or extrachromosomal elements (Sha et al., 1995). The evolutionary history of these viruses is unclear, but there is some evidence for virus and plasmid co-evolution in the group I Spiroplasma species (Gasparich et al., 1993a) and indications of potentially widespread horizontal transmission (Vaughn and de Vos, 1995). Virus infection of spiroplasma cells can pose problems in cultures. For example,

Spiralin.  Spiralin, encoded by the spi gene, is the major membrane protein of Spiroplasma citri (Wróblewski et al., 1977, 1989). The deduced amino acid sequence of the protein (Bové et al., 1993; Chevalier et al., 1990; Saillard et al., 1990) corresponds well with the experimentally determined amino acid composition (Wróblewski et  al., 1984). In particular, spiralin lacks tryptophan and, thus, has no UGG and/or UGA codons, which facilitates gene expression in Escherichia coli. Detailed analyses showed that all Spiroplasma citri spiralins were 241–242 amino acids long (Foissac et  al., 1996). A conserved central

Genus I. Spiroplasma

lyophilized early passages of Spiroplasma citri R8-A2T proved difficult to grow and electron microscopy revealed that these cells carried large numbers of virions of virus SpV1-R8A2 (Cole et al., 1974). Likewise, SpV1 viruses have been found in Spiroplasma poulsonii (Cohen et al., 1987) and Spiroplasma melliferum (Liss and Cole., 1981); the Spiroplasma melliferum SpV1-KC3 virus forms plaques on various strains of Spiroplasma melliferum, including the type strain BC-3T. A second virus, reminiscent of a type B tailed bacterial virus, occurs in a small number of Spiroplasma citri strains (Cole et al., 1973). This SCV2 (= SpV2) virus is a polyhedron with a long, noncontractile tail. It may be associated with lytic infection. Infections in which large numbers of virions of SpV2 viruses are produced tend to be irregular and difficult to maintain under experimental conditions, so this is the least studied of the spiroplasma viruses. A third virus (SpV3) forms polyhedral virions with short tails and has been found in many strains of Spiroplasma citri (Cole, 1979, 1977, 1974). The SpV3 genome is a linear double-stranded DNA molecule of 16 kbp, which can circularize to form a covalently closed molecule with single-stranded gaps, indicating that the linear molecule has cohesive ends. There is significant diversity among SpV3 viruses, extending even to major differences in genome sizes. Virus SpV3-AV9/3 was isolated from Spiroplasma citri strain ASP-9 (Stephens, 1980). Dickinson and Townsend (1984) isolated the SpV3 virus from plants infected with Spiroplasma citri. This virus, when plated on cells of Spiroplasma citri, had a plaque morphology typical of temperate phages. In spiroplasma cells that have been lysogenized, complete virus genomes may be integrated into the spiroplasma chromosome. These cells are then immune to superinfection by the lysogenizing virus, but susceptible to other SpV3 viruses. It is possible that lysogenization of Spiroplasma citri by SpV3-ai affects spiroplasma pathogenicity, particularly with respect to attenuation. Drosophila spiroplasmas, male-lethal or nonlethal, usually carry SpV3 viruses. Each strain of Drosophila spiroplasma carries an associated virus that is lytic to certain other strains (Oishi et al., 1984). A fourth virus (SpV4), with a naked, icosahedral nucleocapsid 25 nm in diameter, was discovered (Ricard et al., 1982) in the B63 strain of Spiroplasma melliferum. SpV4 has a circular, single-stranded DNA genome (Renaudin and Bové, 1994; Renaudin et  al., 1984a, b) and is a lytic Spiromicrovirus within the Microviridae (Chipman et al., 1998). Infection with this virus results in very clear plaques, indicating a lytic process. Host range studies (Renaudin et al., 1984a, b) have shown that only Spiroplasma melliferum is susceptible to SpV4. Two strains of Spiroplasma melliferum, including the type strain BC-3T and B63, are not susceptible, as no plaques were formed on lawns of these spiroplasmas. These strains could be infected by transfection suggesting that resistance to the whole virus occurred at the level of adsorption or penetration of the virus (Renaudin and Bové, 1994; Renaudin et al., 1984b). Genome sequencing.  Genomic DNA sequencing efforts for two Spiroplasma species are in progress. For Spiroplasma citri GII3 (Carle et al., 2010; Saillard et al., 2008), assembly of 20,000 sequencing reads obtained from shotgun and chromosome specific libraries yielded: (1) 39 chromosomal contigs totalling 1525 kbp of the 1820 kbp Spiroplasma citri GII3 chromosome as well as (2) 8 circular contigs, which proved to represent seven

659

plasmids: pSciA (7.8 kbp), pSci1 to pSci6 (12.9 to 35.3 kbp), and one viral RF DNA (SVTS2). The chromosomal contigs contained 1905 putative genes or coding sequences (CDS). Of the CDS-encoded proteins, 29% are involved in cellular processes, cell metabolism, or cell structure. CDS for viral proteins and mobile elements represented 24% of the total, whereas 47% of the CDS were for hypothetical proteins with no known function; 21% of the total CDS appeared truncated as compared to their bacterial orthologs. Families of paralogs were mainly clustered in a large region of the chromosome opposite the origin of replication. Eighty-four CDS were assigned to transport functions, including phosphoenolpyruvate phosphotransferase systems (PTS), ATP binding cassette (ABC) transporters, and ferritin. In addition to the general enzymes EI and HPr, glucose- fructoseand trehalose-specific PTS permeases, and glycolytic and ATP synthesis pathways, Spiroplasma citri possesses a Sec-dependent protein export system and a nearly complete pathway for terpenoid biosynthesis. The sequencing of the Spiroplasma kunkelii CR2-3x genome (1.55 Mb) is also nearing completion (http:// www.genome.ou.edu/spiro.html); the physical and genetic maps have been published (Dally et al., 2006). Several studies have begun to focus on gene content and genomic organization (Zhao et al., 2003, 2004a, b). Results show that, in addition to virus SpV1 DNA insertions, the Spiroplasma kunkelii genome harbors more purine and amino acid biosynthesis, transcriptional regulation, cell envelope, and DNA transport/binding genes than Mycoplasmataceae (e.g., Mycoplasma genitalium and Mycoplasma pneumoniae) genomes (Bai and Hogenhout, 2002). Plasmids.  Several plasmids have been discovered in spiroplasmas (Archer et  al., 1981; Gasparich and Hackett, 1994; Gasparich et al., 1993a; Mouches et al., 1984a; Ranhand et al., 1980). They are especially common in spiroplasmas of group I. Eight extrachromosomal elements, including seven plasmids, were discovered during the Spiroplasma citri GII3 genome sequencing project. The six largest plasmids, pSci1 to pSci6, range from 12.9 to 35.3 kb (Saillard et  al., 2008). In silico analyses of plasmid sequences revealed that they share extensive regions of homology and display a mosaic gene organization. Genes encoding proteins of the TraD-TraG, TrsE-TraE, and Soj-ParA protein families, were predicted in most of the pSci sequences. The presence of such genes, usually involved in chromosome integration, cell to cell DNA transfer, or DNA element partitioning; suggests that these molecules could be inherited vertically as well as horizontally. The largest plasmid, pSci6, encodes P32 (Killiny et  al., 2006), a membrane-associated protein interestingly absent in all insect non-transmissible strains tested so far. The five remaining plasmids (pSci1 to pSci5) encode eight different Spiroplasma citri adhesion-related proteins. The complete sequences of plasmids pSKU146 from Spiroplasma kunkelii CR2-3x and pBJS-O from Spiroplasma citri BR3 have been reported (Davis et al., 2005; Joshi et al., 2005). These large plasmids, like the above Spiroplasma citri plasmids, encode an adhesin and components of a type IV translocationrelated conjugation system. Characterizing the replication and stability regions of Spiroplasma citri plasmids resulted in the identification of a novel replication protein, suggesting that Spiroplasma citri plasmids belong to a new plasmid family and that the soj gene is involved in segregational stability of these plasmids (Breton et al., 2008a). Similar replicons were detected in various spiroplasmas of group I, such as Spiroplasma ­melliferum,

660

Family II. Spiroplasmataceae

Spiroplasma kunkelii, Spiroplasma sp. 277F, and Spiroplasma phoeniceum, showing that they are not restricted to plant pathogenic spiroplasmas. Tools for molecular genetics of Spiroplasma citri.  Recent recombinant DNA tools are described in this section. Several reports have been published concerning the use of SpV1 viruses as tools to introduce recombinant DNA into spiroplasmas, including optimization of transfection conditions (Gasparich et  al., 1993b). The replicative form of SpV1 was used to clone and express the Escherichia coli-derived chloramphenicol acetyltransferase (cat) gene in Spiroplasma citri. Both the replicative form (RF) and the virion DNA produced by the transfected cells contained the cat gene sequences (Stamburski et al., 1991). The G fragment of the Mycoplasma pneumoniae cytadhesin P1 gene could also be expressed in Spiroplasma citri (Marais et  al., 1993) using a similar method. However, the recombinant RF proved unstable, resulting in the loss of the DNA insert (Marais et al., 1996). Recombinant plasmids have also been developed to introduce genes into Spiroplasma citri cells. The introduced genes include antibiotic resistance markers and wild-type genes to complement auxotrophic mutants. Most recombinant plasmids contain the origin of DNA replication (oriC) of the Spiroplasma citri chromosome (Ye et al., 1994b). One such plasmid is pBOT1 (Renaudin, 2002; Renaudin et al., 1995). This plasmid contains a 2 kbp oriC region, a tetracycline resistance gene (tetM) from Tn916, and the linearized Escherichia coli plasmid pBS with a colE1 origin of replication. Because of its two origins of replication, oriC and colE1, pBOT1 is able to shuttle between Spiroplasma citri and Escherichia coli. When introduced into Spiroplasma citri, pBOT1 replicates first as a free extrachromosomal element, but later integrates into the chromosome via homologous recombination involving a single crossover event in the oriC region. Once integrated into the host chromosome, the whole plasmid is stably maintained. Recent studies suggest that the broad host range Spiroplasma citri GII3 plasmids and their shuttle derivatives may have significant advantages over oriC plasmids for gene transfer and expression in spiroplasmas (Breton et al., 2008a). They transform Spiroplasma citri (as well as Spiroplasma kunkelii and Spiroplasma phoeniceum) strains at relatively high efficiencies, the growth of the transformants is not significantly affected, they do not integrate into the chromosome, and their stability/loss can be modulated depending upon the presence/absence of the soj gene. Spiroplasma citri mutants have been produced by random and targeted approaches. The transposon Tn4001 has been used successfully for random mutagenesis of Spiroplasma citri (Foissac et al., 1997c). For targeted gene inactivation, plasmids derived from pBOT1 have been used to disrupt genes (e.g., fructose operon, motility gene scm1) through homologous recombination involving a single crossover event (Duret et  al., 1999; Gaurivaud et al., 2000c). More recently, Lartigue et al. (2002) developed vector pC2, in which the oriC fragment was reduced to the minimal sequence needed to promote plasmid replication; this vector increases recombination frequency at the target gene. To avoid the extensive passaging that was required for recombination prior to transformant screening, vector pC55 was designed using a selective tetracycline resistance marker that is only expressed after the plasmid has integrated into the chromosome at the target gene. This approach was used to inactivate the spiralin gene (spi) and the gene encoding the

IICB component of the glucose phosphotransferase system permease (ptsG) (André et  al., 2005; Duret et  al., 2003; Lartigue et al., 2002). Another series of recombinant plasmids, the pGOT vectors, allow for selection of rare recombination events by using two distinct selective markers. First, transformants are screened for their resistance to gentamicin and next, site-specific recombinants are selected for based on their resistance to tetracycline, which can only be expressed through recombination at the target gene. In this way, inactivation of the crr gene, encoding the glucose phosphotransferase permease IIA component, was obtained (Duret et al., 2005). The use of the transposon gd TnpR/res recombination system to produce unmarked mutations (i.e., without insertion of antibiotic markers) in Spiroplasma citri was demonstrated by the production of a disrupted arcA mutant (Duret et al., 2005); arcA encodes arginine deiminase. In this system, the target gene is disrupted by integration of a plasmid containing target gene sequences along with the tetM gene flanked by binding-specific recombination (res) sites. After integration of the plasmid, a second plasmid is introduced that encodes the resolvase TnpR. TnpR mediates the resolution of the cointegrate at the res sites, thereby removing tetM but leaving behind a mutated version of the target gene. The TnpRencoding plasmid is lost spontaneously when selective pressure is removed. Antigenic structure.  Growth inhibition tests (Whitcomb et al., 1982) were used in the early years to identify spiroplasma species or groups, but metabolism inhibition (Williamson et al., 1979b; Williamson and Whitcomb, 1983) and deformation tests (Williamson et al., 1978) are now used almost exclusively (see below). Antigenic variability, which has been described for some Mycoplasma species (Rosengarten and Wise, 1990; Yogev et al., 1991), has not been demonstrated in spiroplasmas (R. Rosengarten, personal communication). Group classification.  The classification of spiroplasmas was first proposed by Junca et al. (1980) and has been revised periodically (Tully et al., 1987; Williamson et al., 1998). These classifications are based on serological reactions of the organisms in growth inhibition, deformation and metabolism inhibition tests and/or characteristics of their genomes. Development of a classification scheme has resulted in the delineation of spiroplasma groups and subgroups (Table 142). In the scheme, “groups” have been defined as clusters of similar organisms, all of which possess negligible DNA–DNA hybridization with representatives of other groups, but moderate to high levels of hybridization (20–100%) with each other. Groups are, therefore, putative species. This level of genomic differentiation correlates well with substantial differences in serology. Thirty-four groups were presented in a revised classification of spiroplasmas in 1998 (Williamson et al., 1998). Four additional groups (XXXV–XXXVIII) were proposed recently as the result of a global spiroplasma environmental survey (Whitcomb et  al., 2007) and more are anticipated (Jandhyam et al., 2008). Subgroups have been defined by the International Committee on Systematics of Bacteria (ICSB) Subcommittee on the Taxonomy of Mollicutes (ICSB, 1984) as clusters of spiroplasma strains showing intermediate levels of intragroup DNA–DNA hybridization (10–70%) and possessing corollary serological relationships. Three spiroplasma groups [group I (Junca et  al., 1980; Saillard et al., 1987), group VIII (Gasparich et al., 1993c), and

661

Genus I. Spiroplasma Table 142.  Biological properties of spiroplasmasa

Group

Spiroplasma

Strain

ATCC no.

Morphologyb

Genomec

G+Cd

Arge

Dtf

OptTg

Host

I-1 I-2 I-3 I-4 I-5 I-6 I-7 I-8 I-9

S. citri S. melliferum S. kunkelii Spiroplasma sp. Spiroplasma sp. S. insolitum Spiroplasma sp. S. phoeniceum S. penaei

R8-A2 BC-3T E275T 277F LB-12 M55T N525 P40T SHRIMPT

Long helix Long helix Long helix Long helix Long helix Long helix Long helix Long helix Helix

1820 1460 1610 1620 1020 1810 1780 1860 nd

26 26 26 26 26 28 26 26 29

+ + + + − − + + +

4.1 1.5 27.3 2.3 26.3 7.2 4.7 16.8 nd

32 37 30 32 30 30 32 30 28

Phloem/leafhopper Honey bee Phloem/leafhopper Rabbit tick Plant bug Flower surface Green June beetle Phloem/vector Pacific white shrimp

DW-1T

27556 33219T 29320T 29761 33649 33502T 33287 43115T BAA-1082T (CAIM 1252T) 43153T

II

S. poulsonii

Long helix

1040

26

nd

15.8

30

S. floricola S. apis S. mirum S. ixodetis S. monobiae S. syrphidicola Spiroplasma sp. S. chrysopicola S. clarkii S. culicicola S. velocicrescens S. diabroticae S. sabaudiense S. corruscae Spiroplasma sp. S. cantharicola Spiroplasma sp. Spiroplasma sp. S. turonicum S. litorale S. lampyridicola S. leptinotarsae

OMBG B31T SMCAT Y32T MQ-1T EA-1T TAAS-1 DF-1T CN-5T AES-1T MQ-4T DU-1T Ar-1343T EC-1T I-25 CC-1T CB-1 Ar-1357 Tab4cT TN-1T PUP-1T LD-1T

29989T 33834T 29335T 33835T 33825T 33826T 51123 43209T 33827T 35112T 35262T 43210T 43303T 43212T 43262 43207T 43208 51126 700271T 43211T 43206T 43213T

Helix Helix Helix Tight coil Helix Minute helix Minute helix Minute helix Helix Short helix Short helix Helix Helix Helix Wave-coil Helix Helix Helix Helix Helix Unstable helix Motile funnel

1270 1300 1300 2220 940 1230 1170 1270 1720 1350 1480 1350 1175 nd 1380 nd 1320 nd 1305 1370 1375 1085

26 30 30 25 28 30 31 29 29 26 26 25 29 26 26 26 26 26 25 25 25 25

− + + − − + + + + − − + + − − − − − − − − +

0.9 1.1 7.8 9.2 1.9 1.0 1.4 6.4 4.3 1.0 0.6 0.9 4.1 1.5 3.4 2.6 2.6 3.4 nd 1.7 9.8 7.2

37 34.5 37 30 32 32 37 30 30 37 37 32 30 32 30 32 32 30 30 32 30 30

XXI XXII XXIII XXIV XXV XXVI XXVII XXVIII XXIX XXX XXXI XXXII XXXIII XXXIV XXXV XXXVI XXXVII XXXVIII Nd

Spiroplasma sp. S. taiwanense S. gladiatoris S. chinense S. diminutum S. alleghenense S. lineolae S. platyhelix Spiroplasma sp. Spiroplasma sp. S. montanense S. helicoides S. tabanidicola Spiroplasma sp. Spiroplasma sp. Spiroplasma sp. Spiroplasma sp. Spiroplasma sp. S. atrichopogonis

W115 CT-1T TG-1T CCHT CUAS-1T PLHS-1T TALS-2T PALS-1T TIUS-1 BIUS-1 HYOS-1T TABS-2T TAUS-1T B1901 BARC 4886 BARC 4900 BARC 4908 GSU5450 GNAT3597T

980 1195 nd 1530 1080 1465 1390 780 840 nd 1225 nd 1375 1295 nd nd nd nd nd

24 26 26 29 26 31 25 29 28 28 28 27 26 25 nd nd nd nd 28

− − − − − + − + − − + − − − − − − − +

4.0 4.8 4.1 0.8 1.0 6.4 5.6 6.4 3.6 0.9 0.7 3.0 3.7 nd 0.6 1.0 1.2 1.5 nd

30 30 31 37 32 30 30 30 30 37 32 32 30 nd 32 30 32 32 30

Drosophila hemolymph Plant surface Honey bee Rabbit tick Ixodid tick Monobia wasp Syrphid fly Horse fly Deer fly Green June beetle Mosquito Monobia wasp Beetle Mosquito Horse fly/beetle Leafhopper Cantharid beetle Cantharid beetle Mosquito Horse fly Horse fly Firefly Colorado potato beetle Flower surface Mosquito Horse fly Flower surface Mosquito Scorpion fly Horse fly Dragonfly Tiphiid wasp Flower surface Horse fly Horse fly Horse fly Horse fly Horse fly Horse fly Horse fly Horse fly Biting midge

III IV V VI VII VIII-1 VIII-3 VIII-2 IX X XI XII XIII XIV XV XVI-1 XVI-2 XVI-3 XVII XVIII XIX XX

Nd

S. leucomae

nd

24

+

nd

30

Satin moth

T

SMAT

nd, Not determined.

a

For descriptions of morphotypes, see text.

b

Genome size (kbp).

c

DNA G+C content (mol%).

d

, Catabolizes arginine.

e+

Doubling time (h) (Konai et al., 1996a).

f

Optimum growth temperature (°C).

g

T

43260 Helix 43302T Helix 43525T Helix 43960T Helix 49235T Short helix 51752T Helix 51749T Helix 51748T Wave-coil 51751 Rare helices 51750 Late helices 51745T Helix 51746T Helix 51747T Helix 700283 Helix BAA-1183 Helix BAA-1184 Helix BAA-1187 Helix BAA-1188 Helix BAA-520T (NBRC Helix 100390T) BAA-521T (NBRC Helix 100392T)

662

Family II. Spiroplasmataceae

group XVI (Abalain-Colloc et al., 1993)] have been divided into a total of 15 subgroups. “Serovars” have been defined as genotypic clusters varying substantially in metabolism inhibition and deformation serology, but that are insufficiently differentiated from members of existing groups or subgroups to warrant separation. However, with the discovery of a large number of strains for some groups (e.g., group VIII), the serovar/subgroup picture has become very confused (Regassa et al., 2004; see Phylogeny, below). Procedures for species descriptions and minimal standards.  ­ pecies descriptions of spiroplasmas have been in accord with S recommendations of minimum standards proposed by the ICSP (International Committee on Systematics of Prokaryotes) Subcommittee on the Taxonomy of Mollicutes (Brown et al., 2007). Cloning.  Production of spiroplasma lineages produced from a single cell or clonings are performed largely by serial dilution of filtered cultures using 96-well microtiter plates (Whitcomb et  al., 1986; Whitcomb and Hackett, 1987). At a certain dilution, which varies from plating to plating, the mean number of cells per well decreases so that fewer than about 8 of the 96-wells support growth of a spiroplasma clone. Very probably, such clones arise from a single spiroplasmal cell. Cellular morphology.  Using dark-field microscopy, cultures should appear helical and motile during at least one growth phase (see “Morphology” and “Motility” above). However, morphological exceptions do occur (see “Differentiating characters” below and reviewed by Gasparich et al., 2004). 16S rRNA gene sequence analysis.  Preliminary identification is performed by PCR amplification using universal 16S rRNA (Gasparich et al., 2004) or other described primers (e.g., Fukatsu and Nikoh, 1998; Jandhyam, 2008). DNA sequence analysis using a blast search provides preliminary placement within the genus Spiroplasma. Those strains showing close phylogenetic relationships based on 16S rRNA gene sequence analyses should then be screened using serological tests. Serological tests.  The deformation test (Williamson et al., 1978) is used routinely for serological analyses. Reciprocal titers of ³320 are generally required for definitive group placement. Deformation is defined as entire or partial loss of helicity. At the end point, cells are often seen in which an unaffected part of the helical filament exhibits flexing motility despite the presence of a bleb on another part of the cell. The deformation titer is the reciprocal of the final antiserum dilution that exhibits deformation of ³50% of the cells. Antiserum should be produced for any strain thought to represent a novel serogroup and any positive test against characterized groups requires a recriprocal test using the newly prepared antiserum. The high levels of specificity and sensitivity of the metabolism inhibition test make it especially useful for defining groups and subgroups (Williamson et al., 1979b; Williamson and Whitcomb, 1983). Other serological tests have also been employed for characterization of spiroplasmas. Growth inhibition tests were used for delineation of spiroplasma groups I through XI (Whitcomb et al., 1982), but were not used thereafter. Growth inhibition tests are problematic for spiroplasmas because they require development of procedures for obtaining colonies. The spiroplasma motility inhibition test (Hackett et  al., 1997) has proved useful for determination of intraspecific variation in

Spiroplasma leptinotarsae. ELISA has been used for detection of Spiroplasma kunkelii (Gordon et  al., 1985) and Spiroplasma citri (Saillard and Bové, 1983). Optimum growth temperature.  Optimal growth temperatures between 10 and 41°C have been determined (Konai et al., 1996a). Substrate metabolism.  The ability to ferment glucose and produce acid must be examined (Aluotto et  al., 1970). The ability to hydrolyze arginine and produce ammonia should be assessed (Barile, 1983). See the section on “Biochemical reactions” above for more details. Ecology.  The species description must include ecological information such as isolation site within the host and cultivation conditions, common and binomial host name, geographical location of host (with GPS), any known interaction between the spiroplasma and its host, and, in the case of a pathogen, disease symptoms observed. Antibiotic sensitivities.  In early studies (Bowyer and Calavan, 1974; Liao and Chen, 1981b), spiroplasmas proved to be especially sensitive in vitro to tetracycline, erythromycin, tylosin, tobramycin, and lincomycin. Strains have been isolated that are permanently resistant to kanamycin, neomycin, gentamicin, erythromycin, and several tetracycline antibiotics (Liao and Chen, 1981b). Insensitivity to rifampicin has been studied in relation to its inhibition of transcription (see “RNA polymerase and spiroplasmal insensitivity to rifampicin” above) and penicillin insensitivity is seen for all spiroplasmas due to the lack of a cell wall. Natural amphipathic peptides such as Gramicidin S alter the membrane potential of spiroplasma cells and induce the loss of cell motility and helicity (Bévén and Wróblewski, 1997). The toxicity of the lipopeptide antibiotic globomycin was found to be correlated with an inhibition of spiralin processing (Bévén et  al., 1996). As with Gramicidin S, the antibiotic was effective against spiroplasmas, but not Mycoplasma mycoides. Natural 18-residue peptaibols (trichorzins PA) are bacteriocidal to spiroplasmas (Bévén et al., 1998). The mode of action appears to be permeabilization of the host cell membrane. Hosts, ecology, and pathogenicity Hosts.  Almost all spiroplasmas have been found to be associated with arthropods or an arthropod connection is strongly suspected. Hackett et  al. (1990) searched for mollicutes in a wide variety of insect orders. Isolates were obtained from six orders and 14 insect families. Only one of these orders, Odonata (dragonflies), was primitive (heterometabolous) and it was speculated that the spiroplasma from a dragonfly host might have been acquired via predation. Hackett et  al. (1990) suggested that the Spiroplasma/Entomoplasma clade may have arisen in a paraneopteran-holometabolan ancestor, coevolved with these orders, and never adapted to more primitive insect orders. Some insect families have an especially rich spiroplasma, entomoplasma, and mesoplasma flora. Insect gut.  The majority of spiroplasmas appear to be maintained in an insect gut/plant surface cycle. Clark (1984) hypothesized several types of gut infection in which persistence in the gut and the ability to invade hemolymph varied among spiroplasma species. It has been hypothesized (Hackett and Clark, 1989) that the gut cycle was primitive and that other cycles were derived from it. Spiroplasmas have been isolated from guts of

Genus I. Spiroplasma

tabanids (Diptera: Tabanidae) worldwide (French et al., 1997, 1990, 1996; Jandhyam et  al., 2008; Le Goff et  al., 1991, 1993; Regassa and Gasparich, 2006; Vazeille-Falcoz et al., 1997; Whitcomb et al., 1997a). Examination of diversity trends among the tabanid isolates suggests that spiroplasma diversity increases with temperature, resulting in more diversity in southern climes in the Northern Hemisphere (Whitcomb et al., 2007). Although evidence points strongly to multiple cycles of horizontal transmission, the sites where such transmission occurs remain unknown. However, some tabanids utilize honeydew (excreta of sucking insects) deposited on leaf surfaces, suggesting a possible transmission mechanism. Mosquitoes (Chastel and Humphery-Smith, 1991) are also common spiroplasma hosts (Lindh et al., 2005). Additionally, spiroplasmas inhabit the gut of ground beetles (Harpalus pensylvanicus and Anisodactylus sanctaecrucis) as evidenced by 16S rRNA gene sequence analysis of the digestive tract bacterial flora (Lundgren et al., 2007). Plant surfaces.  Flowers and other plant surfaces represent a major site where spiroplasmas and other microbes are transmitted from insect to insect (Clark, 1978; Davis, 1978; McCoy et al., 1979). Members of several spiroplasma groups have been isolated only from flowers and strains of several other spiroplasmas have been isolated from both insects and flowers. Biological evidence suggests that mosquito spiroplasmas are transmitted from insect to insect on flowers (Chastel et al., 1990; Le Goff et al., 1990). It is not known whether any of the so-called “flower spiroplasmas” can exist as true epiphytes. Isolations of spiroplasmas from a variety of insects (Clark, 1982; Hackett et al., 1990) suggest that it is likely that many or most of these flower isolates are deposited passively by visiting arthropods. Plant phloem and sucking insects.  Several spiroplasmas possess a life cycle that involves infection of plant phloem and homopterous insects (Bové, 1997; Fletcher et al., 1998; Garnier et al., 2001; Saglio and Whitcomb, 1979). In the course of passage through the insect, spiroplasmas pass through, accumulate, or multiply in gut epithelial cells and salivary cells. They also accumulate in the insect neurolemma. Large accumulations of spiroplasma cells occur frequently in the hemolymph, where they undoubtedly multiply (Whitcomb and Williamson, 1979). Spiroplasmas may multiply in a number of sucking insect species that have been exposed to diseased plants, but often only a single vector or several vector species transmit spiroplasmal pathogens from plant to plant (summarized in Calavan and Bové, 1989; Whitcomb, 1989; Kersting and Sengonca, 1992). Sex ratio organisms.  Once thought to be a genetic factor, the sex ratio trait in Drosophila was shown by Poulson and Sakaguchi (1961) to be induced by a micro-organism, Spiroplasma poulsonii (Williamson et al., 1999). A number of other spiroplasmas in a variety of insect hosts have been identified that also cause sex ratio distortions, including isolates from the chrysomelid beetle Adalia bipunctata (Hurst and Jiggins, 2000; Hurst et  al., 1999) and the butterfly Danaus chrysippus (Jiggins et  al., 2000). In addition, 16S rRNA gene sequence analysis identified spiroplasmas as the causative agent for male-killing: in a population of Harmonia axyridis (ladybird beetle) in Japan (Nakamura et al., 2005); in populations of Drosophila neocardini, Drosophila ornatifrons and Drosophila paraguayensis from Brazil (Montenegro et al., 2006, 2005); in populations of Anisosticta novemdecimpunctata (ladybird beetle) in Britain (Tinsley and Majerus, 2006); in

663

a population of Adalia bipunctata (Sokolova et al., 2002); in several strains from the Tucson Drosophila stock culture collection (Mateos et al., 2006); and in Drosophila melanogaster populations from Uganda and Brazil (Pool et  al., 2006). Other organisms closely associated with their insect hosts were discovered inferentially by PCR studies (Fukatsu and Nikoh, 2000, 2001) and also appear to be related to Spiroplasma mirum. They also cause preferential male killing in an infected Drosophila population (Anbutsu and Fukatsu, 2003). Natural infection rates of malekilling spiroplasmas in Drosophila melanogaster are about 2.3%, as determined for a Brazilian population (Montenegro et  al., 2005), and vary between 0.1 and 3% for Japanese populations of Drosophila hydei (Kageyama et  al., 2006). The male-killing spiroplasma strain isolated from Adalia bipunctata was used to artificially infect eight different coccinellid beetle species. The data suggest that host range could serve to limit horizontal transfer to closely related host species (Tinsley and Majerus, 2007). Supporting this hypothesis was the study that showed the interspecific lateral transmission of spiroplasmas from Drosophila nebulosa to Drosophila willistoni via ectoparasitic mites (Jaenike et al., 2007). A recent multilocus analysis by Haselkorn et al. (2009) showed that Drosophila species are infected with at least four distinct spiroplasma haplotypes. Studies on Drosophila infections by the sex-ratio organism showed that it did not induce the innate immunity of the insect (Hurst et al., 2003). The sex-ratio spiroplasmas have been shown to be vertically transmitted through female hosts, with spiroplasmas present during oogenesis (Anbutsu and Fukatsu, 2003). Although the exact mechanism of male-killing has not been determined, studies have shown that male killing occurs shortly after formation of the host dosage compensation complex (Bentley et  al., 2007) and that male Drosophila melanogaster mutants lacking any of the five genes involved in the dosage compensation complex are not killed (Veneti et al., 2005). In the Kenyan butterfly Danaus chrysippus, a correlation between male killing and a recessive allele for a gene controlling infection susceptibility has been reported. Moreover, infections seemed to have a negative effect on body size (Herren et al., 2007). Ticks.  Three Spiroplasma species have been isolated from ticks. Two of these, Spiroplasma mirum and Spiroplasma sp. 277F, are from the rabbit tick Haemaphysalis leporispalustris (Tully et al., 1982; Williamson et  al., 1989). The third species was isolated from Ixodes pacificus ticks and named Spiroplasma ixodetis (Tully et al., 1995). 16S rRNA gene sequence analysis of spiroplasmas originally isolated from Ixodes ticks and growing in a Buffalo Green Monkey mammalian cell culture line showed a high degree of identity with the Spiroplasma ixodetis 16S rRNA gene (Henning et al., 2006). Analysis of the 16S rRNA gene sequence from DNA extracted from unfed Ixodes ovatus from Japan indicated the presence of spiroplasmas that were also closely related to Spiroplasma ixodetis (Taroura et al., 2005). The ability of tick spiroplasmas, including Spiroplasma ixodetis, to multiply at 37°C reflects the role of vertebrates as tick hosts. The ability of Spiroplasma ixodetis to grow at 32°C as well as 37°C (Tully et al., 1982) may reflect the ecology of some of the cold-blooded vertebrate hosts of these ticks. There is no evidence that any of these spiroplasmas are transmitted to vertebrate hosts of the ticks. Crustaceans.  Spiroplasma sp. have recently been isolated in both freshwater and salt-water crustaceans.

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Spiroplasma penaei (strain SHRIMPT) was isolated from the hemolymph of Pacific white shrimp (Penaeus vannamei) after high mortalities were observed in an aquaculture pond in Columbia, South America (Nunan et  al., 2004). The pathogenic agent was the spiroplasma (Nunan et al., 2005). Although not cultivated, 16S rRNA gene sequence analysis also revealed the presence of spiroplasmas in the gut of the hydrothermal shrimp Rimicaris exoculata (Zbinden and Cambon-Bonavita, 2003). In another outbreak, Chinese mitten crab (Eriocheir sinensis) reared in aquaculture ponds in China became infected with tremor disease. The causative agent was determined to be a spiroplasma with 99% 16S rRNA gene sequence identity to Spiroplasma mirum (Wang et al., 2004a, b). However, recent studies suggest that the infective agent may be a species similar to, but distinct from, Spiroplasma mirum (Bi et al., 2008). The same organism also infects red swamp crayfish (Procambarus clarkii) that are co-reared with the Chinese mitten crab (Bi et al., 2008; Wang et al., 2005) as well as the shrimp Penaeus vannamei (Bi et al., 2008). Other hosts.  Spiroplasmas have been identified in a variety of other hosts, although not necessarily linked to the gut habitat. The first spiroplasma isolated from a lepidopteran came from the hemolymph of white satin moth larvae (Leucoma salicis L.) from Poland (designated strain SMAT) and was serologically distinct from any previously described spiroplasma group (Oduori et  al., 2005). Another novel spiroplasma (designated strain GNAT3597T) was isolated from biting midges from the genus Atrichopogon (Koerber et al., 2005). Spiroplasmas that are closely related to the male-killing spiroplasmas in ladybird beetles (Majerus et al., 1999; Tinsley and Majerus, 2006) have also been identified in the predatory mite Neoseiulus californicus using 16S rRNA gene sequence analysis (Enigl and Schausberger, 2007). A broad survey of 16 spider families for the presence of endosymbionts using 16S rRNA gene sequence analysis revealed that six families contained spiroplasmas, including Agelenidae, Araneidae, Gnaphosidae, Linyphiidae, Lycosidae, and Tetragnathidae (Goodacre et al., 2006). Biogeography.  Spiroplasmas have been identified from hosts in Africa, Asia, Australia, Europe, South America, and North America. While they are worldwide in distribution, studies suggest that biodiversity may be greatest in warm climates (Whitcomb et al., 2007). Because spiroplasmas are host-associated, it seems reasonable that Spiroplasma species distribution would be limited by host biogeography. Early studies indicated that some spiroplasmas have discrete geographic distributions (Whitcomb et  al., 1990). As the diversity of sampling sites increases, the view of spiroplasma biogeography is likely to shift (Regassa and Gasparich, 2006). Distinct distributions may exist, but probably on a larger geographic scale. While it is not clear what factors account for spiroplasma ranges, the level of host specificity and host overwintering ranges may contribute to the biogeography of Spiroplasma species (Whitcomb et al., 2007). Pathogenicity.  Symptoms of infection and confirmation of Koch’s postulates have been reported for the etiologic roles of: Spiroplasma citri in “stubborn” disease of citrus (Calavan and Bové, 1989; Markham et  al., 1974); corn stunt spiroplasma (Chen and Liao, 1975; Nault and Bradfute, 1979; Williamson and Whitcomb, 1975); Spiroplasma phoeniceum in aster, an experimental host (Saillard et  al., 1987); Spiroplasma poulsonii

in Drosophila pseudoobscura (Williamson et al., 1989); Spiroplasma penaei in Penaeus vannamei (Nunan et al., 2005); and Spiroplasma ­eriocheiris (Wang et al., 2010) in the Chinese mitten crab, ­Eriocheir sinensis (Wang et  al., 2004b). Recent studies have focused on spiroplasma infection and replication in the midgut and Malpighian tubules of leafhoppers (Özbek et  al., 2003). The use of immunofluorescence confocal laser scanning microscopy has revealed the presence of Spiroplasma kunkelii in the midgut, filter chamber, Malpighian tubules, hindgut, fat tissues, hemocytes, muscle, trachea, and salivary glands of leafhopper hosts, but not in the nerve cells of the brain or nerve ganglia (Ammar and Hogenhout., 2005). Plant spiroplasmas may also be pathogenic for unusual vectors (Whitcomb and Williamson, 1979), but are much less so for their usual host (Madden and Nault, 1983; Nault et al., 1984). In fact, some spiroplasmas are beneficial to their leafhopper hosts (Ebbert and Nault, 1994) and it has been hypothesized that infection plays an important role in the host’s overwintering strategies (Moya-Raygoza et al., 2007a, b; Summers et al., 2004). Spiroplasma mirum is experimentally pathogenic for a variety of suckling animals, causing cataract and other ocular symptoms, neural pathology (Clark and Rorke, 1979), and malignant transformation in cultured cells (Kotani et  al., 1990). Spiroplasma melliferum also persists and causes pathology in suckling mice (Chastel et  al., 1990, 1991). Spiroplasma eriocheiris is neurotropic to brain tissue in experimentally injected chicken embryos (Wang et al., 2003). There are two recent reports of spiroplasmas in aquatic invertebrates. Nunan et al. (2005) characterized a spiroplasma in commercially raised shrimp that led to a lethal disease. Spiroplasma melliferum and Spiroplasma apis cause disease in honey bees (Clark, 1977; Mouches et al., 1982a, 1983a). Intrathoracic inoculation of Spiroplasma taiwanense reduced the survival and impaired the flight capacity of inoculated mosquitoes (Humphery-Smith et al., 1991a), and inoculation of Spiroplasma taiwanense per os decreased the survival of mosquito larvae in laboratory trials (Humphery-Smith et  al., 1991b). Spiroplasma poulsonii causes sex ratio abnormalities (male-killing) in Drosophila (Williamson and Poulson, 1979). Male-killing spiroplasma strains related to Spiroplasma poulsonii cause necrosis in neuroblastic and fibroblastic cells (Kuroda et al., 1992). The significance of some biological properties of spiroplasmas is incompletely understood. For example, membranes of Spiroplasma monobiae are potent inducers of tumor necrosis factor alpha secretion and of blast transformation (Sher et al., 1990a, b) in insect cell culture. Spiroplasmas are implicated by circumstantial evidence, in the view of some workers, to be associated with human disease. Bastian first claimed in 1979 that spiroplasmas were associated with Creutzfeldt–Jakob Disease (CJD), an extremely rare scrapie-like disease of humans (Bastian, 1979). Bastian and Foster (2001) reported finding spiroplasma 16S rRNA genes in CJD- and scrapie-infected brains that were not observed in controls. More recent studies (Bastian et al., 2004) presented evidence to show that spiroplasma 16S rRNA genes were found in brain tissue samples from scrapie-infected sheep, chronic wasting disease-infected cervids, and CJD-infected humans. All the brain tissues from non-infected controls were negative for spiroplasmal DNA. These authors further showed that the sequence of the PCR products from the infected brains was 96% identical to the Spiroplasma mirum 16S rRNA gene. However, these

Genus I. Spiroplasma

results could not be replicated in an independent blind study of uninfected and Scrapie-infected hamster brains using the same primers (Alexeeva et al., 2006). A recent study to fulfill Koch’s postulate reported the transfer of spiroplasma from transmissible spongiform encephalopathy (TSE) brains and Spiroplasma mirum to induce spongiform encephalopathy in ruminants (Bastian et al., 2007). The current status of the involvement of spiroplasmas in TSE is the subject of recent reviews (Bastian, 2005; Bastian and Fermin, 2005). Other proposed connections between mollicutes and human disease have been evaluated by Baseman and Tully (1997). Mechanism of Spiroplasma citri phytopathogenicity.  Transposon (Tn4001) mutants have been examined extensively to elucidate the molecular mechanisms associated with Spiroplasma citri phytopathogenicity. One of these mutants, GMT553, highlighted the involvement of selective carbohydrate utilization in Spiroplasma citri pathogenicity (see review by Bové et al., 2003). When introduced into periwinkle plants via injected leafhoppers (Circulifer haematoceps), GMT553 multiplied in the plants as actively as wild-type Spiroplasma citri strain GII3, but did not induce symptoms (Foissac et  al., 1997b, c; Gaurivaud et  al., 2000b). In this mutant, the transposon was found to be inserted in fruR, a transcriptional activator of the fructose operon (fruRAK; Gaurivaud et al., 2000a). The second gene of the operon, fruA, encodes fructose permease, which enables uptake of fructose; and the third gene, fruK, encodes 1-phosphofructokinase. In mutant GMT553, transcription of the fructose operon is abolished and, hence, the mutant cannot utilize fructose as a carbon or energy source (Gaurivaud et  al., 2000a). Mutant GMT553 was functionally complemented for fructose utilization and phytopathogenicity in trans by a recombinant fruR– fruA–fruK operon, fruA–fruK partial operon, or fruA alone, but not fruR or fruR–fruA (Gaurivaud et al., 2000a, b). It should be pointed out that both fructose+ and fructose− spiroplasmas are able to utilize glucose. Further insight into Spiroplasma citri phytopathogenicity in relation to sugar metabolism comes from the production of a spiroplasma mutant unable to use glucose (André et al., 2005). The import of glucose into Spiroplasma citri cells involves a phosphotransferase (PTS) system composed of two distinct polypeptides encoded by (1) crr (glucose PTS permease IIAGlc component) and (2) ptsG (glucose PTS permease IICBGlc component). A ptsG mutant (GII3-glc1) proved unable to import glucose. When introduced into periwinkle (Catharanthus roseus) plants through leafhopper transmission, the mutant induced severe symptoms similar to those obtained with wild-type GII3, in strong contrast to the fructose operon mutant, GMT553, which was virtually non-pathogenic. These results indicated that fructose and glucose utilization were not equally involved in pathogenicity and are consistent with biochemical data showing that, in the presence of both sugars, Spiroplasma citri preferentially used fructose. NMR analyses of carbohydrates in plant extracts revealed the accumulation of soluble sugars, particularly glucose, in plants infected by wild-type Spiroplasma citri GII3 or GII3-glc1, but not in those infected by GMT553. In the infected plant, Spiroplasma citri cells are restricted to the sieve tubes. In the companion cell, sucrose is cleaved by invertase to fructose and glucose. In the sieve tube, wild-type Spiroplasma citri cells will use fructose preferentially over glucose leading to a decreased fructose concentration and, consequently, to an increase of invertase activity, which

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in turn results in glucose accumulation. Glucose accumulation is known to induce stunting and repression of photosynthesis genes in Arabidopsis thaliana. Such symptoms are precisely those observed in periwinkle plants infected by wild-type Spiroplasma citri (André et al., 2005). Genes that are up- or down-regulated in plants following infection with Spiroplasma citri have been studied by differential display analysis of mRNAs in healthy and symptomatic periwinkle plants (Jagoueix-Eveillard et  al., 2001). Expression of the transketolase gene was inhibited in plants infected by the wildtype spiroplasma, but not by the non-phytopathogenic mutant GMT553, further indicating that sugar metabolism and transport are important factors in pathogenicity. Sugar PTS system permeases have been shown to be important in rapid adaptation to sugar differences between plant host and insect vector (André et al., 2003). Leafhopper transmission of Spiroplasma citri.  Spiroplasmas are acquired by leafhopper vectors that imbibe sap from the sieve tubes of infected plants. However, in order to be transmitted to a plant, the mollicutes need first to multiply in the insect vector after crossing the gut barrier (Wayadande and Fletcher, 1995). They multiply to high titers (106–107/ml) in the insect hemolymph, but only when they have reached the salivary glands can they be inoculated into a plant. One gene required for efficient transmission, sc76, was inactivated in a transposon mutant (G76) with reduced transmissibility (Boutareaud et al., 2004); sc76 encodes a putative lipoprotein. Plants infected with the G76 mutant showed symptoms 4–5 weeks later than those infected with wild-type GII3, but when they appeared, the symptoms induced were severe. Mutant G76 multiplied in plants and leafhoppers as efficiently as the wild-type strain. However, leafhoppers injected with the wild-type spiroplasma transmitted the spiroplasma to 100% of exposed plants. In contrast, those injected with mutant G76 infected only 50% of the plants. This inefficiency was shown to be associated with a numerical decrease in spiroplasma cells in the salivary glands that correlated with reduced output from the stylets of transmitting leafhoppers; the number of mutant cells transmitted through Parafilm membranes was less than 5% of numbers of wild-type cells transmitted based on colony-forming units. Functional complementation of the G76 mutant with the sc76 gene restored the wild-type phenotype. Because both wild-type and mutant cells multiplied to equally high titers in the hemolymph, the results suggest that the mutant is inefficiently passed from the hemolymph into the salivary glands or that it may multiply to a lower titer in the glands. Transmission of Spiroplasma citri by leafhopper vectors must involve adherence to and invasion of insect host cells. Electron microscopic studies of leafhopper midgut by Ammar et al. (2004) have demonstrated the attachment of Spiroplasma kunkelii cells by a tip structure to the cell membrane between microvilli of epithelial cells. Spiroplasma citri surface protein P89 was shown to mediate adhesion of the spiroplasma to cells of the vector Circulifer tenellus and was designated SARP1 (Berg et al., 2001; Yu et al., 2000). The gene encoding SARP1, arp1, was cloned and characterized from Spiroplasma citri BR3-T. The putative gene product SARP1 contains a novel domain at the N terminus, called “sarpin” (Berg et al., 2001). The arp1 gene is located on plasmid pBJS-O in Spiroplasma citri (Joshi et  al., 2005). The Spiroplasma kunkelii plasmid pSKU146 encodes an

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Family II. Spiroplasmataceae

adhesin that is a homolog of SARP1 (Davis et al., 2005). Other spiroplasma plasmids encode additional adhesin-related proteins. As indicated above (see Plasmids), Spiroplasma citri GII3 contains six large plasmids, pSci1 to pSci6 (Saillard et  al., 2008). Although plasmids pSci1 to pSci5 encode eight different Spiroplasma citri adhesin-related proteins (ScARPs), they are not required for insect transmission (Berho et al., 2006b). One of the ScARPs, protein P80, shared 63% similarity and 45% identity with SARP1. Protein P80 is carried by plasmid pSci4 and has been named ScARP4a. The ScARP-encoding genes could not be detected in DNA from non-transmissible strains (Berho et  al., 2006b). Sequence alignments of ScARP proteins revealed that they share common features including a conserved signal peptide followed by six to eight repeats of 39–42 amino acids each, a central conserved region of 330 amino acids, and a transmembrane domain at the C terminus (Saillard et al., 2008). Plasmid pSci6 carries the gene for protein P32, which is present in all Spiroplasma citri strains capable of being transmitted by the leafhopper vector Circulifer haematoceps, but absent from all non-transmissible strains (Killiny et  al., 2006). Complementation studies with P32 alone did not restore transmissibility (Killiny et al., 2006). However, if the pSci6 plasmid was transferred to an insect-non-transmissible Spiroplasma citri strain, then the phenotype could be converted to insect-transmissible, indicating the likely presence of additional transmissibility factors on pSci6 (Berho et al., 2006a). Indeed, recent data indicates that factors essential for transmissibility are encoded by a 10 kbp fragment of pSci6 (Breton et  al., 2010). The finding that the insect-transmissible strain Spiroplasma citri Alc254 contains only a single plasmid, pSci6 (S. Richard and J. Renaudin, unpublished) also reinforces the hypothesis that pSci6-encoded determinants play a key role in insect transmission of Spiroplasma citri by its leafhopper vector.

Enrichment and isolation procedures Isolation.  Success in the isolation of fastidious spiroplasmas is influenced strongly by the titer of the inoculum. Spiroplasmas have been isolated from salivary glands, gut, and nerve tissues of their insect hosts. Many spiroplasmas envisioned by darkfield microscopy have proved to be noncultivable (Hackett and Clark, 1989). Initial insect extracts in growth media are passed through a 0.45 mm filter. The filtrate is then observed daily for pH indicator change. An alternative to filtration involves the use of antibiotics or other inhibitors (Grulet et al., 1993; Markham et al., 1983; Whitcomb et al., 1973). Spiroplasma isolations from infected plants are best obtained from sap expressed from vascular bundles of hosts showing early disease symptoms. Plant sap often contains spiroplasmal substances (Liao et  al., 1979) whose presence in primary cultures may necessitate blind passage or serial dilution. Isolation media.  M1D medium (Whitcomb, 1983) has been used for primary isolations of the large proportion of spiroplasma species. SP-4 medium, a rich formulation derived from experiments with M1D, is necessary for isolation of Spiroplasma mirum from fluids of the embryonated egg (Tully et al., 1982). SP-4 medium is also required for isolation of Spiroplasma ixodetis (Tully et al., 1981). Some very fastidious spiroplasmas such as Spiroplasma poulsonii (Hackett et  al., 1986) and ­Spiroplasma

l­eptinotarsae (Hackett and Lynn, 1985) were isolated by cocultivation with insect cells. However, the requirement for cocultivation of Spiroplasma leptinotarsae can be circumvented by placing the primary cultures in BBL anaerobic GasPak jar systems with low redox potential and enhanced CO2 atmosphere (Konai et al., 1996b). By lowering the pH of the growth medium from 7.4 to 6.2 and using bromocresol purple as a pH indicator (pH 5.2 yellow to pH 6.8 purple), it was possible to perform metabolism inhibition tests involving Spiroplasma leptinotarsae as the antigen. The same low-pH medium containing 2.0% Noble agar permitted the growth of colonies (Williamson, unpublished data). Cohen and Williamson (1988) reported that a fortuitous contamination of H-2 medium by a slow-growing, pink-colored yeast (Rhodotorula rubra) permitted primary isolation of the non-male-lethal variant of the Dorsophila willistoni spiroplasma. After 10–12 passages with yeast, the spiroplasmas were able to grow in yeast-free H-2 medium. Maintenance procedures.  Adaptation.  Most spiroplasmas can be adapted to a wide variety of media formulations. Spiroplasmas commonly grow more slowly upon transfer to new media. Initial reduction in growth rate is probably related to a combination of differences in nutrients, pH, osmolality, etc. Isolates may grow at only slightly reduced rates during the first 1–5 passages in a new medium. However, if the new medium is markedly deficient, the growth rate may decrease precipitously after 5–10 passages. Continuous careful passaging may result in growth rate recovery to levels similar to that in the initial medium. For such adaptations, best results are achieved by starting with a 1:1 ratio of old and new media and gradually withdrawing the old formulation. Spiroplasma clarkii, after continuous passage for hundreds of generations, finally adapted to extremely simple media (Hackett et al., 1994). Adaptation may involve mutation and/or activation of adaptive enzymes, or, possibly, other mechanisms. Growth rates in such simple media were much slower than those in rich media. Maintenance media.  Spiroplasma citri can be cultivated in a relatively simple medium that utilizes sorbitol to maintain osmolality (Saglio et al., 1971). A modification of this medium (BSR) has been used extensively for Spiroplasma citri (Bové and Saillard, 1979), in which the horse serum content was lowered to 10% and the fresh yeast extract was omitted. Other simple media, such as C-3G (Liao and Chen, 1977), are suitable for maintenance or large-batch cultivation of fast-growing spiroplasmas. This medium is also adequate for primary isolation of Spiroplasma kunkelii (Alivizatos, 1988). However, cultivation of more fastidious spiroplasmas is best achieved in M1D medium (Hackett and Whitcomb, 1995; Whitcomb, 1983; Williamson and Whitcomb, 1975) if they derive from plant or insect habitats. SP-4 medium (Tully et al., 1977) is very suitable if spiroplasmas derive from tick habitats. SM-1 medium (Clark, 1982) has also been successfully employed for many insect spiroplasmas. Defined media.  Spiroplasma floricola and some strains of Spiroplasma apis have been cultivated in chemically defined media (Chang, 1989, 1982). Preservation.  Spiroplasmas are routinely preserved by lyophilization (FAO/WHO, 1974). Most spiroplasmas can be maintained at −70°C indefinitely. Preservation success at −20°C is irregular and uncertain.

Genus I. Spiroplasma

Differentiation of the genus Spiroplasma from other closely related taxa Spiroplasmas can be clearly differentiated from all other microorganisms by their unique properties of helicity and motility, combined with the complete absence of periplasmic fibrils, cell walls, or cell wall precursors. However, spiroplasmas may be nonhelical under some environmental conditions or when cultures are in the stationary phase of growth. Morphological study of the organisms in the exponential phase of growth usually reveals characteristic helical forms. However, the existence of spiroplasmas that appear entirely or largely as nonhelical forms (e.g., Spiroplasma ixodetis and group XXIII strain TIUS1) raises the theoretical possibility that an organism situated at an apomorphic (advanced) position on the spiroplasma phylogenetic tree could totally lack helicity or motility. In fact, the clade containing Mycoplasma mycoides and the Entomoplasmataceae has apparently done exactly that. Spiroplasma floricola produces nonhelical, but viable, cells early in stationary phase, which can begin within 24 h of medium inoculation. For reasons such as this, it is necessary to examine cultures throughout the growth cycle to ensure that an adequate search for helical cells has been made.

Taxonomic comments Early history.  The term “spiroplasma” was first coined as a trivial term to describe helical organisms shown to be associated with corn stunt disease (Davis et al., 1972a, b) that could not, at that time, be cultivated (Davis and Worley, 1973). Shortly thereafter, when similar organisms associated with citrus stubborn disease were characterized (Saglio et al., 1973), the trivial term was adopted as the generic name and the stubborn organism was named Spiroplasma citri. This species was the first cultured spiroplasma and the first cultured mollicute of plant origin. Shortly after the stubborn agent was named, the genus Spiroplasma was elevated to the status of a family (Skripal, 1974) and added to the Approved Lists of Bacterial Names (Skripal, 1983). The organism that was eventually named Spiroplasma mirum (Tully et al., 1982) was isolated by Clark (1964) in embryonated chicken eggs soon after the discovery of the organism later named Spiroplasma poulsonii. Because Spiroplasma mirum readily passed through filters, it was first mistaken for a virus. The subgroup I-4 277F spiroplasma was cultivated in 1968, but was mistaken for a spirochete (Pickens et al., 1968). The first organism to be initially recognized as a spiroplasma was Spiroplasma kunkelii, which was envisioned by dark-field and electron microscopy in 1971–1972 and cultivated in 1975 (Liao and Chen, 1977; Williamson and Whitcomb, 1975). More than a decade passed before Clark (1982) showed that spiroplasmas, many of them fast-growing, occurred principally in insects. Species concept.  The species concept in spiroplasmas, as in all bacteria, was based on DNA–DNA reassociation (ICSB Subcommittee on the Taxonomy of Mollicutes, 1995; Johnson, 1994; Rosselló-Mora and Amann, 2001; Stackebrandt et  al., 2002; Wayne et al., 1987). In practice, DNA–DNA reassociation results with spiroplasmas have proven difficult to standardize. Estimates of reassociation between Spiroplasma citri (subgroup I-1) and Spiroplasma kunkelii (subgroup I-3) varied between 30 and 70%, depending on the method employed and the degree of stringency (Bové and Saillard, 1979; Christiansen et  al., 1979; Lee

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and Davis, 1980; Liao and Chen, 1981a; Rahimian and Gumpf, 1980). Given these challenges, an alternative method was identified in serology. Surface serology of spiroplasmas has proven to be a robust surrogate for DNA–DNA hybridization assays. Phylogeny.  Phylogenetic studies of Spiroplasma became possible when Carl Woese and colleagues, searching for a molecular chronometer by which microbial evolution could be reconstructed, found that rRNA met most or all of the desired criteria (reviewed by Woese, 1987). Today, sequencing of rRNA genes has become a universal tool for phylogenetic reconstruction. Early phylogenetic analyses involved distance estimates (DeSoete, 1983). Later, neighbor-joining (Saitou and Nei, 1987) was introduced into mollicute phylogeny (Maniloff, 1992) and several mollicute workers have used maximum-likelihood (Felsenstein, 1993). The extensive and classical studies of K.-E. Johansson’s group (Johansson et  al., 1998; Pettersson et  al., 2000) were completed using neighbor-joining, but selectively confirmed by maximum-likelihood and maximum-parsimony (Swofford, 1998). Gasparich et al. (2004) studied the phylogeny of Spiroplasma and its nonhelical descendants using parsimony, maximum-likelihood, distance, and neighbor-joining analyses, which generated 24 phylogenetic inferences that were common to all, or almost all, of the trees. More recently, Bayesian analysis [MrBayes (http://mrbayes.csit.fsu.edu/index.php)] was used to examine an expanded Spiroplasma Apis clade based on 16S rRNA and 16S–23S ITS sequences; the analyses showed congruency between Bayesian and maximum-parsimony trees (Jandhyam et al., 2008). Woese et al. (1980) presented a 16S rRNA gene-based phylogenetic tree for Mollicutes, including Spiroplasma, indicating that these wall-less bacteria were related to members of the phylum Firmicutes such as Lactobacillus spp. and Clostridium innocuum. The tree suggested that Mollicutes might be monophyletic. However, a later study by Weisburg et al. (1989) with 40 additional species of Mollicutes including ten spiroplasmas, failed to confirm the monophyly of Mollicutes at the deepest branching orders. The Woese et al. (1980) model also suggested that the genus Mycoplasma might not be monophyletic, in that the type species, Mycoplasma mycoides, and two related species, Mycoplasma capricolum and Mycoplasma putrefaciens, appeared to be more closely related to the Apis clade of Spiroplasma than to the other Mycoplasma species. This conclusion was supported by analyses of the 5S rRNA genes (Rogers et al., 1985). All trees so far obtained indicate that the acholeplasma-anaeroplasma (Acholeplasmatales–Anaeroplasmatales) and spiroplasma-mycoplasma (Mycoplasmatales–Entomoplasmatales) lineages are monophyletic, but are separated by an ancient divergence. In-depth analysis of characterized spiroplasmas and their nonhelical descendants indicates the existence of four major clades within the monophyletic spiroplasma-mycoplasma lineage (Gasparich et al., 2004; Figure 113). One of the four clades consists of the nonhelical species of the mycoides group (as defined by Johansson, 2002) as well as the six species of Entomoplasma and twelve species of Mesoplasma (the Entomoplasmataceae); this assemblage was designated the Mycoides-Entomoplasmataceae clade. The analyses indicated that the remaining three clades represented Spiroplasma species. One of these clades, the Apis clade, was found to be a sister to the Mycoides-Entomoplasmataceae clade. The Apis clade contains a large number of species from diverse insect hosts, many of which possess life cycles

Spiroplasma chrysopicola

* Spiroplasma syrphidicola

*

*

*

Scale:

Spiroplasma sp. TAAS-1 Spiroplasma mirum Spiroplasma sp. LB-12 Spiroplasma sp. 277F Spiroplasma sp. N525 Spiroplasma poulsonii * Spiroplasma penaei Spiroplasma insolitum Spiroplasma phoeniceum P40 Spiroplasma kunkelii CR2-3x Spiroplasma citri ** Spiroplasma melliferum Entomoplasma freundtii * Mycoplasma mycoides Mesoplasma seiffertii Spiroplasma monobiae ** Spiroplasma diabroticae Spiroplasma floricola Spiroplasma sp. BIUS-1 Spiroplasma sp. W115 * Spiroplasma cantharicola CC-1 * Spiroplasma sp. CB-1 Spiroplasma sp. Ar-1357 Spiroplasma diminutum Spiroplasma taiwanense Spiroplasma gladiatoris Spiroplasma lineolae TALS-2 * Spiroplasma sp. BARC 1901 Spiroplasma helicoides Spiroplasma clarkii Spiroplasma apis * ** Spiroplasma montanense Spiroplasma litorale Spiroplasma turonicum * Spiroplasma corruscae Spiroplasma culicicola Spiroplasma velocicrescens Spiroplasma chinense Spiroplasma leptinotarsae * Spiroplasma lampyridicola Spiroplasma sabaudiense Spiroplasma alleghenense Spiroplasma ixodetis

Mycoplasma pneumoniae Ureaplasma urealyticum

Acholeplasma laidlawii ’Candidatus Phytoplasma’ sp. vigna Il Anaeroplasma bactoclasticum Clostridium innocuum Bacillus subtilis TB11 Asteroleplasma anaerobium Escherichia coli

0.1 substitutions/site

FIGURE 113.  Phylogenetic relationships of members of the class Mollicutes and selected members of the phylum Firmicutes. The phylogram was

based on a Jukes-Cantor corrected distance matrix and weighted neighbor-joining analysis of the 16S rRNA gene sequences of the type strains, except where noted. Escherichia coli was the outgroup. Bootstrap values (100 replicates) 99% (Gasparich et al., 1993c). The strains of this group, including not only the subgroups, but a plethora of isolates from the same ecological context, appear to form a matrix of interrelated strains. Boundaries that seemed clear when the subgroups were initially described, eventually eroded beyond recognition. The 16S rRNA gene sequence similarities are too high to permit cladistic analysis and even 16S– 23S rRNA spacer region sequence analysis failed to resolve the

669

existing subgroups (Regassa et al., 2004). Over time, the concept of the microbial species has undergone a subtle change. It is now recognized (Rosselló-Mora and Amann, 2001; Stackebrandt et al., 2002) that microbial species must at times consist of strain clusters that may contain species with 0.99, so this gene is insufficient for distinguishing species in group VIII. The genome size is 1270 kbp (PFGE). Pathogenicity for insects has not been determined. Source: isolated from the gut of a deer fly (Chrysops sp.) in Maryland, USA. Other strains from deer flies have been collected from as far west as Wyoming, from New England, and very rarely, as far south as Georgia, USA. DNA G+C content (mol%): 30 ± 1 (Bd). Type strain: ATCC 43209, DF-1. Sequence accession no. (16S rRNA gene): AY189127. 8. Spiroplasma clarkii Whitcomb, Vignault, Tully, Rose, Carle, Bové, Hackett, Henegar, Konai and Williamson 1993c, 264VP clar¢ki.i. N.L. masc. gen. n. clarkii of Clark, in honor of Truman B. Clark, a pioneer spiroplasma ecologist. The morphology is as described for the genus. The helical motile filaments remain stable throughout exponential growth. Colonies on solid medium containing 0.8% Noble agar are diffuse, without fried-egg morphology. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, this species is placed in the classical Apis cluster of spiroplasmas, but it does not have an especially close neighbor in trees constructed using 16S rRNA gene sequences. The genome size is 1720 kbp (PFGE). Pathogenicity for insects has not been determined. Source: isolated from the gut of a larval scarabaeid beetle (Cotinus nitida) in Maryland, USA. DNA G+C content (mol%): 29 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 33827, CN-5. Sequence accession no. (16S rRNA gene): M24474. 9. Spiroplasma corruscae Hackett, Whitcomb, French, Tully, Gasparich, Rose, Carle, Bové, Henegar, Clark, Konai, Clark and Williamson 1996c, 949VP cor.rus¢cae. N.L. gen. n. corruscae of corrusca, referring to the species of firefly beetle (Ellychnia corrusca) from which the organism was first isolated. The morphology is as described for the genus. Cells are helical and motile. Colonies on solid medium containing

671

2.25% Noble agar are slightly diffuse to discrete and generally without the characteristic fried-egg morphology. Biological properties are listed in Table 142. Serologically distinct from previously established Spiroplasma species, groups, and subgroups. Phylogenetically, closely related to Spiroplasma turonicum and Spiroplasma litorale in trees constructed using 16S rRNA gene sequences. The genome size has not been determined. Source: isolated from the gut of an adult lampyrid beetle (Ellychnia corrusca) in Maryland in early spring, but found much more frequently in horse flies in summer months. Other strains have been collected from Canada and Georgia, Connecticut, South Dakota, and Texas, USA. DNA G+C content (mol%): 26 ± 1 (Tm, Bd). Type strain: ATCC 43212, EC-1. Sequence accession no. (16S rRNA gene): AY189128. 10. Spiroplasma culicicola Hung, Chen, Whitcomb, Tully and Chen 1987, 368VP cu.li.ci′co.la. L. n. culex, -icis a gnat, midge, and also a genus of mosquitoes (Culex, family Culicidae); L. suffix -cola (from L. n. incola) inhabitant, dweller; N.L. n. culicicola intended to mean an inhabitant of the Culicidae. Cells are pleomorphic, but are commonly very short motile helices, 1–2 mm in length. Colonies on solid medium containing 1% Noble agar have a fried-egg appearance with satellites. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, this species is placed in the classical Apis cluster of spiroplasmas, but does not have an especially close neighbor in trees constructed using 16S rRNA gene sequences. The genome size is 1350 kbp (PFGE). Source: isolated from a triturate of a salt marsh mosquito (Aedes sollicitans) collected in New Jersey, USA. DNA G+C content (mol%): 26 ± 1 (Tm, Bd). Type strain: ATCC 35112, AES-1. Sequence accession no. (16S rRNA gene): AY189129. 11. Spiroplasma diabroticae Carle, Whitcomb, Hackett, Tully, Rose, Bové, Henegar, Konai and Williamson 1997, 80VP di.a.bro.ti′cae. N.L. gen. n. diabroticae of Diabrotica, referring to Diabrotica undecimpunctata, the chrysomelid beetle from which the organism was isolated. The morphology is as described for the genus. Cells are helical, motile filaments, 200–300 nm in diameter. Colonies on solid medium containing 0.8% Noble agar are diffuse, without fried-egg morphology. Biological properties are listed in Table 142. Serologically distinct from other established Spiroplasma species, groups, and subgroups. Phylogenetically, closely related to Spiroplasma floricola in trees constructed using 16S rRNA gene sequences. The genome size is 1350 kbp (PFGE). Source: isolated from the hemolymph of an adult chrysomelid beetle, Diabroticae undecimpunctata howardi. DNA G+C content (mol%): 25 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 43210, DU-1. Sequence accession no. (16S rRNA gene): M24482.

672

Family II. Spiroplasmataceae

12. Spiroplasma diminutum Williamson, Tully, Rosen, Rose, Whitcomb, Abalain-Colloc, Carle, Bové and Smyth 1996, 232VP di.min.u¢tum. L. v. deminuere to break into small pieces, make smaller; L. neut. part. adj. diminutum made smaller, reflecting a smaller size. The morphology is as described for the genus. Cells are short (1–2 mm), helical filaments, 100–200 nm in diameter that appear to be rapidly moving, irregularly spherical bodies when exponential phase broth cultures are examined under dark-field illumination. Colonies on solid medium containing 1.6% Noble agar have dense centers, granular perimeters, and nondistinct edges with satellite colonies. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, closely related to group XVI spiroplasmas in trees constructed using 16S rRNA gene sequences. The genome size is 1080 kbp (PFGE). Source: isolated from a frozen triturate of adult female Culex annulus mosquitoes collected in Taishan, Taiwan. DNA G+C content (mol%): 26 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 49235, CUAS-1. Sequence accession no. (16S rRNA gene): AY189130. 13. Spiroplasma floricola Davis, Lee and Worley 1981, 462VP flor.i¢co.la. L. n. flos, -oris a flower; L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. floricola flower-dweller. The morphology is as described for the genus. Helical cells are 150–200 nm in diameter and 2–5 mm in length. Colonies on solid media have granular central regions surrounded by satellite colonies that probably form after migration of cells from the central focus. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, closely related to Spiroplasma diabroticae and various flower spiroplasmas in trees constructed using 16S rRNA gene sequences. The genome size of strain OBMG is 1270 kbp. Experimentally pathogenic for insects and embryonated chicken eggs. Source: isolated from flowers of tulip tree and magnolia trees in Maryland, USA. Other strains have been collected from coleopterous insects. DNA G+C content (mol%): 25 (Tm). Type strain: ATCC 29989, 23-6. Sequence accession no. (16S rRNA gene): AY189131. 14. Spiroplasma gladiatoris Whitcomb, French, Tully, Gasparich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Clark and Williamson 1997b, 718VP gla.di.a¢to.ris. L. gen. n. gladiatoris of a gladiator, reflecting the initial isolation of the organism from the horse fly Tabanus gladiator. Morphology is as described for the genus. Cells are motile helical filaments. Colonies on solid medium containing 3% Noble agar are granular with dense centers and diffuse edges, do not have satellites, and never have a fried-egg appearance. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. This species has a specific antigen,

common to several spiroplasmal inhabitants of horse flies, that confers a high level of one way cross-reactivity when it is used as an antigen. Phylogenetically, closely related to two other tabanid spiroplasmas, Spiroplasma helicoides and group XXXIV strain B1901, in phylogenetic trees constructed using 16S rRNA gene sequences. The genome size has not been determined. Source: isolated from the gut of a horse fly (Tabanus gladiator) in Maryland, USA. Other strains have been collected at various locations in the southeastern United States. DNA G+C content (mol%): 26 ± 1 (Bd). Type strain: ATCC 43525, TG-1. Sequence accession no. (16S rRNA gene): M24475. 15. Spiroplasma helicoides Whitcomb, French, Tully, Gasparich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Clark and Williamson 1997b, 718VP he.li.co.i¢des. Gr. n. helix spiral; Gr. suff. -oides like, resembling, similar; N.L. neut. adj. helicoides spiral-like. The morphology is as described for the genus. Cells are motile helical filaments that lack a cell wall. Colonies on solid medium containing 2.25% Noble agar have dense centers and smooth edges, do not have satellites, and have a perfect fried-egg appearance. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. This species has a specific antigen, common to several spiroplasmal inhabitants of horse flies, that confers a high level one-way cross-reaction when it is used as antigen. Phylogenetically, closely related to two other tabanid spiroplasmas, Spiroplasma gladiatoris and Spiroplasma sp. BARC 1901, in trees constructed using 16S rRNA gene sequences. Genome size has not been determined. Source: isolated from the gut of a horse fly Tabanus abactor collected in Oklahoma, USA. Other strains have been collected in Georgia, USA. DNA G+C content (mol%): 26 ± 1 (Bd). Type strain: ATCC 51746, TABS-2. Sequence accession no. (16S rRNA gene): AY189132. 16. Spiroplasma insolitum Hackett, Whitcomb, Tully, Rose, Carle, Bové, Henegar, Clark, Clark, Konai, Adams and Williamson 1993, 276VP in.so′li.tum. L. neut. adj. insolitum unusual or uncommon, to denote unusual base composition. Cells in exponential phase are long, motile, helical cells that lack true cell walls and periplasmic fibrils. Colonies on solid SP-4 medium containing 0.8 or 2.25% Noble agar are diffuse, with small central zones of growth surrounded by small satellite colonies. Colonies on solid SP-4 medium containing horse serum and 0.8% Noble agar show fried-egg morphology. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species and groups, but cross-reacts reciprocally in complex patterns of relatedness with group I subgroups and Spiroplasma poulsonii. DNA–DNA renaturation experiments confirm that the differences between the type strain and other subgroups of group I are great enough to warrant its designation as a distinct species. Has close phylogenetic affinities with other group I members and with Spiroplasma poulsonii

Genus I. Spiroplasma

in trees ­constructed using 16S rRNA gene sequences. The genome size is 1850 kbp (PFGE). Pathogenicity for insects has not been determined. Source: the type strain was isolated from a fall flower (Asteraceae: Bidens sp.) collected in Maryland, USA. Similar isolates have been found in the hemocoel of click beetles. Also isolated from other composite and onagracead flowers and from the guts of many insects visiting these flowers, including cantharid and meloid beetles; syrphid flies; andrenid and megachilid bees; and four families of butterflies. DNA G+C content (mol%): 28 ± 1 (Tm, Bd). Type strain: ATCC 33502, M55. Sequence accession no. (16S rRNA gene): AY189133. 17. Spiroplasma ixodetis Tully, Rose, Yunker, Carle, Bové, Williamson and Whitcomb 1995, 27VP ix.o.de¢tis. N.L. gen. n. ixodetis of Ixodes, the genus name of Ixodes pacificus ticks, from which the organism was first isolated. Cells are coccoid forms, 300–500 nm in diameter, straight and branched filaments, or tightly coiled helical organisms. Motility is flexional, but not translational. Colonies on solid medium containing 2.25% Noble agar usually have the appearance of fried eggs. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically unique; occurs at base of spiroplasma lineage in trees constructed using 16S rRNA gene sequences. The genome size is 2220 kbp (PFGE). Source: isolated from macerated tissue suspensions prepared from pooled adult Ixodes pacificus ticks (Ixodidae) collected in Oregon, USA. DNA G+C content (mol%): 25 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 33835, Y32. Sequence accession no. (16S rRNA gene): M24477. 18. Spiroplasma kunkelii Whitcomb, Chen, Williamson, Liao, Tully, Bové, Mouches, Rose, Coan and Clark 1986, 175VP kun.kel¢i.i. N.L. masc. gen. n. kunkelii of Kunkel, named after Louis Otto Kunkel (1884–1960), to honor his major and fundamental contributions to the study of plant mollicutes. Cells in exponential phase are helical, motile filaments, 100–150 nm in diameter and 3–10 mm long to nonhelical filaments or spherical cells, 300–800 nm in diameter. Colonies on solid medium containing 0.8% Noble agar are usually diffuse, rarely exhibiting central zones of growth into agar. Colonies on solid C-3G medium containing 5% horse serum or on media containing 2.25% Noble agar frequently have a fried-egg morphology. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares complex patterns of reciprocal cross-reactivity with members of other group I subgroups and Spiroplasma poulsonii. DNA–DNA renaturation experiments confirm that the serological differences between the type strain and other subgroups of group I are great enough to warrant its designation as a distinct species. Has close phylogenetic affinities with other group I

673

members and with Spiroplasma poulsonii in trees constructed using 16S rRNA gene sequences. The genome size is 1610 kbp (PFGE). Pathogenicity for plants and insects has been experimentally verified. Source: isolated from maize displaying symptoms of corn stunt disease and from leafhoppers associated with diseased maize, largely in the neotropics. DNA G+C content (mol%): 26 ± 1 (Tm, Bd). Type strain: ATCC 29320, E275. Sequence accession no. (16S rRNA gene): DQ319068 (strain CR2-3x). 19. Spiroplasma lampyridicola Stevens, Tang, Jenkins, Goins, Tully, Rose, Konai, Williamson, Carle, Bové, Hackett, French, Wedincamp, Henegar and Whitcomb 1997, 711VP lam.py.ri.di¢co.la. N.L. n. Lampyridae the firefly beetle family; L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. lampyridicola an inhabitant of members of the Lampyridae. The morphology is as described for the genus. Cells are motile helical filaments. Colonies on solid medium containing 3.0% Noble agar are small and granular with dense centers, but do not have a true fried-egg appearance. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. When tested as antigen, cross-reacts (one-way) with many specific spiroplasma antisera. Phylogenetically, a sister to Spiroplasma leptinotarsae in trees constructed using 16S rRNA gene sequences. The genome size is 1375 kbp (PFGE). Source: isolated from the gut fluids of a firefly beetle (Photuris pennsylvanicus) collected in Maryland, USA. Also known from Georgia and New Jersey, USA. DNA G+C content (mol%): 26 ± 1 (Tm, Bd). Type strain: ATCC 43206, PUP-1. Sequence accession no. (16S rRNA gene): AY189134. 20. Spiroplasma leptinotarsae Hackett, Whitcomb, Clark, Henegar, Lynn, Wagner, Tully, Gasparich, Rose, Carle, Bové, Konai, Clark, Adams and Williamson 1996b, 910VP lep.ti.no.tar¢sae. N.L. gen. n. leptinotarsae of Leptinotarsa, referring to Leptinotarsa decemlineata, the Colorado potato beetle. Cells in vivo are usually seen in the resting stage, in which they consist of coin-like compressed coils. When placed in fresh medium, these bodies turn immediately into “spring”or “funnel”-shaped spirals, which are capable of very rapid translational motility. After a relatively small number of passes in vitro, this spectacular morphology is lost and the cells return to the modal morphology as described for the genus. Colonies on solid medium containing 2.0% Noble agar are slightly diffuse to discrete and produce numerous satellites. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. When tested as antigen, cross-reacts with many spiroplasma antisera (one-way). Phylogenetically, a sister to Spiroplasma lampyridicola in trees constructed using 16S rRNA gene sequences. The genome size is 1,085 kbp (PFGE). Source: isolated from the gut of Colorado potato beetle (Leptinotarsa decemlineata) larvae in Maryland, USA. Also

674

Family II. Spiroplasmataceae

isolated from beetles collected in Maryland, Michigan, New Mexico, North Carolina, Texas, Canada, and Poland. DNA G+C content (mol%): 25 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 43213, LD-1. Sequence accession no. (16S rRNA gene): AY189305. 21. Spiroplasma leucomae Oduori, Lipa and Gasparich 2005, 2449VP leu.co¢mae. N.L. gen. n. leucomae of Leucoma, systematic genus name of the white satin moth (Lepidoptera: Lymantriidae), the source of the type strain. Morphology is as described for the genus. Cells are filamentous, helical, motile, and approximately 150 nm in diameter. They freely pass through filters with pores of 450 and 220 nm, but do not pass through filters with 100 nm pores. Biological properties are listed in Table 142. Serologically distinct from previously established Spiroplasma species, groups, and subgroups. The genome size has not been determined. Pathogenicity for the moth larvae is not known. Source: isolated from fifth instar satin moth larvae (Leucoma salicis). DNA G+C content (mol%): 24 ± 1 (Tm). Type strain: ATCC BAA-521, NBRC 100392, SMA. Sequence accession no. (16S rRNA gene): DQ101278. 22. Spiroplasma lineolae French, Whitcomb, Tully, Carle, Bové, Henegar, Adams, Gasparich and Williamson 1997, 1080VP lin.e.o¢lae. N.L. n. lineola a species of tabanid fly; N.L. gen. n. lineolae of Tabanus lineola, from which the organism was isolated. The morphology is as described for the genus. Cells are motile, helical filaments, 200–300 nm in diameter. Colonies on solid medium containing 3% Noble agar are small, granular, and never have a fried-egg appearance. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetic position has not been determined, but its other taxonomic properties suggest that it may be related to other tabanid spiroplasmas of the Apis cluster. The genome size is 1390 kbp (PFGE). Source: type strain isolated from the viscera of the tabanid fly Tabanus lineola collected in coastal Georgia. A strain from Tabanus lineola has been collected in Costa Rica (Whitcomb et al., 2007). DNA G+C content (mol%): 25 ± 1 (Tm, Bd). Type strain: ATCC 51749, TALS-2. Sequence accession no. (16S rRNA gene): DQ860100. 23. Spiroplasma litorale Konai, Whitcomb, French, Tully, Rose, Carle, Bové, Hackett, Henegar, Clark and Williamson 1997, 361VP li.to.ra¢le. L. neut. adj. litorale of the shore or coastal area. The morphology is as described for the genus. Cells are motile, helical filaments. Colonies on solid medium containing 2.25% Noble agar are granular with dense centers, uneven margins, and multiple satellites, and never have fried-egg appearance. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, closely related to

two other tabanid spiroplasmas, Spiroplasma turonicum and Spiroplasma litorale, in trees constructed using 16S rRNA gene sequences. The genome size is 1370 kbp (PFGE). Source: isolated from the gut of a female green-eyed horse fly (Tabanus nigrovittatus) from the Outer Banks of North Carolina. Also collected from coastal Georgia and both Atlantic and Pacific coasts of Costa Rica. DNA G+C content (mol%): 25 ± 1 (Bd). Type strain: ATCC 34211, TN-1. Sequence accession no. (16S rRNA gene): AY189306. 24. Spiroplasma melliferum Clark, Whitcomb, Tully, Mouches, Saillard, Bové, Wróblewski, Carle, Rose, Henegar and Williamson 1985, 305VP mel.li′fe.rum. L. adj. mellifer, -fera, -ferum honey-bearing, honey-producing; L. neut. adj. melliferum intended to mean isolated from the honey bee (Apis mellifera). Morphology is as described for the genus. Cells are pleomorphic, varying from helical filaments that are 100–150 nm in diameter and 3–10 mm in length to nonhelical filaments or spherical cells that are 300–800 nm in diameter. The motile cells lack true cell wells and periplasmic fibrils. Colonies on solid medium supplemented with 0.8% Noble agar are usually diffuse, rarely exhibiting central zones of growth into agar. Colonies on solid medium containing 2.25% Noble agar are smaller, but frequently have a friedegg morphology. Physiological and genomic properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares complex patterns of reciprocal cross-reactivity with members of other group I subgroups and Spiroplasma poulsonii. Has close phylogenetic affinities with other group I members and with Spiroplasma poulsonii in trees constructed using 16S rRNA gene sequences. DNA–DNA renaturation experiments confirm that the serological differences between the type strain and other subgroups of group I are great enough to warrant its designation as a distinct species. The genome size is 1460 kbp (PFGE). Pathogenic for honey bees in natural and experimental oral infections. Source: isolated from hemolymph and gut of honey bees (Apis mellifera) in widely separated geographic regions. Also recovered from hemolymph of bumble bees, leafcutter bees, and a robber fly, and the intestinal contents of sweat bees, digger bees, bumble bees, and a butterfly. Also recovered from a variety of plant surfaces (flowers) in widely separated geographic regions. DNA G+C content (mol%): 26–28 (Tm, Bd). Type strain: ATCC 33219, BC-3. Sequence accession no. (16S rRNA gene): AY325304. 25. Spiroplasma mirum Tully, Whitcomb, Rose and Bové 1982, 99VP mi′rum. L. neut. adj. mirum extraordinary. The morphology is as described for the genus. Helical filaments measure 100–200 nm in diameter and 3–8 mm in length. Colonies on solid media containing fetal bovine serum and 0.8–2.25% Noble agar (Difco) are diffuse and without central zones of growth into the agar. Solid media prepared with 1.25% agar and in which fetal bovine serum

Genus I. Spiroplasma

has been replaced with bovine serum fraction yield colonies with central zones of growth into the agar and no peripheral growth on the surface of the medium. Moderate turbidity is produced during growth in liquid media. Biological properties are listed in Table 142. This species has been cultivated in a defined medium. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, in trees constructed using 16S rRNA gene sequences, this species is basal to group I and group VIII spiroplasmas on the one hand, and to the Apis cluster and Entomoplasmataceae on the other. It is the most primitive (plesiomorphic) spiroplasma with modal helicity. The genome size is 1300 kbp (PFGE). Produces experimental ocular and nervous system disease and death in intracerebrally inoculated suckling animals (rats, mice, hamsters, and rabbits). Pathogenic for chicken embryos via yolk sac inoculation. Experimentally pathogenic for the wax moth (Galleria mellonella). Source: the type strain was isolated from rabbit ticks (Haemaphysalis leporispalustris) collected in Georgia, USA. Other strains have been collected in Georgia, Maryland, and New York, USA. DNA G+C content (mol%): 30–31 (Tm). Type strain: ATCC 29335, SMCA. Sequence accession no. (16S rRNA gene): M24662. 26. Spiroplasma monobiae Whitcomb, Tully, Rose, Carle, Bové, Henegar, Hackett, Clark, Konai, Adams and Williamson 1993b, 259VP mo.no.bi′ae. N.L. n. Monobia a genus of vespid wasps; N.L. gen. n. monobiae of the genus Monobia, from which the organism was isolated. The morphology is as described for the genus, with motile helical filaments. Colonies on solid medium containing 2.25% Noble agar are diffuse and never have a fried-egg appearance. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, a member of the Apis clade, but with no especially close neighbors in trees constructed using 16S rRNA gene sequences. The genome size is 940 kbp (PFGE). Source: isolated from the hemolymph of an adult vespid wasp (Monobia quadridens) collected in Maryland, USA. Based on its residence in the gut of a flower-visiting insect, this species is thought to be transmitted on flowers. DNA G+C content (mol%): 28 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 33825, MQ-1. Sequence accession no. (16S rRNA gene): M24481. 27. Spiroplasma montanense Whitcomb, French, Tully, Rose, Carle, Bové, Clark, Henegar, Konai, Hackett, Adams and Williamson 1997c, 722VP mon.ta.nen¢se. N.L. neut. adj. montanense pertaining to Montana, where the species was first isolated. The morphology is as described for the genus. Cells are motile, helical filaments that lack a cell wall. Colonies on solid medium containing 2.25% Noble agar are granular and have dense centers, irregular margins, and numerous small satellites. Biological properties are listed in Table 142.

675

Serologically distinct from other Spiroplasma species, groups, and subgroups. Reacts reciprocally in deformation serology at very low levels in deformation tests with Spiroplasma apis. “Bridge strains” have been isolated in Georgia with substantial cross-reactivity with both Spiroplasma montanense and Spiroplasma apis. Sister to Spiroplasma apis in trees constructed using 16S rRNA gene sequences. The genome size is 1225 kbp (PFGE). Source: isolated from the gut of the tabanid fly Hybomitra opaca, in southwestern Montana. Other isolates have been obtained from New England, Connecticut, and southeastern Canada. DNA G+C content (mol%): 28 ± 1 (Bd). Type strain: ATCC 51745, HYOS-1. Sequence accession no. (16S rRNA gene): AY189307. 28. Spiroplasma penaei Nunan, Lightner, Oduori and Gasparich 2005, 2320VP pe.na′e.i. N.L. n. Penaeus a species of shrimp; N.L. gen. penaei of Penaeus, referring to Penaeus vannamei, from which the organism was isolated. The morphology is as described for the genus. Cells are helical and motile. Biological properties are listed in Table 142. Serologically distinct from previously characterized Spiroplasma species, groups, and subgroups, but shares some cross-reactivity with members of other group I subgroups. Has close phylogenetic affinities with other group I members and with Spiroplasma poulsonii in trees constructed using 16S rRNA gene sequences. The genome size has not been determined. Pathogenicity has been indicated by injection into Penaeus vannamei. Source: isolated from the hemolymph of the Pacific white shrimp, Penaeus vannamei. DNA G+C content (mol%): 29 ± 1 (Tm). Type strain: CAIM 1252, SHRIMP, ATCC BAA-1082. Sequence accession no. (16S rRNA gene): AY771927. 29. Spiroplasma phoeniceum Saillard, Vignault, Bové, Raie, Tully, Williamson, Fos, Garnier, Gadeau, Carle and Whitcomb 1987, 113VP phoe.ni¢ce.um. N.L. neut. adj. phoeniceum (from L. neut. adj. phonicium) of Phoenice, an ancient country that was located on today’s Syrian coast, referring to the geographical origin of the isolates. Morphology is as described for the genus. Colonies on solid medium containing 0.8% Noble agar show fried-egg morphology. Physiological and genomic properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares some cross-reactivity with members of other group I subgroups and Spiroplasma poulsonii. Has close phylogenetic affinities with other group I members and with Spiroplasma poulsonii in trees constructed using 16S rRNA gene sequences. Has been shown to be transmissible to leafhoppers by injection and experimentally pathogenic to aster inoculated by the injected leafhoppers. DNA–DNA renaturation experiments confirm that the differences between the type strain and other subgroups of group I are great enough to warrant its designation as a distinct species. The genome size is 1860 kbp (PFGE).

676

Family II. Spiroplasmataceae

Source: isolated from periwinkles that were naturally infected in various locations along the Syrian coastal area. DNA G+C content (mol%): 26 ± 1 (Tm, Bd). Type strain: ATCC 43115, P40. Sequence accession no. (16S rRNA gene): AY772395.

32. Spiroplasma sabaudiense Abalain-Colloc, Chastel, Tully, Bové, Whitcomb, Gilot and Williamson 1987, 264VP

30. Spiroplasma platyhelix Williamson, Adams, Whitcomb, Tully, Carle, Konai, Bové and Henegar 1997, 766VP

The morphology is as described for the genus. Cells are helical filaments, 100–160 nm in diameter and 3.1–3.8 mm long. Motile. Colonies on solid medium containing 1.6% Noble agar are diffuse, rarely exhibiting fried-egg morphology, with numerous satellite colonies. Physiological and genomic properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, related to Spiroplasma alleghenense and Spiroplasma sp. TIUS-1 in trees constructed using 16S rRNA gene sequences. The genome size is 1175 kbp (PFGE). Source: isolated from a triturate of female Aedes spp. mosquitoes in Savoy, France. DNA G+C content (mol%): 30 ± 1 (Tm, Bd). Type strain: ATCC 43303, Ar-1343. Sequence accession no. (16S rRNA gene): AY189308.

pla.ty.he¢lix. Gr. adj. platys flat; Gr. fem. n. helix a coil or spiral; N.L. fem. n. platyhelix flat coil, referring to the flattened nature of the helical filament. Cells are flattened, helical filaments, 200–300 nm in diameter. They show no rotatory or translational motility, but exhibit contractile movements in which tightness of coiling moves along the axis of the filament. Colonies on solid medium containing 2.25% Noble agar form perfect friedegg colonies with dense centers, smooth edges, and without satellites. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. The genome size is 780 kbp (PFGE). Source: isolated from the gut of a dragonfly, Pachydiplax longipennis, collected in Maryland, USA. DNA G+C content (mol%): 29 ± 1 (Bd). Type strain: ATCC 51748, PALS-1. Sequence accession no. (16S rRNA gene): AY800347. 31. Spiroplasma poulsonii Williamson, Sakaguchi, Hackett, Whitcomb, Tully, Carle, Bové, Adams, Konai and Henegar 1999, 616VP poul.so′ni.i. N.L. masc. gen. n. poulsonii of Poulson, named in memory of Donald F. Poulson, in whose laboratory at Yale University this spiroplasma was discovered and studied intensively. Morphology is as described for the genus. Long, motile, helical filaments, 200–250 nm in diameter occur in  vivo in Drosophila hemolymph and in  vitro. Colonies on solid medium containing 1.8% Noble agar are small (60–70 mm in diameter), have dense centers and uneven edges, and are without satellites. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares some reciprocal crossreactivity with members of group I subgroups. Phylogenetically related to group I spiroplasmas in trees constructed using 16S rRNA gene sequences. The genome size is 2040 kbp (PFGE). Spiroplasmas causing sex-ratio abnormalities occur naturally in Drosophila spp. collected in Brazil, Colombia, Dominican Republic, Haiti, Jamaica, and the West Indies. Non-male-lethal spiroplasmas also occur in natural populations of Drosophila hydei in Japan. Pathogenicity (lethality to male progeny) has been confirmed by injection into Drosophila pseudoobscura female flies. Vertical transmissibility is lost after cultivation and cloning. Source: isolated from the hemolymph of Drosophila pseudoobscura females infected by hemolymph transfer of the Barbados-3 strain of Drosophila willistoni SR organism. DNA G+C content (mol%): 26 ± 1 (Tm, Bd). Type strain: ATCC 43153, DW-1. Sequence accession no. (16S rRNA gene): M24483.

sa.bau.di.en¢se. L. neut. adj. sabaudiense of Sabaudia, an ancient country of Gaul, corresponding to present day Savoy, referring to the geographic origin of the isolate.

33. Spiroplasma syrphidicola Whitcomb, Gasparich, French, Tully, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Clark and Williamson 1996, 799VP syr.phi.di¢co.la. N.L. pl. n. Syrphidae a family of flies; L. suff. -cola (from L. masc. or fem. n. incola) inhabitant, dweller; N.L. masc. n. syrphidicola inhabitant of syrphid flies, the insects from which the organism was isolated. Helical motile filaments are short and thin, passing a 220 nm filter quantitatively. Grows to titers as high as 1011/ ml. These short, thin, abundant cells are provisionally diagnostic for group VIII. Colonies on solid medium containing 2.25% Noble agar are irregular with satellites, diffuse, and never have a fried-egg appearance. Growth on solid medium containing 1.6% Noble agar is diffuse. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares some reciprocal crossreactivity with members of other group VIII subgroups. Placement of group VIII strains into subgroups has become increasingly difficult as more strains have accumulated. Phylogenetically, this species is closely related to other group VIII strains in trees constructed using 16S rRNA gene sequences. The 16S rRNA gene sequence similarity coefficients of group VIII spiroplasmas are >0.99, so this gene is insufficient for species separations in group VIII. DNA–DNA renaturation experiments confirm that the differences between the type strain and other subgroups of group VIII are great enough to warrant its designation as a distinct species. Genome size is 1230 kbp (PFGE). Source: isolated from the hemolymph of the syrphid fly Eristalis arbustorum in Maryland, USA. Strains that are provisionally identified as Spiroplasma syrphidicola have been obtained from horse flies collected from several locations in the southeastern United States. DNA G+C content (mol%): 30 ± 1 (Bd). Type strain: ATCC 33826, EA-1. Sequence accession no. (16S rRNA gene): AY189309.

Genus I. Spiroplasma

677

34. Spiroplasma tabanidicola Whitcomb, French, Tully, Gasparich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Clark and Williamson 1997b, 718VP

36. Spiroplasma turonicum Hélias, Vazeille-Falcoz, Le Goff, Abalain-Colloc, Rodhain, Carle, Whitcomb, Williamson, Tully, Bové and Chastel 1998, 460VP

ta.ba.ni.di¢co.la. N.L. n. Tabanidae family name for horse flies; L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. tabanidicola an inhabitant of horse flies.

tu.ro¢ni.cum. L. neut. adj. turonicum of Touraine, the province in France from which the organism was first isolated.

The morphology is as described for the genus. Cells are motile, helical filaments that lack a cell wall. Colonies on solid medium containing 3% Noble agar are uneven and granular with dense centers and irregular edges, do not have satellites, and never have a fried-egg appearance. Physiological and genomic properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. However, some strains may show a very low level reciprocal serological cross-reaction in deformation serology with Spiroplasma gladiatoris. This species has a specific antigen, common to several spiroplasmal inhabitants of horse flies, that confers a high level one-way cross-reaction when it is used as antigen. The genome size is 1375 kbp (PFGE). Source: isolated from the gut of a horse fly belonging to the Tabanus abdominalis-limbatinevris complex. DNA G+C content (mol%): 26 ± 1 (Bd). Type strain: ATCC 51747, TAUS-1. Sequence accession no. (16S rRNA gene): DQ004931.

The morphology is as described for the genus. Cells are motile, helical filaments. Colonies on solid medium containing 3% Noble agar exhibit a “cauliflower-like” appearance and do not have a fried-egg morphology. Biological properties are listed in Table 142. Serologically distinct from previously established Spiroplasma species. Phylogenetically, related to two other tabanid spiroplasmas, Spiroplasma corruscae and Spiroplasma litorale, in trees constructed using 16S rRNA gene sequences. The genome size is 1305 kbp (PFGE). Source: isolated from a triturate of a single horse fly (Haematopota pluvialis) collected in France. DNA G+C content (mol%): 25 ± 1 (Bd). Type strain: ATCC 700271, Tab4c. Sequence accession no. (16S rRNA gene): AY189310.

35. Spiroplasma taiwanense Abalain-Colloc, Rosen, Tully, Bové, Chastel and Williamson 1988, 105VP tai.wan.en¢se. N.L. neut. adj. taiwanense of or belonging to Taiwan, referring to the geographic origin of the isolate. The morphology is as described for the genus. Cells are motile, helical filaments, 100–160 nm in diameter and 3.1– 3.8 mm long. Colonies on solid medium containing 1.6% Noble agar have fried-egg morphology. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, this species is in the classical Apis cluster of spiroplasmas, but does not have an especially close neighbor in trees constructed using 16S rRNA gene sequences. The genome size is 1195 kbp (PFGE). Source: isolated from a triturate of female mosquitoes (Culex tritaeniorhynchus) at Taishan, Taiwan, Republic of China. DNA G+C content (mol%): 25 ± 1 (Tm, Bd). Type strain: ATCC 43302, CT-1. Sequence accession no. (16S rRNA gene): M24476.

References Abalain-Colloc, M.L., C. Chastel, J.G. Tully, J.M. Bové, R.F. Whitcomb, B. Gilot and D.L. Williamson. 1987. Spiroplasma sabaudiense sp. nov. from mosquitos collected in France. Int. J. Syst. Bacteriol. 37: 260– 265. Abalain-Colloc, M.L., L. Rosen, J.G. Tully, J.M. Bové, C. Chastel and D.L. Williamson. 1988. Spiroplasma taiwanense sp. nov. from Culex tritaeniorhynchus mosquitos collected in Taiwan. Int. J. Syst. Bacteriol. 38: 103–107. Abalain-Colloc, M.L., D.L. Williamson, P. Carle, J.H. Abalain, F. Bonnet, J.G. Tully, M. Konai, R.F. Whitcomb, J.M. Bové and C. Chastel. 1993. Division of group XVI spiroplasmas into subgroups. Int. J. Syst. Bacteriol. 43: 342–346.

37. Spiroplasma velocicrescens Konai, Whitcomb, Tully, Rose, Carle, Bové, Henegar, Hackett, Clark and Williamson 1995, 205VP ve.lo.ci.cres¢cens. L. adj. velox, -ocis fast, quick; L. part. adj. crescens growing; N.L. n. part. adj. velocicrescens fastgrowing. The morphology is as described for the genus. Cells are helical, motile filaments, 200–300 nm in diameter. Colonies on solid medium containing 0.8% Noble agar are diffuse and never have a fried-egg appearance. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, this species is sister to Spiroplasma chinense in trees constructed using 16S rRNA gene sequences. The genome size is 1480 kbp (PFGE). Source: isolated from the gut of a vespid wasp, Monobia quadridens, collected in Maryland, USA. Based on its residence in the gut of a flower-visiting insect, this species is thought to be transmitted on flowers. DNA G+C content (mol%): 27 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 35262, MQ-4. Sequence accession no. (16S rRNA gene): AY189311.

Adams, J.R., R.F. Whitcomb, J.G. Tully, E.A. Clark, D.L. Rose, P. Carle, M. Konai, J.M. Bové, R.B. Henegar and D.L. Williamson. 1997. Spiroplasma alleghenense sp. nov., a new species from the scorpion fly Panorpa helena (Mecoptera: Panorpidae). Int. J. Syst. Bacteriol. 47: 759–762. Alexeeva, I., E.J. Elliott, S. Rollins, G.E. Gasparich, J. Lazar and R.G. Rohwer. 2006. Absence of Spiroplasma or other bacterial 16S rRNA genes in brain tissue of hamsters with scrapie. J. Clin. Microbiol. 44: 91–97. Alivizatos, A.S. 1988. Isolation and culture of corn stunt Spiroplasma in serum-free medium. J. Phytopathol. 122: 68–75. Aluotto, B.B., R. G. Wittler, C.O.Williams and J. E. Faber. 1970. Standardized bacteriologic techniques for characterization of Mycoplasma species. Int. J. Syst. Bacteriol. 20: 35–58.

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Family II. Spiroplasmataceae

nov., from a syrphid fly (Diptera: Syrphidae). Int. J. Syst. Bacteriol. 46: 797–801. Whitcomb, R.F., F. E. French, J. G. Tully, P. Carle, R. Henegar, K. J. Hackett, G. E. Gasparich and D.L. Williamson. 1997a. Spiroplasma species, groups, and subgroups from North American Tabanidae. Curr. Microbiol. 35: 287–293. Whitcomb, R.F., F.E. French, J.G. Tully, G.E. Gasparich, D.L. Rose, P. Carle, J. Bové, R.B. Henegar, M. Konai, K.J. Hackett, J.R. Adams, T.B. Clark and D.L. Williamson. 1997b. Spiroplasma chrysopicola sp. nov., Spiroplasma gladiatoris sp. nov., Spiroplasma helicoides sp. nov., and Spiroplasma tabanidicola sp. nov., from tabanid (Diptera: Tabanidae) flies. Int. J. Syst. Bacteriol. 47: 713–719. Whitcomb, R.F., F.E. French, J.G. Tully, D.L. Rose, P.M. Carle, J.M. Bove, E.A. Clark, R.B. Henegar, M. Konai, K.J. Hackett, J.R. Adams and D.L. Williamson. 1997c. Spiroplasma montanense sp. nov., from Hybomitra horseflies at northern latitudes in north America. Int. J. Syst. Bacteriol. 47: 720–723. Whitcomb, R.F., J. G. Tully, G. E. Gasparich, L. B. Regassa, D. L. Williamson and F.E. French. 2007. Spiroplasma species in the Costa Rican highlands: implications for biogeography and biodiversity. Biodivers. Conserv. 16: 3877–3894. Williamson, D.L. 1969. The sex ratio spirochete in Drosophila robusta. Jpn. J. Genet. 44: 36–41. Williamson, D.L. 1974. Unusual fibrils from the spirochete-like sex ratio organism. J. Bacteriol. 117: 904–906. Williamson, D.L. and R.F. Whitcomb. 1974. Helical wall-free prokaryotes in Drosophila, leafhoppers and plants. Colloq. Inst. Natl. Santé Rech. Med. 33: 283–290. Williamson, D.L. and R.F. Whitcomb. 1975. Plant mycoplasmas: a cultivable spiroplasma causes corn stunt disease. Science 188: 1018–1020. Williamson, D.L., R. F. Whitcomb and J.G. Tully. 1978. The Spiroplasma deformation test, a new serological method. Curr. Microbiol. 1: 203– 207. Williamson, D.L., D.I. Blaustein, R.J.C. Levine and M.J. Elfvin. 1979a. Anti-actin-peroxidase staining of the helical wall-free prokaryote Spiroplasma citri. Curr. Microbiol. 2: 143–145. Williamson, D.L., J. G. Tully and R.F. Whitcomb. 1979b. Serological relationships of spiroplasmas as shown by combined deformation and metabolism inhibition tests. Int. J. Syst. Bacteriol. 29: 345–351. Williamson, D.L. and D.F. Poulson. 1979. Sex ratio organisms (spiroplasmas) of Drosophila. In The Mycoplasmas, vol. 3 (edited by Whitcomb and Tully). Academic Press, New York, pp. 175–208. Williamson, D.L. and R.F. Whitcomb. 1983. Special serological tests for spiroplasma identification. In Methods in Mycoplasmology, vol. 2 (edited by Razin and Tully). Academic Press, New York, pp. 249–259. Williamson, D.L., P.R. Brink and G.W. Zieve. 1984. Spiroplasma fibrils. Isr. J. Med. Sci. 20: 830–835. Williamson, D.L., J. G. Tully and R.F. Whitcomb. 1989. The genus Spiroplasma. In The Mycoplasmas, vol. 5 (edited by Whitcomb and Tully). Academic Press, San Diego, pp. 71–111. Williamson, D.L., J. Renaudin and J.M. Bové. 1991. Nucleotide sequence of the Spiroplasma citri fibril protein gene. J. Bacteriol. 173: 4353–4362. Williamson, D.L., J.G. Tully, L. Rosen, D.L. Rose, R.F. Whitcomb, M.L. Abalain-Colloc, P. Carle, J.M. Bové and J. Smyth. 1996. Spiroplasma diminutum sp. nov., from Culex annulus mosquitoes collected in Taiwan. Int. J. Syst. Bacteriol. 46: 229–233. Williamson, D.L., J.R. Adams, R.F. Whitcomb, J.G. Tully, P. Carle, M. Konai, J.M. Bove and R.B. Henegar. 1997. Spiroplasma platyhelix sp. nov., a new mollicute with unusual morphology and genome size from the dragonfly Pachydiplax longipennis. Int. J. Syst. Bacteriol. 47: 763–766. Williamson, D.L., R.F. Whitcomb, J.G. Tully, G.E. Gasparich, D.L. Rose, P. Carle, J.M. Bové, K.J. Hackett, J.R. Adams, R.B. Henegar, M. Konai,

C. Chastel and F.E. French. 1998. Revised group classification of the genus Spiroplasma. Int. J. Syst. Bacteriol. 48: 1–12. Williamson, D.L., B. Sakaguchi, K.J. Hackett, R.F. Whitcomb, J.G. Tully, P. Carle, J.M. Bové, J.R. Adams, M. Konai and R.B. Henegar. 1999. Spiroplasma poulsonii sp. nov., a new species associated with malelethality in Drosophila willistoni, a neotropical species of fruit fly. Int. J. Syst. Bacteriol. 49: 611–618. Woese, C.R., J. Maniloff and L.B. Zablen. 1980. Phylogenetic analysis of the mycoplasmas. Proc. Natl. Acad. Sci. U. S. A. 77: 494–498. Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev. 51: 221–271. Wolgemuth, C.W. and N.W. Charon. 2005. The kinky propulsion of Spiroplasma. Cell 122: 827–828. Wróblewski, H., K.E. Johansson and S. Hjérten. 1977. Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane. Biochim. Biophys. Acta 465: 275–289. Wróblewski, H., S. Nyström, A. Blanchard and A. Wieslander. 1989. Topology and acylation of spiralin. J. Bacteriol. 171: 5039–5047. Wróblewski, H., D. Robic, D. Thomas and A. Blanchard. 1984. Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas. Ann. Microbiol. (Paris) 135A: 73–82. Ye, F., F. Laigret, J.C. Whitley, C. Citti, L.R. Finch, P. Carle, J. Renaudin and J.M. Bové. 1992. A physical and genetic map of the Spiroplasma citri genome. Nucleic Acids Res. 20: 1559–1565. Ye, F., F. Laigret and J.M. Bové. 1994a. A physical and genomic map of the prokaryote Spiroplasma melliferum and its comparison with the Spiroplasma citri map. C. R. Acad. Sci. Ser. III 317: 392–398. Ye, F., J. Renaudin, J.M. Bové and F. Laigret. 1994b. Cloning and sequencing of the replication origin (oriC) of the Spiroplasma citri chromosome and construction of autonomously replicating artificial plasmids. Curr. Microbiol. 29: 23–29. Ye, F., F. Laigret, P. Carle and J.M. Bové. 1995. Chromosomal heterogeneity among various strains of Spiroplasma citri. Int. J. Syst. Bacteriol. 45: 729–734. Ye, F., U. Melcher, J.E. Rascoe and J. Fletcher. 1996. Extensive chromosome aberrations in Spiroplasma citri strain BR3. Biochem. Genet. 34: 269–286. Yogev, D., R. Rosengarten, R. Watson-McKown and K.S. Wise. 1991. Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5¢ regulatory sequences. EMBO J. 10: 4069–4079. Yu, J., A.C. Wayadande and J. Fletcher. 2000. Spiroplasma citri surface protein P89 implicated in adhesion to cells of the vector Circulifer tenellus. Phytopathology 90: 716–722. Zaaria, A., C. Fontenelle, M. Le Henaff and H. Wróblewski. 1990. Antigenic relatedness between the spiralins of Spiroplasma citri and Spiroplasma melliferum. J. Bacteriol. 172: 5494–5496. Zbinden, M. and M.A. Cambon-Bonavita. 2003. Occurrence of Deferribacterales and Entomoplasmatales in the deep-sea Alvinocarid shrimp Rimicaris exoculata gut. FEMS Microbiol. Ecol. 46: 23–30. Zhao, Y., R.W. Hammond, R. Jomantiene, E.L. Dally, I.M. Lee, H. Jia, H. Wu, S. Lin, P. Zhang, S. Kenton, F.Z. Najar, A. Hua, B.A. Roe, J. Fletcher and R.E. Davis. 2003. Gene content and organization of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii. Mol. Genet. Genomics 269: 592–602. Zhao, Y., R.W. Hammond, I.M. Lee, B.A. Roe, S. Lin and R.E. Davis. 2004a. Cell division gene cluster in Spiroplasma kunkelii: functional characterization of ftsZ and the first report of ftsA in mollicutes. DNA Cell Biol. 23: 127–134. Zhao, Y., H. Wang, R.W. Hammond, R. Jomantiene, Q. Liu, S. Lin, B.A. Roe and R.E. Davis. 2004b. Predicted ATP-binding cassette systems in the phytopathogenic mollicute Spiroplasma kunkelii. Mol. Genet. Genomics 271: 325–338.

Family I. Acholeplasmataceae

687

Order III. Acholeplasmatales Freundt, Whitcomb, Barile, Razin and Tully 1984, 348VP Daniel R. Brown, Janet M. Bradbury and Karl-Erik Johansson A.cho.le.plas.ma.ta¢les. N.L. neut. n. Acholeplasma type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Acholeplasmatales the Acholeplasma order. This order in the class Mollicutes is assigned to a group of wallless prokaryotes that do not require sterol for growth and occur in a wide variety of habitats, including many vertebrate hosts, insects, and plants. A single family, Acholeplasmataceae, and a single genus, Acholeplasma, recognize the prominent and distinct characteristics of the assigned organisms. Type genus: Acholeplasma Edward and Freundt 1970, 1AL.

Further descriptive information The trivial name acholeplasma(s) is commonly used when reference is made to species of this order. The initial proposal for elevation of the acholeplasmas to ordinal rank ­(Freundt et  al., 1984) was based primarily on the universal lack of a sterol requirement for growth of Acholeplasma ­species, in addition to other major genetic, nutritional, biochemical, and physiological characteristics that distinguish them from other members of the class Mollicutes. A subsequent proposal for an additional order, Entomoplasmatales (Tully et al., 1993), within the class to distinguish a group of mollicutes that are phylogenetically more closely related to the Mycoplasmatales than to acholeplasmas necessitated ­further revisions within the class.

References Edward, D.G. and E.A. Freundt. 1970. Amended nomenclature for strains related to Mycoplasma laidlawii. J. Gen. Microbiol. 62: 1–2. Edward, D.G. 1971. Determination of sterol requirement for Mycoplasmatales. J. Gen. Microbiol. 69 : 205–210. Freundt, E.A., R.F. Whitcomb, M.F. Barile, S. Razin and J.G. Tully. 1984. Proposal for elevation of the family Acholeplasmataceae to ordinal rank: Acholeplasmatales. Int. J. Syst. Bacteriol. 34: 346–349. IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group. 2004. Description of the genus ‘Candidatus Phytoplasma’, a taxon for the wall-less non-helical prokaryotes that colonize plant phloem and insects. Int. J. Syst. Evol. Microbiol. 54: 1243–1255. Lim, P.O. and B.B. Sears. 1992. Evolutionary relationships of a plantpathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences. J. Bacteriol. 174: 2606–2611. Razin, S. and J.G. Tully. 1970. Cholesterol requirement of mycoplasmas. J. Bacteriol. 102: 306–310.

Although most mollicutes require exogenous cholesterol or serum for growth, all species within the genus Acholeplasma and some assigned to the genera Asteroleplasma, Spiroplasma, and Mesoplasma do not have that requirement. The species that do not have a sterol requirement can easily be excluded from the sterolrequiring taxa by tests that measure growth responses to cholesterol or to a number of serum-free broth preparations (Edward, 1971; Razin and Tully, 1970; Rose et al., 1993; Tully, 1995). For instance, the Acholeplasmatales grow through end-point dilutions in serum-containing medium and in serum-free preparations, indicating the absence of a growth requirement for cholesterol. Analyses of rRNA and other genes have shown that a large group of uncultured, plant-pathogenic organisms referred to by the trivial name phytoplasmas (Sears and Kirkpatrick, 1994) are closely related to acholeplasmas (Lim and Sears, 1992; Toth et al., 1994). The 16S rRNA gene sequences for members of the genus Acholeplasma that have been determined so far show that the acholeplasmas form two clades, one of which is a sister ­lineage to the phytoplasmas, although the formal taxonomic assignment of “Candidatus Phytoplasma” proposed gen. nov. (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004) currently remains incertae sedis.

Rose, D.L., J.G. Tully, J.M. Bové and R.F. Whitcomb. 1993. A test for measuring growth responses of Mollicutes to serum and polyoxyethylene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532. Sears, B.B. and B.C. Kirkpatrick. 1994. Unveiling the evolutionary relationships of plant pathogenic mycoplasmalike organisms. ASM News 60: 307–312. Toth, K.F., N. Harrison and B.B. Sears. 1994. Phylogenetic relationships among members of the class Mollicutes deduced from rps3 gene sequences. Int. J. Syst. Bacteriol. 44: 119–124. Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised taxonomy of the class Mollicutes - proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378–385. Tully, J.G. 1995. Determination of cholesterol and polyoxyethylene sorbitan growth requirements of mollicutes. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, San Diego, pp. 381–389.

Family I. Acholeplasmataceae Edward and Freundt 1970, 1AL Daniel

R. Brown, Janet M. Bradbury and Karl-Erik Johansson

A.cho.le.plas.ma.ta.ce¢ae. N.L. neut. n. Acholeplasma, -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Acholeplasmataceae the Acholeplasma family.

Further descriptive information Type genus: Acholeplasma Edward and Freundt 1970, 1AL.

This family is monotypic, so its properties are essentially those of the genus Acholeplasma.

688

Family I. Acholeplasmataceae

Genus I. Acholeplasma Edward and Freundt 1970, 1AL Daniel R. Brown, Janet M. Bradbury and Karl-Erik Johansson A.cho.le.plas¢ma. Gr. pref. a not; Gr. n. chole bile; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Acholeplasma name intended to indicate that cholesterol, a constituent of bile, is not required.

Cells are spherical, with a diameter of about 300  nm, or filamentous, 2–5 µm long. Nonmotile. Colonies have a “fried-egg” appearance and may reach 2–3 mm in diameter. Facultatively anaerobic; most strains grow readily in simple media. All members lack a sterol requirement for growth. Chemo-­organotrophic, most species utilizing glucose and other sugars as the major energy sources. Many strains are capable of fatty acid biosynthesis from acetate. Arginine and urea are not hydrolyzed. Pigmented carotenoids occur in some species. All species are resistant, or only slightly susceptible, to 1.5% digitonin. Saprophytes found in soil, compost, wastewaters, or commensals of vertebrates, insects, or plants. None are known to be a primary pathogen, but they may cause cytopathic effects in tissue cultures. The genome sizes range from about 1500 to 2100 kbp. All species examined utilize the universal genetic code in which UGA is a stop codon. DNA G+C content (mol%): 27–38. Type species: Acholeplasma laidlawii (Sabin 1941) Edward and Freundt 1970, 1AL (Sapromyces laidlawi Sabin 1941, 334).

Further descriptive information Cells of acholeplasmas typically appear as pleomorphic coccoid, coccobacillary, or short filamentous forms when grown in mycoplasma broth containing 20% horse serum or 1% bovine serum fraction. Viable spherical cells usually have a minimum diameter of about 300 nm. Filaments may be as much as 500 nm in length, but some longer filaments and branching filaments occur in some strains. Filaments often show beading with eventual development of coccoid forms. Cellular morphology may also depend upon the ratio of unsaturated to saturated fatty acids in the medium. Adjustment of preparative materials to the osmolarity of the culture medium is necessary for proper morphological examination. Most acholeplasmas exhibit heavy turbidity when grown aerobically in broth containing 5–20% serum, usually of horse or fetal bovine origin, or when grown in 1% bovine serum fraction broth at 37°C. Less turbidity is evident when most acholeplasmas are cultured in serum-free broth and some species may be inhibited in media containing 20% horse serum. Strains of some acholeplasmas (Acholeplasma morum, Acholeplasma modicum, and Acholeplasma axanthum) may not grow well in serumfree medium unless glucose and some fatty acids (Tween 80 and palmitic acid) are included. Colonies on solid medium containing serum or bovine serum fraction are usually large (100– 200 nm in diameter) with the classical “fried-egg” appearance after 24–72  h at 37°C (Figure 114). Colonies of Acholeplasma axanthum and several other acholeplasmas may show only central zones of growth into the agar or other unusual colony forms, such as mulberry-like colonies. Most acholeplasmas display optimum growth at 37°C. Growth is much slower at 25–27°C and strains may require 7–10 d to reach the turbidity observed after 24 h at 37°C. Species of plant origin (Acholeplasma brassicae and Acholeplasma palmae) have an optimum growth temperature of 30°C.

FIGURE 114.  Colonies of Acholeplasma laidlawii PG8T (=NCTC 10116T;

diameter 0.15–0.25 mm) after 3 d growth on Mycoplasma Experience Solid Medium at 36°C in 95% nitrogen/5% carbon dioxide. Original magnification = 25×. Image provided by Helena Windsor and David Windsor.

Most species in the genus are strong fermenters and produce acid from glucose metabolism, although a few species such as Acholeplasma parvum may not ferment glucose or other carbohydrates (Table 143). Fermentation of mannose is usually negative, although several species do catabolize this carbohydrate. All Acholeplasma species examined possess a fructose 1,6-diphosphate-activated lactate dehydrogenase, which is a property shared with certain streptococci. Gourlay (1970) found that a fresh isolate of Acholeplasma laidlawii from a bovine source was infected with a filamentous, single-stranded DNA virus designated L1 (Bruce et  al., 1972; Maniloff, 1992). Later, L2 and L3 viruses were also isolated from Acholeplasma laidlawii (Gourlay, 1971, 1972, 1973; ­Gourlay et  al., 1973). L2 virus is a quasi-spherical, doublestranded DNA virus (Maniloff et al., 1977), and L3 is a shorttailed phage with double-stranded DNA (Garwes et al., 1975; Gourlay, 1974; Haberer et  al., 1979; Maniloff et  al., 1977). Another virus isolated from Acholeplasma laidlawii is L172, a single-stranded DNA, quasi-spherical virus that is different from L1 (Liska, 1972). Two viruses have been isolated from other acholeplasmas, including one from Acholeplasma modicum, designated M1 (Congdon et al., 1979), and from Acholeplasma oculi strain PG49 (designated O1) (Ichimaru and Nakamura, 1983). The nucleic acid structure of the last two viruses has not been defined. Antisera to filter-cloned whole-cell antigens are utilized in several serological techniques to assess the antigenic structure of acholeplasmas and to provide identification of the organism to the species level (Tully, 1979). The three most useful ­techniques are growth inhibition (Clyde, 1983), plate ­immunofluorescence

689

Genus I. Acholeplasma

A. brassicae

A. cavigenitalium

A. equifetale

A. granularum

A. hippikon

A. modicum

A. morum

A. multilocale

A. oculi

A. palmae

A. parvum

A. pleciae

A. vituli

Glucose fermentation Mannose fermentation Arbutin hydrolysis Esculin hydrolysis Film and spots Benzyl viologen reduction DNA G+C content (mol%)

A. axanthum

Characteristic

A. laidlawii

TABLE 143.  Differential characteristics of the species of the genus Acholeplasma a

+ − + + + +

+ − + + − +

+ − − nd nd +

+ − − nd − +

+ + nd nd + +

+ − − − − +

+ + nd nd + +

+ − − − − +

+ − + + − +

+ + nd nd + −

+ − + + − +

+ − − nd nd +

− nd nd − nd +

+ nd nd nd nd nd

+ + − − − nd

31–36

31

35.5

36

30.5

30–32

33

29

34

31

27

30

29

31.6

37.6–38.3

Symbols: +, >85% positive; −, 0–15% positive; nd, not determined.

a

(Gardella et al., 1983; Tully, 1973), and metabolism inhibition (Taylor-Robinson, 1983). Acholeplasmas may be the most common mollicutes in vertebrate animals and they are found frequently in the upper respiratory tract and urogenital tract of such hosts (Tully, 1979, 1996). Eukaryotic cells in continuous culture are frequently contaminated with acholeplasmas, primarily from the occurrence of acholeplasmas in animal serum used in tissue culture media. At least five Acholeplasma species have been identified on plant surfaces (Acholeplasma axanthum, Acholeplasma brassicae, Acholeplasma laidlawii, Acholeplasma oculi, and Acholeplasma palmae), possibly representing contamination from insects. However, with the exception of Acholeplasma pleciae (Knight, 2004), the only acholeplasmas identified from insects have been from mosquitoes. Acholeplasma laidlawii was identified in a pool of Anopheles sinensis, and a strain of Acholeplasma morum was present in a pool of Armigeres subalbatus (D.L. Williamson and J.G. Tully, unpublished). Little evidence exists for a pathogenic role of acholeplasmas in natural diseases. The widespread distribution of acholeplasmas in both healthy and diseased animal tissues and of antibodies against acholeplasmas in most animal sera complicates experimental pathogenicity studies. However, Acholeplasma axanthum was pathogenic for goslings and young goose embryos (Kisary et  al., 1975, 1976). Inoculation into leafhoppers, ­including those known to be vectors of plant mycoplasma diseases, shows multiplication and prolonged persistence of acholeplasmas in host tissues (Eden-Green and Markham, 1987; Whitcomb et al., 1973; Whitcomb and Williamson, 1975), but there is no evidence that the few Acholeplasma species found on plant surfaces play any role in plant or insect disease. A few recent reports are available on the antibiotic sensitivity of acholeplasmas and whether the actions of these drugs are inhibitory to growth or kill cells. Acholeplasmas are sensitive to the following antibiotics (minimum inhibitory concentration range in µg/ml): tetracycline, 0.5–25.0; erythromycin, 0.03–1.0; lincomycin, 0.25–1.0; tylosin tartarate, 0.1–12.5; and kanamycin, 20–200 (Kato et al., 1972; Lewis and Poland, 1978; Ogata et al., 1971).

Enrichment and isolation procedures Typical steps in isolation of all mollicutes were outlined in the recently revised minimal standards for descriptions of novel

species (Brown et al., 2007). Techniques for isolation of acholeplasmas from animal tissues and from cell cultures have been described (Tully, 1983). Although colonies are occasionally first detected on blood agar, complex undefined media such as American Type Culture Collection medium 988 (SP-4) are usually required for primary isolation and maintenance. Cell wall-targeting antibiotics are included to discourage growth of other bacteria. Phenol red facilitates detection of species that excrete acidic or alkaline metabolites. Commonly used alternatives such as Frey’s, Hayflick’s and Friis’ media differ from SP-4 mainly in the proportions of inorganic salts, amino acids, serum sources, and types of antibiotics included. Broths are incubated aerobically at 37°C for 14 d and examined periodically for turbidity or pH changes, either to acid or alkaline levels. Tubes showing turbidity are plated to agar prepared from the same medium formulation, and the plates are incubated at 37°C in an atmosphere of 95% N2, 5% CO2, as in the GasPak system. Tubes without obvious turbidity should be plated at the end of the 14-d incubation period. Initial isolates may contain a mixture of species, so cloning by repeated filtration through membrane filters with a pore size of 450 or 220 nm is essential. The initial filtrate and dilutions of it are cultured on solid medium and an isolated colony is subsequently picked from a plate on which only a few colonies have developed. This colony is used to found a new cultural line, which is then expanded, filtered, plated, and picked two additional times. Identification is confirmed by additional biochemical and serological tests.

Maintenance procedures Stock acholeplasma cultures can be maintained in either mycoplasma broth medium containing 5–20% serum or in the serum-fraction broth formulation at room temperature (25–30°C) with only weekly transfer (Tully, 1995). Maintenance is best in broth medium devoid of glucose, since excess acid production reduces viability. Stock cultures can also be maintained indefinitely when frozen at −70°C. Agar colonies can also be maintained for 1–2 weeks at 25°C if plates are sealed to prevent drying. For optimum preservation, acholeplasmas should be lyophilized directly in the culture medium when the broth cultures reach a mid-exponential phase, usually 1–2 d at 37°C. Lyophilized cultures should be sealed under vacuum and stored at 4°C (Leach, 1983).

690

Family I. Acholeplasmataceae

Differentiation of the genus Acholeplasma from other genera Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et  al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. They usually possess two 16S rRNA operons. ­Aerobic or facultative anaerobic growth in artificial media and the absence of a requirement for sterols or cholesterol for growth exclude assignment to the genera Anaeroplasma, Asteroleplasma, “Candidatus Phytoplasma”, Mycoplasma, or Ureaplasma. Absence of a spiral cellular morphology, regular association with a vertebrate host or fluids of vertebrate origin, and regular use of the codon UGG to encode tryptophan (Knight, 2004) and UGA as a stop codon (Tanaka et  al., 1989, 1991) support exclusion from the genera Spiroplasma, Entomoplasma, or Mesoplasma. Reduction of the redox indicator benzyl viologen has been reported to be fairly specific for differentiation of the genus Acholeplasma from other mollicutes (Pollack et al., 1996a). Only Acholeplasma multilocale failed to give a positive reaction, although several Mesoplasma and Entomoplasma species yielded variable responses to the test (Pollack et al., 1996a). Most acholeplasmas have membrane-localized NADH oxidase activity, in comparison to the NADH oxidase activity located in the cytoplasm of other genera within the class. Another special characteristic is the occurrence in most acholeplasmas of unique pyrimidine enzymic activities, especially a dUTPase enzyme, with the possible exception again of Acholeplasma multilocale (Pollack et  al., 1996b). Acholeplasmas may possess a number of other biological characteristics that may distinguish them from other genera within the class Mollicutes, including polyterpenol synthesis (Smith and Langworthy, 1979), positional distribution of fatty acids (Rottem and Markowitz, 1979), the presence of superoxide dismutase (Kirby et al., 1980; Lee and Kenny, 1984; Lynch and Cole, 1980; O’Brien et al., 1981), and the presence of spacer tRNA (­Nakagawa et  al., 1992). However, most of these features have not been established for even a majority of Acholeplasma species.

Taxonomic comments Acholeplasma genome sizes range from 1215 to 2095  kbp by pulsed-field gel electrophoresis or complete DNA sequencing, but most are in a more narrow range of 1215–1610 kbp (Carle et  al., 1993; Neimark et  al., 1992) that overlaps with genome sizes of many Spiroplasma species. Tests of eight Acholeplasma species showed less than 8% DNA–DNA hybridization between type strains and surprisingly extensive genomic heterogeneity within species (Aulakh et  al., 1983; Stephens et  al., 1983a, b). The highest level of relatedness, 21% DNA–DNA hybridization, was between the type strains of Acholeplasma laidlawii and Acholeplasma granularum. Some strain pairs, such as within Acholeplasma laidlawii, shared as little as 40% DNA–DNA hybridization, differences that in other genera would have justified subdivision of an apparently diverse strain complex into component species. However, no polyphasic taxonomic basis was found to support such designations. Restriction endonuclease digest patterns also reflect heterogeneity within some species (Razin et al., 1983). The DNA–DNA hybridization and restriction digest patterns of eight Acholeplasma axanthum strains isolated from a variety of hosts and habitats differed markedly

from each other and some heterogeneity occurred among six different Acholeplasma oculi strains. Mesoplasma pleciae was first isolated from the hemolymph of a larva of a Plecia corn root maggot and assigned to the genus Mesoplasma because sustained growth occurred in serum-free mycoplasma broth only when the medium contained 0.04% Tween 80 fatty acid mixture (Tully et al., 1994a). However, 16S rRNA gene sequence similarities and its preferred use of UGG rather than UGA to encode tryptophan support proper reclassification as Acholeplasma pleciae comb. nov. (Knight, 2004); the type strain is PS-1T (Tully et al., 1994a). Mycoplasma feliminutum was first described during a time when the only named genus of mollicutes was Mycoplasma. Its publication coincided with the first proposal of the genus Acholeplasma (Edward and Freundt, 1969, 1970), with which Mycoplasma feliminutum is properly affiliated through established phenotypic (Heyward et al., 1969) and 16S rRNA gene sequence (Brown et al., 1995; Johansson and Pettersson, 2002) similarities. This explains the apparent inconsistencies with its assignment to the genus Mycoplasma. The name Mycoplasma feliminutum should therefore be revised to Acholeplasma feliminutum comb. nov.; the type strain is BenT (=ATCC 25749T; Heyward et al., 1969). The lack of signature enzymic activities cast serious doubt on the status of Acholeplasma multilocale PN525T as an authentic member of the genus Acholeplasma (Pollack et  al., 1996b). It may be affiliated with an unrecognized metabolic subgroup, but it seems more likely to be a strain of Mycoplasma or Entomoplasma.

Acknowledgements The major contributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged.

Further reading Taylor-Robinson, D. and J.G. Tully. 1998. Mycoplasmas, ureaplasmas, spiroplasmas, and related organisms. In Topley and Wilson, Principles and Practice of Microbiology, 9th edn, vol. 2 (edited by Balows and Duerden). Arnold Publishers, London, pp. 799–827. Tully, J.G. 1989. Class Mollicutes: new perspectives from plant and arthropod studies. In The Mycoplasmas (edited by Whitcomb and Tully). Academic Press, San Diego, pp. 1–31.

Differentiation of the species of the genus Acholeplasma Esculin hydrolysis by a b-d-glucosidase and arbutin hydrolysis are sometimes useful diagnostic tests for differentiation of some acholeplasmas (Bradbury, 1977; Rose and Tully, 1983). The production of carotenoid pigments, principally neurosporene, has been used to differentiate some acholeplasmas, especially Acholeplasma axanthum and Acholeplasma modicum (Mayberry et  al., 1974; Smith and Langworthy, 1979; Tully and Razin, 1970). Carotenoids are also synthesized in some strains of Acholeplasma laidlawii under certain growth conditions (Johansson, 1974). The “film and spots” reaction, which occurs in a number of Mycoplasma and several Acholeplasma species, relates to the production of crystallized calcium soaps of fatty acids on the surface of agar plates (Edward, 1954; ­Fabricant and Freundt, 1967). Fatty acids are liberated from the serum or

Genus I. Acholeplasma

supplemental egg yolk (Fabricant and Freundt, 1967; Thorns and Boughton, 1978) in the agar medium by the lipolytic activity of the organisms. Failure to cross-react with antisera against previously recognized species provides evidence for species novelty. For this reason, deposition of antiserum against a novel type strain into a recognized collection is still mandatory for new species descriptions (Brown et al., 2007). Prelimi-

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nary differentiation can be by PCR and DNA sequencing using primers specific for bacterial 16S rRNA genes or the 16S–23S intergenic region. A similarity matrix relating the candidate strain to its closest neighbors, usually species with >0.94 16S rRNA gene sequence similarity, will suggest an assemblage of related species that should be examined for serological crossreactivities.

List of species of the genus Acholeplasma 1. Acholeplasma laidlawii (Sabin 1941) Edward and Freundt 1970, 1AL (Sapromyces laidlawi Sabin 1941, 334) laid.law¢i.i. N.L. gen. masc. n. laidlawii of Laidlaw, named after Patrick P. Laidlaw, one of the microbiologists who first isolated this species. This is the type species of the genus Acholeplasma. Filaments, usually relatively short, although much longer branched filaments may develop in media with certain ratios of saturated to unsaturated fatty acids. Coccoid forms may predominate in certain cultures including co-culture with eukaryotic cells. Agar colonies are large for a mollicute and exhibit well-developed central zones and peripheral growth on horse serum agar. On serum-free agar, colonies are smaller and may show only the central zone of growth into the agar. Relatively strong turbidity is produced during growth in broth containing serum. Temperature range for growth is 20–41°C with optimum at 37°C, even for strains recovered from plant or non-animal sources. Usually produces large amounts of carotenoids when cultivated in the presence of PPLO serum fraction (Difco). Serologically distinct from most established species in the genus, but partial cross-reactions may occur with Acholeplasma granularum strains. DNA–DNA hybridization between strains of this species range from 40 to >80%. Acholeplasma granularum strain BTS-39T showed 20% hybridization with Acholeplasma laidlawii strain PG8T. Pathogenicity has not been established. Source: isolated from sewage, manure, humus, soil, and many animal hosts and their tissues, including some isolates from the human oral cavity, vagina, and wounds. Has been recovered from the surfaces of some plants, although few isolations have been reported from insect hosts. Frequent contaminant of eukaryotic cell cultures. DNA G+C content (mol%): 31.7–35.7 (Bd, Tm). Type strain: ATCC 23206, PG8, NCTC 10116, CIP 75.27, NBRC 14400. Sequence accession no. (16S rRNA gene): U14905. Further comment: on the Approved Lists of Bacterial Names and on the Approved Lists of Bacterial Names (Amended Edition), this taxon is incorrectly cited as Acholeplasma laidlawii [Freundt 1955 (sic)] Edward and Freundt (1970). 2. Acholeplasma axanthum Tully and Razin 1970, 754AL a.xan¢thum. Gr. pref. a not, without; Gr. adj. xanthos -ê -on yellow; N.L. neut. adj. axanthum without yellow (pigment). Predominantly coccobacillary and coccoid with a few short myceloid elements. Large colonies with clearly marked centers form on horse serum agar; colonies on serum-free agar are smaller and usually lack the peripheral

growth around their center. Agar colonies produce zones of b-hemolysis by the overlay technique. Growth in media devoid of serum or serum fraction is much poorer than for other acholeplasmas. Minimal nutritional requirements are poorly defined, but marked stimulation of growth with polyoxyethylene sorbitan (Tween 80) suggests a requirement for fatty acids. Temperature range for growth is 22–37°C with optimum growth at 37°C. Synthesis of carotenoid pigments can be demonstrated only when large volume cultures are tested. Produces sphingolipids. No evidence for pathogenicity. Source: originally isolated from murine leukemia tissue culture cells, but numerous subsequent isolations of the organism from bovine serum and a variety of bovine tissue sites (nasal cavity, lymph nodes, kidney) suggest cell-culture contamination was of bovine serum origin. Also isolated from variety of other animals and surfaces of some plants. DNA G+C content (mol%): 31 (Bd). Type strain: S-743, ATCC 25176, NCTC 10138. Sequence accession no. (16S rRNA gene): AF412968. 3. Acholeplasma brassicae Tully, Whitcomb, Rose, Bové, Carle, Somerson, Williamson and Eden-Green 1994b, 683VP bras.si¢cae. L. fem. gen. n. brassicae of cabbage, referring to the plant origin of the organism. Cells are primarily coccoid. Temperature range for growth is 18–37°C. Optimal growth occurs at 30°C. No evidence for pathogenicity. Source: isolated as a surface contaminant from broccoli (Brassica oleracea var. italica). DNA G+C content (mol%): 35.5 (Bd, Tm, HPLC). Type strain: 0502, ATCC 49388. Sequence accession no. (16S rRNA gene): AY538163. 4. Acholeplasma cavigenitalium Hill 1992, 591VP ca.vi.ge.ni.ta¢li.um. N.L. n. cavia guinea pig (Cavia cobaya); L. pl. n. genitalia -ium the genitals; N.L. pl. gen. n. cavigenitalium of guinea pig genitals. Pleomorphic cells, mostly coccoid. Grows on broth or agar medium under aerobic conditions, with optimum temperature between 35 and 37°C. Colonies on agar medium have typical fried-egg appearance. Originally described as a non-fermenter, but the type strain ferments glucose. Does not grow well on SP-4 broth or in horse serum broth, but grows well on simple base medium with additions of 10–15% fetal bovine serum. No evidence for pathogenicity. Source: isolated from the vagina of guinea pigs. DNA G+C content (mol%): 36 (Bd). Type strain: GP3, NCTC 11727, ATCC 49901. Sequence accession no. (16S rRNA gene): AY538164.

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Family I. Acholeplasmataceae

5. Acholeplasma entomophilum Tully, Rose, Carle, Bové, Hackett and Whitcomb 1988, 166VP en.to.mo.phi¢lum. Gr. n. entomon insect; N.L. neut. adj. philum (from Gr. neut. adj. philon) friend, loving; N.L. neut. adj. entomophilum insect-loving. Cells are pleomorphic, but primarily coccoid. Colonies on solid medium usually have a fried-egg appearance. Acid is produced from glucose, but not mannose. Carotenoids are not produced. “Film and spot” reaction is negative. Agar colonies hemadsorb guinea pig erythrocytes. Strains require 0.4% Tween 80 or fatty acid supplements for growth in serum-free media. Temperature range for growth is 23–32°C, with optimum growth at about 30°C. Pathogenicity has not been established. Source: isolated from gut contents of tabanid flies, beetles, butterflies, honey bees, and moths, and from flowers. DNA G+C content (mol%): 30 (Bd). Type strain: TAC, ATCC 43706. Sequence accession no. (16S rRNA gene): M23931. Further comment: with the proposal of the order Entomoplasmatales (Tully et  al., 1993), Acholeplasma entomophilum was transferred to the family Entomoplasmataceae. The name Acholeplasma entomophilum was therefore revised to Mesoplasma entomophilum comb. nov. The type strain is TACT (=ATCC 43706T; Tully et al., 1988). 6. Acholeplasma equifetale Kirchhoff 1978, 81AL eq.ui.fe.ta¢le. L. n. equus horse; N.L. adj. fetalis -is -e pertaining to the fetus; N.L. neut. adj. equifetale pertaining to the horse fetus. Cells are pleomorphic, but predominantly coccoid. Colonies on solid medium containing serum usually have a fried-egg appearance; on serum-free medium, colonies are similar, but usually smaller. Growth temperature range is 22–37°C. Pathogenicity has not been established. Source: isolated from the lung and liver of aborted horse fetuses. Also recovered from the respiratory tract of apparently normal horses and the respiratory tract and cloacae of broiler chickens (Bradbury, 1978). DNA G+C content (mol%): 30.5 (Bd). Type strain: C112, ATCC 29724, NCTC 10171. Sequence accession no. (16S rRNA gene): AY538165. Further comment: Kirchhoff is incorrectly cited as “­Kirchoff” on the Approved Lists of Bacterial Names. 7. Acholeplasma florum McCoy, Basham, Tully, Rose, Carle and Bové 1984, 14VP flo¢rum. L. gen. p1. n. florum of flowers, indicating the recovery site of the organism. Cells are ovoid. Colonies on agar are umbonate. Films and spots are produced on serum-containing media. Glucose is utilized, but mannose is not. Carotenes are not produced, nor is b-d-glucosidase. Pathogenicity has not been established. Source: the known strains were isolated from flower surfaces. DNA G+C content (mol%): 27.3 (Bd). Type strain: L1, ATCC 33453. Sequence accession nos: AF300327 (16S rRNA gene), NC_006055 (strain L1T complete genome).

Further comment: with the proposal of the order Entomoplasmatales (Tully et  al., 1993), Acholeplasma florum was transferred to the family Entomoplasmataceae. The name Acholeplasma florum was therefore revised to Mesoplasma florum comb. nov. The type strain is L1T (=ATCC 33453T; McCoy et al., 1984). 8. Acholeplasma granularum (Switzer 1964) Edward and Freundt 1970, 2AL (Mycoplasma granularum Switzer 1964, 504) gra.nu.la¢rum. N.L. fem. n. granula (from L. neut. n. granulum) a small grain, a granule; N.L. gen. pl. n. granularum of small grains, made up of granules, granular. Cells are pleomorphic, with short filaments and coccoid cells. Colonies on solid medium are large with clearly marked central zones and a fried-egg appearance. Colonies on serum-free medium are smaller and may lack the peripheral zone of growth around central core. Temperature range for growth is 22–37°C, with optimum around 37°C. Agar colonies produce a zone of b-hemolysis by the overlay technique using sheep erythrocytes. DNA–DNA hybridization studies showed 20–22% hybridization with Acholeplasma laidlawii, but none with other acholeplasmas. Pathogenicity has not been established. Aerosol challenge of specific pathogen-free pigs did not induce clinical or histological evidence of disease. Source: isolated frequently from the nasal cavity of swine, with occasional isolates from swine lung and feces. Also isolated from the conjunctivae and nasopharynx of horses, and the genital tract of guinea pigs. Occasional contaminant of eukaryotic cell cultures. DNA G+C content (mol%): 30.5–32.4 (Tm, Bd). Type strain: BTS-39, ATCC 19168, NCTC 10128. Sequence accession no. (16S rRNA gene): AY538166. 9. Acholeplasma hippikon Kirchhoff 1978, 81AL hip.pi¢kon. Gr. neut. adj. hippikon pertaining to the horse. Cells are pleomorphic with predominantly coccoid forms. Colonies on solid medium containing horse serum typically have a fried-egg appearance, with smaller colonies on serum-free agar medium. Growth occurs over a temperature range of 22–37°C, with optimal growth at 35–37°C. Agar colonies produce b-hemolysis with the overlay technique, using a variety of animal red blood cells. ­Pathogenicity has not been established. Source: isolated from the lung of aborted horse fetuses. DNA G+C content (mol%): 33.1 (Bd). Type strain: C1, ATCC 29725, NCTC 10172. Sequence accession no. (16S rRNA gene): AY538167. Further comment: Kirchhoff is incorrectly cited as “­Kirchoff” on the Approved Lists of Bacterial Names. 10. Acholeplasma modicum Leach 1973, 147AL mo¢di.cum. L. neut. adj. modicum moderate, referring to moderate growth. Cells are pleomorphic, with spherical, ring-shaped, and coccobacillary forms. Colonies on solid medium are distinctly smaller than those of most other acholeplasmas. Very small colonies without peripheral zones of growth are noted on serum-free solid medium. Very light turbidity is observed in serum-free broth, but more turbidity is found

Genus I. Acholeplasma

in broth containing serum. Growth temperature range is 22–37°C, with optimum growth around 35–37°C. Can be shown to produce carotenoids when large volumes of cells are examined. Agar colonies produce a- or b-hemolysis by the overlay technique using sheep, ox, or guinea pig red blood cells. Pathogenicity has not been established. Source: isolated from various tissues of cattle, including blood, bronchial lymph nodes, thoracic fluids, lungs, and semen. Also isolated from nasal secretions of pigs, and occasionally from chickens, turkeys, and ducks. DNA G+C content (mol%): 29.3 (Tm). Type strain: PG49, ATCC 29102, NCTC 10134. Sequence accession no. (16S rRNA gene): M23933. 11. Acholeplasma morum Rose, Tully and Del Giudice 1980, 653VP mor¢um. L. n. morum a mulberry, denoting the mulberrylike appearance of agar colonies of the organism. Cells are pleomorphic, predominantly coccoid or coccobacillary forms, but with some beaded filaments. Colonies on solid medium without serum supplements are very small in size and have only central zones without any peripheral growth. Optimal growth on solid medium occurs with a 10% serum concentration and colony growth appears to be suppressed in a medium with 20% serum. Optimal growth in broth is apparent when 5–10% serum is added or when 1% bovine serum fraction supplements are added, but poor growth occurs in broth containing 20% horse serum. Growth in serum-free broth usually requires some fatty acid supplements, such as palmitic acid or polyoxyethylene sorbitan (Tween 80). Temperature range for growth is 23–37°C, with optimum growth at about 35–37°C. Pathogenicity has not been established. Calf kidney cell cultures containing the organism show cytopathogenic effects. Source: originally recovered from commercial fetal bovine serum and from calf kidney cultures containing fetal bovine serum. One isolation, in broth containing horse serum, was from a pool of Armigeres subalbatus mosquitoes collected by Leon Rosen in Taiwan in 1978 (strain SP7; D.L. Williamson and J.G. Tully, unpublished). DNA G+C content (mol%): 34.0 (Tm). Type strain: 72-043, ATCC 33211, NCTC 10188. Sequence accession no. (16S rRNA gene): AY538168. 12. Acholeplasma multilocale Hill, Polak-Vogelzang and ­Angulo 1992, 516VP mul.ti.lo.ca¢le. L. adj. multus many, numerous; L. adj. localis -is -e of or belonging to a place, local; N.L. neut. adj. multilocale referring to more than one location. Pleomorphic cells. Colonies on agar medium have a typical fried-egg appearance. Organisms grow well in broth medium at 35–37°C. No evidence for pathogenicity. Source: isolated from the nasopharynx of a horse and the feces of a rabbit. DNA G+C content (mol%): 31 (Bd). Type strain: PN525, NCTC 11723, ATCC 49900. Sequence accession no. (16S rRNA gene): AY538169. 13. Acholeplasma oculi corrig. al-Aubaidi, Dardiri, Muscoplatt and McCauley 1973, 126AL o¢cu.li. L. n. oculus the eye; L. gen. n. oculi of the eye.

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Cells are pleomorphic, including spherical, ring-shaped, and coccobacillary forms. Medium-sized colonies with typical fried-egg appearance are formed on horse serum agar. Colonies on serum-free agar are smaller and may lack the peripheral growth around the central core. Growth occurs at temperatures of 25–37°C. Agar colonies produce zones of hemolysis by the overlay technique using sheep red blood cells. Pathogenicity is not well established. Intravenous inoculation of goats produced signs of pneumonia and death within 6 d. Conjunctival inoculation of goats produced mild conjunctivitis. Source: isolated from the conjunctiva of goats with keratoconjunctivitis; porcine nasal secretions; equine nasopharynx, lung, spinal fluid, joint, and semen; the urogenital tract of cattle; and the external genitalia of guinea pigs. Present in amniotic fluid of pregnant women (Waites et al., 1987). Occasionally isolated from ducks and turkeys, with unreported isolations from an ostrich. Also several isolations from palm trees and other plants (Eden-Green and Tully, 1979; Somerson et al., 1982). Isolations from eukaryotic cell cultures may represent contamination of bovine origin. DNA G+C content (mol%): 27 (Tm). Type strain: 19-L, ATCC 27350, NCTC 10150. Sequence accession no. (16S rRNA gene): U14904. Further comment: originally named Acholeplasma oculusi by al-Aubaidi et al. (1973); the orthographic error was corrected by al-Aubaidi (1975). 14. Acholeplasma palmae Tully, Whitcomb, Rose, Bové, Carle, Somerson, Williamson and Eden-Green 1994b, 683VP pal¢mae. L. fem. gen. n. palmae of a palm tree, referring to the plant from which the organism was isolated. Cells are primarily coccoid. Colonies on solid medium usually have a fried-egg appearance. The temperature range for growth is 18–37°C, with optimal growth occurring at 30°C. No evidence for pathogenicity. It is one of the closest phylogenetic relatives of the phytoplasmas. Source: isolated from the crown tissues of a palm tree (Cocos nucifera) with lethal yellowing disease. DNA G+C content (mol%): 30 (Bd, Tm, HPLC). Type strain: J233, ATCC 49389. Sequence accession no. (16S rRNA gene): L33734. 15. Acholeplasma parvum Atobe, Watabe and Ogata 1983, 348VP par¢vum. L. neut. adj. parvum small, intended to refer to the poor biochemical activities and tiny agar colonies of the organism. Pleomorphic coccobacillary cells. Colonies on agar medium present a typical fried-egg appearance under both aerobic and anaerobic conditions. Initial reports of growth in the absence of cholesterol or serum have been made, but growth on serum-free medium is not well confirmed. The organism does not grow in most standard media for acholeplasmas or in most other medium formulations for sterolrequiring mycoplasmas. Needs special growth factor of 1% phytone or soytone peptone supplements; growth is sometimes better with the addition of 15% fetal bovine serum. Organisms grow on agar better than in broth; growth is

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Family I. Acholeplasmataceae

­ etter under aerobic conditions than under anaerobic b ­conditions and better at 22–30°C than at 37°C. No evidence of fermentation of any carbohydrate, including glucose, salicin, and esculin. No evidence for pathogenicity. Source: isolated from the oral cavities and vagina of healthy horses. DNA G+C content (mol%): 29.1 (Tm). Type strain: H23M, ATCC 29892, NCTC 10198. Sequence accession no. (16S rRNA gene): AY538170. 16. Acholeplasma pleciae (Tully, Whitcomb, Hackett, Rose, ­Henegar, Bové, Carle, Williamson and Clark 1994a) Knight 2004, 1952VP (Mesoplasma pleciae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 690) ple.ci¢ae. N.L. gen. n. pleciae of Plecia, referring to the genus of corn maggot (Plecia sp.) from which the organism was first isolated. Cells are primarily coccoid. Colonies on solid media incubated under anaerobic conditions at 30°C have a friedegg appearance. Supplements of 0.04% polyoxyethylene sorbitan (Tween 80) are required for growth in serum-free media. Temperature range for growth is 18–32°C, with optimal growth at 30°C. Agar colonies do not hemadsorb guinea pig erythrocytes. No evidence for pathogenicity. Source: originally isolated from the hemolymph of a larva of the corn root maggot (Plecia sp.). DNA G+C content (mol%): 31.6 (Bd, Tm, HPLC). Type strain: PS-1, ATCC 49582. Sequence accession no. (16S rRNA gene): AY257485. 17. Acholeplasma seiffertii Bonnet, Saillard, Vignault, Garnier, Carle, Bové, Rose, Tully and Whitcomb 1991, 48VP seif.fer¢ti.i. N.L. gen. masc. n. seiffertii of Seiffert, in honor of Gustav Seiffert, a German microbiologist who performed

References al-Aubaidi, J.M. 1975. Orthographic errors in the name Acholeplasma oculusi. Int. J. Syst. Bacteriol. 25: 221. al-Aubaidi, J.M., A.H. Dardiri, C.C. Muscoplatt and E.H. McCauley. 1973. Identification and characterization of Acholeplasma oculusi spec. nov. from the eyes of goats with keratoconjunctivitis. Cornell Vet. 63: 117–129. Angulo, A.F., R. Reijgers, J. Brugman, I. Kroesen, F.E.N. Hekkens, P. Carle, J.M. Bové, J.G. Tully, A.C. Hill, L.M. Schouls, C.S. Schot, P.J.M. Roholl and A.A. Polak-Vogelzang. 2000. Acholeplasma vituli sp. nov., from bovine serum and cell cultures. Int. J. Syst. Evol. Microbiol. 50: 1125–1131. Atobe, H., J. Watabe and M. Ogata. 1983. Acholeplasma parvum, a new species from horses. Int. J. Syst. Bacteriol. 33: 344–349. Aulakh, G.S., E.B. Stephens, D.L. Rose, J.G. Tully and M.F. Barile. 1983. Nucleic acid relationships among Acholeplasma species. J. Bacteriol. 153: 1338–1341. Bonnet, F., C. Saillard, J.C. Vignault, M. Garnier, P. Carle, J.M. Bové, D.L. Rose, J.G. Tully and R.F. Whitcomb. 1991. Acholeplasma seiffertii sp. nov., a mollicute from plant surfaces. Int. J. Syst. Bacteriol. 41: 45–49. Bradbury, J. 1977. Rapid biochemical tests for characterization of the Mycoplasmatales. J. Clin. Microbiol. 5: 531–534. Bradbury, J.M. 1978. Acholeplasma equifetale in broiler chickens. Vet. Rec. 102: 516.

pioneering studies of sterol-nonrequiring mollicutes that occur in soil and compost. Cells are primarily coccoid. Colonies on solid medium ­ sually have the appearance of fried-eggs. Acid produced u from glucose and mannose. Colonies on agar hemadsorb guinea pig erythrocytes. Temperature range for growth is 20–35°C; optimum growth occurs at 28°C. No evidence for pathogenicity. Source: isolated from floral surfaces of a sweet orange (Citrus sinensis) and wild angelica (Angelica sylvestris). DNA G+C content (mol%): 30 (Bd). Type strain: F7, ATCC 49495. Sequence accession no. (16S rRNA gene): AY351331. Further comment: with the proposal of the order Entomoplasmatales (Tully et  al., 1993), Acholeplasma seiffertii was transferred to the family Entomoplasmataceae. The name Acholeplasma seiffertii was therefore revised to Mesoplasma seiffertii comb. nov. The type strain is F7T (=ATCC 49495T; Bonnet et al., 1991). 18. Acholeplasma vituli Angulo, Reijgers, Brugman, Kroesen, Hekkens, Carle, Bové, Tully, Hill, Schouls, Schot, Roholl and Polak-Vogelzang 2000, 1130VP vi.tu¢li. L. n. vitulus calf; L. gen. n. vituli of calf, referring to the provenance or occurrence of the organism in fetal calf serum. Cells are predominantly coccoid in shape. Colonies on solid media demonstrate a fried-egg appearance under both aerobic and anaerobic conditions. Temperature range for growth is 25–37°C. No evidence for pathogenicity. Source: isolated from fetal bovine serum or contaminated eukaryotic cell cultures containing serum. DNA G+C content (mol%): 38.3 (Bd), 37.6 (Tm). Type strain: FC 097-2, ATCC 700667, CIP 107001. Sequence accession no. (16S rRNA gene): AF031479.

Brown, D., G. McLaughlin and M. Brown. 1995. Taxonomy of the feline mycoplasmas Mycoplasma felifaucium, Mycoplasma feliminutum, Mycoplasma felis, Mycoplasma gateae, Mycoplasma leocaptivus, Mycoplasma leopharyngis, and Mycoplasma simbae by 16S rRNA gene sequence comparisons. Int. J. Syst. Bacteriol. 45: 560–564. Brown, D., R. Whitcomb and J. Bradbury. 2007. Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. Bruce, J., R.N. Gourlay, R. Hull and D.J. Garwes. 1972. Ultrastructure of Mycoplasmatales virus laidlawii I. J. Gen. Virol. 16: 215–221. Carle, P., D.L. Rose, J.G. Tully and J.M. Bové. 1993. The genome size of spiroplasmas and other mollicutes. Int. Org. Mycoplasmol. Lett. 2: 263. Clyde, W.A., Jr. 1983. Growth inhibition tests. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 405–410. Congdon, A.L., E.S. Boatman and G.E. Kenny. 1979. Mycoplasmatales virus MV-M1: discovery in Acholeplasma modicum and preliminary characterization. Curr. Microbiol. 3: 111–115. Eden-Green, S.J. and P.G. Markham. 1987. Multiplication and persistence of Acholeplasma spp. in leafhoppers. J. Invertebr. Pathol. 49: 235–241. Eden-Green, S.J. and J.G. Tully. 1979. Isolation of Acholeplasma spp. from coconut palms affected by lethal yellowing disease in Jamaica. Curr. Microbiol. 2: 311–316.

Genus I. Acholeplasma Edward, D.G. 1954. The pleuropneumonia group of organisms: a review, together with some new observations. J. Gen. Microbiol. 10: 27–64. Edward, D.G. and E.A. Freundt. 1969. Proposal for classifying organisms related to Mycoplasma laidlawii in a family Sapromycetaceae, genus Sapromyces, within the Mycoplasmatales. J. Gen. Microbiol. 57: 391–395. Edward, D.G. and E.A. Freundt. 1970. Amended nomenclature for strains related to Mycoplasma laidlawii. J. Gen. Microbiol. 62: 1–2. Fabricant, J. and E.A. Freundt. 1967. Importance of extension and stand­ardization of laboratory tests for the identification and classification of mycoplasma. Ann. N. Y. Acad. Sci. 143: 50–58. Gardella, R.S., R.A. Del Giudice and J.G. Tully. 1983. Immunofluorescence. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 431–439. Garwes, D., B. Pike, S. Wyld, D. Pocock and R. Gourlay. 1975. Characterization of Mycoplasmatales virus-laidlawii 3. J. Gen. Virol. 29: 11–24. Gourlay, R.N. 1970. Isolation of a virus infecting a strain of Mycoplasma laidlawii. Nature 225: 1165. Gourlay, R.N. 1971. Mycoplasmatales virus-laidlawii 2, a new virus isolated from Acholeplasma laidlawii. J. Gen. Virol. 12: 65–67. Gourlay, R.N. 1972. Isolation and characterization of mycoplasma viruses. Proceedings of the CIBA Found. Symp., pp. 145–156. Gourlay, R.N. 1973. Mycoplasma viruses: isolation, physicohemical, and biological properties. Ann. N. Y. Acad. Sci. 225: 144–148. Gourlay, R.N. 1974. Mycoplasma viruses: isolation, physicochemical, and biological properties. CRC Crit. Rev. Microbiol. 3: 315–331. Gourlay, R.N., J. Brownlie and C.J. Howard. 1973. Isolation of T-­mycoplasmas from goats, and the production of subclinical mastitis in goats by the intramammary inoculation of human T-mycoplasmas. J. Gen. Microbiol. 76: 251–254. Haberer, K., G. Klotz, J. Maniloff and A. Kleinschmidt. 1979. Structural and biological properties of mycoplasmavirus MVL3: an unusual virus-procaryote interaction. J. Virol. 32: 268–275. Heyward, J.T., M.Z. Sabry and W.R. Dowdle. 1969. Characterization of Mycoplasma species of feline origin. Am. J. Vet. Res. 30: 615–622. Hill, A. 1992. Acholeplasma cavigenitalium sp. nov., isolated from the vagina of guinea pigs. Int. J. Syst. Bacteriol. 42: 589–592. Hill, A., A. Polak-Vogelzang and A. Angulo. 1992. Acholeplasma multilocale sp. nov., isolated from a horse and a rabbit. Int. J. Syst. Bacteriol. 42: 513–517. Ichimaru, H. and M. Nakamura. 1983. Biological properties of a plaqueinducing agent obtained from Acholeplasma oculi. Yale J. Biol. Med. 56: 761–763. Johansson, K-E. 1974. Fractionation of membrane proteins from Acholeplasma laidlawii by preparative agarose suspension electrophoresis. In Protides of the Biological Fluids – 21st Colloquium (edited by Peeters). Pergamon Press, Oxford, pp. 151–156. Johansson, K.E., Pettersson B. 2002. Taxonomy of Mollicutes. In Molecular Biology and Pathogenicity of Mycoplasmas (edited by Razin and Herrmann). Kluwer Academic/Plenum Press, New York, pp. 1–30. Kato, H., T. Murakami, S. Takase and K. Ono. 1972. Sensitivities in vitro to antibiotics of Mycoplasma isolated from canine sources. Jpn. J. Vet. Sci. 34: 197–206. Kirby, T., J. Blum, I. Kahane and I. Fridovich. 1980. Distinguishing between manganese-containing and iron-containing superoxide dismutases in crude extracts of cells. Arch. Biochem. Biophys. 20: 551–555. Kirchhoff, H. 1978. Acholeplasma equifetale and Acholeplasma hippikon, two new species from aborted horse fetuses. Int. J. Syst. Bacteriol. 28: 76–81. Kisary, J. and L. Stipkovits. 1975. Effect of Mycoplasma gallinarum on the replication in  vitro of goose parvovirus strain “B”. Acta Microbiol. Acad. Sci. Hung. 22: 305–307. Kisary, J., A. El-Ebeedy and L. Stipkovits. 1976. Mycoplasma infection of geese. II. Studies on pathogenicity of mycoplasmas in goslings and goose and chicken embryos. Avian Pathol. 5: 15–20.

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Knight, T.F., Jr. 2004. Reclassification of Mesoplasma pleciae as Acholeplasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene sequence data. Int. J. Syst. Evol. Microbiol. 54: 1951–1952. Leach, R.H. 1973. Further studies on classification of bovine strains of Mycoplasmatales, with proposals for new species, Acholeplasma modicum and Mycoplasma alkalescens. J. Gen. Microbiol. 75: 135–153. Leach, R.H. 1983. Preservation of Mycoplasma cultures and culture collections. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 197–204. Lee, G.Y. and G.E. Kenny. 1984. Immunological heterogeneity of superoxide dismutases in the Acholeplasmataceae. Int. J. Syst. Bacteriol. 34: 74–76. Lewis, J. and J. Poland. 1978. Sensitivity of mycoplasmas of the respiratory tract of pigs and horses to erythromycin and its use in selective media. Res. Vet. Sci. 24: 121–123. Liska, B. 1972. Isolation of a new Mycoplasmatales virus. Stud. Biophys. 34: 151–155. Lynch, R.E. and B.C. Cole. 1980. Mycoplasma pneumoniae: a prokaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase. Biochem. Biophys. Res. Commun. 96: 98–105. Maniloff, J. 1992. Mycoplasma viruses. In Mycoplasmas: Molecular Biology and Pathogenesis (edited by Maniloff, McElhaney, Finch and Baseman). American Society for Microbiology, Washington, DC, pp. 41–59. Maniloff, J., J. Das and J.R. Christensen. 1977. Viruses of mycoplasmas and spiroplasmas. Adv. Virus Res. 21: 343–380. Mayberry, W.R., P.F. Smith and T.A. Langworthy. 1974. Heptose-containing pentaglycosyl diglyceride among the lipids of Acholeplasma modicum. J. Bacteriol. 118: 898–904. McCoy, R.E., H.G. Basham, J.G. Tully, D.L. Rose, P. Carle and J.M. Bové. 1984. Acholeplasma florum, a new species isolated from plants. Int. J. Syst. Bacteriol. 34: 11–15. Nakagawa, T., T. Uemori, K. Asada, I. Kato and R. Harasawa. 1992. Acholeplasma laidlawii has tRNA genes in the 16S–23S spacer of the rRNA operon. J. Bacteriol. 174: 8163–8165. Neimark, H.C., J.G. Tully, D. Rose and C. Lange. 1992. Chromosome size polymorphism among mollicutes. Int. Org. Mycoplasmol. Lett. 2: 261. O’Brien, S.J., J.M. Simonson, M.W. Grabowski and M.F. Barile. 1981. Analysis of multiple isoenzyme expression among twenty-two species of Mycoplasma and Acholeplasma. J. Bacteriol. 146: 222–232. Ogata, M., H. Atobe, H. Kushida and K. Yamamoto. 1971. In vitro sensitivity of mycoplasmas isolated from various animals and sewage to antibiotics and nitrofurans. J. Antibiot. (Tokyo) 24: 443–451. Pollack, J.D., J. Banzon, K. Donelson, J.G. Tully, Jr, J.W. Davis, K.J. ­Hackett, C. Agbanyim and R.J. Miles. 1996a. Reduction of benzyl viologen distinguishes genera of the class Mollicutes. Int. J. Syst. Bacteriol. 46: 881–884. Pollack, J.D., M.V. Williams, J. Banzon, M.A. Jones, L. Harvey and J.G. Tully. 1996b. Comparative metabolism of Mesoplasma, Entomoplasma, Mycoplasma, and Acholeplasma. Int. J. Syst. Bacteriol. 46: 885–890. Razin, S., J. Tully, D. Rose and M. Barile. 1983. DNA cleavage patterns as indicators of genotypic heterogeneity among strains of Acholeplasma and Mycoplasma species. J. Gen. Microbiol. 129: 1935–1944. Rose, D.L. and J.G. Tully. 1983. Detection of b-d-glucosidase: hydrolysis of esculin and arbutin. In Methods in Mycoplasmology (edited by Tully). Academic Press, New York, pp. 385–389. Rose, D.L., J.G. Tully and R.A. Del Giudice. 1980. Acholeplasma morum, a new non-sterol-requiring species. Int. J. Syst. Bacteriol. 30: 647– 654. Rottem, S. and O. Markowitz. 1979. Unusual positional distribution of fatty acids in phosphatidylglycerol of sterol-requiring mycoplasmas. FEBS Lett. 107: 379–382. Sabin, A.B. 1941. The filterable microorganisms of the pleuropneumonia group. Bacteriol. Rev. 5: 1–66.

696

Family II. Incertae sedis

Smith, P. and T. Langworthy. 1979. Existence of carotenoids in Acholeplasma axanthum. J. Bacteriol. 137: 185–188. Somerson, N., J. Kocka, D. Rose and R. Del Giudice. 1982. Isolation of acholeplasmas and a mycoplasma from vegetables. Appl. Environ. Microbiol. 43: 412–417. Stephens, E.B., G.S. Aulakh, D.L. Rose, J.G. Tully and M.F. Barile. 1983a. Intraspecies genetic relatedness among strains of Acholeplasma laidlawii and of Acholeplasma axanthum by nucleic acid hybridization. J. Gen. Microbiol. 129: 1929–1934. Stephens, E.B., G.S. Aulakh, D.L. Rose, J.G. Tully and M.F. Barile. 1983b. Interspecies and intraspecies DNA homology among established species of Acholeplasma: a review. Yale J. Biol. Med. 56: 729–735. Switzer, W.P. 1964. Mycoplasmosis. In Diseases of Swine, 2nd edn (edited by Dunne). Iowa State University Press, Ames, IA, pp. 498–507. Tanaka, R., A. Muto and S. Osawa. 1989. Nucleotide sequence of ­tryptophan tRNA gene in Acholeplasma laidlawii. Nucleic Acids Res. 17: 5842. Tanaka, R., Y. Andachi and A. Muto. 1991. Evolution of tRNAs and tRNA genes in Acholeplasma laidlawii. Nucleic Acids Res. 19: 6787–6792. Taylor-Robinson, D. 1983. Metabolism inhibition tests. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 411–421. Thorns, C. and E. Boughton. 1978. Studies on film production and its specific inhibition, with special reference to Mycoplasma bovis (M. agalactiae var. bovis). Zentralbl. Veterinarmed. B 25: 657–667. Tully, J.G. 1973. Biological and serological characteristics of the acholeplasmas. N. Y. Acad. Sci. 225: 74–93. Tully, J.G. 1979. Special features of the acholeplasmas. In The Mycoplasmas, vol. 1 (edited by Barile and Razin). Academic Press, New York, pp. 431–449. Tully, J.G. 1983. Methods in mycoplasmology, vol. 2, Diagnostic Mycoplasmology. Academic Press, New York. Tully, J.G. 1995. Determination of cholesterol and polyoxyethylene sorbitan growth requirements of mollicutes. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, San Diego, pp. 381–389.

Tully, J.G. 1996. Mollicute-host interrelationships: current concepts and diagnostic implications. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, San Diego, pp. 1–21. Tully, J. and S. Razin. 1970. Acholeplasma axanthum, sp. n.: a new sterol-nonrequiring member of the Mycoplasmatales. J. Bacteriol. 103: 751–754. Tully, J.G., D.L. Rose, P. Carle, J.M. Bové, K.J. Hackett and R.F. Whitcomb. 1988. Acholeplasma entomophilum sp. nov. from gut contents of a wide-range of host insects. Int. J. Syst. Bacteriol. 38: 164–167. Tully, J.G., J.M. Bove, F. Laigret and R.F. Whitcomb. 1993. Revised taxonomy of the class Mollicutes – proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. ­Bacteriol. 43: 378–385. Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Rose, R.B. Henegar, J.M. Bové, P. Carle, D.L. Williamson and T.B. Clark. 1994a. Taxonomic descriptions of eight new non-sterol-requiring Mollicutes assigned to the genus Mesoplasma. Int. J. Syst. Bacteriol. 44: 685–693. Tully, J.G., R.F. Whitcomb, D.L. Rose, J.M. Bové, P. Carle, N.L. Somerson, D.L. Williamson and S. Eden-Green. 1994b. Acholeplasma brassicae sp. nov. and Acholeplasma palmae sp. nov., two non-sterolrequiring mollicutes from plant surfaces. Int. J. Syst. Bacteriol. 44: 680–684. Waites, K.B., J.G. Tully, D.L. Rose, P.A. Marriott, R.O. Davis and G.H. Cassell. 1987. Isolation of Acholeplasma oculi from human amniotic fluid in early pregnancy. Curr. Microbiol. 15: 325–327. Whitcomb, R.F. and D.L. Williamson. 1975. Helical wall-free prokaryotes in insects: multiplication and pathogenicity. Ann. N. Y. Acad. Sci. 266: 260–275. Whitcomb, R.F., J.G. Tully, J.M. Bové and P. Saglio. 1973. Spiroplasmas and acholeplasmas: multiplication in insects. Science 182: 1251–1253.

Family II. Incertae sedis This family includes the phytoplasma strains of the order Acholeplasmatales. Although never ­cultured in cell-free media,

these plant pathogens and symbionts have been well studied by culture-­independent methods.

Genus I. “Candidatus Phytoplasma” gen. nov. IRPCM Phytoplasma/Spiroplasma Working Team 2004, 1244 Nigel A. Harrison, Dawn Gundersen-Rindal and Robert E. Davis Phy.to.plas¢ma. Gr. masc. n. phytos a plant; Gr. neut. n. plasma something formed or molded, a form.

Phytoplasmas (Sears and Kirkpatrick, 1994) are wall-less, nutritionally fastidious, phytopathogenic prokaryotes 0.2–0.8 µm in diameter that morphologically resemble members of the Mollicutes. Sequencing of nearly full-length PCR-amplified 16S rRNA genes (Gundersen et al., 1994; Namba et al., 1993; Seemüller et al., 1994), combined with earlier studies (Kuske and Kirkpatrick, 1992b; Lim and Sears, 1989), provided the first comprehensive phylogeny of the organisms and showed that they constitute a unique, monophyletic clade within the Mollicutes. These organisms are most closely related to members of the genus Acholeplasma within the Anaeroplasma clade as defined by Weisburg et al. (1989). Sustained culture in cell-free

media has not yet been demonstrated for any phytoplasma. Their genome sizes have been estimated to range from 530 to 1350 kb, and the G+C content of phytoplasma DNA is about 23–30 mol%. The presence of a characteristic oligonucleotide sequence in the 16S rRNA gene, CAA GAY BAT KAT GTK TAG CYG GDC T, and standard codon usage indicate that phytoplasmas represent a distinct taxon for which the name “Candidatus Phytoplasma” has been adopted by specialists in the molecular biology and pathogenicity of these and similar phytopathogenic organisms (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004). At present, the designation “Candidatus” must still be used for new types.

Genus I. “Candidatus Phytoplasma”

Further descriptive information Phytoplasma cells typically have a diameter less than 1 µm and are polymorphic. Viewed in ultra-thin section by electron microscopy Figure 115, they appear ovoid, oblong, or filamentous in plant and insect hosts (Doi et al., 1967; Hearon et al., 1976). Transmission electron microscopy of semi-thick (0.3 µm) sections (Thomas, 1979) and serial sections (Chen and Hiruki, 1978; Florance and Cameron, 1978; Waters and P. Hunt., 1980), and scanning electron microscopy studies (Bertaccini et  al., 1999; Haggis and Sinha, 1978; Marcone et al., 1996) have done much to clarify the gross cellular morphology of phytoplasmas. They range from spherical to filamentous, often with extensive branching reminiscent of that seen in Mycoplasma mycoides. Small dense rounded forms ~0.1 µm in diameter, formerly considered to be “elementary bodies” when seen in thin section, were shown to represent constrictions in filamentous forms. Dumbbell-shaped forms once thought to be “dividing cells” are actually branch points of filamentous forms, whereas forms thought to have internal vesicles have been shown to have involuted membranes oriented such that the plane of the sections cut through the cell membrane twice (McCoy, 1979). Phytoplasma cell membranes are resistant to digitonin and sensitive to hypotonic salt solutions, and, as such, are similar to those of non-sterol requiring mollicutes (Lim et al., 1992). Phytoplasmas are consistently observed within phloem sieve elements (Christensen et al., 2004; McCoy et al., 1989; Oshima et  al., 2001b; Webb et  al., 1999) and occasionally have been reported in both companion cells (Rudzinska-Langwald and Kaminska., 1999; Sears and Klomparens, 1989) and parenchyma cells (Esau et  al., 1976; Siller et  al., 1987) of infected plants. Sieve elements are specialized living cells that lack nuclei when mature and transport photosynthate from leaves not only to growing tissues, but also to other tissues unable to photosynthesize (Oparka and Turgeon, 1999; Sjölund, 1997). This applies particularly to roots that require considerable energy

697

for the uptake of water and nutrients (Flores et  al., 1999). Phloem sap is unique in that it contains from 12 to 30% sucrose and is under high hydrostatic (turgor) pressure (Evert, 1977). Sieve elements have pores in their end plates and lateral walls, allowing passage of photosynthate to adjacent sieve tube elements. The sieve pores, which have an average diameter of ~0.2 µm, are of sufficient size to allow passage of spherical and filamentous phytoplasma cells from one sieve element to another (McCoy, 1979). The chemical composition of sieve sap is complex, containing sugars, minerals, free amino acids, proteins, and ATP (Van Helden et  al., 1994). This rich milieu, with its high osmotic and hydrostatic pressures, serves to support extensive multiplication of phytoplasmas in planta. Phytoplasmas also multiply in the internal tissues and organs of their insect vectors (Kirkpatrick et al., 1987; Lefol et al., 1994; Marzachi et al., 2004; Nasu et al., 1970), which are primarily leafhoppers, planthoppers, and psyllids (D’Arcy and Nault, 1982; Jones, 2002; Weintraub and Beanland, 2006). In many respects, the composition of insect hemolymph is similar to that of plant phloem sap, as both contain high levels of complex and simple organic compounds (Moriwaki et al., 2003; Saglio and Whitcomb, 1979). Physical maps of several phytoplasma genomes have been constructed (Firrao et  al., 1996; Lauer and Seemüller, 2000; Marcone and Seemüller, 2001; Padovan et al., 2000). The presence of extrachromosomal DNAs or plasmids in numerous phytoplasmas has also been reported (Davis et al., 1988; Denes and Sinha, 1991; Kuboyama et  al., 1998; Kuske and Kirkpatrick, 1990; Liefting et al., 2004; Lin et al., 2009; Nakashima and Hayashi, 1997; Nishigawa et al., 2003; Oshima et al., 2001a; TranNguyen and Gibb, 2006) and suggested as a potential means of intermolecular recombination (Nishigawa et al., 2002b). Phytoplasma-associated extrachromosomal DNAs have been shown to contain genes encoding a putative geminivirus-related replication (Rep) protein (Liefting et  al., 2006; Nishigawa et  al., 2001; Rekab et  al., 1999) and a single-stranded DNA-binding

FIGURE 115.  Electron micrographs of ultrathin sections of leaf petiole from a sunnhemp (Crotalaria juncea L.) plant

displaying Crotalaria phyllody disease symptoms. (a) Polymorphic phytoplasma cells occluding the lumen of adjacent leaf phloem sieve tube elements. Bar = 2 µm. (b) Ultratructural morphology indicates phytoplasma cells are bounded by a unit membrane and contain DNA fibrils and ribosomes. Bar = 200 nm. Images provided by Phil Jones.

698

Family II. Incertae sedis

protein (Nishigawa et al., 2002a), as well as a putative gene similar to DNA primase of other bacterial chromosomes (Liefting et  al., 2004) and still other genes of as yet unknown identity. Moreover, heterogeneity in extrachromosomal DNAs has been associated with reduced pathogenicity and loss of insect vector transmissibility (Denes and Sinha, 1992; Nishigawa et al., 2002a, 2003). Onion yellows mild strain (OY-M) was the first phytoplasma genome to be completely sequenced. The genome of this aster yellows group strain consists of a circular chromosome of 860,632 bp. It also contains two extrachromosomal DNAs, EcOYM (5025 bp) and plasmid pOYM (3932 bp) (Nishigawa et  al., 2003; Oshima et  al., 2002), representing two different classes based on the type of replication protein encoded. While EcOYM contains a rep gene homologous to that of the geminiviruses, pOYM has a rep gene that encodes a unique protein with characteristics of both viral-rep and plasmid-rep (Namba, 2002). The chromosome is a circular DNA molecule with a G+C content of 28 mol% and contains 754 open reading frames (ORFs), comprising 73% of the chromosome. Of these, 66% of ORFs exhibit significant homology to gene sequences currently archived in the GenBank database. Putative proteins encoded by ORFs could be assigned to one of six different functional categories: (1) information storage and processing (260 ORFs); (2) metabolism (107 ORFs); (3) cellular processes (77 ORFs); (4) poorly characterized, i.e., with homology to uncharacterized proteins of other organisms (50 ORFs); or (5) others, i.e., without homology to any known proteins (260 ORFs). Like mycoplasmal genomes, the OY-M phytoplasma genome lacks many genes related to amino acid and fatty acid biosynthesis, the tricarboxylic acid cycle, and oxidative phosphorylation. However, OY-M phytoplasma differs from mycoplasma in that it lacks genes for the phosphotransferase system and for metabolizing UDP-galactose to glucose 1-phosphate, suggesting that it possesses a unique sugar intake and metabolic system. Furthermore, OY-M phytoplasma lacks most of the genes needed to synthesize nucleotides and ATP suggesting that it probably assimilates these and other necessary metabolites from host cytoplasm. Many genes, such as those for glycolysis, are present as multiple redundant copies representing 18% of the total genome. Twenty-seven genes encoding transporter systems such as malate, metal-ion and amino acid transporters, some of which have multiple copies, were identified, suggesting that phytoplasmas aggressively import many metabolites from the host cell. Other than genes encoding glucanase and hemolysinlike proteins, no other genes presently known to be related to bacterial pathogenicity were evident in the OY-M phytoplasma genome, suggesting novel mechanisms for virulence. Annotation of the OY-M phytoplasma genome has been followed by three other phytoplasma genome annotations. Aster yellows witches’-broom phytoplasma (“Candidatus Phytoplasma asteris”-related strain AY-WB) possesses a circular 706,569 nucleotide chromosome and plasmids AYWB-pI (3872 bp), -pII (4009 bp), -pIII (5104 bp), and -pIV (4316 bp) (Bai et al., 2006). Australian tomato big bud phytoplasma (“­Candidatus Phytoplasma australiense”-related strain TBB) has a circular 879,324 bp chromosome and a 3700 bp plasmid (Tran-Nguyen et  al., 2008), whereas apple proliferation phytoplasma (“Candidatus Phytoplasma mali”-related strain AT) has a linear 601,943 bp chromosome (Kube et al., 2008).

The ­chromosome of “­Candidatus Phytoplasma mali” is characterized by large terminal inverted repeats and covalently closed hairpin ends. Analysis of protein-coding genes revealed that glycolysis, the major energy-yielding pathway supposed for OY-M phytoplasma, is incomplete in AT phytoplasma. It also differs from OY-M and AY-WB phytoplasmas by a lower G+C content (21.4 mol%), fewer paralogous genes, a strongly reduced number of ABC transporters for amino acids, and an extended set of genes for homologous recombination, excision repair, and SOS response. Comparative genomics have also recently identified ORFs shared by AY-WB phytoplasma and the distantly-related corn stunt pathogen Spiroplasma kunkelii that are absent from obligate animal and human pathogenic mollicutes. These proteins were identified as polynucleotide phosphorylase (PNPase), cmpbinding factor (CBF), cytosine deaminase, and Y1xR protein and could be important for insect transmission or plant pathogenicity. Also identified were four additional proteins, ppGpp synthetase, HAD hydrolase, AtA (AAA type ATPase), and P-type Mg2+ transport ATPase, that seemed to be more closely related between AY-WB and Spiroplasma kunkelii than to their mycoplasmal counterparts (Bai et al., 2004). Phytoplasmas possess a unique genome architecture that is characterized by multiple, nonrandomly distributed sequencevariable mosaics (SVMs) of clustered genes, originally recognized in a study of closely related “Candidatus Phytoplasma asteris”-related strains CPh and OY-M (Jomantiene and Davis, 2006). Targeted genome sequencing and comparative genomics indicated that this genome architecture is a common characteristic among phytoplasmas, leading to the proposal that the origin of SVMs was an ancient event in the evolution of the phytoplasma clade (Jomantiene et al., 2007), perhaps as a result of recurrent targeted attacks by mobile elements such as phages (Wei et al., 2008a). Jomantiene and Davis (2006) proposed that sizes and numbers of SVMs could account in part for the known variation in genome size among phytoplasma strains; this concept was independently suggested by Bai et  al. (2006) on the basis of results from a comparative study of two completely sequenced phytoplasma genomes. Nucleotide sequences within SVMs included full length or pseudogene forms of fliA, an ATP­dependent Zn protease gene, tra5, smc, himA, tmk, and ssb (encoding single-stranded DNA-binding protein), genes potentially encoding hypothetical proteins of unknown function, genes exhibiting similarities to transposase, and a phage-related gene (­Jomantiene et al., 2007). A similar set of nucleotide sequences occurs in AY-WB genomic regions termed potential mobile units (PMUs) by Bai et al. (2006). The presence of sequences encoding putatively secreted and/or transmembrane, cell surfaceinteracting proteins indicates that these genomic features are likely to be significant for phytoplasma/host interactions (Bai et  al., 2006; Jomantiene and Davis, 2006; Jomantiene et  al., 2007). Short (17–35 bases) conserved, imperfect palindromic DNA sequences (PhREPs) that are present in SVMs possibly play a role in phytoplasma genome plasticity and targeting of mobile genetic elements. SVMs can be viewed as composites formed by the acquisition of genes through horizontal transfer, recombination, and rearrangement, and capture of mobile elements recurrently targeted to SVMs, leading Jomantiene et al. (2007) to suggest that SVMs provide loci for acquisition of new genes

Genus I. “Candidatus Phytoplasma”

and targeting of mobile genetic elements to specific regions in phytoplasma chromosomes. The chromosomes of avirulent, mildly, moderately, and highly virulent strains of “Candidatus Phytoplasma mali” (­Seemüller and Schneider, 2007) differ from one another in size and exhibit distinct restriction endonuclease patterns when cleaved with rare cutting enzymes. PCR-based DNA amplifications, primed separately by eight primer pairs, revealed target sequence heterogeneity among all “Candidatus Phytoplasma mali”-related strains tested, but no correlations linked ­molecular markers with strain virulence or the maximum titer obtained upon infection of apple trees. In a separate study, a comparison of mild (OY-M) and severe (OY-W) strains of onion yellows (OY) phytoplasma indicated that severe symptoms were associated with higher populations of OY-W in infected host plants (Oshima et al., 2001b). A cluster of eight genes, considered essential for glycolysis, were subsequently identified within a similar 30 kb genomic region of both strains (Oshima et al., 2007). Of these, five genes (smtA, greA, osmC, eno, and pfkA) were randomly duplicated in OY-W, possibly influencing glycolytic activitiy. A higher consumption of metabolites such as sugars in the intracellular environment of the phloem may explain differences between OY-W and OY-M in growth rate, which in turn may be linked, directly or indirectly, to symptom severity. Cloned fragments of phytoplasma DNA have been widely employed as probes in dot and Southern blot hybridization assays to identify and characterize phytoplasmas (reviewed by Lee and Davis, 1992; Lee et al. (2000). Southern blot restriction fragment length polymorphism (RFLP) analysis has enabled investigations of genetic relationships among phytoplasmas associated with similar hosts or with symptomatologically similar diseases (Kison et al., 1997, 1994; Kuske et al., 1991; Schneider and Seemüller, 1994b). Several discrete phytoplasma groups, each comprising strains that shared extensive sequence homology and little or no apparent homology with other phytoplasmas, were identified by this type of analysis. Lee and co-workers (1992) coined the term “genomic strain cluster” to denote each of seven discrete genotypic groups resolved by employing a selection of phytoplasma genomic DNA probes (reviewed by Lee and Davis, 1992). Of these, aster yellows (AY) was the largest group, represented by 15 genetically variable strains that were further delineated into three distinct genomic types (types I, II, and III) or subclusters (Lee et  al., 1992). Significantly, major groupings later revealed by RFLP analysis of 16S rRNA genes were consistent with those defined by monoclonal antibody typing (Lee et al., 1993a) and other molecular methods (Lee et al., 1998b), but differed from distinctions made in traditional classification based solely on biological properties such as plant host range, symptomatology, and insect vector specificity (­Chiykowski and Sinha, 1990). Polyclonal antibodies (PAbs) have been produced against phytoplasma-enriched extracts (intact organisms or membrane fractions) partially purified from plants (reviewed by Chen et al., 1989) and against vector leafhopper-derived immunogens (Errampelli and Fletcher, 1993; Kirkpatrick et al., 1987). Most PAbs exhibit relatively high background reactions with healthy host antigens; thus, generation of useful polyclonal antisera has been limited so far to a few phytoplasmas maintained at high titer in host tissues. Phytoplasmas can be differentiated on the basis of their antigenic properties through the use of PAbs in

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enzyme-linked immunosorbent (ELISA), immunofluorescence, or Western blot assays. Antigenic similarity revealed among phytoplasmas by these assays is often in agreement with ­relationships demonstrated by vector transmission ­studies. Detection of antigenically distinct phytoplasmas in plants exhibiting very similar disease symptoms attests to the unreliability of symptom expression alone as a means of differentiating phytoplasmas. Improvements in phytoplasma extraction methods have provided a source of immunogens for monoclonal antibody (MAb) production (Chang et al., 1995; Hsu et al., 1990; Jiang et al., 1989; Loi et al., 2002, 1998; Shen and Lin, 1993; Tanne et  al., 2001). Used in ELISA, dot or tissue blot immunoassay, ­immunocapture PCR (Rajan et al., 1995), immunofluorescence microscopy, or immunosorbent electron microscopy (ISEM) (Clark, 1992; Shen and Lin, 1994), MAbs have demonstrated considerable promise for detection and differentiation of phytoplasmas infecting a broad range of host plants, including woody perennials (Guo et al., 1998). Due to their high degree of specificity, monoclonals seem most suited for differentiating very closely related strains (Lee et al., 1993a). Isolation, cloning, and expression of immunodominant protein genes have identified putative proteins that account for a major portion of the membrane proteins of several phytoplasmas (Arashida et al., 2008; Barbara et al., 2002; Berg et al., 1999; Blomquist et al., 2001; Galetto et al., 2008; Kakizawa et al., 2004, 2009; Morton et  al., 2003; Suzuki et  al., 2006; Yu et  al., 1998). When these purified proteins were used as immunogens, the resulting polyclonal antisera exhibited high specific titers and low background reactions in ELISA and Western blot analyses that were designed to detect phytoplasma proteins in infected hosts. Similarly, the secA gene was cloned from an onion yellows (OY-M) strain of aster yellows phytoplasma (Kakizawa et  al., 2001) and used to raise an anti-SecA rabbit antibody against a purified partial SecA protein expressed in Escherichia coli. Light microscopy of thin sections of garland chrysanthemum (Chrysanthemum coronarium) treated by immunohistochemical straining revealed that the SecA protein was present in phloem of OY-M-infected but not healthy host plants. In addition, antisera against both OY-M phytoplasma SecA protein and GyrA protein of Acholeplasma laidlawii reacted with proteins of several unrelated phytoplasmas extracted from plant tissues (Koui et al., 2002; Wei et al., 2004a). Phytoplasmas are the apparent etiological agents of diseases of at least 1000 plant species worldwide (McCoy et  al., 1989; Seemüller et al., 1998). Although they can be transmitted from infected to healthy plants by scion or root grafts, most plant to plant spread occurs naturally via phloem-feeding insect vectors primarily of the family Cicadellidae (leafhoppers) and, less commonly, by planthoppers (Fulgoroidea) of the family Ciixidae and psyllids (Psylloidea) (D’Arcy and Nault, 1982; Tsai, 1979; Weintraub and Beanland, 2006). Phytoplasmas are transmitted in a circulative-propagative manner that typically involves a transmission latent period from 2 to 8 weeks (Carraro et al., 2001; Webb et  al., 1999). The insect vector becomes infected upon ingesting phytoplasmas in phloem sap of infected plants. After an incubation period of one to several weeks, the phytoplasma multiplies to high titer in the salivary glands and the insect becomes capable of infecting the phloem of the healthy plants on which it feeds (Kunkel, 1926; Lee et al., 1998a; Nasu et al., 1970). Generally, phytoplasma infection does not appear to significantly affect the activity, weight, longevity, or fecundity

700

Family II. Incertae sedis

of vector insects (Garnier et al., 2001). Some phytoplasmas can be vectored by many species of leafhoppers (McCoy et al., 1989; Nielson, 1979) and different insect species may serve as vectors in different geographic regions. Several vectors also have the ability to transmit more than one type of phytoplasma, whereas other phytoplasmas are transmitted by one or a few vector species to a narrow range of plant species (Lee et al., 1998a). There is mounting evidence also for transovarial transmission of some phytoplasmas (Alma et  al., 1997; Hanboonsong et  al., 2002; Kawakita et al., 2000; Tedeschi et al., 2006). Plants may serve as both natural and experimental hosts to several different phytoplasmas. Dual or mixed infections involving related or unrelated phytoplasmas are known to occur naturally in plants and appear to be more common in perennial than annual plants (Bianco et al., 1993; Lee et al., 1995). Also, closely related phytoplasma strains are capable of inducing dissimilar symptoms on the same plant species (White et al., 1998), whereas similar symptoms on the same host plant may be induced by unrelated phytoplasmas (Harrison et al., 2003). The ability to accurately identify phytoplasmas by using DNA-based methods has shown that these organisms are more genetically diverse than was once thought (Davis and Sinclair, 1998). The geographic occurrence of phytoplasmas is determined largely by geographic ranges and feeding behavior (mono-, oligo-, or polyphagous) of the vector species, the relative susceptibility of the preferred host plant species, and the native host ranges of plant and insect hosts (Lee et al., 1998a). Phytoplasmas can be introduced into new geographic regions by long-distance dispersal of infectious vectors (Lee et al., 2003) and by movement of infected plants or vegetative plant parts. Most recently, phytoplasma DNA has been detected in embryos of aborted seed from diseased plants (Cordova et al., 2003; Nipah et al., 2007) and seed transmission of phytoplasmas infecting alfalfa (Medicago sativa L.) has been demonstrated (Khan et al., 2002). An array of characteristic symptoms is associated with phytoplasma infection of several hundred plant species worldwide. Symptoms vary according to the particular host species, stage of host infection and the associated phytoplasma strain (reviewed by Davis and Lee, 1992; Hogenhout et  al., 2008; Kirkpatrick, 1989, 1992; Lee et  al., 2000; McCoy et  al., 1989; Seemüller et  al., 2002; Sinclair et  al., 1994). Some symptoms indicative of profound disturbances in the normal balance of growth ­regulators in plants include virescence (greening of petals), phyllody (conversion of floral organs into leafy structures), big bud, floral proliferation, sterility of flowers, proliferation of adventitious or axillary shoots, internode elongation and ­etiolation, generalized stunting (small flowers, leaves and fruits or shortened internodes), unseasonal discoloration of leaves or shoots (yellow to purple discoloration), leaf curling, cupping or crinkling, witches’-brooms (bunchy growth at stem apices), vein clearing, vein enlargement, phloem discoloration, and general plant decline such as die-back of twigs, branches and trunks (Lee and Davis, 1992; Lee et al., 2000; McCoy et al., 1989). Infection of herbaceous host plants is followed by rapid intraphloemic spread of phytoplasma from leaves to roots, often accompanied by six-fold increases in phytoplasma populations in these tissues between 14 and 28 d post-inoculation (Kuske and Kirkpatrick, 1992a; Wei et  al., 2004b). Phytoplasma concentrations ranging from 2.2 × 108 to 1.5 × 109 cells per gram of tissue have been measured in high titer herbaceous hosts such

as periwinkle (Catharanthus roseus) and in certain woody perennial hosts such as alder (Alnus) and most poplar (Populus) species. Lowest phytoplasma concentrations, from 370 to 34,000 cells per gram of tissue, were detected in apple trees that were grafted on resistant rootstocks and in oak (Quercus robur) or hornbeam (Carpinus betulus) trees exhibiting nonspecific leaf yellowing symptoms (Berg and Seemüller, 1999). Colonization is usually marked by phloem dysfunction and a reduction in photosynthetic capacity. Alterations in phloem function have been correlated with structural degeneration of sieve elements due possibly to physical blockage by colonizing phytoplasma or the action of a phytotoxin (Guthrie et al., 2001; Siddique et al., 1998). The onset of symptoms may be accompanied by substantial impairment of the photosynthetic rate of mature leaves and by fluctuations in carbohydrate and amino acid levels in source versus sink leaves (Lepka et al., 1999; Tan and Whitlow, 2001). Leaf yellowing is associated with: decreases in chlorophyll content, carotenoids, and soluble proteins (Bertamini and Nedunchezhian, 2001); abnormal stomatal function (Martinez et al., 2000); histopathological changes such the amount of total polyphenols; loss of cellular integrity (Musetti et  al., 2000); fluctuations in hydrogen peroxide; peroxidase activity and glutathione content in diseased versus healthy plant tissues (Musetti et  al., 2004); and increases in calcium (Ca2+) ions in cells (Musetti and Favali, 2003; Rudzinska-Langwald and Kaminska, 2003). Such adverse changes are accompanied by differential regulation of genes encoding proteins involved in floral development (Pracros et  al., 2006), photosynthesis, sugar transport, and response to stress or in pathways of lipid and phenylpropanoid or phytosterol synthesis (Albertazzi et al., 2009; Carginale et al., 2004; Hren et al., 2009; Jagoueix­Eveillard et al., 2001). The organisms degenerate and lose their cellular contents following treatment of infected plants with tetracycline antibiotics (Kamin´ska and S´liwa, 2003; Sinha and Peterson, 1972). Tetracycline sensitivity and the lack of sensitivity to cell wallinhibiting antibiotics such as penicillin (Davis and Whitcomb, 1970; Ishii et al., 1967) also support their inclusion in the Mollicutes. Protective or therapeutic treatments with tetracycline antibiotics for phytoplasma disease control have been extended to a few high-value crop plants such as coconut for control of palm lethal yellowing, and to cherries and peaches for control of X-disease (McCoy, 1982; Nyland, 1971; Raju and Nyland, 1988). Administered by trunk injection, treatment of each tree with 1.0 g (protective dose) or 3.0 g (therapeutic dose) three times per year was sufficient for control of coconut lethal yellowing disease (McCoy, 1982).

Enrichment and isolation procedures Isolation of phytoplasma-enriched fractions from plant and insect host tissues is possible by differential centrifugation and filtration after first disrupting tissues in osmotically-augmented buffers (Kirkpatrick et al., 1995; Lee et al., 1988; Sinha, 1979; Thomas and Balasundaran, 2001). Further purification of phytoplasmas is possible by centrifugation of enriched preparations in discontinuous Percoll density gradients (Davis et  al., 1988; Gomez et al., 1996; Jiang and Chen, 1987) or by affinity chromatography using phytoplasma-specific antibodies coupled to Protein A-Sepharose columns (Jiang et al., 1988; Seddas et al., 1995). Viability of these enriched preparations may be assessed

Genus I. “Candidatus Phytoplasma”

by infectivity tests in which aliquots of the phytoplasma preparations are micro-injected into vector insects, which are then fed on healthy indicator plants (Nasu et al., 1974; Sinha, 1979; Whitcomb et al., 1966a, b). Separation of enriched phytoplasma DNA from mixtures with host DNA is also possible by use of cesium chloride-bisbenzimide buoyant density gradient centrifugation (Kollar and Seemüller, 1989). Present as an uppermost band in final gradients, phytoplasma DNA fractionated by this means was suitable for endonuclease digestion and cloning for DNA probe development (Harrison et  al., 1992, 1991; Kollar and Seemüller, 1990).

Maintenance procedures Viable phytoplasmas have been maintained for at least 6 years in intact vector insects frozen at −70°C (Chiykowski, 1983). Viable X-disease phytoplasmas have been maintained for 2 weeks in salivary glands suspended in a tissue culture medium (Nasu et  al., 1974). Extracts of phytoplasma-infected insects prepared in a MgCl2/glycine buffer, osmotically adjusted to 800 milliosmoles/kg with sucrose, retained their infectivity for up to 3 d (Smith et al., 1981). Phytoplasma strains have been routinely maintained in diseased plants kept in an insect-proof greenhouse or in plantlets grown in tissue culture (Bertaccini et  al., 1992; Davis and Lee, 1992; Jarausch et  al., 1996; Sears and Klomparens, 1989; Wongkaew and Fletcher, 2004). While plant to plant transmission is accomplished naturally by vector insects and, in some cases, through grafts, experimental transmissions commonly include the use of plant parasitic dodders (Cuscuta  sp.) (Marcone et  al., 1999a). Although phytoplasma strains are commonly maintained in plants by periodic graft inoculation, maintenance of phytoplasmas exclusively in plants can result in strain attenuation over time and an associated loss of transmissibility by vector insects (Chiykowski, 1988; Denes and Sinha, 1992).

Differentiation of the genus “Candidatus Phytoplasma” from other genera Phytoplasma-specific nucleic acid probes and PCR technology have largely supplanted traditional methods of electron microscopy and biological criteria for sensitive detection, identification, and genetic characterization of phytoplasmas. Molecular-based analyses have shown phytoplasma genomes to be A+T rich (Kollar and Seemuller, 1989; Oshima et al., 2004) and to range from 530 to 1350 kbp in size (Marcone et al., 1999b, 2001; Neimark and Kirkpatrick, 1993). Before any phytoplasma genomes were sequenced, phytoplasmas were shown to contain two rRNA operons (Davis, 2003a; Harrison et al., 2002; Ho et al., 2001; Jomantiene et al., 2002; Jung et al., 2003a; Lee et al., 1998b; Liefting et  al., 1996; Marcone et  al., 2000; Schneider and Seemüller, 1994a). Other genes that have been identified include ribosomal protein genes (Gundersen et al., 1994; Lee et al., 1998b; Lim and Sears, 1992; Martini et al., 2007; Miyata et  al., 2002a; Toth et  al., 1994) of the S10-spc operon (Miyata et  al., 2002a), a nitroreductase gene (Jarausch et  al., 1994), DNA gyrase genes (Chuang and Lin, 2000), genes encoding elongation factors G and Tu (An et al., 2006; Berg and Seemüller, 1999; Koui et al., 2003; Marcone et al., 2000; Miyata et al., 2002b; Schneider et  al., 1997), secA, secY, and secE genes of a functional Sec protein translocation system (Kakizawa et  al., 2001, 2004), gidA, potB, potC, and potD (Mounsey et al., 2006),

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a gene encoding an RNase P ribozyme (Wagner et al., 2001), recA (Chu et  al., 2006), rpoC (Lin et  al. 2006), polC (Chi and Lin., 2005), and insertion sequence (IS)-like elements (Lee et  al., 2005). Numerous other putative genes or pseudogenes have been identified recently after partially or fully sequencing random fragments of genomic DNA cloned from phytoplasmas by various methods (Bai et  al., 2004; Cimerman et  al., 2006, 2009; Davis et al., 2003b, 2005; Garcia-Chapa et al., 2004; Liefting and Kirkpatrick, 2003; Melamed et al., 2003; Miyata et al., 2003; Streten and Gibb, 2003). Development of phytoplasma-specific rRNA gene primers has permitted PCR-mediated amplification of various regions of the rRNA operons (Ahrens and Seemüller, 1992; Baric and Dalla-Via, 2004; Davis and Lee, 1993; Deng and Hiruki, 1991; ­Gundersen and Lee, 1996; Lee et  al., 1993b) (Namba et  al., 1993; Smart et al., 1996). RFLP analysis of PCR-amplified rDNA provided a practical solution to the problem of phytoplasma identification and classification (Lee et al., 2000, 1998b). Pairwise comparisons of disparate strains were marked by considerable differences in RFLP patterns, whereas strains that were considered closely related on the basis of similar biological properties were often, although not always, indistinguishable on the basis of RFLP patterns. Alternatively, heteroduplex mobility analysis has demonstrated greater sensitivity than RFLP analysis for detecting minor variability in 16S rRNA genes of closely related phytoplasma strains (Cousin et  al., 1998; Wang and Hiruki, 2000), since RFLP analysis is limited to detection of recognition sites for restriction endonucleases. Cluster analysis of rDNA RFLP patterns provided the first means to differentiate between known and unknown phytoplasmas from a wide range of plant hosts and geographic locations, and to resolve phytoplasmas into well-defined phylogenetic groups and ­subgroups (Ahrens and Seemüller, 1992; Lee et  al., 1993b; Schneider et al., 1993, 1995).

Taxonomic comments The inability to cultivate phytoplasmas outside of their plant and insect hosts has thus far rendered traditional methods impractical as aids for taxonomy of these organisms. Unlike their culturable Mollicute relatives, which were originally classified based only upon biological and phenotypic properties in pure culture, phytoplasmas cannot be classified by these criteria. Through application of DNA-based methods, it is now possible to accurately identify and characterize phytoplasmas and to assess their genetic interrelationships. These capabilities have assisted development of classification systems, first based on hybridization data, later based on 16S rDNA RFLPs, and ultimately on phylogenetic analysis of 16S rRNA genes and other conserved gene sequences. Classification schemes founded upon these molecular criteria have been refined and expanded upon over time, with the goal of defining a taxonomy for these unique organisms. In a phytoplasma classification scheme proposed by Lee et al. (1993b), based on analyses of rDNA RFLPs, a total of nine primary 16S rDNA groups (termed 16Sr groups) and 14 subgroups were initially recognized. Phytoplasma groups delineated by these analyses were consistent with genomic strain clusters previously identified by DNA hybridization analysis (Lee et al., 1992), although a greater diversity among strains comprising group 16SrI (aster yellows and related strains) was indicated by the earlier hybridization data. Subgroups within a

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Family II. Incertae sedis

given 16Sr group were distinguished by the presence of one or more restriction sites in a phytoplasma strain that differed from those in all existing members of a given subgroup. For those strains in which intra-rRNA operon heterogeneity was detected, subgroup designations were assigned according to the combined patterns of both 16S rRNA genes. RFLP analysis of more variable ribosomal protein genes (Gundersen et  al., 1994; Lee et  al., 2004a, b) or tuf genes (Marcone et  al., 2000; Schneider et  al., 1997) has provided a means for more detailed subdivision of phytoplasma primary groups delineated by 16S rDNA RFLP data. This strategy for finer subgroup differentiation has been used to modify and expand upon earlier classifications and to incorporate many newly identified phytoplasma strains. Based on RFLP analysis of nearly full-length 16S rRNAs, at least 15 primary 16Sr groups have been recognized (Lee et al., 1998b; Montano et al., 2001): 16SrI, Aster yellows; 16SrII, Peanut witches’-broom; 16SrIII, X-disease; 16SrIV, Coconut lethal yellows; 16SrV, Elm yellows; 16SrVI, Clover proliferation; 16SrVII, Ash yellows; 16SrVIII, Loofah witches’-broom; 16SrIX, Pigeonpea witches’broom; 16SrX, Apple proliferation; 16SrXI, Rice yellow dwarf; 16SrXII, Stolbur; 16SrXIII, Mexican periwinkle virescence; 16SrXIV, Bermuda grass white leaf; and 16SrXV Hibiscus witches’broom. A total of 45 subgroups were identified when ribosomal protein gene RFLP data was also considered in the analyses. Sequencing of 30 nearly full-length amplified 16S rRNA genes was undertaken by Namba et  al. (1993), Gundersen et  al. (1994), and Seemüller et  al. (1994) from a diversity of strains previously characterized by rDNA RFLP analysis. These collective efforts, combined with earlier studies (Kuske and Kirkpatrick, 1992b; Lim and Sears, 1989), provided the first comprehensive phytoplasma phylogeny. In recognition of their unique phylogenetic status, the trivial name “phytoplasma” was initially proposed (Sears and Kirkpatrick, 1994) and has since been adopted formally (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004) to collectively name these fastidious, phytopathogenic mollicutes previously known as mycoplasma-like organisms. Within the phytoplasma clade, major subclades (primary groups representing “Candidatus” species) include: (1) Stolbur; (2) Aster yellows; (3) Apple proliferation; (4) Coconut lethal yellowing; (5) Pigeonpea witches’-broom; (6) X-disease; (7) Rice yellow dwarf; (8) Elm yellows; (9) Ash yellows; (10) Sunnhemp witches’-broom; (11) Loofah witches’-broom; (12) Clover proliferation; and (13) Peanut witches’-broom (Kirkpatrick et al., 1995; Schneider et al., 1995; White et al., 1998). Primary phytoplasma groups including 19 novel groups, namely Australian grapevine yellows (AUSGY), Italian bindweed stolbur (IBS), Buckthorn witches’-broom (BWB), Spartium witches’-broom (SpaWB), Galactia little leaf (GaLL), Vigna little leaf (ViLL), Clover yellow edge (CYE), Hibiscus witches’-broom (HibWB), Pear decline (PD), European stone fruit yellows (ESFY), Japanese hydrangea phyllody (JHP), Psammotettix cephalotes-borne (BVK), Italian alfalfa witches’-broom (IAWB), Cirsium phyllody (CirP), ­Bermuda grass white leaf (BGWL), Sugarcane white leaf (SCWL), Tanzanian lethal decline (TLD), Stylosanthes little leaf (StLL), and Pinus sylvestris yellows (PinP), that were absent from previous classification schemes have been subsequently defined. These new taxonomic entities were delineated on the basis of phylogenetic tree branching patterns, differences in 16S rRNA

gene sequence similarities that were 1.2–2.3% or greater and, in some instances, by additional considerations such as plant host and vector specificity, primer specificity, and RFLP comparisons of ribosomal and nonribosomal DNA, as well as serological comparisons (Seemüller et al., 2002, 1998). Most recently, Wei et al. (2007) applied computer-simulated RFLP analysis for classification of phytoplasma strains. Through comparisons of virtual RFLP patterns of 16S rRNA genes and calculations of coefficients of RFLP similarity, the authors classified all available 16S rRNA gene sequences, including sequences from 250 previously unclassified phytoplasma strains, into a total of 28 16Sr RFLP groups. These included ten new groups and dozens of new subgroup lineages (Cai et  al., 2008; Wei et al., 2008b). Each new group represents a potential “Candidatus Phytoplasma” species level taxon. This information was used to augment the 16Sr RFLP classification system (Lee et al., 2000, 1998b, 1993b) with the following additional groups: 16SrXVI, Sugarcane yellow leaf syndrome; 16SrXVII, Papaya bunchy top group; 16SrXVIII, American potato purple top wilt group; 16SrXIX, Japanese chestnut witches’-broom group; 16SrXX, Buckthorn witches’-broom group; 16SrXXI, Pine shoot proliferation group; 16SrXXI, Nigerian coconut lethal decline (LDN) group; 16SrXXIII, Buckland valley grapevine yellows group; 16SrXXIV, Sorghum bunchy shoot group; 16SrXXV, Weeping tea witches’-broom group; 16SrXXVI, Mauritius sugarcane yellow D3T1 group; 16SrXXVII, Mauritius sugarcane yellow D3T2 group; and 16SrXXVIII, Havana derbid phytoplasma group. The virtual RFLP patterns are available for online use as reference patterns at http://www.ba.ars.usda. gov/data/mppl/virtualgel.html. The spacer region (SR) separating the 16S from the 23S rRNA gene of phytoplasmas was also shown to be a reliable phylogenetic marker. Phylogenetic trees derived from the entire 16S–23S SR (Gibb et  al., 1998; Kenyon et  al., 1998) or variable regions flanking the tRNAile gene (Kirkpatrick et  al., 1995; Schneider et al., 1995) differentiated phytoplasmas into groups that were concordant with the major groups established previously from analyses of 16S rRNA genes. Phytoplasmas collectively differ in their 16S rRNA gene sequence by no more than 14%, whereas their respective 16S–23S SR sequences differ by as much as 22%. This added variation has contributed to improved accuracy of phytoplasma classification at the subgroup level. Similarly, phylogenetic analysis of ribosomal protein genes, secY, secA, or 23S rRNA genes has been employed to differentiate closely related phytoplasma strains, as well as to aid the group and subgroup classification of diverse phytoplasmas (Daire, 1993; Hodgetts et al., 2008; Lee et al., 1998b, 2004a, 2006b; Martini et  al., 2007; Reinert, 1999). Such studies have led to finer differentiation among phytoplasma subgroups and to enriched descriptions of “Candidatus Phytoplasma” species (Lee et al., 2004a, 2006a, b). A polyphasic system for taxonomy based on integration of genotypic, phenotypic, and phylogenetic information employed for bacterial classification (Murray et  al., 1990; Stackebrandt and Goebel, 1994) has proved problematic for nonculturable phytoplasmas. In response to a rapidly growing database of phylogenetic markers, even in the absence of species-defining biological or phenotypic characters, the Working Team on Phytoplasmas of the International Research Programme of Comparative Mycoplasmology (IRPCM Phytoplasma/Spiroplasma

Genus I. “Candidatus Phytoplasma”

Working Team – Phytoplasma Taxonomy Group, 2004) proposed that taxonomy of phytoplasmas be based primarily upon phylogenetic analyses. This proposal was agreed to and adopted as policy by the ICSB Subcommittee on the Taxonomy of ­Mollicutes (1993, 1997), which also recommended that the provisional taxonomic status of “Candidatus”, originally proposed by Murray and Schleifer (1994), be used for assigning genera names as follows: “Candidatus Phytoplasma” (from phytos, Greek for plant; plasma, Greek for thing molded) [(Mollicutes) NC; NA; O; NAS (GenBank no. M30790); oligonucleotide sequence of unique region of the 16S rRNA gene is CAA GAY BAT KAT GTK TAG CYG GDC T; P (Plant, phloem; Insect, salivary gland); M]. (IRPCM Phytoplasma/Spiroplasma Working Team –­ Phytoplasma Taxonomy Group, 2004). By this same approach, major groups within the genus also delineated by phylogenetic analysis of near full-length 16S rRNA gene sequences were considered to represent one or more distinct species. Current guidelines for “Candidatus Phytoplasma” species descriptions (Anonymous, 2000; Firrao et  al., 2005; IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004) are based upon identification of a significantly unique 16S rRNA gene sequence >1200 bp in length. The strain from which the sequence is obtained should be designated as the reference strain. Strains with minimal differences in the 16S rRNA sequence, relative to the reference strain, should be referred to as related strains. In general, a strain can be described as a new “Candidatus Phytoplasma” species if its 16S rRNA gene sequence has less than 97% identity to any previously described “Candidatus Phytoplasma” species (ICSB Subcommittee on the Taxonomy of Mollicutes, 2001). There are cases in which phytoplasmas may share more than 97% of their 16S rRNA gene sequence, but clearly represent ecologically distinct populations and, thus, they may warrant description as

703

separate species. In such cases, the description of two different species is recommended when all of the following conditions apply: (1) the two phytoplasmas are transmitted by different vector species; (2) the two phytoplasmas have a different natural plant host, or at least their symptomatology is significantly different in the same plant host; (3) there is evidence of significant molecular diversity between phytoplasmas as determined by DNA hybridization assays with cloned nonribosomal DNA markers, serological reactions, or by PCR-based assays. The taxonomic rank of subspecies should not be used. Reference strains should be available to the scientific community in graftinoculated or in vitro micropropagated host plants or as DNA if perpetuation of strains in infected host plants is not feasible. Descriptions of “Candidatus Phytoplasma” species should be preferably submitted to the International Journal of Systematic and Evolutionary Microbiology (http://ijs.­sgmjournals.org/). Recent phylogenetic investigations, including the present analyses (Figure 116), suggest 97.5% 16S rRNA gene sequence similarity may represent a more suitable upper threshold for “Candidatus Phytoplasma” species separation, in that taxonomic subgroups designated based on 16S rRNA gene sequence similarities of £97.5% more consistently define species that are phylogenetically distinct from nearest related species. Regardless of homology criteria, a taxonomy is emerging for the phytoplasmas in the absence of cultivability where species and related strains of a species are clearly recognized with due consideration of the genetic, ecological, and environmental constraints unique to this group of plant- and insectassociated Mollicutes. To a large extent, the present taxonomy employs vernacular names based on associated diseases, but is constantly shifting towards a traditional taxonomy as more and more “Candidatus Phytoplasma” species continue to be ­recognized and proposed.

List of species of the genus “Candidatus Phytoplasma” In accordance with the current guidelines for “Candidatus ­Phytoplasma” species descriptions, the following species have been designated. Proposed assignments to the class Mollicutes are based on nucleic acid sequences. None of these species have been cultivated independently of their host, and their metabolism and growth temperatures are unknown.

Morphology: other. Sequence accession no. (16S rRNA gene): DQ174122. Unique regions of 16S rRNA gene: 5¢-GTTTCTTCGGAAA-3¢ (68–80), 5¢-GTTAGAAATGACT-3¢ (142–153), 5¢-GCTGGTGGCTT-3¢ (1438–1448). Habitat, association, or host: Solanum tuberosum phloem.

1. “Candidatus Phytoplasma allocasuarinae” Marcone, Gibb, Streten and Schneider 2004a, 1028 Vernacular epithet: Allocasuarina yellows phytoplasma, strain AlloYR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AY135523. Unique region of 16S rRNA gene: 5¢-TTTATTCGAGAGGGCG-3¢. Habitat, association, or host: phloem of Allocasuarina muelleriana (Slaty she-oak).

3. “Candidatus Phytoplasma asteris” Lee, Gundersen-Rindal, Davis, Bottner, Marcone and Seemüller 2004a, 1046 Vernacular epithet: Aster yellows (AY) phytoplasma, strain OAY R. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): M30790. Unique regions of 16S rRNA gene: 5¢-GGGAGGA-3¢, 5¢-CTGACGGTACC-3¢, and 5¢-CACAGTGGAGGTTATCAGTTG-3¢. Habitat, association, or host: phloem of Oenothera hookeri (Evening primrose).

2. “Candidatus Phytoplasma americanum” Lee, Bottner, Secor and Rivera-Varas 2006a, 1596 Vernacular epithet: Potato purple top, strain APPTW12NER. Gram reaction: not applicable.

4. “Candidatus Phytoplasma aurantifolia” Zreik, Carle, Bové and Garnier 1995, 452 Vernacular epithet: Witches’-broom disease of lime phytoplasma, strain WBDLR.

704

Family II. Incertae sedis

P.trifoli (CP)

77

P. fraxini (AshY1) P. ulmi (EY1)

79 100

100

P. ziziphi (JWB-G1) LfWB StLL

93 56

LDG LDT LY-c2 SBS P. castaneae (CnWB)

72 88

b

P. pini (PinP) CIRP GaLL 98

BVK

100

P. oryzae (RYD) SCWL P. cynodontis (BGWL) P. phoenecium (AlmWB-A4)

98 100

ViLL WX GLL-eth

59

P. brasiliense (HibWB) IAWB SPLL

100

P. australasia (PpYC) P. aurantifolia (WBDL)

91

WTWB P. mali (AP15) P. prunorum (ESFY-G1)

a

P. pyri (PD1) 100

P. spartii (SPAR) P. allocasuarinae (AlloY) P. rhamni (BWB) STOL P. australiense (AusGY)

100

P. japonicum (JHP) IBS 100

P. asteris (MIAY) MPV

A. palmae A. laidlawii 10 changes FIGURE 116.  Phylogenetic analysis of the phytoplasmas. Phylogenetic trees were constructed by parsimony analy-

ses of phytoplasma 16S rRNA gene sequences using the computer program PAUP (Swofford, 1998). The closely related culturable Acholeplasma palmae was employed as the outgroup. Because phytoplasma taxa are too numerous to present in a single inclusive tree, a global phylogeny of representative phytoplasmas is first presented. The global tree is divided into lower (a) and upper (b) regions. Each region of the global phylogeny is then expanded into inclusive trees, a and b, which collectively include 145 phytoplasmas from diverse geographic origins. Taxonomic subgroups, representing phytoplasmas sharing at least 97.5% 16S rRNA gene sequence similarity, are identified on each inclusive tree. Each phylogenetically distinct subgroup is equivalent to a subclade (or putative species) within the genus “Candidatus Phytoplasma”. In all trees, branch lengths are proportional to the number of inferred character state transformations. Bootstrap (confidence) values greater than or equal to 50 are shown on the branches. Phytoplasmas for which 16S rRNA gene sequences of at least 1200 bp in length have been determined (312 total) are listed by subgroup in Table 144 along with their sequence accession numbers.

705

Genus I. “Candidatus Phytoplasma”

a

84 100 100

52

100

87

SUNHP SPWB PnWB AlWB GPh P. australasia (PpYC) CoAWB IAWB PEP SPLL GLL-eth P. brasiliense (HibWB) CaM CaWB1 FBP P. aurantifolia (WBDL) BoLL

WTWB P. mali (AP15) AP1/93 AP2 AT P. pyri (PD1) PD 100 PPER P. prunorum (ESFY-G1) 82 ESFY-142 ESFY4 100 ESFY5 P. spartii (Spar) P. allocasuarinae (AlloY) 100 P. rhamni (BWB) P. asteris (MIAY) PPT MBS PaWB MD Bstv2M.f12 HyPH APWB IOWB PRIVC RPh ApSL AAY SAY ValY WcWB OY-M CabD3 AY-WB 100 BB HYDP CPh STRAWB2 BBS ACLR CWL SY PY 100 VK STOL STOL2 80 PpDB PYL 65 SLY SV3101 95 P. australiense (AusGY) P. japonicum (JHP) 51 IBS MPV 100 STRAWB1 CbY1 A. palmae

P. australasia

IAWB SPLL GLL-eth P. brasiliense P. aurantifolia BoLL WTWB P. mali P. pyri P. prunorum P. spartii P. allocasuarinae P. rhamni

P. asteris

STOL

P. australiense P. japonicum IBS MPV

5 changes

FIGURE 116.  (Continued)

Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): U15442. Unique region of 16S rRNA gene: 5¢-GCAAGTGGTGAACCATTTGTTT-3¢. Habitat, association, or host: phloem of Citrus; hemolymph and salivary glands of Hishimonus phycitis (Cicadellidae). 5. “Candidatus Phytoplasma australasia” White, Blackall, Scott and Walsh 1998, 949 Vernacular epithet: Papaya yellow crinkle phytoplasma, strain PpYCR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): Y10097. Unique regions of 16S rRNA gene: 5¢-TAAAAGGCATCTTTTATC-3¢ and 5¢-CAAGGAAGAAAAGCAAATGGCGAACCATTTGTTT-3¢.

Habitat, association, or host: phloem of Carica papaya and Lycopersicon esculentum. 6. “Candidatus Phytoplasma australiense” Davis, Dally, Gundersen, Lee and Habili 1997, 268 Vernacular epithet: Australian grapevine yellows phytoplasma, strain AUSGYR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): L76865. Unique regions of 16S rRNA gene: 5¢-CGGTAGAAATAT­ CGT-3¢ and 5¢-TTTATCTTTAAAAGACCTCGCAAGA-3¢. Habitat, association, or host: Vitis phloem.  

7. “Candidatus Phytoplasma brasiliense” Montano, Davis, ­Dally, Hogenhout, Pimentel and Brioso 2001, 1117 Vernacular epithet: Hibiscus witches’-broom (HibWB) ­phytoplasma, strain HibWB26R.

706

Family II. Incertae sedis

b

56

51

97 88

100

100

P. trifolii (CP) PWB BLL-bd BLL 100 CSV BLTVA 88 VR ArAWB EriWB 100 P. fraxini (AshY1) ASHY LWB3 AshY3 ALY 86 FD HD1 SpaWB229 VC RuS P. ulmi (EY1) ULW 100 100 JWB-ch P. ziziphi (JWB-G1) CLY-5 NecY-In1 JWB-Ka LfWB 100 LfWB-t StLL CY LY-c2 PanD ScY LDY 81 LfY1 LDG 72 LDN LDT SBS SGS-v1 SCWL BVK 100 CIRP GaLL P. oryzae (RYD-J) RYD-Th BGWL-2 CWL P. cynodontis (BGWL-C1) BGWL P. casteneae (CnWB) P. pini (Pin127) PinG KAP PPWB-f P. phoenecium (AlmWB-A4) ViLL BBP TWB VAC CYE-C DanVir-a GDIII CbY18 WWB-a BLWB PoiBI VGYIII CX ScYPI-Afr WX LP A. palmae

P. trifolii

EriWB

P. fraxini

P. ulmi

P. ziziphi

LfWB StLL

LY

LDG LDT SBS SCWL BVK CIRP GaLL P. oryzae P. cynodontis P. casteneae P. pini P. phoenecium ViLL

WX

5 changes FIGURE 116.  (Continued)

Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AF147708. Unique regions of 16S rRNA gene: 5¢-GAAAAAGAAAG-3¢, 5¢-TCTTTCTTT-3¢, 5¢-CAG-3¢, 5¢-ACTTTG-3¢, and 5¢-GTCA AAAC-3¢. Habitat, association, or host: Hibiscus phloem.

8. “Candidatus Phytoplasma caricae” Arocha, López, Piñol, Fernández, Picornell, Almeida, Palenzuela, Wilson and Jones 2005, 2462 Vernacular epithet: Cuban papaya phytoplasma, strain PAYR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AY725234.

707

Genus I. “Candidatus Phytoplasma”

Table 144.  Provisional groupings, strain designations, associated plant disease, geographic origin and accession numbers of 16S rRNA gene

sequences derived from phytoplasmasa Subgroup

Strain

AlloY AP           ESFY             AshY       EriWB  

AlloY AP15R AT AP2 AP1/93 D365/04 APSb ESFY-G1R ESFY5 ESFY4 PPER ESFY-142 ESFY-173 ESFY-215 AshY1 AshY3 ASHY LWB3 EriWB ArAWB

AusGY           AY      

AusGY PpDB PYL PYLb SLY SV3101 MIAY OY-M MBS APWB

 

IOWB

           

HyPH HYPh RPh MD GDS AYWB_ro4

                                       

PaWB PY1 BVGY AAY CabD4 AY-BW ApSL SAY ValY PRIVC WcWB CabD3 AY-sb Bstv2Mf12 ACLR-AY ACLR BBS3 STRAWB2 PoY KVG

Associated plant disease Allocasuarina yellows Apple proliferation Apple proliferation Apple proliferation Apple proliferation Apple proliferation Apple proliferation European stone fruit yellows European stone fruit yellows European stone fruit yellows European stone fruit yellows European stone fruit yellows European stone fruit yellows European stone fruit yellows Ash yellows Ash yellows Ash yellows Lilac witches’-broom Erigeron witches’-broom Argentinian alfalfa witches’broom Australian grapevine yellows Papaya die-back Phormium yellow leaf, rrnA Phormium yellow leaf Strawberry lethal yellows Strawberry virescence Oenothera virescence Onion yellows Maize bushy stunt Aphanamixis polystachya witches’-broom Ipomoea obscura witches’broom Hydrangea phyllody Hydrangea phyllody Oilseed rape phyllody Mulberry dwarf   Aster yellows witches’broom Paulownia witches’-broom Periwinkle yellows   American aster yellows Cabbage proliferation Aster yellows Apple sessile leaf Severe aster yellows Valeriana yellows, rrnA Primrose virescence Watercress witches’-broom Cabbage proliferation Sugar beet aster yellows   Apricot chlorotic leafroll Apricot chlorotic leafroll Blueberry stunt Strawberry green petal Populus yellows Clover phyllody

“Candidatus Phytoplasma species”

Geographic origin

16S accession no.a

P. allocasuarinae P. mali           P. prunorum             P. fraxini          

Australia Italy Germany Germany France Slovenia Italy Germany Austria Czech Republic Germany Spain Spain Spain USA, New York USA, Utah Germany USA, Massachusetts Brazil Argentina

AY135523* AJ542541* X68375* AF248958* AJ542542* EF025917 EF193361 AJ542544* AY029540* Y11933* X68374 AJ575108* AJ575106 AJ575105 AF092209* AF105315* X68339* AF105317* AY034608 AY147038

P. australiense           P. asteris      

Australia Australia New Zealand New Zealand Australia Tonga USA, Michigan Japan Mexico Bangladesh

L76865 Y10095* U43569 U43570* AJ243045* AY377868* M30790* NC005303* AY265208* AY495702*

 

Taiwan

AY265205*

           

Italy France Czech Republic South Korea   Ohio, USA

AY265207* AY265219 U89378* AY075038* DQ112021 NC007716

                                       

Korea China   Southern USA USA, Texas USA, Ohio Lithuania USA, California Lithuania  Germany USA, Hawaii USA, Texas Hungary   Spain Europe USA, Michigan USA, Florida Croatia Germany

AF279271* AF453328 AY083605 X68373* AY180932 AY389820 AY734454 M86340* AY102274* AY265210* AY665676* AY180947* AF245439 AY180951 AY265211 X68338* AY265213 U96616* AF503568 X83870 (continued)

708

Family II. Incertae sedis

Table 144.  (continued)

Subgroup

Strain

          THP   BWB BGWL       BVK

CPh HYDP AY-WB BB PPT THP Derbid BWB BGWL-C1 BGWL BGWL-2 CWL BVK

CIRP CnWB CP    

CIRP CnWB CPR BLL BLTVA

     

VR PWB CSV

EY          

EY1 ULW FD RuS HD1 VC

SpaWB JWB

SpaWB229 JWB-G1T

 

JWB-Ka

 

JWB-ch

    FBP

NecY-In1 CLY-5 WBDL

      BoLL GaLL GLL-eth HibWB IAWB   IBS StrawY JHP

CaWB-YNO1 FBP PPLL BoLL GaLL GLL-eth HibWB IAWB PEP IBS StrawY JHP

LDG   LDT LfWB   LY  

LDG LDN LDT LfWB LfWB-t CPY LDY

Associated plant disease Clover phyllody Hydrangea phyllody Aster yellows Tomato big bud Potato purple top Tomato ‘hoja de perejil’ Derbid phytoplasma Buckthorn witches’-broom Bermudagrass white leaf Bermudagrass white leaf Bermudagrass white leaf  Cynodon white leaf Psammotettic cephalotesborne Cirsium phyllody Chestnut witches’- broom Clover proliferation Brinjal little leaf Columbia basin potato purple top Vinca virescence Potato witches’-broom Centauria stolstitialis virescence Elm yellows Ulmus witches’-broom Flavescence doree Rubus stunt Hemp dogbane yellows Asymptomatic Virginia creeper Spartium witches’-broom Jujube witches’-broom Gifu isolate 1 Jujube witches’-broom Korea isolate 1 Ziziphus jujube witches’broom Nectarine yellows Cherry lethal yellows Witches’-broom disease of lime Cactus witches’-broom Faba bean phyllody Pigeon pea little leaf Bonamia little leaf Galactia little leaf Gliricidia little leaf Hibiscus witches’-broom Alfalfa witches’-broom Pichris echioides phyllody Italian bindweed yellows Strawberry lethal yellows Japanese hydrangea phyllody Cape St Paul wilt Awka disease of coconut Coconut lethal disease Loofah WB Loofah WB Carludovica palmata yellows Yucatan coconut decline

“Candidatus Phytoplasma species”

Geographic origin

16S accession no.a

          P. lycopersici   P. rhamni P. cynodontis        

Canada Belgium USA, Ohio USA, Arkansas Mexico Bolivia Cuba Germany Italy Italy Thailand Australia Germany

AF222066* AY265215* AY389827* AY180955* AF217247* AY787136 AY744945 X76431* AJ550984* Y16388* AF248961* AF509321* X76429*

  P. castaneae P. trifolii    

Germany Korea Canada India USA, Washington

X83438* AB054986* AY390261* X83431* AY692280*

     

USA, California Canada Italy

AY500817* AY500818* AY270156*

P. ulmi          

USA, New York Italy Italy Italy USA, New York USA, Florida

AY197655* X68376* X76560* AY197648* AY197654* AF305198*

P. spartii P. ziziphi

Italy Japan

AY197652* AB052876*

 

Korea

AB052879*

 

China

AF305240

    P. aurantifolia

India China Oman

AY332659* AY197659* U15442*

            P. brasiliense       P. fragariae P. japonicum

China Sudan Australia Australia Australia Ethiopia Brazil Italy Italy Southern Italy Lithuania Japan

AJ293216 X83432* AJ289191 Y15863* Y15865* AF361018* AF147708* Y16390* Y16393* Y16391* DQ086423 AB010425

             

Ghana Nigeria Tanzania Taiwan Taiwan Mexico Mexico

Y13912* Y14175* X80177* L33764* AF086621* AF237615 U18753* (continued)

709

Genus I. “Candidatus Phytoplasma” Table 144.  (continued)

Subgroup

Strain

            ScY   MPV

LfY1 LfY5(PE65) LY-c2 LY-JC8 PanD ScY SCD3T2 SCD3T1 MPV

      PD       PinP   PPWB     RYD   SBS SCWL  

PerWB-FL CbY1 STRAWB1 PD1 PD PYLR EPC Pin127R PinG AlmWB-A4 KAP PPWB-f RYD-J RYD-Th SBS SCWL SGS-v1

SpaWB SPLL SPWB               StLL STOL     ViLL CIWB WTWB WX                        

Spar SPLL PpYC GPh PnWB CoAWB SPLL SUNHP AlWB TBB StLL STOL VK 2642BN ViLL IM-3 WTWB BBP BLWB CbY18 CX CYE DanVir-a LP PoiBI ScYP I-Afr TWB VAC VGYIII WWB-a

 

WX

Associated plant disease Coconut leaf yellowing Coconut leaf yellowing Coconut lethal yellows Coconut lethal yellows Pandanus decline Sugarcane yellows, group 4 Sugarcane yellows, group 3 Sugarcane yellows, group 3 Mexican periwinkle virescence Periwinkle witches’-broom Chinaberry yellows Strawberry green petal Pear decline Pear decline Peach yellow leafroll Pear decline Pinus halepensis yellows Pinus sylvestris yellows Almond witches’-broom Knautia arvensis phyllody Pigeonpea witches’-broom Rice yellow dwarf Rice yellow dwarf Sorghum bunchy shoot Sugarcane white leaf Sorghum grassy shoot, variant 1 Spartium witches’-broom Sweet potato little leaf Papaya yellow crinkle Gerbera phyllody Peanut witches’ broom Cocky apple witches’-broom Sweet potato little leaf Sunnhemp phyllody Alfalfa witches’-broom Australian tomato big bud Stylosanthes little leaf Stolbur of Capsicum annum Grapevine yellows Grapevine yellows Vigna little leaf Cassia italica witches’-broom Weeping tea witches’-broom Blueberry proliferation Black locust witches’-broom Chinaberry yellows Canadian peach X Clover yellow edge Dandelion virescence, rrnA Little peach Poinsettia branch-inducing Sugarcane yellows Tsuwabuki WB Vaccinium witches’-broom Virginia grapevine yellows Walnut witches’-broom, rrnA Western X

“Candidatus Phytoplasma species”

Geographic origin

16S accession no.a

                 

Mexico Mexico USA, Florida Jamaica USA, Florida Mauritius Mauritius Mauritius Mexico

AF500329* AF500334 AF498309* AF498307 AF361020* AJ539178* AJ539180 AJ539179 AF248960*

      P. pyri       P. pini   P. phoenecium     P. oryzae        

USA, Florida Bolivia USA, Florida Italy Germany USA, California Iran Spain Germany Lebanon Italy USA, Florida Japan Thailand Australia Thailand Australia

AY204549 AF495882* U96614* AJ542543* X76425* Y16394 DQ471321 AJ632155* AJ310849* AF515636* Y18052* AF248957* D12581 AB052873* AF509322* X76432* AF509324*

P. spartii   P. australasia                     P. solani   P. omaniense                            

Italy Australia Australia Japan? Taiwan Australia Australia Thailand Oman Australia Australia Europe Europe France Australia Oman Australia Lithuania USA, Maryland Bolivia Canada Canada Lithuania USA, S. Carolina Southern USA Africa Japan Germany USA, Virginia USA, Georgia

X92869* X90591* Y10097* AB026155* L33765* AJ295330* AJ289193 X76433* AY169322* Y08173 AJ289192* X76427* X76428* AJ964960 Y15866* EF666051 AF521672* AY034090* AF244363* AF495657* L33733* AF175304* AF370119* AF236122* AF190223* AF056095* D12580* X76430* AF060875* AF190226*

 

USA, California

L04682*

Accession numbers denoted by an asterisk were used as sources of 16S rRNA gene sequences for comprehensive phylogenetic analysis of subgroup phytoplasmas from diverse geographic origins. a

710

Family II. Incertae sedis

Unique regions of 16S rRNA gene: 5¢-AAA-3¢ (196–198), 5¢ATT-3¢ (600–603), 5¢-AGGCGCC-3¢ (1089–1095), 5¢-GCGGATTTAGTCACTTTTCAGGC-3¢ (1379–1401). Habitat, association, or host: Carica papaya phloem. 9. “Candidatus Phytoplasma castaneae” Jung, Sawayanagi, Kakizawa, Nishigawa, Miyata, Oshima, Ugaki, Lee, Hibi and Namba 2002, 1548 Vernacular epithet: Chestnut witches’ broom phytoplasma, strain CnWBR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AB054986. Unique regions of 16S rRNA gene: 5¢-CTAGTTTAAAAACAATGCTC-3¢ and 5¢-CTCATCTTCCTCCAATTC-3¢. Habitat, association, or host: Castanea crenata phloem. 10. “Candidatus Phytoplasma cynodontis” Marcone, Schneider and Seemüller 2004b, 1081 Vernacular epithet: Bermuda grass white leaf (BGWL) phytoplasma, strain BGWl-C1R. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AJ550984. Unique region of 16S rRNA gene: 5¢-AATTAGAAGGCAT­ CTTTTAAT-3¢. Habitat, association, or host: phloem of Cynodon dactylon (Bermuda grass). 11. “Candidatus Phytoplasma fragariae” Valiunas, Staniulis and Davis 2006, 280 Vernacular epithet: Strawberry yellows phytoplasma, strain StrawY R. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): DQ086423. Unique regions of 16S rRNA gene: 5¢-GTGCAATGCTCAACGTTGTGAT-3¢, 5¢-AATTGCA-3¢, and 5¢-TGAGTAATCAAGAGGGAG-3¢. Habitat, association, or host: phloem of Fragaria x ananassa. 12. “Candidatus Phytoplasma fraxini” Griffiths, Sinclair, Smart and Davis 1999, 1613 Vernacular epithet: Ash yellows phytoplasma, strain AshYR and lilac witches’-broom (LWB) phytoplasma. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AF092209. Unique regions of 16S rRNA gene: 5¢-CGGAAACCCCTCAAAAGGTTT-3¢ and 5¢-AGGAAAGTC-3¢. Habitat, association, or host: phloem of Fraxinus and Syringa. 13. “Candidatus Phytoplasma graminis” Arocha, López, Piñol, Fernández, Picornell, Almeida, Palenzuela, Wilson and Jones 2005, 2462 Vernacular epithet: Sugarcane yellow leaf phytoplasma, strain SCYPR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AY725228. Unique regions of 16S rRNA gene: 5¢-TTTG-3¢ (465–468), 5¢-TTG-3¢ (478–480), 5¢-GGG-3¢ (1552–1554), 5¢-TAA-3¢

(1381–1383), and 5¢-ATTTACGTTTCTG-3¢ (1392–1404). Habitat, association, or host: Saccharum officinarum phloem. 14. “Candidatus Phytoplasma japonicum” Sawayanagi, ­Horikoshi, Kanehira, Shinohara, Bertaccini, Cousin, Hiruki and Namba 1999, 1284 Vernacular epithet: Japanese Hydrangea phyllody phytoplasma, strain JHPR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AB010425. Unique regions of 16S rRNA gene: 5¢-GTGTAGCCGGGCTGAGAGGTCA-3¢ and 5¢-TCCAACTCTAGCTAAACAGTTTCTG-3¢. Habitat, association, or host: Hydrangea phloem. 15. “Candidatus Phytoplasma lycopersici” Arocha, Antesana, Montellano, Franco, Plata and Jones 2007, 1709 Vernacular epithet: Tomato “hoja de perejil” phytoplasma, strain THPR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AY787136. Unique regions of 16S rRNA gene: 5¢-CTTA-3¢ (positions 175–178), 5¢-AATGGT-3¢ (198–203), 5¢-ATA-3¢ (229–231), 5¢-TGGAGGAA-3¢ (234–242), 5¢-CACG-3¢ (302–305), 5¢-TCT-3¢ (315–317), 5¢-GCT-3¢ (334–336), 5¢-TAT-3¢ (336–338), 5¢-TAC-3¢ (413–415), and 5¢-AGC-3¢ (434–436). Habitat, association, or host: Lycopersicon esculentum phloem. 16. “Candidatus Phytoplasma mali” Seemüller and Schneider 2004, 1224 Vernacular epithet: Apple proliferation (AP) phytoplasma, strain AP15R. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AJ542541. Unique region of 16S rRNA gene: 5¢-AATACTCGAAACCAGTA-3¢. Habitat, association, or host: Malus phloem. 17. “Candidatus Phytoplasma omanense” Al-Saady, Khan, ­Calari, Al-Subhi and Bertaccini 2008, 464 Vernacular epithet: Cassia witches’-broom (CWB) phytoplasma, strain IM-1R. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): EF666051. Unique regions of 16S rRNA gene: 5¢-AAAAAACAGT-3¢ (467– 474), 5¢-TTGC-3¢ (642–645), 5¢-GTTAAAG-3¢ (853–861), 5¢-TAATT-3¢ (1010–1014), and 5¢-AAATT-3¢ (1052–1056). Habitat, association, or host: Cassia italica phloem. 18. “Candidatus Phytoplasma oryzae” Jung, Sawayanagi, Wongkaew, Kakizawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Hibi and Namba 2003c, 1928 Vernacular epithet: Rice yellow dwarf (RYD) phytoplasma, strain RYD-ThR. Gram reaction: not applicable. Morphology: other. Sequence accession nos (16S rRNA gene): D12581, AB052873 (RYD-Th).

Genus I. “Candidatus Phytoplasma”

Unique regions of 16S rRNA gene: 5¢-AACTGGATAGGAAATTAAAAGGT-3¢ and 5¢-ATGAGACTGCCAATA-3¢. Habitat, association, or host: Oryza sativa phloem. 19. “Candidatus Phytoplasma phoenicium” Verdin, Salar, Danet, Choueiri, Jreijiri, El Zammar, Gélie, Bové and Garnier 2003, 837 Vernacular epithet: Almond witches’-broom (AlmWB) phyto­plasma, strain AlmWB-A4R. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AF515636. Unique region of 16S rRNA gene: 5¢-CCTTTTTCGGAAGGTATG-3¢. Habitat, association, or host: Prunus amygdalus phloem. 20. “Candidatus Phytoplasma pini” Schneider, Torres, Martín, Schröder, Behnke and Seemüller 2005, 306 Vernacular epithet: Pinus halepensis yellows (Pin) phytoplasma, strain Pin127SR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AJ632155. Unique regions of 16S rRNA gene: 5¢-GGAAATCTTTCGGGATTTTAGT-3¢ and 5¢-TCTCAGTGCTTAACGCTGTTCT-3¢. Habitat, association, or host: Pinus phloem. 21. “Candidatus Phytoplasma prunorum” Seemüller and Schneider 2004, 1224 Vernacular epithet: European stone fruit yellows (ESFY) phytoplasma, strain ESFY-G1R. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AJ542544. Unique regions of 16S rRNA gene: 5¢-AATACCCGAAACCAGTA-3¢ and 5¢-TGAAGTTTTGAGGCATCTCGAA-3¢. Habitat, association, or host: Prunus phloem. 22. “Candidatus Phytoplasma pyri” Seemüller and Schneider 2004, 1224 Vernacular epithet: Pear decline (PD) phytoplasma, strain PD1R. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AJ542543. Unique regions of 16S rRNA gene: 5¢-AATACTCAAAACCAGTA-3¢ and 5¢-ATACGGCCCAAACTCATACGGA-3¢. Habitat, association, or host: Pyrus phloem. 23. “Candidatus Phytoplasma rhamni” Marcone, Gibb, Streten and Schneider 2004a, 1028 Vernacular epithet: Buckthorn witches’-broom phytoplasma, strain BWBR. Gram reaction: not applicable. Morphology: other. Sequence accession nos (16S rRNA gene): X76431, AJ583009. Unique regions of 16S rRNA gene: 5¢-CGAAGTATTTCGATAC-3¢. Habitat, association, or host: phloem of Rhamnus catharticus (buckthorn). 24. “Candidatus Phytoplasma solani” Firrao, Gibb and Streton 2005, 251

711

Vernacular epithet: Stolbur phytoplasma; subgroup A reference type of the stolbur phytoplasma taxonomic group 16SrXII (Lee et al., 2000). Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AJ970609 (strain PO; Cimerman et al., 2006). Unique region of 16S rRNA gene: not reported. Habitat, association, or host: many species of Solanaceae plus several species in other plant families, and Fulguromorpha spp. planthopper vectors. 25. “Candidatus Phytoplasma spartii” Marcone, Gibb, Streten and Schneider 2004a, 1028 Vernacular epithet: Spartium witches’-broom phytoplasma, strain SpaWBR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): X92869. Unique region of 16S rRNA gene: 5¢-TTATCCGCGTTAC-3¢. Habitat, association, or host: phloem of Spartium junceum (Spanish broom). 26. “Candidatus Phytoplasma tamaricis” Zhao, Sun, Wei, Davis, Wu and Liu 2009, 2496 Vernacular epithet: Salt cedar witches’-broom phytoplasma, strain SCWB1R. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): FJ432664. Unique regions of 16S rRNA gene: 5¢-ATTAGGCATCTAGTAACTTTG-3¢, 5¢-TGCTCAACATTGTTGC-3¢, 5¢-AGCTTTGCAAAGTTG-3¢, and 5¢-TAACAGAGGTTATCAGAGTT-3¢. Habitat, association, or host: phloem of Tamarix chinensis (salt cedar). 27. “Candidatus Phytoplasma trifolii” Hiruki and Wang 2004, 1352 Vernacular epithet: Clover proliferation phytoplasma, strain CPR. Gram reaction: not applicable. Morphology: other. Sequence accession no. (16S rRNA gene): AY390261. Unique regions of 16S rRNA gene: 5¢-TTCTTACGA-3¢ and 5¢-TAGAGTTAAAAGCC-3¢. Habitat, association, or host: Trifolium phloem. 28. “Candidatus Phytoplasma ulmi” Lee, Martini, Marcone and Zhu 2004b, 345 Vernacular epithet: Elm yellows phytoplasma (EY) phytoplasma, strain EY1R. Gram reaction: not applicable. Morphology: other. Sequence accession nos (16S rRNA gene): AY197655, AY197675, and AY197690. Unique regions of 16S rRNA gene: 5¢-GGAAA-3¢ and 5¢-CGTTAGTTGCC-3¢. Habitat, association, or host: Ulmus americana phloem. 29. “Candidatus Phytoplasma vitis” Firrao, Gibb and Streton 2005, 251 Vernacular epithet: Flavescence dorée phytoplasma; strains are genetically heterogenous and vary in degree of virulence, but all are referable to subgroups C or D of the elm yellows

712

Family II. Incertae sedis

phytoplasma taxonomic group 16SrV (Lee et al., 2000). Gram reaction: not applicable. Morphology: other. Sequence accession nos (16S rRNA gene): AY197645 (16SrV subgroup C), AY197644 (16SrV subgroup D1). Unique regions of 16S rRNA gene: not reported. Habitat, association, or host: grapevines (Vitis vinifera) and the leafhopper vector Scaphoideus titanus. 30. “Candidatus Phytoplasma ziziphi” Jung, Sawayanagi, Kakizawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Hibi and Namba 2003b, 1041

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Order IV. Anaeroplasmatales Wei, W., S. Kakizawa, H.Y. Jung, S. Suzuki, M. Tanaka, H. Nishigawa, S. Miyata, K. Oshima, M. Ugaki, T. Hibi and S. Namba. 2004a. An antibody against the SecA membrane protein of one phytoplasma reacts with those of phylogenetically different phytoplasmas. Phytopatho­ logy 94: 683–686. Wei, W., S. Kakizawa, S. Suzuki, H.Y. Jung, H. Nishigawa, S. Miyata, K. Oshima, M. Ugaki, T. Hibi and S. Namba. 2004b. In planta dynamic analysis of onion yellows phytoplasma using localized inoculation by insect transmission. Phytopathology 94: 244–250. Wei, W., R.E. Davis, I.M. Lee and Y. Zhao. 2007. Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups. Int. J. Syst. Evol. Microbiol. 57: 1855–1867. Wei, W., R.E. Davis, R. Jomantiene and Y. Zhao. 2008a. Ancient, recurrent phage attacks and recombination shaped dynamic sequencevariable mosaics at the root of phytoplasma genome evolution. Proc. Natl. Acad. Sci. U. S. A. 105: 11827–11832. Wei, W., I.M. Lee, R.E. Davis, X. Suo and Y. Zhao. 2008b. Automated RFLP pattern comparison and similarity coefficient calculation for rapid delineation of new and distinct phytoplasma 16Sr subgroup lineages. Int. J. Syst. Evol. Microbiol. 58: 2368–2377. Weintraub, P.G. and L. Beanland. 2006. Insect vectors of phytoplasmas. Annu. Rev. Entomol. 51: 91–111. Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. Mandelco, J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic analysis of the mycoplasmas: basis for their classification. J. Bacteriol. 171: 6455–6467.

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Whitcomb, R.F., D.D. Jensen and J. Richardson. 1966a. The infection of leafhoppers by western X-disease. virus: II. Fluctuation of virus concentration in the hemolymph after injection. Virology 28: 454–458. Whitcomb, R.F., D.D. Jensen and J. Richardson. 1966b. The infection of leafhoppers by the western X-disease virus: I. Frequency of transmission after injection or acquisition feeding. Virology 28: 448–453. White, D.T., L.L. Blackall, P.T. Scott and K.B. Walsh. 1998. Phylogenetic positions of phytoplasmas associated with dieback, yellow crinkle and mosaic diseases of papaya, and their proposed inclusion in ‘Candidatus Phytoplasma australiense’ and a new taxon, ‘Candidatus Phytoplasma australasia’. Int. J. Syst. Bacteriol. 48: 941–951. Wongkaew, P. and J. Fletcher. 2004. Sugarcane white leaf phytoplasma in tissue culture: long-term maintenance, transmission, and oxytetracycline remission. Plant Cell Rep. 23: 426–434. Yu, Y.L., K.W. Yeh and C.P. Lin. 1998. An antigenic protein gene of a phytoplasma associated with sweet potato witches’ broom. Microbiology 144: 1257–1262. Zhao, Y., Q. Sun, W. Wei, R.E. Davis, W. Wu and Q. Liu. 2009. ‘Candidatus Phytoplasma tamaricis’, a novel taxon discovered in witches’broom-diseased salt cedar (Tamarix chinensis Lour.). Int. J. Syst. Evol. Microbiol. 59: 2496–2504. Zreik, L., P. Carle, J.M. Bové and M. Garnier. 1995. Characterization of the mycoplasmalike organism associated with witches’ broom disease of lime and proposition of a Candidatus taxon for the organism, “Candidatus Phytoplasma aurantifolia”. Int. J. Syst. Bacteriol. 45: 449–453.

Order IV. Anaeroplasmatales Robinson and Freundt 1987, 81VP Daniel R. Brown, Janet M. Bradbury and Karl-Erik Johansson A.na.e.ro.plas.ma.ta¢les. N.L. neut. n. Anaeroplasma, -atos type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Anaeroplasmatales the Anaeroplasma order. This order in the class Mollicutes represents a unique group of strictly anaerobic, wall-less prokaryotes (trivial name, anaeroplasmas) first isolated from the bovine and ovine rumen. Other than their anaerobiosis, the description of organisms in the order is essentially the same as for the class. A single family, Anaeroplasmataceae, with two genera, was proposed to recognize the two most prominent characteristics of the organisms: a requirement of sterol supplements for growth by those strictly anaerobic organisms now assigned to the genus Anaeroplasma; and strictly anaerobic growth in the absence of sterol supplements by those now assigned to the genus Asteroleplasma. Genome sizes range from 1542 to 1794 kbp as estimated by renaturation kinetics. The DNA G+C content ranges from 29 to 40 mol%. All species examined utilize the universal genetic code in which UGA is a stop codon. Phylogenetic studies indicate that members of the Anaeroplasmatales are much more closely related to the Acholeplasmatales than to the Mycoplasmatales or Entomoplasmatales (Weisburg et al., 1989). Type genus: Anaeroplasma Robinson, Allison and Hartman 1975, 179AL.

Further descriptive information The initial proposal for elevation of the anaeroplasmas to an order of the class Mollicutes (Robinson and Freundt, 1987) was based upon the description of three novel species and the observation that some anaeroplasmas did not have a sterol requirement for growth. The obligate requirement for anaerobic growth conditions is the single most important property in distinguishing members of the Anaeroplasmatales from other mollicutes. The

anaeroplasmas exist in a natural environment where the oxidation potential is maintained at a low level by the metabolism of associated micro-organisms. Anaerobic methods for preparing media and culture techniques for the organisms are essentially those described by Hungate (1969), with media and inocula maintained in closed vessels and exposure to air avoided during inoculation and incubation. A primary isolation medium and clarified rumen fluid broth have been described (Bryant and Robinson, 1961; Robinson, 1983; Robinson et al., 1975).

References Bryant, M.P. and I.M. Robinson. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44: 1446–1456. Hungate, R.E. 1969. A roll tube method for cultivation of strict anaerobes. In Methods in Microbiology, vol. 3B (edited by Norris and ­Ribbons). Academic Press, London, pp. 117–132. Robinson, I.M., M.J. Allison and P.A. Hartman. 1975. Anaeroplasma abactoclasticum gen. nov., sp. nov., obligately anaerobic mycoplasma from rumen. Int. J. Syst. Bacteriol. 25: 173–181. Robinson, I.M. 1983. Culture media for anaeroplasmas. In Methods in Mycoplasmology, vol. 1, (edited by Razin and Tully). Academic Press, New York, pp. 159–162. Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended classification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81. Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. Mandelco, J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic analysis of the mycoplasmas: basis for their classification. J. Bacteriol. 171: 6455–6467.

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Family I. Anaeroplasmataceae

Family I. Anaeroplasmataceae Robinson and Freundt 1987, 80VP Daniel R. Brown, Janet M. Bradbury and Karl-Erik Johansson A.na.e.ro.plas.ma.ta.ce¢ae. N.L. neut. n. Anaeroplasma, -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Anaeroplasmataceae the Anaeroplasma family. All members have an obligate requirement for anaerobiosis. Organisms assigned to the genus Anaeroplasma require sterol supplements for growth. Organisms assigned to the genus Asteroleplasma grow in the absence of sterol supplements. Other characteristics are as described for the type genus. Type genus: Anaeroplasma Robinson, Allison and Hartman 1975, 179AL.

Further descriptive information The obligate requirement for anaerobic growth conditions and for growth only in media containing cholesterol is established with the methods described by Hungate (1969), with media and inocula maintained in closed vessels and exposure to air avoided during inoculation and incubation. The primary isolation medium and the clarified rumen fluid broth supplemented with cholesterol have been described (Robinson, 1983).

Genus I. Anaeroplasma Robinson, Allison and Hartman 1975, 179AL Daniel R. Brown, Janet M. Bradbury and Karl-Erik Johansson A.na.e.ro.plas¢ma. Gr. prefix an without; Gr. masc. n. aer air; Gr. neut. n. plasma a form; N.L. neut. n. Anaeroplasma intended to denote “anaerobic mycoplasma”.

Cells are predominantly coccoid, about 500 nm in diameter; clusters of up to ten coccoid cells may be joined by short filaments. Older cells have a variety of pleomorphic forms. Cells lack a cell wall and are bound by a single plasma membrane. Gram-stain-negative due to absence of cell wall. Obligately anaerobic; the inhibitory effect of oxygen on growth is not alleviated during repeated subcultures. Require sterol supplements for growth. Nonmotile. Optimal temperature, 37°C; no growth at 26 or 47°C. Optimal pH, 6.5–7.0. Surface colonies have a dense center with a translucent periphery, or “fried-egg” appearance. Subsurface colonies are golden, irregular, and often multilobed. Strains vary in their ability to ferment various carbohydrates. The products of carbohydrate fermentation include acids (generally acetic, formic, propionic, lactic, and succinic), ethanol, and gases (primarily CO2, but some strains also produce H2). Bacteriolytic and nonbacteriolytic strains have been described. Commensals in the bovine and ovine rumen. DNA G+C content (mol%): 29–34 (Tm, Bd). Type species: Anaeroplasma abactoclasticum Robinson, Allison and Hartman 1975, 179AL.

Further descriptive information Cells of Anaeroplasma examined by phase-contrast microscopy appear as single cells, clumps, dumbbell forms, and clusters of coccoid forms joined by short filaments. In electron micrographs of negatively stained preparations, pleomorphic forms are observed; these include filamentous cells, budding cells, and cells with bleb-like structures. All species examined have similar fermentation products of acetate, formate, lactate, ethanol, and carbon dioxide (Robinson et al., 1975). Anaeroplasma abactoclasticum is the only species known not to digest casein. Anaeroplasma abactoclasticum strains are the only ones known to produce succinate through fermentation. Anaeroplasma bactoclasticum, Anaeroplasma intermedium, and Anaeroplasma varium are the only species known to produce hydrogen and propionate during their fermentation. The roll-tube anaerobic culture technique (Hungate, 1969), with pre-reduced medium maintained in a system for exclusion of oxygen, is used to culture the organisms (Robinson, 1983; Robinson and Allison, 1975; Robinson et al., 1975;

Robinson and Hungate, 1973). Anaerobic mollicutes in a sewage sludge digester were cultured in an anaerobic cabinet (Rose and Pirt, 1981). Although it is possible that other types of anaerobic culture techniques might be acceptable (anaerobic culture jar or GasPak system), the effective use of such equipment has not been demonstrated (Robinson, 1983). Strains with bacteriolytic activity are detected with the addition of autoclaved Escherichia coli cells to the Primary Isolation Medium (PIM) described below. Clear zones around colonies of anaeroplasmas, when viewed by a stereoscopic microscope, are suggestive of bacteriolytic anaeroplasmas. Colonies can be subcultured to clarified rumen fluid broth (CRFB) medium described below. A slide agglutination test was first used to show that the antigens of anaerobic mollicutes were not related to established Mycoplasma or Acholeplasma species found in cattle (Robinson and Hungate, 1973). Later, the agglutination test was adapted to either a plate or tube test and combined with an agar gel diffusion test and a modified growth inhibition procedure to examine the antigenic interrelationships among the anaerobic mollicutes (Robinson and Rhoades, 1977). On the basis of these tests, a serological grouping of anaerobic mollicutes appeared compatible with the group separations based upon cultural, biochemical, and biophysical properties of the organisms (Robinson, 1979; Robinson and Rhoades, 1977). There is no current evidence for the pathogenicity of any of the Anaeroplasma species described so far. Obligately anaerobic mollicutes appear to be a heterogeneous group that has been found so far only in the rumen of cattle and sheep (Robinson, 1979; Robinson et al., 1975). Each new isolated group of these organisms seems to have different properties, suggesting that additional undescribed species are likely to exist. The ecological role of these organisms in the rumen has not been determined. Although the titer of these organisms in the rumen appears to be low when compared to titers of other rumen organisms, the mollicutes probably contribute to the pool of microbial fermentation products at that site. Growth of anaeroplasmas is inhibited by thallium acetate (0.2%), bacitracin (1000 mg/ml), streptomycin (200 mg/ml), and d-cycloserine (500 mg/ml), but not by benzylpenicillic acid (1000 U/ml).

Genus I. Anaeroplasma

Enrichment and isolation procedures The PIM medium used to grow and detect anaerobic mycoplasmas (Robinson, 1983; Robinson et al., 1975; Robinson and Hungate, 1973) contains: 40% (v/v) rumen fluid strained through cheesecloth, autoclaved, and clarified by centrifugation; 0.05% (w/v) glucose; 0.05% (w/v) cellobiose; 0.05% (w/v) starch; 3.75% (v/v) of a mineral solution consisting of 1.7 × 10−3 M K2HPO4, 1.3 × 10−3 M KH2PO4, 7.6 × 10−4 M NaCl, 3.4 × 10−3 M (NH4)2SO4, 4.1 × 10−4 M CaCl2, and 3.8 × 10−4 M MgSO4·7H2O; 0.2% (w/v) trypticase; 0.1% (w/v) yeast extract; 0.0001% (w/v) resazurin; 0.5% (w/v) autoclaved Escherichia coli cells; 0.4% (w/v) Na2CO3; 0.05% (w/v) cysteine hydrochloride; 1.5% (w/v) agar; and 0.0006% (w/v) benzylpenicillic acid. Pure cultures are established by picking individual colonies from PIM roll tubes and subculturing into CRFB medium. CRFB medium contains the same ingredients and concentrations as PIM, except: glucose, cellobiose, and starch concentrations are 0.2% (w/v), and autoclaved Escherichia coli cells, agar, and benzylpenicillic acid are omitted. Growth also occurs in a rumen fluid-free medium (Medium D) in which growth factors supplied in rumen fluid are replaced by lipopolysaccharide (Boivin; Difco) and cholesterol (Robinson, 1983; Robinson et al., 1975), or in a completely defined medium in which the trypticase, yeast extract, and lipopolysaccharide of Medium D are replaced by amino acids, vitamins, and phosphatidylcholine esterified with unsaturated fatty acids (Robinson, 1979, 1983). An agar-overlay plating technique carried out in an anaerobic hood has also been reported to be an effective isolation procedure (Robinson, 1979). Anaerobic mollicutes grow only in a prereduced medium maintained in a system for exclusion of oxygen. When resazurin is used in the test medium and becomes oxidized, mollicutes will fail to grow.

Maintenance procedures Cultures are viable after storage for as long as 5 years at −40°C in CRFB medium. They may also be preserved by lyophilization using standard techniques for other mollicutes (Leach, 1983). However, the type strains of several species of Anaeroplasma are no longer available from the American Type Culture Collection because it was impossible to revive the cultures sent by the depositors.

Differentiation of the genus Anaeroplasma from other genera Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et  al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. The obligately anaerobic nature of Anaeroplasma species is a distinctive and stable characteristic among these organisms. Strictly anaerobic growth plus the requirement for sterol supplements for growth exclude assignment to any other taxon in the class. Moreover, the bacteriolytic capability possessed by some of the anaeroplasmas has not been reported for other mycoplasmas. Plasmalogens (alkenyl-glycerol ethers), which are found in various anaerobic bacteria but not in aerobic bacteria, are major components of polar lipids from anaeroplasmas (Langworthy et al., 1975); this further supports the contention that anaeroplasmas are distinct from other mollicutes.

Taxonomic comments The first organism in the group to be described was referred to as Acholeplasma bactoclasticum (type strain JRT = ATCC 27112T; Robinson and Hungate, 1973) because the organism was

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thought to lack a sterol requirement for growth. Later, when other obligately anaerobic mollicutes were isolated, these and the JRT strain were found to require sterol for growth. A proposal was then made to form the new genus Anaeroplasma to accommodate strain JRT (as Anaeroplasma bactoclasticum; Robinson and Allison, 1975) and a second anaerobic mollicute ­designated Anaeroplasma abactoclasticum (Robinson et al., 1975). These developments prompted a proposal for an amended classification of anaerobic mollicutes, which included descriptions of Anaeroplasma varium and Anaeroplasma intermedium, the family Anaeroplasmataceae, and the order Anaeroplasmatales within the class Mollicutes (Robinson and Freundt, 1987). Early serological studies suggested the existence of several distinct species of anaeroplasmas. Subsequent reports on DNA– DNA hybridization, DNA base composition, and genome size comparisons of organisms in the group also indicated the existence of a number of species in two distinct genera of anaerobic mollicutes (Christiansen et al., 1986; Stephens et al., 1985). Strains initially assigned to Acholeplasma abactoclasticum [serovar 3, type strain 6-1T (=ATCC 27879T)] were found to be a single species with about 80% interstrain DNA–DNA hybridization. However, strains A-2T (serovar 1) and 7LAT (serovar 2), previously included in the description of Acholeplasma bactoclasticum, are the type strains of separate species designated Anaeroplasma varium and Anaeroplasma intermedium, respectively (Robinson and Freundt, 1987). Strains of Anaeroplasma all have DNA G+C contents in the range 29.3–33.7 mol%, whereas the base composition of serovar 4 strains 161T, 162, and 163 clustered above 40 mol%. These were assigned to the new genus Asteroleplasma [type strain 161T (=ATCC 27880T)] whose members are ­anaerobic, but do not require sterol for growth. Genome sizes ranged from 1542 to 1715 kbp for Anaeroplasma species, as determined by renaturation kinetics (Christiansen et al., 1986). Although the genome sizes reported were in the expected range for members of the class Mollicutes, no data are currently available on genome sizes estimated by the more accurate pulsed-field gel electrophoresis technique. A phylogenetic analysis of members of the Anaeroplasmatales, based upon 16S rRNA gene sequence comparison, was carried out by Weisburg et  al. (1989). Anaeroplasma and Acholeplasma are sister genera basal on the mollicute tree.

Acknowledgements The major contributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged.

Further reading Johansson, K.-E. 2002. Taxonomy of Mollicutes. In Molecular Biology and Pathogenicity of Mycoplasmas (edited by Razin and Herrmann). Kluwer Academic/Plenum Publishers, New York, pp. 1–29.

Differentiation of the species of the genus Anaeroplasma The technical challenges of cultivating these anaerobic mollicutes have led to a current reliance principally on the combination of 16S rRNA gene sequencing and reciprocal serology for species differentiation. Serological characterization of anaeroplasmas has been performed with agglutination, ­modified metabolism inhibition, and immunodiffusion tests (Robinson and Rhoades, 1977). Failure to cross-react with antisera against previously recognized species provides substantial evidence for species novelty. DNA–DNA hybridization values between species

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Family I. Anaeroplasmataceae

examined are less than 5%. Bacteriolytic and nonbacteriolytic organisms occur within the genus. When grown on agar media containing a suspension of autoclaved Escherichia coli cells, bacteriolytic strains form colonies surrounded by a clear zone

due to lysis of the suspended cells by a diffusible enzyme(s). On media lacking suspended cells, bacteriolytic and nonbacteriolytic strains of Anaeroplasma cannot be distinguished from each other on the basis of colonial or cellular morphology.

List of species of the genus Anaeroplasma 1. Anaeroplasma abactoclasticum Robinson, Allison and Hartman 1975, 179AL a.bac.to.clas¢ti.cum. Gr. pref. a without; Gr. bakt- (L. transliteration bact-) part of the stem of the Gr. dim. n. bakterion (L. transliteration bacterium) a small rod; N.L. adj. clasticus, -a, um (from Gr. adj. klastos, -ê, -on broken in pieces) breaking; N.L. neut. adj. abactoclasticum intended to denote “not bacteriolytic”. This is the type species of the genus Anaeroplasma. Cells are coccoid, about 500 nm in diameter, sometimes joined into short chains by filaments. Colonies on solid medium are subsurface, but nevertheless present a typical fried-egg appearance. Growth is inhibited by 20 mg/ml digitonin. A major distinguishing factor is the lack of the extracellular bacterioclastic and proteolytic enzymes that characterize the lytic species. No evidence of a role in pathogenicity. Source: occurs primarily in the bovine and ovine rumen. DNA G+C content (mol%): 29.3 (Bd). Type strain: 6-1, ATCC 27879. Sequence accession no. (16S rRNA gene): M25050. 2. Anaeroplasma bactoclasticum (Robinson and Hungate 1973) Robinson and Allison 1975, 186AL (Acholeplasma bactoclasticum Robinson and Hungate 1973, 180) bac.to.clas¢ti.cum. Gr. bakt- (L. transliteration bact-) part of the stem of the Gr. dim. n. bakterion (L. transliteration bacterium) a small rod; N.L. adj. clasticus, -a, um (from Gr. adj. klastos, -ê, -on broken in pieces) breaking; N.L. neut. adj. bactoclasticum bacteria-breaking. Pleomorphic and coccoid cells ranging in size from 550 to 2000 nm in diameter. Cells cluster and sometimes form short chains. Colonies on solid medium have a typical friedegg appearance. Optimal temperature for growth is between 30 and 47°C. Growth is inhibited by 20 mg/ml digitonin. Skim milk is cleared by a proteolytic, extracellular enzyme and certain bacteria are lysed by an extracellular enzyme

that attacks the peptidoglycan layer of the cell wall. Shares some serological relationship to other established species in the genus, but can be distinguished by agglutination, modified metabolism inhibition, and agar gel immunodiffusion precipitation tests. No evidence of pathogenicity. Source: occurs in the bovine and ovine rumen. DNA G+C content (mol%): 32.5 to 33.7 (Tm, Bd). Type strain: JR, ATCC 27112. Sequence accession no. (16S rRNA gene): M25049. 3. Anaeroplasma intermedium Robinson and Freundt 1987, 79VP in.ter.me¢di.um. L. neut. adj. intermedium intermediate. Cellular morphology and colonial features are similar to those of Acholeplasma bactoclasticum. Serologically distinct from other species in the genus by agglutination, metabolism inhibition, and agar gel immunodiffusion precipitation tests (Robinson and Rhoades, 1977). No evidence of pathogenicity. Source: occurs in the bovine and ovine rumen. DNA G+C content (mol%): 32.5 (Bd). Type strain: 7LA, ATCC 43166. Sequence accession no. (16S rRNA gene): not available. 4. Anaeroplasma varium Robinson and Freundt 1987, 79VP va¢ri.um. L. neut. adj. varium diverse, varied, intended to mean different from Anaeroplasma bactoclasticum. Cellular morphology and colonial features are similar to those of Acholeplasma bactoclasticum. Serologically distinct from other species in the genus by agglutination, metabolism inhibition, and agar gel immunodiffusion precipitation tests (Robinson and Rhoades, 1977). No evidence of pathogenicity. Source: occurs in the bovine and ovine rumen. DNA G+C content (mol%): 33.4 (Tm). Type strain: A-2, ATCC 43167. Sequence accession no. (16S rRNA gene): M23934.

Genus II. Asteroleplasma Robinson and Freundt 1987, 79VP Daniel R. Brown, Janet M. Bradbury and Karl-Erik johansson A.ste.rol.e.plas¢ma. Gr. pref. a not; N.L. neut. n. sterolum sterol; e combining vowel; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Asteroleplasma name intended to indicate that sterol is not required for growth.

Cellular and colonial morphology similar to species of the genus Anaeroplasma. Nonmotile. The three strains that form the new genus and species are obligately anaerobic and capable of growth in the absence of cholesterol or serum supplements. Temperature optimum for growth is 37°C. No evidence of bacteriolytic activity. The organisms are serologically distinct from other members in the family Anaeroplasmataceae. Occur in the ovine rumen. DNA G+C content (mol%): about 40 (Tm, Bd).

Type species: Asteroleplasma anaerobium Robinson and Freundt 1987, 79VP.

Further descriptive information The most prominent characteristics of the organisms are strictly anaerobic growth and growth in the absence of sterol supplements. The G+C contents of strains analyzed to date are higher than the values for Anaeroplasma species (Stephens et al., 1985). DNA–DNA reassociation values clearly show that strains 161T,

Genus II. Asteroleplasma

162, and 163 of Asteroleplasma anaerobium are genetically related and distinct from established species in the genera Acholeplasma or Anaeroplasma (Stephens et  al., 1985). Tube agglutination tests and gel diffusion precipitation tests showed that strains assigned to Asteroleplasma anaerobium are serologically distinct from Anaeroplasma species (Robinson and Rhoades, 1977). No data have been reported on antibiotic sensitivity or pathogenicity of asteroleplasmas. Strains have been isolated only from sheep rumen. Isolation and maintenance techniques are similar to those reported for Anaeroplasma species.

Differentiation of the genus Asteroleplasma from other genera Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et  al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. The obligately anaerobic nature of Asteroleplasma is a distinctive and stable characteristic. Strictly anaerobic growth plus growth in the absence of sterol supplements exclude assignment to any other taxon in the class. Extracellular bacteriolytic and proteolytic enzymes are absent. Growth is not inhibited by 20 mg/ml digitonin.

Taxonomic comments The taxonomic position of strains 161T, 162, and 163 of obligately anaerobic mollicute serovar 4 was delineated through observations that they did not require sterol for growth (Robinson

723

et al., 1975), were serologically distinct (Robinson and Rhoades, 1977), and had G+C contents that were much higher than those of Anaeroplasma species (Christiansen et al., 1986). Less than 5% DNA–DNA relatedness existed between these strains and species assigned to the genus Anaeroplasma (Stephens et al., 1985). Lastly, the significance of the group and the need to clarify its taxonomic status was emphasized when it was demonstrated that a significant proportion of the anaerobic mollicute population in the bovine and ovine rumen does not require sterol for growth (Robinson and Rhoades, 1982). The phylogenetic analysis of Weisburg et al. (1989) indicated that Asteroleplasma anaerobium had branched from the Firmicutes lineage independently of Acholeplasma and Anaeroplasma. Further, Asteroleplasma shared two of three important synapomorphies that united the Mycoplasma and Spiroplasma lineages. Thus, the question of possible monophyly and the true phylogenetic position of Asteroleplasma with respect to other mollicutes remains open.

Acknowledgements The major contributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged.

Further reading Johansson, K.-E. 2002. Taxonomy of Mollicutes. In Molecular Biology and Pathogenicity of Mycoplasmas (edited by Razin and Herrmann). Kluwer Academic/Plenum Publishers, New York, pp. 1–29.

List of species of the genus Asteroleplasma 1. Asteroleplasma anaerobium Robinson and Freundt 1987, 79VP a.na.e.ro¢bi.um. Gr. pref. an not; Gr. n. aer air; Gr. n. bios life; N.L. neut. adj. anaerobium not living in air. This is the type species of the genus Asteroleplasma. Cell morphology and colonial characteristics are similar to those of other members of the order Anaeroplasmatales. Strains 161T, 162, and 163 form a homogeneous and distinct serological group, as judged by about 80% DNA–DNA ­hybridization and

References Brown, D., R. Whitcomb and J. Bradbury. 2007. Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. Christiansen, C., E.A. Freundt and I.M. Robinson. 1986. Genome size and deoxyribonucleic acid base composition of Anaeroplasma abactoclasticum, Anaeroplasma bactoclasticum, and a sterol-nonrequiring anaerobic mollicute. Int. J. Syst. Bacteriol. 36: 483–485. Hungate, R.E. 1969. A roll tube method for cultivation of strict anaerobes. In Methods in Microbiology, vol. 3B (edited by Norris and ­Ribbons). Academic Press, London, pp. 117–132. Langworthy, T., W. Mayberry, P. Smith and I. Robinson. 1975. Plasmalogen composition of Anaeroplasma. J. Bacteriol. 122: 785–787. Leach, R.H. 1983. Preservation of Mycoplasma cultures and culture collections. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 197–204. Robinson, I.M. and M.J. Allison. 1975. Transfer of Acholeplasma bactoclasticum Robinson and Hungate to genus Anaeroplasma (Anaeroplasma bactoclasticum Robinson and Hungate comb. nov.), emended description of species. Int. J. Syst. Bacteriol. 25: 182–186. Robinson, I.M., M.J. Allison and P.A. Hartman. 1975. Anaeroplasma abactoclasticum gen. nov., sp. nov., obligately anaerobic mycoplasma from rumen. Int. J. Syst. Bacteriol. 25: 173–181.

serological agglutination, metabolism inhibition, and agar gel immunodiffusion precipitation tests. No evidence of pathogenicity. Source: all isolates have been identified from the bovine or ovine rumen. DNA G+C content (mol%): 40.2–40.5 (Bd, Tm). Type strain: 161 (the type strain ATCC 27880 no longer exists). Sequence accession no. (16S rRNA gene): M22351. Robinson, I.M. and K.R. Rhoades. 1977. Serological relationships between strains of anaerobic mycoplasmas. Int. J. Syst. Bacteriol. 27: 200–203. Robinson, I.M. 1979. Special features of anaeroplasmas. In The Mycoplasmas, vol. 1 (edited by Barile and Razin). Academic Press, New York, pp. 515–528. Robinson, I.M. and K.R. Rhoades. 1982. Serologic relationships between strains of anaerobic mycoplasmas. Rev. Infect. Dis. 4: S271. Robinson, I.M. 1983. Culture media for anaeroplasmas. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 159–162. Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended classification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81. Robinson, J.P. and R.E. Hungate. 1973. Acholeplasma bactoclasticum sp. n., an anaerobic mycoplasma from the bovine rumen. Int. J. Syst. Bacteriol. 23: 171–181. Rose, C. and S. Pirt. 1981. Conversion of glucose to fatty acids and methane: roles of two mycoplasmal agents. J. Bacteriol. 147: 248–254. Stephens, E., I. Robinson and M. Barile. 1985. Nucleic acid relationships among the anaerobic mycoplasmas. J. Gen. Microbiol. 131: 1223–1227. Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. ­Mandelco, J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic analysis of the mycoplasmas: basis for their classification. J. Bacteriol. 171: 6455–6467.

Phylum XVII. Acidobacteria phyl. nov. J. Cameron Thrash and John D. Coates A.ci¢do.bac.ter¢i.a. Acidobacteriales type order of the phylum; removing the -ales ending and inserting -a to denote phylum; N.L. neut. n. Acidobacteria the phylum of Acidobacteriales.

The phylum currently contains two classes, three orders, three families, and six described genera. The phylum is identified on the basis of phylogenetic analysis of 16S rRNA gene sequences (Figure 117). Culture-independent methods have identified significant diversity within the phylum based on 16S rRNA gene sequences (Barns et al., 1999; Hugenholtz et al., 1998; Kuske et al., 1997; Ludwig et  al., 1997; Meisinger et  al., 2007; Quaiser  et  al., 2003; Zimmermann et  al., 2005). Currently, more than 3000 ­Acidobacteria sequences exist in public databases making up 26 coherent groups within the phylum (Barns et  al., 2007). Acidobacteria sequences have been detected in a wide variety of habitats including soils, sediments, hot springs, marine

snow, feces (Barns et al., 1999), a variety of cave environments (Meisinger et al., (2007), and references therein), and metalcontaminated soils (Barns et al., 2007). Although few of these representatives have been cultivated, many new isolates have recently been reported ( Janssen et  al., 2002; Joseph et  al., 2003; McCaig et  al., 2001; Sait et  al., 2006, 2002; Stevenson et al., 2004). Future analysis of these additional isolates should improve understanding of the various functional roles played by members of this phylum, supplementing that for the six genera already described. Table 145 provides characteristics that can be used to distinguish the six genera. Type order: Acidobacteriales ord. nov.

Aquifex aeolicus Verrucomicrobium spinosum Magnetospirillum magnetotacticum Agrobacterium tumefaciens Dechloromonas agitata Escherichia coli Pseudomonas aeruginosa Geobacter metallireducens Acanthopleuribacterales

Holophagales

Acanthopleuribacter pedis FYK2218 (AB303221) Geothrix fermentans H5 (U41563)

Holophagae

Holophaga foetida TMBS4 (X77215) Edaphobacter modestus Jbg-1 (DQ528760) Edaphobacter aggregans Wbg-1 (DQ528761) Acidobacterales

Acidobacteria Acidobacteriia

Terriglobus roseus KBS 63 (DQ660892) Acidobacterium capsulatum 161 (D26171) Chloroflexus aggregans Brevibacterium equis

FIGURE 117.  Phylogenetic tree of the Acidobacteria based on the 16S rRNA gene. The phylum contains six described

genera in two classes, three orders, and three families. The class Acidobacteriia contains the genera Acidobacterium, Edaphobacter, and Terriglobus in a single family, Acidobacteriaceae, in the order Acidobacteriales. The class Holophagae contains the genera Holophaga, and Geothrix in the family Holophagaceae in the order Holophagales, and the genus Acanthopleuribacter in the family Acanthopleuribacteraceae in the order Acanthopleuribacterales. Bar = 0.07 substitutions per site. Tree was constructed using Bayesian analysis (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). 725

d-Glucose, l-aspartate, l-glutamate, l-glutamine, l-ornithine, l-arabinose, d-fructose, d-glucosamine, d-lactose, d-lyxose, trehalose, d-xylose, d-glucuronic acid, l-rhamnose, d-sorbitol, l-lyxitol, d-mannitol, myo-inositol, xylitol, Casamino acids, yeast extract, peptone 30 15–37 5.5 4.0–7.0 na 55.8–56.9

− −

+/− na − + Oxygen

0.5–0.9 × 1–2.1 Beige

Edaphobacter

12–23 12–23 5–6 4.5–7.0 na 59.8

d-Glucose, d-fructose, d-galactose, d-mannose, d-xylose, d-arabinose, sucrose, d-maltose, d-raffinose, d-cellobiose, succinate, d-glucuronate, d-gluconic acid

– –

– – na + Oxygen

0.5–0.7 × 0.9–1.4 White or pink

Terriglobus

28–32 10–35 6.8–7.5 5.5–8.0 0.1–1.5 62.5

+ 3,4,5-Trimethoxybenzoate, syringate, 5-hydroxyvanillate, phloroglucinolmonomethylether, sinapate, ferulate, caffeate, gallate, 2,4,6-trihydroxybenzoate, pyrogallol, phloroglucinol, and pyruvate –

– – – – –

0.5–0.7 × 1–3 Beige

Holophaga

b

ASW, Artificial Seawater, Nagai Chemical Products.

Symbols: +, present or confirmed capability; -, absent/none or confirmed incapability; na, not available; aqds, anthraquinone-2,6-disulfonate.

na 25–37 na 3–6 50 are shown on the left or above corresponding branches and are based on 1000 replicates. 737

738

Phylum XVIII. Fibrobacteres

TABLE 146.  Taxonomic divisions within the phylum Fibrobacteres

Delineation [Max. (mean ± SD)]a Within lineage

To Fibrobacter succinogenes str. S85c

0.29 (0.18 ± 0.06) 0.18 (0.10 ± 0.06) 0.11 (0.07 ± 0.03)

0.25 (0.15 ± 0.07) 0.16 (0.08 ± 0.06) 0.09 (0.05 ± 0.03)

0.06 (0.05 ± 0.01)

0.06 (0.04 ± 0.02)

0.04

0.09 (0.09 ± 0.00)

Unclassified Fibrobacteria

0.18 (0.14 ± 0.03)

0.16 (0.13 ± 0.02)

Class II. Fibrobacteria-2 Class III. Environmental clones

0.14 (0.09 ± 0.02) 0.19 (0.14 ± 0.04)

0.22 (0.20 ± 0.01) 0.25 (0.22 ± 0.02)

Lineage Phylum. Fibrobacteres Class I. Fibrobacteria Order I. Fibrobacterales Family I. Fibrobacteraceae Genus I. Fibrobacter F. succinogenes F. intestinalis

Isolate sourcesb Environmental clones

Cultivated isolates

Equine cecum Bovine rumen na

Ovine rumen Bovine rumen Ovine rumen Rat cecum Porcine cecum na

Termite gut Sulfidic cave stream biofilm Acid-impacted lake Termite gut Soil and waterd

na na

a Delineation values are derived from a distance matrix of all sequences in Figure 118, based on Jukes–Cantor-corrected distances. Maximum delineation values are the maximum pairwise distances between any two sequences within a given lineage; mean delineation values are the mean distances between any two sequences from Figure 118 within a given lineage.

na, Not applicable.

b

Pairwise distances between sequences from all lineages were compared to that from Fibrobacter succinogenes str. S85, used as a reference.

c

Water source was 10 m downstream from manure application site.

d

98 98

98 100

Pericapritermes sp. gut clone Pn-Fib-2 Termes comi s gut clone Tc-Fib-6 Reticulitermes sp. gut clone Ram-Fib-2 California grassland soil clone FCPN637 Oklahoma mixed grass prairie soil clone FFCH12636 water (10 m downstream manure) clone 258ds10 Gemmatimonas aurantiaca

Fibrobacteria-2

88 100

Fibrobacteria

95

Fibrobacterales

Fibrobacter succinogenes subsp. succinogenes str. BL2 Fibrobacter succinogenes subsp. succinogenes str. S85 Fibrobacter succinogenes subsp. elongatus str. HM2 97 Fibrobacter succinogenes str. MC1 100 Fibrobacter succinogenes str. GC5 Fibrobacter intestinalis str. NR9 100 Fibrobacter intestinalis str. JG1 acid-impacted lake clone ADK-MOh02-47 100 sulfidic cave stream biofilm clone PC06110-B11 Macrotermes sp. gut clone MgMjR-014 Nasutitermes sp. gut clone Nt2-109 Nasutitermes sp. gut clone Nt2-023 Microtermes sp. gut clone M1NP2 42 58 Microtermes sp. gut clone M1PT4 22 52 Microtermes sp. gut clone M1PT4 02 86 Microtermes sp. gut clone M2PT2 01 98 Microtermes sp. gut clone M2PT2 90 95

100

98

Environmental Clones

0.10 FIGURE 119.  Distance phylogram of species within the three classes of the phylum Fibrobacteres. Environmental clone sequences are only included in lineages with no cultivated representatives. The tree was constructed from nearly full-length (>1300 bp) 16S rRNA gene sequences using the neighbor-joining algorithm with Jukes–Cantor corrected distances. Bootstrap values >50 are shown above corresponding branches and are based on 1000 replicates.

Phylum XVIII. Fibrobacteres

References Garrity, G.M. and J.G. Holt. 2001. The Road Map to the Manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 1, The Archaea and the Deeply Branching and Phototrophic Bacteria (edited by Boone, Castenholz and Garrity). Springer, New York, pp. 119–166. Griffiths, E. and R.S. Gupta. 2001. The use of signature sequences in different proteins to determine the relative branching order of bacterial divisions: evidence that Fibrobacter diverged at a similar time to Chlamydia and the Cytophaga–Flavobacterium–Bacteroides division. Microbiology 147: 2611–2622. Gupta, R.S. 2004. The phylogeny and signature sequences characteristics of Fibrobacteres, Chlorobi, and Bacteroidetes. Crit. Rev. Microbiol. 30 : 123–143. Gupta, R.S. and E. Lorenzini. 2007. Phylogeny and molecular signatures (conserved proteins and indels) that are specific for the Bacteroidetes and Chlorobi species. BMC Evol. Biol. 7: 71.

739

Hongoh, Y., P. Deevong, T. Inoue, S. Moriya, S. Trakulnaleamsai, M. Ohkuma, C. Vongkaluang, N. Noparatnaraporn and T. Kudo. 2005. Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Appl. Environ. Microbiol. 71: 6590–6599. Hongoh, Y., P. Deevong, S. Hattori, T. Inoue, S. Noda, N. Noparatnaraporn, T. Kudo and M. Ohkuma. 2006a. Phylogenetic diversity, localization, and cell morphologies of members of the candidate phylum TG3 and a subphylum in the phylum Fibrobacteres, recently discovered bacterial groups dominant in termite guts. Appl. Environ. Microbiol. 72: 6780–6788. Hongoh, Y., L. Ekpornprasit, T. Inoue, S. Moriya, S. Trakulnaleamsai, M. Ohkuma, N. Noparatnaraporn and T. Kudo. 2006b. Intracolony variation of bacterial gut microbiota among castes and ages in the fungus-growing termite Macrotermes gilvus. Mol. Ecol. 15: 505–516.

Class I. Fibrobacteria class. nov. Anne M. Spain, Cecil W. Forsberg and Lee R. Krumholz Fi.bro.bac.te¢ri.a. N.L. masc. n. Fibrobacter type genus of the type order of the class; suff. -ia ending proposed by Gibbons and Murray and by Stackebrandt et al. to denote a class; N.L. neut. pl. n. Fibrobacteria the Fibrobacter class. The class Fibrobacteria is circumscribed here on the basis of 16S rRNA sequences. The class contains the single type order Fibrobacterales ord. nov.

Order I. Fibrobacterales ord. nov. Anne M. Spain, Cecil W. Forsberg and Lee R. Krumholz Fi.bro.bac.ter.a¢les. N.L. masc. n. Fibrobacter type genus of the order; -ales ending to denote order; N.L. fem. pl. n. Fibrobacterales the Fibrobacter order. The order Fibrobacterales is circumscribed herein on the basis of phylogenetic sequences. The order contains the sole family, Fibrobacteraceae fam.nov. Cells are rod-shaped of varying lengths and are capable of growth on cellulose. Members of the genus Fibrobacter are anaerobic. Type genus: Fibrobacter Montgomery, Flesher and Stahl 1988, 434VP.

Reference Montgomery, L., B. Flesher and D. Stahl. 1988. Transfer of Bacteroides succinogenes (Hungate) to Fibrobacter gen. nov. as Fibrobacter succinogenes comb. nov. and description of Fibrobacter intestinalis sp. nov. Int. J. Syst. Bacteriol. 38: 430–435.

Family I. Fibrobacteraceae fam. nov. Anne M. Spain, Cecil W. Forsberg and Lee R. Krumholz Fi.bro.bac.ter.a¢ceae. N.L. masc. n. Fibrobacter type genus of the family, -aceae ending to denote family; N.L. fem. pl. n. Fibrobacteraceae the Fibrobacter family. Cells are Gram-stain-negative obligately anaerobic and rod-toovoid in shape depending on the strain and the culture conditions. Older cultures form sphaeroplasts. Motility appears to occur by gliding, but cannot be observed microscopically. Flagella have not been detected. Membranes are composed of straight-chain fatty acids and phospholipids are predominantly ethanolamine plasmalogens.

All of the strains grow by degrading cellulose and cellulose containing plant compounds. Cells will also grow on the soluble sugars, glucose, or cellobiose. The major fermentation products are succinate and acetate. Although bacteria will grow on soluble forms of cellulose, they are thought to exist primarily as cells that are directly attached to decomposing plant material. Type genus: Fibrobacter Montgomery, Flesher and Stahl 1988, 434VP.

740

Family I. Fibrobacteraceae

Genus I. Fibrobacter Montgomery, Flesher and Stahl 1988, 434VP Anne M. Spain, Cecil W. Forsberg and Lee R. Krumholz Fi¢bro.bac¢ter. L. fem. n. fibra fiber or filament in plants or animals; N.L. masc. n. bacter rod or staff; N.L. masc n. Fibrobacter bacterial rod that subsists on fiber.

Cells are rod-shaped (0.3–0.5 mm × 0.8–1.6 mm) or ovoid (0.8– 1.6 mm × 0.8–1.6 mm). Obligately anaerobic non-sporing and not detectably motile by microscopy. They are able to migrate through agar when growing on cellulose, suggesting a gliding form of motility. Ferment a narrow range of carbohydrates including glucose, cellobiose, and cellulose. Other sugars including lactose or maltose are fermented by a few species. The fermentation products are acetate and succinate, and sometimes formate at low levels. Cells require CO2, straight-chain and branched-chain fatty acids in the media as well as ammonia as the N source. Membranes are composed of straight-chain fatty acids, and phospholipids are predominantly ethanolamine plasmalogens. Habitat is the mammalian gastrointestinal tract. DNA G+C content (mol%): 45–51. Type species: Fibrobacter succinogenes Montgomery, Flesher and Stahl 1988, 434VP.

Taxonomic comments and further descriptive information The first pure culture of Fibrobacter was described as Bacteroides succinogenes by (Hungate, 1950). Prévot (1966) considered Bacteroides succinogenes to belong in the genus Ruminobacter, but the name Ruminobacter succinogenes was not validly published. More recently, phylogenetic analysis showed that strains of Bacteroides succinogenes were phylogenetically distinct from other species of Bacteroides necessitating the formation of a new genus (Montgomery et al., 1988). Sequences of small-subunit rRNA of several strains were shown to have less than 72% similarity with Bacteroides fragilis providing evidence that these organisms constituted a distinct evolutionary line of descent at the phylum level. There are currently no phenotypic characteristics that are useful for distinguishing the two species Fibrobacter intestinalis and Fibrobacter succinogenes. Small-subunit rRNA analysis must be used. Fibrobacter succinogenes subsp. succinogenes can be distinguished from Fibrobacter succinogenes subsp. elongatus based on cell morphology, the former being ovoid and the latter more slender and rod-shaped. Within the genus, strains of Fibrobacter succinogenes subsp. succinogenes and Fibrobacter succinogenes subsp. elongatus have a 16S rRNA sequence similarity of 95.3–98.1% (Amann et al., 1992) and DNA hybridization of less than 20%. Between the two species, the 16S rRNA similarity is 91.8–92.9%, with a DNA hybridization of less than 10%.

Enrichment and isolation procedures All of the isolates to date have been obtained from the gastrointestinal (GI) tract and have been described as two species within Fibrobacter. These organisms were isolated by directly diluting GI tract contents into roll tubes containing media with milled filter paper as substrate. Hungate originally isolated Fibrobacter succinogenes using an agar mineral medium containing clarified rumen fluid (a source of nutrients) and acid treated milled cotton (Hungate, 1950) in a mineral solution containing bicarbonate and a reducing agent. This original medium contained 1–2% agar and involved shake or roll tubes. Bryant and Doetsch (1954) later isolated a variety of strains from the rumen, most

likely using similar techniques. Much more recently, an effective technique was described by Macy et al. (1982), which involves diluting the contents of the rat cecum using a mineral solution buffered with 0.5% NaHCO3 and reduced with cysteine and ­sulfide. Dilutions were then used as inoculum for a similar agar medium containing 0.5% agar and 0.5% pebble-milled Whatman no. 1 filter paper. Each serum tube (1.6 × 15 cm) contained a low volume (3 ml) of agar medium. Tubes were then cooled on a tube roller producing an extremely thin agar layer. Tubes were then incubated at 39°C for several days. Cells do not form colonies under these conditions, but the clearings due to cellulose hydrolysis can be easily visualized in this soft thin agar. Cells were picked from the cleared areas and transferred to a rich sugar based medium. Alternatively, Fibrobacter succinogenes can be isolated from enrichment cultures (Stewart et  al., 1981). The enrichment media contained minerals, vitamins, fatty acids, and dewaxed cotton (0.25%). After approximately five transfers in the enrichment media, colonies of Fibrobacter succinogenes are isolated by diluting into roll tubes containing a cellobiose-based medium. The above isolation techniques are tedious and also technically challenging. As such, they have not been used commonly outside of the rumen microbiology field. It is likely that this is the main reason why there are no isolates from any environment other than the GI tract.

Miscellaneous comments Accessibility to cellulose digestion sites in the cell-wall matrix has been suggested as the rate-limiting factor in cellulose digestion (Dehority and Tirabasso, 1998). It has been proposed that access to cell-wall polymers is limited by the small pore size between polymers, which is on the order of 2–4 nm. This size is not sufficient to allow free diffusion of simple globular enzymes with masses greater than 20 kDa into the wall matrix (Gardner et  al., 1999). Since the plant cell wall is a matrix of different polymers, a combination of cellulase and hemicellulase enzymes acting simultaneously is therefore essential. It was originally assumed that Fibrobacter succinogenes cells contained an array of the key enzymes for plant cell-wall biodegradation because it can burrow into the plant cell-wall matrix (Cheng et al., 1983). Insight into this conclusion was clearly documented (Matulova et al., 2005) by growth of Fibrobacter succinogenes strain S85 on 13 C enriched wheat straw and by assessment of the products of hydrolysis using a combination of nuclear magnetic resonance spectroscopy, and sugar linkage and compositional analysis. They observed the absence of acetylated xylooligosaccharides among the hydrolysis products despite the highly acetylated state of wheat straw cell-wall materials (Bourquin and Fahey, 1994), thereby documenting extensive enzymic deacetylation. Free sugars and polymers that accumulated in the culture fluid were xylose, arabinose, and arabinoglucuronoxylan oligosaccharides indicating extensive hemicellulase action. They also observed simultaneous degradation of hemicellulose and cellulose, and furthermore, amorphous and crystalline regions of cellulose were degraded at the same rate, which supported the

Genus I. Fibrobacter

concept of the concerted action of numerous enzymes in the surface degradation of the cell walls. Glucose did not accumulate in the medium, indicating rapid utilization of plant cell oligosaccharides with minimal cellodextrin export and recycling as previously suggested (Wells et al., 1995). The accumulation of xylose and arabinose occurred as was expected since they are not used as a carbon source (Matte and Forsberg, 1992; Matte et al., 1992). However, 4-OMe-a-glucuronic acid was not detected despite the presence of a-glucuronidase (Smith and Forsberg, 1991), which may be explained by low activity of the enzyme. This research clearly documented that digestion of the cell-wall matrix by Fibrobacter succinogenes involves the interaction of a complex array of fibrolytic enzymes. Enzymology and cloning studies prior to sequencing the genome of Fibrobacter succinogenes S85 documented the presence of seven endoglucanases, as well as a cellodextrinase, a chloride-stimulated cellobiosidase, a lichenase, and a a-glucuronidase (Forsberg et  al., 2000). Added to this array of enzymes were at least three xylanases (Jun et  al., 2003), an arabinose debranching xylanase (Matte and Forsberg, 1992), three acetyl xylan esterases (Kam et al., 2005; McDermid et al., 1990a), an arabinofuranosidase, and a ferulic acid esterase (McDermid et al., 1990b). The debranching nature of both the acetylxylan esterase and the xylanase clearly have very important roles in plant cell-wall biodegradation because they improve access of other enzymes to previously unavailable substrate. Bera-Maillet et  al. (2004) conducted experiments to determine whether ten glycosyl hydrolase genes, previously cloned from Fibrobacter succinogenes S85, were present in other strains of Fibrobacter succinogenes and in Fibrobacter intestinalis strain NR9. Almost all of the glycosyl hydrolase genes were detected in strains of Fibrobacter succinogenes closely related to strain S85, and a few were present in Fibrobacter intestinalis NR9. Only celF, a member of glycosyl hydrolase family 51, a 118-kDa endoglucanase of carbohydrate binding module families 11 and 30, was detected in all strains of Fibrobacter succinogenes and in Fibrobacter intestinalis NR9. A concluding remark from this study was that Fibrobacter succinogenes strain S85 is a representative model of the species for studying the mechanism of cellulose and plant cellwall digestion. To elucidate the role of adhesion of Fibrobacter cells to microcrystalline cellulose in the digestion of cellulose, adhesion defective mutants were isolated from both Fibrobacter succinogenes S85 (Gong and Forsberg, 1989) and Fibrobacter intestinalis DR7 (Miron and Forsberg, 1998). In both studies it was observed that the mutants either grew more slowly on cellulose or not at all, but their growth on glucose was unaffected, which suggested that adhesion was essential for cellulose digestion. Sequencing of the genome of Fibrobacter succinogenes strain S85 has dramatically modified the approach to studies on the mechanism of plant cell-wall digestion. The genome of Fibrobacter succinogenes S85 is 3.8 Mbp and contains 3252 open reading frames putatively encoding proteins. Of these, 120 appear to encode enzymes involved in plant cell-wall biodegradation on the basis of amino acid sequence similarity to plant cell-wall-degrading enzymes of other bacteria and fungi. The degradative enzymes include 91 glycosyl hydrolases, 14 carbohydrate esterases, and nine pectate hydrolases (Morrison et  al., 2003). The glycosyl hydrolases include 33 cellulases (in families 5, 8, 9, 10, 44, 45, 51, and 74), and 27 xylanases (mainly in families 8, 10, 11, 30,

741

39, and 43). Only five of these cellulases have cellulose binding modules (CBMs) while 13 of the xylanases have CBMs. Hemicellulose debranching enzymes include 14 cinnamoyl esterases (ten containing CBMs) and nine pectate lyases (four containing CBMs). From a count of putative plant cell-wall-degrading enzymes, the number present in Fibrobacter succinogenes exceeds those present in Thermobifida fusca, Clostridium thermocellum, and Cytophaga hutchinsonii. However, no proteins were found with similarity to scaffoldin, cohesin, or dockerin proteins characteristic of cellulosomal cellulase complexes present in the ruminal bacteria Ruminococcus albus and Ruminococcus flavefaciens and the cellulolytic ruminal fungi (Doi and Kosugi, 2004; Lynd et  al., 2002). Furthermore, there were no genes encoding cellulases from families 6 and 48, which typically contain exoglucanases found in both cellulosomal and non-cellulosomal cellulase systems that degrade crystalline cellulose, for example, those of Thermobifida fusca and Clostridium thermocellum. Furthermore, in contrast to the cellulase systems of Thermobifida fusca and Clostridium thermocellum, which once synthesized remain active, that of Fibrobacter succinogenes is inactivated upon suspension of growth (Maglione et al., 1997). This further supports the conclusion that the mechanism of cellulose digestion by Fibrobacter succinogenes is very different from that of other organisms in nature. The availability of the genome has not led to an immediate solution of the mechanism of cellulose digestion by Fibrobacter succinogenes. Nonetheless, it has greatly enhanced our ability to dissect the cellulase system through the application of one- and two-dimensional electrophoresis and mass spectroscopy of tryptic digests of nanogram quantities of proteins. A total of 11 novel cellulose binding proteins were identified as important candidates for roles in adhesion of cells to cellulose as all were absent from the outer membrane of a mutant strain of Fibrobacter succinogenes S85 incapable of binding to cellulose. None of these proteins contained a classic CBM (Jun et  al., 2007). Some of these proteins have affinity specifically for amorphous cellulose, as documented, with a second non-adherent mutant that bound to amorphous cellulose, but not to crystalline cellulose, while others are specific for crystalline cellulose. The binding properties of the proteins were determined by mixing detergent-solubilized outer membranes from the two mutants and the wild-type bacterium with amorphous and crystalline cellulose, and analysis of the bound proteins by SDS-PAGE and mass spectrometry. One of these proteins was a pilin, and a role for pili in binding of Ruminococcus albus to cellulose has been documented (Pegden et  al., 1998). In the same study, 16 proteins induced by growth of Fibrobacter succinogenes on cellulose were also identified, and this included Cel10A (FSU0257) a previously characterized chloride-stimulated cellobiosidase (Huang et  al., 1988; Jun et  al., 2007). A critical question is whether all of the putative glycosyl hydrolases are expressed at a level sufficiently high to have a role in plant cell-wall digestion, or whether only selected hydrolase enzymes are responsible for this phenotype. Several published studies (Jun et al., 2007; Qi et al., 2007, 2008a) and unpublished data have led to the identification of a short list of glycoside hydrolases that are expressed by Fibrobacter succinogenes S85 (Table 147). However, this is not the complete list since the nucleotide sequences of several biochemically characterized xylanases (Matte and Forsberg, 1992), acetyl xylan esterases (McDermid et al., 1990a), feruloyl

7.3 5.0

54.7 62.5

+ −

+ + +

+

+ +

+ + +

+

+

+

+

+

+ + + + + + + + + +

+ +

CD

+ + + + + + + + + +

+ −

SP

− −

6 6 6

−

−

− − 11, 11 − − − 11, 30, 30 11 − −

4 −

CBM

− −

−

−

− − − − + + − − − −

− −

Fn

− +

−

+

− − − − − + − − + +

− −

Ig

Modular structure

− −

10.5 (69 r)

− − 10.8 10.3 9.6 − 9.8 − 11.4 (46 r) 11.6 (46 r)

− −

BTD

+ −

+

+

+ + + −

+

− −

Unknown

Unpublished Bera-Maillet et al. (2000), Broussolle et al. (1994)

Paradis et al. (1993), Zhu et al. (1994) Jun et al. (2003) Jun et al. (2003) Jun et al. (2003)

Qi et al. (2008a)

Qi et al. (2007) Unpublished Qi et al. (2007) Qi et al. (2007) Jun et al. (2007) Unpublished Malburg et al. (1997) McGavin et al. (1989) Qi et al. (2007) Malburg et al. (1996) Iyo and Forsberg (1996) Teather and Erfle (1990), Wen et al. (2005)

Qi et al. (2007) Iyo and Forsberg (1994)

Reference(s)

b

Abbreviations: FSU, Fibrobacter succinogenes; Mw, molecular weight; IP, isoelectric point; SP, signal peptide; CD, catalytic domain; CBM, carbohydrate-binding module; Fd, fibronection-like domain; Ig, immunoglobin-like domain; BTD, basic terminal domain, number of residues (r) in the domain is in parentheses.

a

Symbols: +, present; −, absent.

5.34 5.12 5.25

64.9 68.8 66.7

2294 Xyn10C (XynB) 2292 Xyn10A (XynD) 2292 Xyn10B (XynE) Uncertain level of expression: 1346 Cel5K 451 Cel9G (EGB)

6.2

66.4

5.27

5.2 4.8 5.2 5.4 4.9 5.0 7.3 4.6 5.8 5.9

Xyn11C (XynC)

52.8 40.2 96.8 76.5 231.0 213.6 116.3 70.8 64.7 69.1 60.3 35.2m

Cel45C Cel5B Cel5H Cel8B Cel9H Cel9I Cel51A (CelF) Cel5G (Cel-3) Cel9B (CelE) Cel9C (CelD) Cel5K (CelG) Lic16C (mlg)

9.0 5.3

IP

77m

60.2 40.6

Mw

Cel10A (ClCBase) Cel5C (CedA)

Name

Cel9D

Glucan glucohydrolase: 2558 Xylanases: 777

Exoglucanase-like: 257 2070 Endoglucanases: 1947 2005 2914 2303 809 810 382 2772 2361 2362 1346 226

FSU#

TABLE 147.  Cellulase and hemicellulase genes known to be expressed in Fibrobacter succinogenes S85 a,b

742 Family I. Fibrobacteraceae

Genus I. Fibrobacter

esterase(s) (McDermid et al., 1990b), and an a-glucuronidase (Smith and Forsberg, 1991) have not been determined. It will be important to obtain the sequences of these genes, because of their potentially important roles in hemicellulose digestion. Beginning with highly expressed cellulases, the synergistic interaction of the enzymes was studied (Qi et al., 2007). These experiments led to the conclusion that the two predominant endoglucanases Cel51A (FSU0382) and Cel9B (FSU2361) formerly characterized as endoglucanases 1 and 2, respectively (McGavin and Forsberg, 1988), probably play central roles in cellulose digestion. However, one of the most interesting enzymes recently identified is a glucan glucohydrolase Cel9D (FSU2558) that preferentially cleaves glucose residues from the non-reducing ends of long chains of glucose residues, but does not readily hydrolyze cellobiose (Qi et  al., 2008a). It exhibits a strong synergistic interaction with Cel51A (FSU0382) and Cel8B (FSU2303), both of which produce a mixture of cellooligosaccharides as their cellulose hydrolysis products. Based on our knowledge of the Fibrobacter succinogenes cellulase system, a schematic diagram illustrating the cellulases and CBMs involved in cellulose digestion is presented in Figure 120.

743

Although information about the proteins involved in cellulose digestion is extensive, precise knowledge of the mechanism of decrystallization of highly crystalline cellulose, and the proteins involved in the process, is still lacking. Fibrobacter succinogenes S85 obviously has a very efficient mechanism for the initial amorphization of crystalline cellulose, since it grows as rapidly as any other cellulolytic bacterium with crystalline cellulose as the sole source of carbon and energy. Fibrobacter intestinalis strains are known for their capacity to degrade and utilize cellulose as a source of carbon. The availability of the Fibrobacter succinogenes genome sequence has been valuable in the identification of numerous Fibrobacter intestinalis glycosyl hydrolases with similarity to those in Fibrobacter succinogenes. Application of both forward and reverse suppressive subtractive hybridization (Qi et al., 2005, 2008b) found 37 glycosyl hydrolases in Fibrobacter intestinalis strain DR7 that, on the basis of low stringency hybridization, were similar to those in Fibrobacter succinogenes. In addition, five other cellulase similarities were already known. This genome sequence provides information that will enable the cloning and subsequent enzymic characterization of many Fibrobacter intestinalis cellulases.

List of species of the genus Fibrobacter 1. Fibrobacter succinogenes (Bacteroides succinogenes Hungate 1950, 13AL) Montgomery, Flesher and Stahl 1988, 434VP suc.ci.no¢ge.nes. N.L. n. acidum succinum succinic acid; N.L. suff. -genes (from Gr. v. gennaô to produce), producing; N.L. adj. succinogenes succinic-acid-producing. Cellulose is also used by all strains. Soluble substrates include cellobiose, d-glucose, and sometimes lactose. Other compounds such as starch, pectin, maltose, and trehalose have been used, but consistent results have not been obtained. Habitats include the mammalian GI tract. Several studies have addressed the question of distribution of Fibrobacter within the intestine of cattle, sheep, goats, and horses (Koike et  al., 2004; Lin et  al., 1994; Lin and Stahl, 1995). Fibrobacter succinogenes subsp. succinogenes was present in roughly equal levels in comparison to Fibrobacter succinogenes subsp. elongatus in the rumina of sheep and hay fed cattle. Fibrobacter succinogenes subsp. succinogenes was the dominant Fibrobacter subspecies in rumina of goats. However, this subspecies was only a minor component if observed at all in the lower GI tract of horses and goats and in the rumina of commercial (non-hay fed) steers. These data in conjunction with earlier results support the conjecture that Fibrobacter succinogenes subsp. succinogenes is a critical component of the rumen microbial community during feeding of hay. Source: bovine rumen. DNA G+C content (mol%): 48–49 (Bd). Type strain: S85, ATCC 19169. Sequence accession no. (16S rRNA gene): AJ496032. 1a. Fibrobacter succinogenes subsp. succinogenes (Bacteroides succinogenes Hungate 1950, 13AL) Montgomery, Flesher and Stahl 1988, 434VP suc.ci.no¢ge.nes. N.L. n. acidum succinum succinic acid; N.L. suff. -genes (from Gr. v. gennaô to produce), producing; N.L. adj. succinogenes succinic-acid-producing. Cells are ovoid to lemon shaped (0.8–1.6 mm × 0.9–1.6 mm). Sphaeroplasts form after growth ceases. Colonies on

cellobiose agar are lenticular, translucent, and light brown when examined by transmitted light. Individual colonies poorly clear cellulose contained in agar unless they are cultured with Treponema bryanti (Kudo et  al., 1987). Optimal clearing occurs in roll tubes containing 0.5% each of milled cellulose and purified agar (Montgomery and Macy, 1982). Broth cultures are evenly turbid and produce a smooth sediment. All of the strains tested require biotin, and paraaminobenzoic acid (PABA) is stimulatory. Source: bovine rumen. DNA G+C content (mol%): 48–49 (Bd). Type strain: S85, ATCC 19169 Sequence accession no. (16S rRNA gene): AJ496032. 1b. Fibrobacter succinogenes subsp. elongatus corrig. Montgomery, Flesher and Stahl 1988, 434VP e¢lon.ga¢tus. L. masc. part. adj. elongatus elongated, stretched out. Cells are rod-shaped (0.3–0.5 mm × 0.8–2.0 mm). Sphaeroplasts form after growth ceases. Colonies on cellobiose agar are lenticular, translucent, and light brown when examined by transmitted light. Some strains produce a yellow pigment. Clear zones are formed in cellulose agar, but colonies are indistinct and transient in the absence of fermentable sugars. Clearing is best at 0.5% each of agar and cellulose. Broth cultures are evenly turbid and produce a smooth sediment; some strains produce a yellow pigment. Vitamin requirements are biotin and sometimes cyanocobalamine (B12) and PABA. Habitats include the mammalian intestines, rumina, and ceca. In studies characterizing the distribution of Fibrobacter species in the GI tracts of cattle, sheep, goats, and horses (Koike et al., 2004; Lin et al., 1994; Lin and Stahl, 1995), Fibrobacter succinogenes subsp. elongatus was shown to make up the majority of Fibrobacter cells in commercial (grain fed) steers and in the lower GI tract and cecum of one of three goats and in the rumina of the other two goats. It was present at roughly equal levels relative to

744

Family I. Fibrobacteraceae

Cellulases that also involved in adhesion

Cel45C ? Cel5B ? Cel9G ?

Cel9B/Cel9C

Cel9H

Fn3

Fn3

GP I (F

CBM4

92 BG U25 49) (FS U22 FS

Pyruvate Embden -Meyerhof -Parnas Pathway

Cb P (FS U0 16 2)

Pi

CD CBM Linker Unknown BTD

CBM11

66 5)

Glc-1-P + Glc

Cel5C (CedA)

Cellobiose

SU 1

BG (FSU2249)

Glc

Glc

FSU1565

Cel5H

Porin?

TCA

Cel5G

?

Cel10A

Cellodextrins

Cel51A

CBM11

Glucose

Cel9D

CBM30

Cel8B

CBM30

Crystalline Cellulose

CBM11

G-6-P

ATP ADP CBM11

Other non-catalytic (glyco)CBPs

FIGURE 120.  Putative strategy for cellulose degradation by Fibrobacter succinogenes. This figure shows the cellulases produced by Fibrobacter succino-

genes. Degradation of cellulose could be divided into three steps. In STEP 1, cellulases Cel9B, Cel9C, Cel51A, Cel8B, and Cel5G cleave the cellulose chains probably at amorphous regions and degrade the strand(s) into cellodextrins; STEP 2, cellodextrinases Cel5C and Cl-stimulated cellobiosidase Cel10A degrade the cellodextrins produced in STEP 1 to cellobiose; STEP 2¢, cellodextrins and single glycan chains on cellulose are degraded to glucose by Cel9D; STEP 3, cellobiose is degraded by cell associated b-glucosidase to glucose intra- or extracellularly. Glucose and cellobiose produced from STEP 2 and 3 can be transported into cells by unknown permeases (Maas and Glass, 1991) and metabolized. Cellulases Cel5G, Cel5H, Cel8B, and Cel9D, which contain the BTDII (amino acid Basic C-Terminal Domain), are cell associated. Cel9H, Cel10A, and Cel51A were also known to be cell associated. Adhesion of Fibrobacter succinogenes was shown to be mediated by both catalytic and non-catalytic proteins. Cel5H, Cel51A, Cel10A, and Cel5G contain CBMs (cellulose-binding modules), which are cell associated, and may mediate the attachment of cells to cellulose. Cel9H was known as a cellulose-binding cellulase, but its catalytic properties remain to be studied. Three other cellulases, Cel45C, Cel5B, and Cel9G are also produced by this bacterium. However, since the distribution of these enzymes has not been studied and no CBM or other noncatalytic domains (CDs) could be predicted, the role of these enzymes is unknown (contributed by Dr Meng Qi, University of Guelph).

Fibrobacter succinogenes subsp. succinogenes in the rumina of hay fed cattle. Source: ovine rumen. DNA G+C content (mol%): 51 (Bd) (for strain REH9-1), not determined (for type strain). Type strain: HM2, ATCC 43856. Sequence accession no. (16S rRNA gene): AJ496186, M62689. 2. Fibrobacter intestinalis Montgomery, Flesher and Stahl 1988, 434VP in.tes.tin.a¢lis. L. n. intestinum intestines; L. masc. suff. -alis suffix denoting pertaining to; N.L. masc. adj. intestinalis ­pertaining to the intestines, referring to the original site of isolation.

Cells are rods (0.3–0.4 mm × 0.8–2.0 mm) and form sphaeroplasts after growth ceases. Colonies on cellobiose agar are lenticular, translucent, and light brown when examined by transmitted light. Clear zones are formed in cellulose agar, but colonies are indistinct and transient in the absence of fermentable sugars. Broth cultures are evenly turbid and produce a smooth sediment; some strains produce a yellow pigment. Vitamin requirements are cyanocobalamin (vitamin B12), PABA, and sometimes thiamine and biotin. ­Habitats include mammalian intestines, rumina, and ceca. Source: rat cecum. DNA G+C content (mol%): 45 (Bd). Type strain: NR9, ATCC 43854. Sequence accession no. (16S rRNA gene): AJ496284, M62695.

Genus I. Fibrobacter

References Amann, R.I., C. Lin, R. Key, L. Montgomery and D.A. Stahl. 1992. Diversity among Fibrobacter isolates: towards a phylogenetic classification. Syst. Appl. Microbiol. 15: 23–31. Bera-Maillet, C., V. Broussolle, P. Pristas, J.P. Girardeau, G. Gaudet and E. Forano. 2000. Characterisation of endoglucanases EGB and EGC from Fibrobacter succinogenes. Biochim. Biophys. Acta 1476: 191–202. Bera-Maillet, C., Y. Ribot and E. Forano. 2004. Fiber-degrading systems of different strains of the genus Fibrobacter. Appl. Environ. Microbiol. 70: 2172–2179. Bourquin, L.D. and G.C. Fahey, Jr. 1994. Ruminal digestion and glycosyl linkage patterns of cell wall components from leaf and stem fractions of alfalfa, orchardgrass, and wheat straw. J. Anim. Sci. 72: 1362–1374. Broussolle, V., E. Forano, G. Gaudet and Y. Ribot. 1994. Gene sequence and analysis of protein domains of EGB, a novel family E endoglucanase from Fibrobacter succinogenes S85. FEMS Microbiol. Lett. 124: 439–447. Bryant, M.P. and R.N. Doetsch. 1954. A study of actively cellulolytic rodshaped bacteria of the bovine rumen. J. Dairy Sci. 37: 1176–1183. Cheng, K.J., C.S. Stewart, D. Dinsdale and J.W. Costerton. 1983. Electron microscopy of bacteria involved in the digestion of plant cell walls. Anim. Feed Sci. Technol. 10: 93–120. Dehority, B.A. and P.A. Tirabasso. 1998. Effect of ruminal cellulolytic bacterial concentrations on in situ digestion of forage cellulose. J. Anim. Sci. 76: 2905–2911. Doi, R.H. and A. Kosugi. 2004. Cellulosomes: plant-cell-wall-degrading enzyme complexes. Nat. Rev. Microbiol. 2: 541–551. Forsberg, C.W., E. Forano and A. Chesson. 2000. Microbial adherence to plant cell wall and enzymatic hydrolysis. In Ruminant Physiology Digestion, Metabolism, Growth and Reproduction (edited by Cronje). CABI Publishing, Wallingford, pp. 79–98. Gardner, P.T., T.J. Wood, A. Chesson and T. Stuchbury. 1999. Effect of degradation on the porosity and surface area of forage cell walls of differing lignin content. J. Sci. Food Agric. 79 11–18. Gong, J. and C.W. Forsberg. 1989. Factors affecting adhesion of Fibrobacter succinogenes subsp. succinogenes S85 and adherence-defective mutants to cellulose. Appl. Environ. Microbiol. 55: 3039–3044. Huang, L., C.W. Forsberg and D.Y. Thomas. 1988. Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85. J. Bacteriol. 170: 2923–2932. Hungate, R.E. 1950. The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14: 1–49. Iyo, A.H. and C.W. Forsberg. 1994. Features of the cellodextrinase gene from Fibrobacter succinogenes S85. Can. J. Microbiol. 40: 592–596. Iyo, A.H. and C.W. Forsberg. 1996. Endoglucanase G from Fibrobacter succinogenes S85 belongs to a class of enzymes characterized by a basic C-terminal domain. Can. J. Microbiol. 42: 934–943. Jun, H.S., J.K. Ha, L.M. Malburg, Jr, G.A. Verrinder and C.W. Forsberg. 2003. Characteristics of a cluster of xylanase genes in Fibrobacter succinogenes S85. Can. J. Microbiol. 49: 171–180. Jun, H.S., M. Qi, J. Gong, E.E. Egbosimba and C.W. Forsberg. 2007. Outer membrane proteins of Fibrobacter succinogenes with potential roles in adhesion to cellulose and in cellulose digestion. J. Bacteriol. 189 6806–6815. Kam, D.K., H.S. Jun, J.K. Ha, G.D. Inglis and C.W. Forsberg. 2005. Characteristics of adjacent family 6 acetylxylan esterases from Fibrobacter succinogenes and the interaction with the Xyn10E xylanase in hydrolysis of acetylated xylan. Can. J. Microbiol. 51: 821–832. Koike, S., J. Pan, T. Suzuki, T. Takano, C. Oshima, Y. Kobayashi and K. Tanaka. 2004. Ruminal distribution of the cellulolytic bacterium Fibrobacter succinogenes in relation to its phylogenetic grouping. Anim. Sci. J. 75: 417–422. Kudo, H., K.J. Cheng and J.W. Costerton. 1987. Interactions between Treponema bryantii and cellulolytic bacteria in the in vitro degradation of straw cellulose. Can. J. Microbiol. 33: 244–248.

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Lin, C.Z. and D.A. Stahl. 1995. Taxon-specific probes for the cellulolytic genus Fibrobacter reveal abundant and novel equine-associated populations. Appl. Environ. Microbiol. 61: 1348–1351. Lin, C.Z., B. Flesher, W.C. Capman, R.I. Amann and D.A. Stahl. 1994. Taxon specific hybridization probes for fiber-digesting bacteria suggest novel gut-associated Fibrobacter. Syst. Appl. Microbiol. 17: 418–424. Lynd, L.R., P.J. Weimer, W.H. van Zyl and I.S. Pretorius. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66: 506–577. Maas, L.K. and T.L. Glass. 1991. Cellobiose uptake by the cellulolytic ruminal anaerobe Fibrobacter (Bacteroides) succinogenes. Can. J. Microbiol. 37: 141–147. Macy, J.M., J.R. Farrand and L. Montgomery. 1982. Cellulolytic and non-cellulolytic bacteria in rat gastrointestinal tracts Appl. Environ. Microbiol. 44: 1428–1434. Maglione, G., J.B. Russell and D.B. Wilson. 1997. Kinetics of cellulose digestion by Fibrobacter succinogenes S85. Appl. Environ. Microbiol. 63: 665–669. Malburg, L.M.J., A.H. Iyo and C.W. Forsberg. 1996. A novel family 9 endoglucanase gene (celD), whose product cleaves substrates mainly to glucose, and its adjacent upstream homolog (celE) from Fibrobacter succinogenes S85. Appl. Environ. Microbiol. 62: 898–906. Malburg, S.R., L.M. Malburg, T. Liu, A.Y. Iyo, C. Forsberg. 1997. Catalytic properties of the cellulose-binding endoglucanase F from Fibrobacter succinogenes S85. Appl. Environ. Microbiol. 63: 2449–2453. Matte, A. and C.W. Forsberg. 1992. Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85. Appl. Environ. Microbiol. 58: 157–168. Matte, A., C.W. Forsberg and A.M. Verrinder Gibbins. 1992. Enzymes associated with metabolism of xylose and other pentoses by Prevotella (Bacteroides) ruminicola strains, Selenomonas ruminantium D, and Fibrobacter succinogenes S85. Can. J. Microbiol. 38: 370–376. Matulova, M., R. Nouaille, P. Capek, M. Pean, E. Forano and A.M. Delort. 2005. Degradation of wheat straw by Fibrobacter succinogenes S85: a liquid- and solid-state nuclear magnetic resonance study. Appl. Environ. Microbiol. 71: 1247–1253. McDermid, K.P., C.W. Forsberg and C.R. MacKenzie. 1990a. Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85. Appl. Environ. Microbiol. 56: 3805–3810. McDermid, K.P., C.R. MacKenzie and C.W. Forsberg. 1990b. Esterase activities of Fibrobacter succinogenes subsp. succinogenes S85. Appl. Environ. Microbiol. 56: 127–132. McGavin, M. and C.W. Forsberg. 1988. Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85. J. Bacteriol. 170: 2914–2922. McGavin, M.J., C.W. Forsberg, B. Crosby, A.W. Bell, D. Dignard and D.Y. Thomas. 1989. Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3. J. Bacteriol. 171: 5587–5595. Miron, J. and C.W. Forsberg. 1998. Features of Fibrobacter intestinalis DR7 mutant which is impaired with its ability to adhere to cellulose. Anaerobe 4: 35–43. Montgomery, L. and J.M. Macy. 1982. Characterization of rat cecum cellulolytic bacteria. Appl. Environ. Microbiol. 44: 1435–1443. Montgomery, L., B. Flesher and D. Stahl. 1988. Transfer of Bacteroides succinogenes (Hungate) to Fibrobacter gen. nov. as Fibrobacter succinogenes comb. nov. and description of Fibrobacter intestinalis sp. nov. Int. J. Syst. Bacteriol. 38: 430–435. Morrison, M., K. Nelson, I. Cann, C. Forsberg, R.I. Mackie, J.B. Russell, B.A. White, D.B. Wilson, K. Amya, B. Cheng, S. Qi, H.S. Jun, S. Mulligan, K. Tran, H. Carty, H. Khouri, W. Nelson, S. Daugherty and C. Fraser. 2003. The Fibrobacter succinogenes strain S85 sequencing project. 3rd ASM-TIGR, Microbial Genome Meeting, New Orleans. Paradis, F.W., H. Zhu, P.J. Krell, J.P. Phillips and C.W. Forsberg. 1993. The xynC gene from Fibrobacter succinogenes S85 codes for a xylanase with two similar catalytic domains. J. Bacteriol. 175: 7666–7672.

746

Family I. Fibrobacteraceae

Pegden, R.S., M.A. Larson, R.J. Grant and M. Morrison. 1998. Adherence of the Gram-positive bacterium Ruminococcus albus to cellulose and identification of a novel form of cellulose-binding protein which belongs to the pil family of proteins. J. Bacteriol. 180: 5921–5927. Prévot, A.R. 1966. Manual for the Classification and Determination of the Anaerobic Bacteria. Lea & Febiger, Philadelphia. Qi, M., K.E. Nelson, S.C. Daugherty, W.C. Nelson, I.R. Hance, M. Morrison and C.W. Forsberg. 2005. Novel molecular features of the fibrolytic intestinal bacterium Fibrobacter intestinalis not shared with Fibrobacter succinogenes as determined by suppressive subtractive hybridization. J. Bacteriol. 187: 3739–3751. Qi, M., H.S. Jun and C.W. Forsberg. 2007. Characterization and synergistic interactions of Fibrobacter succinogenes glycoside hydrolases. Appl. Environ. Microbiol. 73: 6098–6105. Qi, M., H.S. Jun and C.W. Forsberg. 2008a. Cel9D, an atypical 1,4-b-dglucan glucohydrolase from Fibrobacter succinogenes: characteristics, catalytic residues, and synergistic interactions with other cellulases. J. Bacteriol. 190: 1976–1984. Qi, M., K.E. Nelson, S.C. Daugherty, W.C. Nelson, I.R. Hance, M. Morrison and C.W. Forsberg. 2008b. Genomic differences between Fibrobacter succinogenes S85 and Fibrobacter intestinalis DR7 identified by suppression subtractive hybridization. Appl. Environ. Microbiol. 74: 987–993.

Smith, D.C. and C.W. Forsberg. 1991. a-Glucuronidase and other hemicellulase activities of Fibrobacter succinogenes S85 grown on crystalline cellulose or ball-milled barley straw. Appl. Environ. Microbiol. 57: 3552–3557. Stewart, C.S., C. Paniagua, D. Dinsdale, K-J. Cheng and S.H. Garrow. 1981. Selective isolation and characteristics of Bacteriodes succinogenes from the rumen of a cow. Appl. Environ. Microbiol. 41: 504–510. Teather, R.M. and J.D. Erfle. 1990. DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3–1,4-b-d-glucan 4-glucanohydrolase) gene. J. Bacteriol. 172: 3837–3841. Wells, J.E., J.B. Russell, Y. Shi and P.J. Weimer. 1995. Cellodextrin efflux by the cellulolytic ruminal bacterium Fibrobacter succinogenes and its potential role in the growth of nonadherent bacteria. Appl. Environ. Microbiol. 61: 1757–1762. Wen, T.N., J.L. Chen, S.H. Lee, N.S. Yang and L.F. Shyur. 2005. A truncated Fibrobacter succinogenes 1,3–1,4-b-d-glucanase with improved enzymatic activity and thermotolerance. Biochemistry 44: 9197–9205. Zhu, H., F.W. Paradis, P.J. Krell, J.P. Phillips and C.W. Forsberg. 1994. Enzymatic specificities and modes of action of the two catalytic domains of the XynC xylanase from Fibrobacter succinogenes S85. J. Bacteriol. 176: 3885–3894.

Phylum XIX. Fusobacteria Garrity and Holt 2001, 140 James T. Staley and William B. Whitman Fu.so.bac.te¢ri.a. N.L. neut. n. Fusobacterium genus of the phylum with ending -ia to denote phylum; N.L. neut. pl. n. Fusobacteriia the phylum of Fusobacterium.

The phylum Fusobacteria is described in part on the basis of phylogenetic analyses of the 16S rRNA gene sequences of its members. The phylum contains rod-shaped bacteria that stain Gram-negative. Described species are fermentative and produce a variety of organic acids when grown on carbohydrates, amino acids or peptides. Some species are pathogenic to humans. Type order: Fusobacteriales Staley and Whitman, this volume.

Reference Garrity, G.M. and J.G. Holt. 2001. The Road Map to the Manual. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 1, The Archaea and the Deeply Branching and Phototrophic Bacteria (edited by Boone, Castenholz and Garrity). Springer, New York, pp. 119–166.

Class I. Fusobacteriia class. nov. James T. Staley and William B. Whitman Fu.so.bac.te¢ri.ia. N L. neut. n. Fusobacterium type genus of the order Fusobacteriales; suff. -ia ending proposed by Gibbons and Murray and by Stackebrandt et al. to denote class; N.L. neut. pl. n. Fusobacteriia the Fusobacterium class. The class Fusobacteria is described in part on the basis of phylogenetic analyses of the 16S rRNA gene sequences of its members. The class contains facultative aerobic to obligately anaerobic organisms that stain as Gram-negative rods and ferment carbohydrates or amino acids and peptides to produce various

organic acids including acetic, propionic, butyric, formic or succinic, depending on the substrate and species. Species occur in sediments as well as the oral or intestinal habitats of animals. Some species are human pathogens. Type order: Fusobacteriales Staley and Whitman, this volume.

Order I. Fusobacteriales ord. nov. James T. Staley and William B. Whitman Fu.so.bac.te.ri.a¢les. N.L. neut. n. Fusobacterium type genus of the order; suff. -ales ending denoting order; N.L. fem. pl. n. Fusobacteriales the Fusobacterium order. The order Fusobacteriales is described in part on the basis of phylogenetic analyses of the 16S rRNA gene sequences of its members. The order contains facultative aerobic to obligately anaerobic organisms that stain as Gram-negative rods. All described species are nonmotile and fermentative. Organisms ferment carbohydrates or amino acids and peptides to produce various organic acids including acetic, propionic, butyric, formic or succinic depending on the substrate and species. Species occur in anoxic environments including sediments as well as the oral or intestinal habitats

of ­animals, including mammals. Some species are ­pathogenic to humans. Type genus: Fusobacterium Knorr 1922, 4AL.

Reference Knorr, M. 1922. Über die fusospirilläre Symbiose, die Gattung Fusobacterium (K.B. Lehmann) und Spirillum sputigenum. Zugleich ein Beiträg zür Bakteriologie der Mundhohle. II. Mitteilung. Die. Gattung Fusobacterium. I Abt. Orig. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. 89: 4–22.

747

748

Family I. Fusobacteriaceae

Family I. Fusobacteriaceae fam. nov. James T. Staley and William B. Whitman Fu.so.bac.te.ri.a.ce¢a.e. N. L. neut. n. Fusobacterium type genus of the family; suff. -aceae ending denoting family; N.L. fem. pl. n. Fusobacteriaceae the Fusobacterium family. The family Fusobacteriaceae is described in part on the basis of phylogenetic analyses of the 16S rRNA gene sequencess of its members. Micro-aerotolerant to obligately anaerobic organisms that stain as Gram-stain-negative rods. All named species are nonmotile and fermentative. Ferment carbohydrates or amino acids and peptides to produce various organic acids including acetic, propionic, butyric, formic or succinic depending on the

substrate and species. Occur in anoxic environments including sediments as well as the oral and intestinal habitats of animals including mammals. Comprises the genera Cetobacterium, Fusobacterium, Ilyobacter, and Propionigenium. Type genus: Fusobacterium Knorr 1922, 4AL.

Genus I. Fusobacterium Knorr 1922, 4AL Saheer E. Gharbia, Haroun N. Shah and Kirstin J. Edwards Fu.so.bac.te¢ri.um. L. n. fusus a spindle; L. neut. n. bacterium a small rod; N.L. neut. n. Fusobacterium a small spindle-shaped rod.

Nonsporeforming rods that are Gram-stain-negative and obligately anaerobic. Metabolize peptone or carbohydrates in PYglucose to produce butyrate, often with acetate and lower levels of lactate, propionate, succinate, and formate. DNA G+C content (mol%): 26–34. Type species: Fusobacterium nucleatum Knorr 1922, 17AL [Bacillus fusiformis Veillon and Zuber 1898, 540 and other combinations using “Fusiformis” except the organism described as Fusobacterium fusiforme by Hoffman in the 7th edition of the Manual; Group I, Spaulding and Rettger 1937, 535; Group III (and probably Fusobacterium polymorphum) Baird-Parker 1960, 458; not Fusobacterium plauti-vincentii Knorr 1922, 5.].

Further descriptive information The genus Fusobacterium includes species that do not ferment adonitol, arabinose, dulcitol, glycerol, glycogen, inocitol, inulin, mannitol, melezitose, rhamnose, ribose, sorbitol, or sorbose. Cellobiose is not fermented and esculin is not hydrolyzed except by Fusobacterium mortiferum and Fusobacterium necrogenes; does not reduce nitrate (except for Fusobacterium ulcerans); and does not produce catalase, lecithinase, or acetylmethycarbinol. In addition to butyric, propionic, and acetic acids, species produce variable amounts of butanol from PY medium. Small amounts of formate, lactate, succinate, and ethanol may be produced. Some species convert threonine or lactate to propionate. Pyruvate is converted to acetate and butyrate and sometimes also to formate, succinate, and lactate. H2S is produced. All species, except Fusobacterium naviforme and Fusobacterium russii, produce propionate from threonine. Lactate is converted to propionate by Fusobacterium necroforum and Fusobacterium equinum. Fusobacteria form volatile sulfur compounds from cysteine and methio­ nine (Claesson et  al., 1990; Pianotti et  al., 1986). All species produce indole except for Fusobacterium mortiferum, Fusobacterium necrogenes, Fusobacterium russii, Fusobacterium ulcerans, and some strains of Fusobacterium varium. Apart from a weak reaction by Fusobacterium necrophorum, only Fusobacterium equinum produces esterases, while Fusobacterium canifelinum is the only fluoroquinolone resistant species of the genus. Additional features are described in Table 148.

Cell morphology.  The cells are pleomorphic, some are arranged into filaments, and are spindle-shaped with pointed ends (fusiform) in a few species, while others are coccobacilli. Width is variable. The cells may be single, in pairs end-to-end, or form long coiled filaments. Staining may be irregular and spheroplasts are common in some species. The cells of Fusobacterium nucleatum are slender, spindle-shaped with tapered or pointed ends 0.4–0.7 mm thick and 4–10 mm long, and appear singly, in tandem pairs, or in bundles of roughly parallel bacilli. Filaments are often seen in old cultures of Fusobacterium periodonticum and Fusobacterium simiae which have similar cellular morphology to Fusobacterium nucleatum. The cells of Fusobacterium necrophorum are pleomorphic, often curved, with rounded and sometimes tapered ends, and they may have spherical enlargements. Free coccoid bodies and especially filaments are common. Cell length may vary from coccobacilli to long threads in clinical samples. Fusobacterium naviforme strains have boat-shaped cells. Gonidial forms may be seen in old cultures of Fusobacterium gonidiaformans. Fusobacterium varium is a small bacillus that does not form filaments. Strains of Fusobacterium mortiferum are extremely pleomorphic with globular forms, swellings, and threads. Short rods are predominant in cultures of Fusobacterium equinum. The other fusobacteria are pleomorphic filamentous organisms. Cell-wall composition.  Fusobacteria have a cell-wall structure based on two membranes separated by a periplasmic space containing a peptidoglycan layer. Meso -lanthioine replaces diaminopimelic acid in Fusobacterium nucleatum, Fusobacterium gonidiaformans, Fusobacterium necrophorum, Fusobacterium russii, Fusobacterium necrogenes, Fusobacterium simiae, and Fusobacterium periodonticum (Gharbia and Shah, 1990; Kato et al., 1981; Vasstrand et al., 1982), while Fusobacterium varium, Fusobacterium naviforme, and Fusobacterium ulcerans retain meso-diaminopimelic acid and Fusobacterium mortiferum contain both versions. Fusobacterium equinium and Fusobacterium canifelinum have not been tested. Information on the fatty acid content and cellwall composition of various Fusobacterium species can be found in Hofstad (1979), Kato et  al. (1981, 1979), Miyagawa et al. (1979), Hofstad and Skaug (1980), Jantzen and Hofstad

w− −w −w − − − − − + − − − − − − + − − − − − − − − − −

w− −w −w − − − − − + − − − − − − + − − − − − − − − − −

F. nucleatum subsp. nucleatum − −

F. nucleatum subsp. fusiforme

− −

F. nucleatum subsp. polymorphum − − − − − − − − − −

w− −w −w − − − − − + − − − − − − +

− −

F. nucleatum subsp. vincentii − − − − − − − − − −

w− −w −w − − − − − + − − − − − − +

− −

F. nucleatum subsp. animalis − − − − − − − − − −

w− −w −w − − − − − + − + − − − − +

− −

F. canifelinum − − − − − − − − − −

w− −w −w − − − − − + − − − − − − +

− −

− − + − − − − − + +

+ + − nt + +

− −w − − − − − − +

− −

F gonidiaformans − − w − − − − − − −

− − w− − − − − − + − − − − + − +

− −

F. mortiferum − + + + + − − +− − −

wa − aw wa w− wa v v − − + − − + − +

w− +−

F. naviforme − − − − − + − − − −

− − −w − − − − − + − − − − − − −

− −

F. necrogenes − + + + + − − − − −

wa − wa − − wa −w −w − − + − − + − +

w− +

F. necrophorum subsp. necrophorum − + + − − − − − w w

−w v −w − − − − − + − v +– + + + +

− −

F. necroforum subsp. funduleforme − − + − − − − − w w

− + + +

−w v −w − − − − − + − v

− −

F. perfoetans + − w − + − − − − −

w − w − − − − w − − − − − + − +

− −

F. periodonticum − − − − − − − − − −

w− − w− − − − − − + − − − − − − +

− −

F. russii − w + − − − − − − −

− − − − − − − − − − − − − − − −

− −

F. simae − − − − − − − − w −

w − w − − − − − + − + w − − − +

− −

F. ulcerans − − + − − − − − − −

− − aw − − aw − − − + + − − + − +

− −

− w w − − − − − − −

wa − wa − − w − − +− − + −w − + − +

− −

F. varium

a

Symbols: w, weakly positive; w−: most strains weakly positive, some negative; −w: most strains negative, some weakly positive; aw: strongly acid with some weakly acid reactions; wa: usually weakly acid with occasional strong acid reactions; nt, not tested. (Adapted from Conrads et al., 2004a.)

Cellobiose Esculin hydrolysis Utilization of: Fructose Gelatin Glucose Lactose Maltose Mannose Raffinose Sucrose Indole Nitrate Bile growth Lipase b-Hemolysis Gas produced in agar Lactate→propionate Threonine→propionate Activity of: N-Acetyl-glucosaminidase Alkaline phosphatase Acid-phosphatase a-Galactosidase b-Galactosidase b-Glucoronidase a-Glucosidase b-Glucosidase Esterase (C4) Esterase (C8)

Characteristic

F. equinum

Table 148.  Characteristics differentiating the species and subspecies of Fusobacteriuma

Genus I. Fusobacterium 749

750

Family I. Fusobacteriaceae

(1981), and Vasstrand (1981). LPS exhibits O-antigenic specificity linked to lipid A through 2-keto-3-deoxyoctonate. The lipid A of Fusobacterium nucleatum is structurally similar to that of Enterobactereaceae and cross-reacts serologically with antibodies to Escherichia coli lipid A (Dahlen and Mattsby-Baltzer, 1983). Fusobacterium necrophorum isolates have a rough-type LPS, whereas that of Fusobacterium necrophorum subsp. necrophorum is of a smooth type (Brown et al., 1997). The repeat unit of the O-antigenic polysaccharide of Fusobacterium necrophorum LPS is an acid identified as a 2-amino-2-deoxy-2-C-methyl-pentonic acid (2-amino-2-methyl-3,4,5-trihydroxypentanoic acid), a novel structure not found before in nature (Hermansson et  al., 1993). The main protein of the outer membrane of Fusobacterium nucleatum, designated FomA, has a molecular mass of 40 kDa (Bakken et al., 1989b). It is a nonspecific porin present in the outer membrane as a trimer (Kleivdal et al., 1995). The gene encoding the FomA porin has been sequenced ­(Bolstad et al., 1994). The deduced topology is similar to that of porin structures from other bacteria. fomA has been cloned and expressed in Escherichia coli (Haake and Wang, 1997; Jensen et  al., 1996). Several other outer-membrane proteins have been predicted from the genome sequence, among them some very-high-molecular-mass-proteins (Kapatral et al., 2002). FomA and outer-membrane proteins with molecular masses of 55, 60 and 70 kDa are the major antigens in Fusobacterium nucleatum (Bakken et al., 1989a). The cellular fatty acids in the Fusobacterium species exa­mined are straight-chain saturated and monoenoic acids of chain lengths C12-C18 and O-3-hydroxy-tetradecanoate is distinctive to the oral species Fusobacterium nucleatum, Fusobacterium simiae, and Fusobacterium periodonticum ( Jantzen and Hofstad, 1981). Comparison of small-subunit rRNA sequences has revealed levels of sequence similarity that are consistent with the single genus, but intrageneric heterogeneity is evident (Lawson et al., 1991; Tanner et al., 1994). Fusobacterium nucleatum, the species isolated most frequently from humans, is divided into five subspecies; Fusobacterium nucleatum subsp. nucleatum, subsp. polymorphum, subsp. vincetii, subsp. fusiforme, and subsp. animalis, described on the basis of electrophoretic patterns of whole-cell proteins and DNA homology (Dzink et al., 1990) and electrophoretic mobility of two enzymes and DNA homology (Gharbia and Shah, 1992). Recent evidence to support the heterogeneity within Fusobacterium nucleatum and the existence of distinct subspecies within Fusobacterium nucleatum was obtained by comparison of their 16S–23S internal transcribed spacer regions (Conrads et al., 2002). Fusobacterium periodonticum is phylogenetically similar to Fusobacterium nucleatum ( Jousimies-Somer and Summanen, 2002). Fusobacterium nucleatum-like isolates from cats and dogs were found to be distinct from Fusobacterium nucleatum both in their 16S rRNA and DNA–DNA hybridization. These have been reclassified as Fusobacterium canifelinum which are resistant to levofloxacin (MIC >4 mg/ml) and other fluoroquinolones. The resistance is due to two substitutions in the quinolone resistance determining region of gyrA relative to Fusobacterium nucleatum. The first replacement is of Ser79 with leucine and the second is Gly83 is replaced with arginine (Conrads et al., 2005). Animal isolates of Fusobacterium necrophorum form two subspecies: Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme (Shinjo et  al., 1991)

corresponding to biovars A and B. A distinctive feature separating the subspecies genetically is the presence of isoleucine and alanine tRNA gene in Fusobacterium necrophorum subsp. necroforum, while Fusobacterium necrophorum subsp. funduliforme contains the isoleucine tRNA gene ( Jin et al., 2002). Genetic differences between the species have been reported in leukotoxin A (lktA) between both subspecies (Narayanan et  al., 2001). Subspecies necrophorum is more frequently isolated, often in pure culture, from liver abscesses than subsp. funduliforme. Leukotoxin, an exotoxin, is a major virulence factor. In Fusobacterium necrophorum subsp. necrophorum, Lkt is a high-molecular-mass protein that is encoded by a tricistronic leukotoxin operon (lktBAC) and induces apoptosis and necrosis of bovine leukocytes in a dose-dependent manner. The subsp. funduliforme produces lower concentration of leukotoxin and hence is less virulent than subsp. necrophorum. The low toxicity associated with subsp. funduliforme leukotoxin, a less virulent subspecies, may in part be due to the differences in the lktA gene and reduced transcription (Tadepalli et al., 2008). Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans share several phenotypic properties similar to Fusobacterium varium, but Fusobacterium ulcerans reduces nitrate to nitrite (Citron, 2002). It also contains unique fragments of 1000 and 550 bp not present in Fusobacterium varium and Fusobacterium mortiferum (Claros et al., 1999). Fusobacterium necrogenes is distantly related to the other Fusobacterium species. Based on 16S–23S rDNA spacer region sequences (Conrads et al., 2002) Fusobacterium gonidiaformans is genealogically related to Fusobacterium necrophorum (Nicholson et al., 1994). Metabolism.  The core metabolism of Fusobacterium nucleatum is similar to that of Clostridium, Lactococcus, and Enterococcus species (Kapatral et al., 2002). More than 137 transporters for the uptake of substrates such as peptides, sugars, metal ions, and cofactors have been detected. Amino acids and small peptides comprise the major sources of energy for all Fusobacterium species (Gharbia and H.N. Shah, 1989), however, peptides influence the uptake of amino acids enhancing the utilization of histidine and glutamate while threonine, methionine, and asparagine were repressed (Gharbia et  al., 1989). Acidic and cationic amino acids are the main acids incorporated. Glutamate, histidine, lysine, and serine appear to be key amino acids in Fusobacterium nucleatum (Gharbia and Shah, 1991a; Rogers et al., 1992). Biosynthetic pathways exist for glutamate, aspartate, and glutamylglutamate to be used as growth substrates for Fusobacterium nucleatum (Takahashi and Sato, 2002). Glutamate is a key catabolic substrate in Fusobacterium species (Gharbia and Shah, 1991b). It is catabolized via the 2-oxoglutarate pathway with the production of acetate and butyrate as end products (Gharbia and Shah, 1991b). Glutamate may also be degraded by the methylaspartate pathway in Fusobacterium varium (Gharbia and Shah, 1991b). Enzymes representative for the mesaconate pathway for catabolism of glutamate have been detected in Fusobacterium varium, Fusobacterium mortiferum, and Fusobacterium ulcerans (Gharbia and Shah, 1991b). Fusobacterium varium and Fusobacterium mortiferum also possess enzymes representative of the 4-aminobutyrate pathway. Amino acids are imported as monomers, di-, or oligopeptides, and an active transport of the dipeptide l-cysteinglycine has been detected in Fusobacterium nucleatum (Carlsson et al., 1994).

Genus I. Fusobacterium

Fusobacterium species differ in their ability to use fermentable carbohydrates as energy sources for growth (Robrish et  al., 1991). Fusobacterium nucleatum and other species utilize glucose for biosynthesis of intracellular glycopolymers that can be degraded to produce energy under conditions of amino-acid deprivation (Robrish et al., 1987). The accumulation of glucose is dependent on energy generated by the fermentation of amino acids (Robrish et al., 1987). Fusobacterium mortiferum is an exception in that accumulation of sugars is independent of a fermentable amino acid. Significantly, Fusobacterium mortiferum has the ability to metabolize various sugars as energy sources for growth (Robrish et al., 1991). Sugars utilized include a- and b-glycosides, which are transported by the phosphoenolpyruvate-dependent sugar: phosphotransferase system. Thus, it utilizes sucrose and its isomeric a-d-glucosyl-d-fructoses as energy sources for growth (Pikis et al., 2002). The genes encoding phospho-b-glucosidase (P-b-glucosidase; EC 3.22.1.86) and 6-phospho-a-d-glucosidase (maltose-6phosphate hydrolase; EC 3.2.1.122) known as pbgA and malH, respectively, have been expressed in Escherichia coli (Bouma et al., 1997; Thompson et al., 1997). Genetics.  The genome of Fusobacterium nucleatum subsp. nucleatum (strain ATCC 25586T) contains 2.17 Mb encoding 2067 open reading frames (ORFs) organized on a single circular chromosome (Kapatral et al., 2002). About 2.3% of the ORFs are unique to Fusobacterium nucleatum. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high­molecular-mass outer-membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H2S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. Analysis of the draft genome sequence of Fusobacterium nucleatum subsp. vincentii (ATCC 49256), and comparison of this sequence to the genome ATCC 25586 sequence resolved that 441  ORFs have no orthologs in strain ATCC 25586. Of these, 118  ORFs have no known function and are unique, whereas 323 ORFs have functional orthologs in other organisms. Genes for eukaryotic serine/threonine kinase and phosphatase, transpeptidase E-transglycosylase Pbp1A were also identified among the ATCC 49256-specific ORFs. Unique ABC transporters, cryptic phages, and three types of restriction-modification systems have been identified in ATCC 49256. ORFs for ethanolamine utilization, thermostable carboxypeptidase, glutamyl-transpeptidase, and deblocking aminopeptidases are absent from ATCC 49256. Both strains lack a catalase-peroxidase system, but possess thioredoxin/glutaredoxin enzymes. Genes for resistance to antibiotics such as acriflavin, bacitracin, bleomycin, daunorubicin, florfenicol, and other general multidrug resistance are present (Kapatral et al., 2003)

751

The genome of Fusobacterium nucleatum subsp. polymorphum ATCC 10953 contains a chromosome of approximately 2.4 Mbp and a plasmid (pFN3) of 11.9 kbp, and there are 2361 proteins encoded in the genome. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes (Fusobacterium nucleatum subsp. nucleatum and Fusobacterium nucleatum subsp. vincentii) 627 ORFs unique to Fusobacterium nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other Gram-stain-positive organisms such as Bacillus and Streptococcus. A 10 kb region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons similar to the CdISt IStrons described in Clostridium difficile. Additionally, three plasmids isolated from Fusobacterium nucleatum strains were sequenced. These are designated pFN1, pPA52, and pKH9 (Bachrach et  al., 2004; Haake et  al., 2000; McKay et al., 1995). Ecology.  Fusobacterium are normal inhabitants of the mucous membrane of humans and animals. The habitat of Fusobacterium nucleatum and Fusobacterium periodonticum is the human oral cavity; the gingival crevice is the niche of both. Strains are isolated from the oral microflora of adults and children and are also found in edentulous infants. The vagina is likely to be the primary endogenous site for Fusobacterium naviforme and Fusobacterium gonidiaformans (Hill, 1993). The habitat of Fusobacterium mortiforum and Fusobacterium varium is the gastrointestinal tract, where they are present in small numbers. Fusobacterium necrogenes, which was originally isolated from a chicken abscess and from cecal contents of ducks, is very rarely isolated from humans. The habitat of Fusobacterium ulcerans, isolated from tropical ulcers, is unknown. Fusobacterium necrophorum is a normal inhabitant of the alimentary tract of cattle, horses, sheep, and pigs and is frequently isolated from cats and dogs. Reports, mainly from the first part of the twentieth century, indicate its presence in a range of wild animals including reptiles. However, the relatively frequent isolation of Fusobacterium necroforum from soft-tissue infections of the oral cavity and the upper respiratory tract compared to elsewhere in the body, suggest that these sites are the principal human habitat of this organism. Fusobacterium russii is normally found in the canine and feline oral flora (Love et al., 1987), but it has also been isolated from human feces. The oral cavity of horses and the stump-tailed macaque (Macaca arctoides) are the habitat of Fusobacterium equinum and Fusobacterium simiae, respectively. Fusobacterium canifelinum was isolated from the microflora of infected cat and dog bite wounds in humans (Conrads et al., 2005).

Maintenance procedures Fusobacteria are not particularly demanding with regard to a low oxidation-reduction potential. They are, however, killed fairly readily by exposure to oxygen. This is possibly due to their susceptibility to peroxides. Growth is best at 35–37°C and at a pH near 7.

752

Family I. Fusobacteriaceae

All Fusobacterium species grow on blood agar based on proteo­se-peptone, tryptone, or trypticase. Good growth is usually obtained in a rich semifluid medium such as brain heart medium supplemented with yeast extract. Batch cultivation is best performed in a fluid medium containing a reducing agent.

Laboratory isolation and identification Fusobacterium species can be separated from other related taxa based on their phylogenetic relatedness and distinctive phenotypic characteristics (Table 149). The isolation of fusobacteria from an aerobic infection requires proper collection of the specimen, the use of an anaerobic transport vial, optimal anaerobiosis (an anaerobic chamber is not necessary), and a rich medium for optimal growth. Fastidious Anaerobe Agar Base (FAA, Lab M) supplemented with 5% sheep or horse blood is recommended for culture. The use of a selective medium is necessary for the enumeration of viable fusobacteria in specimens of normal flora. The addition of josamycin, vancomycin, and norfloxacin plus 5% defibrinated horse blood to FAA supports the growth of ­Fusobacterium species while completely inhibiting most other obligate anaerobes and facultative bacteria (Brazier et  al., 1991). Rifampin blood agar (Sutter et al., 1971) is useful for the selective isolation of Fusobacterium varium and Fusobacterium mortiferum. Diagnostic tables are convenient for the examination of bile resistance, production of alkaline phosphatase, and orthonitrophenol-b-d-galactopyranoside (ONPG)-test. Commercially available kits based on the detection of preformed enzymes and designed for identification of anaerobes may be of help in supporting a suspected identity of a Fusobacterium isolate.

Surface colonies of Fusobacterium nucleatum are 1–2 mm in diameter after incubation for 2 d. They are white to yellow-gray in color, speckled, smooth or breadcrumb-like. The colonies are usually nonhemolytic. Cultures of Fusobacterium nucleatum produce an unpleasant, but not fetid, smell. After incubation for 2 d, colonies of animal isolates of Fusobacterium necrophorum produce a foul, putrid odor. Fluid or semifluid cultures are characterized by abundant production of gas, and colonies are about 2 mm in diameter, flat, circular with scalloped to erose edge, opaque, and white to gray (subsp. necrophorum); or 1 mm wide, circular with entire margins, gray, translucent, and with smooth surfaces (subsp. funduliforme) (Shinjo et al., 1991). The colonial morphology of Fusobacterium necrophorum isolated from human infections is similar to that of Fusobacterium necrophorum subsp. funduliforme. The colonies are small and have been described as creamy, opaque, smooth, umbonate or raised, and with entire edges (Hall et al., 1997). The colonial morphology of most other Fusobacterium species is similar to that of Fusobacterium necrophorum subsp. funduliforme. All Fusobacterium species produce butyric acid. They are catalase and nitrate negative, sensitive to kanamycin (1000 mg disk) and colistin (10 mg disk), resistant to vancomycin (5 mg disk), and produce a rancid odor. Determination of the electrophoretic migration of glutamate dehydrogenase and 2-oxoglutarate reductase provides a rapid method for the identification of most Fusobacterium species (Gharbia and Shah, 1990). Identification based on gas-liquid chromatography of cellular fatty acids may be of value in reference laboratories, provided a database is used as a basis for comparison. A DNA probe has been used successfully to detect

Table 149.  Characteristics differentiating Fusobacterium from genetically related taxaa

Reaction Catalase Oxidase Indole Arginine dihydroylase Nitrate to nitrite Esculin hydrolysis Gelatinase Starch hydrolysis ONPG Acid from: Arabinose Cellobiose Fructose Galactose Glucose Glycogen Lactose Maltose Mannose Mannitol Starch Sucrose Trehalose Xylose

Fusobacterium Cetobacterium nucleatum ceti

Leptotrichia buccalis

Sebaldella termitidis

Sneathia Streptobacillus sanguinegens moniliformis

− nd + nd − − − nd nd

− − + nd v − − − +

− nd − nd − + − v nd

− nd − nd − nd − − nd

− − − + − + nd − nd

− − − + − v − nd −

nd − −w nd −w nd − − − − − − − −

nd nd nd nd nd nd nd nd nd nd nd nd nd nd

− + + d + nd + + + − d + + −

− nd + nd + nd − + nd + − + + +

− nd nd nd + − − − v − − − − nd

− − + + + + − + + − + − − −

a Symbols: +, 90% or more strains are positive; −, 90% or more strains are negative; d, 11–89% of the strains are positive; ONPG, O-nitrophenyl-b-d-galactopyranoside; v, variable; w, weak; nd, no data.

753

Genus I. Fusobacterium

Fusobacterium nucleatum directly in samples of subgingival plaque (Lippke et al., 1991), and checkerboard DNA–DNA hybridization has been used successfully to detect Fusobacterium nucleatum and subspecies in periodontal and endodontic infections (Socransky et al., 1998; Sunde et al., 2000). A PCR-detection of part of the

major 40 kDa outer-membrane protein, the FomA porin of Fusobacterium nucleatum, is specific for Fusobacterium nucleatum when used under condition of high stringency (Bolstad and Jensen, 1993). Sequencing of 16S rRNA from clinical isolates provides a discriminatory tool for separating all species (Figure 121).

List of species of the genus Fusobacterium 1. Fusobacterium nucleatum Knorr 1922, 17AL [Bacillus fusiformis Veillon and Zuber 1898, 540 and other combinations using “Fusiformis” except the organism described as Fusobacterium fusiforme by Hoffman in the 7th edition of the Manual; Group I, Spaulding and Rettger 1937, 535; Group III (and probably Fusobacterium polymorphum) Baird-Parker 1960, 458; not Fusobacterium plauti-vincentii Knorr 1922, 5.] nu.cle.a¢tum. L. neut. adj. nucleatum having a kernel, intended to mean nucleated. Cells from glucose broth cultures are 0.4–0.7 × 3 mm, have tapered to pointed ends, and often have central swellings and intracellular granules. Cell length is variable but is usually fairly uniform within actively growing cultures. Cells do not possess pili or flagella (Dahlen et al., 1978; Falkler and Hawley, 1977). Surface colonies on blood agar are 1–2 mm in diameter, circular to slightly irregular, convex to pulvinate, translucent often with a “flecked” appearance, usually nonhemolytic (horse or rabbit blood), but may be slightly hemolytic under the area of confluent growth or may produce greenish discoloration of the blood agar upon exposure to oxygen. Glucose broth cultures have a flocculent or granular sediment with or without turbidity, a final pH of 5.6–6.2, and a foul “bad breath” odor. Produces DNase (Porschen and Sonntag, 1974). No phosphatase detected (Porschen and Sonntag, 1974). Most strains produce H2S. Capable of hemagglutinating human and animal erythrocytes. Contains the diamino acid lanthionine in cell-wall peptidoglycan as a major component;

no lysine, ­diaminopimelic acid, or ornithine (Gharbia and Shah, 1990; Kato et al., 1979; Vasstrand, 1981). Heptose and KDO are present in the lipopolysaccharide (Hofstad, 1974). Grows in the presence of up to 6% oxygen; survives exposure to air for 100 min (Loesche, 1969). Strains are resistant to 3 mg/ml of erythromycin (broth disc test). Some Fusobacterium nucleatum isolates produce b-lactamase, an activity not reported for other Fusobacterium species. A penicillin-hydrolyzing b-lactamase inhibited by clavulanic acid has been isolated from Fusobacterium nucleatum (Turner et al., 1985). Fusobacterium nucleatum, similar to other species of the genus, are susceptible to amoxycillin/ clavulanate, carbapenems, chloramphenicol, quinolone clinafloxalin, linezolide, and nitroimidazoles (Goldstein et  al., 1999). Resistance to cefoxitin and clindamycin is very low. Resistance to tetracyclines is common in Fusobacterium nucleatum since a high percentage of strains harbor a chromosomal tetM locus. Alternatively, a tetM determinant on a conjugal transposon (Roberts and Lansciardi, 1990) was also detected. Resistance to glycylglycines has not been observed (Downes et  al., 1999). Fusobacterium nucleatum is sensitive to the bactericidal action of protegrins and other antibacterial peptides (Miyasaki et al., 1998). Other characteristics of the species are given in Table 148. DNA G+C content (mol%): 26–28 (Tm). Type strain: ATCC 25586, CCUG 32989, CCUG 33059, CIP 101130, JCM 8532, LMG 13131. Sequence accession no. (16S rRNA gene): AJ133496. Further comments: Fusobacterium nucleatum is divided into Fusobacterium nucleatum (AF543300)

27.7 29.8

Fusobacterium naviforme (AJ006965) Fusobacterium canifelinum (AY162220)

90.7

Fusobacterium simiae (X55407)

87.1

Fusobacterium peridonticum (AJ810271)

68.6

Fusobacterium perfoetens (M58684) Fusobacterium russi (X55409)

73.5

Fusobacterium necrophorum (AF044948)

66.8 43.4

Fusobacterium equinum (EF447428) Fusobacterium gonidiaformans (X55410) Fusobacterium varium (AJ867036)

68.6

Fusobacterium ulcerans (AJ867037)

100.0

Fusobacterium mortiferum (AJ867033)

97.3

Fusobacterium necrogenes (AJ867034) 60

50

40

30

20

10

0

Figure 121.  Unrooted neighbor-joining tree of the 16S rRNA gene sequences of Fusobacterium. GenBank accession numbers are given after species names. The numbers above branches are bootstrap percentages from 1000 resampled datasets. Bar = 0.1 difference per nucleotide.

754

Family I. Fusobacteriaceae

five subspecies: Fusobacterium nucleatum subsp. nucleatum, subsp. animalis, subsp. fusiforme, subsp. polymorphum, and subsp. vincentii, described on the basis of electrophoretic patterns of whole-cell proteins and DNA homology (Dzink et  al., 1990) and electrophoretic mobility of glutamate dehydrogenase and 2-oxoglutarate dehydrogenases and DNA homology (Gharbia and Shah, 1992). The five subspecies differed from each other and from other closely related species by comparisons of their 16S–23S internal transcribed spacer regions (Conrads et al., 2002). 1a. Fusobacterium nucleatum subsp. nucleatum (Knorr 1922) Dzink, Sheenan and Socransky 1990, 77VP (Fusobacterium nucleatum Knorr 1922, 17) nu.cle.a¢tum. L. neut. adj. nucleatum having a kernel, intended to mean nucleated. DNA G+C content (mol%): 26–28 (Tm). Type strain: ATCC 25586, CCUG 32989, CCUG 33059, CIP 101130, JCM 8532, LMG 13131. Sequence accession no. (16S rRNA gene): AJ133496. Further information: the subspecies name Fusobacterium nucleatum subsp. nucleatum Knorr (1922) is automatically created by the valid publication of Fusobacterium nucleatum subsp. polymorphum (ex Knorr (1922) Dzink (1990), and the valid publication of Fusobacterium nucleatum subsp. vincentii (ex Knorr (1922) Dzink (1990) [Rule 40d (formerly Rule 46)]. 1b. Fusobacterium nucleatum subsp. animalis Gharbia and Shah 1992, 297VP a.ni.ma¢lis. L. n. animal, -alis an animal; L. gen. n. animalis of an animal. Type strain: ATCC 51191, CCUG 32879, CIP 104879, JCM 11025, NCTC 12276. Sequence accession no. (16S rRNA gene): X55404. 1c. Fusobacterium nucleatum subsp. fusiforme (ex Veillon and Zuber 1898) Gharbia and Shah 1992, 297VP (“Sphaerophorus fusiformis” Veillon and Zuber 1898; Sebald 1962) fu.si.for¢me. L.n. fusus a spindle; L. neut. suff. forme in shape of; N.L. neut. adj. fusiforme spindle-shaped. Type strain: ATCC 51190, CCUG 32880, CIP 104878, DSM 19508, JCM 11024, NCTC 11326. Sequence accession no. (16S rRNA gene): AM849219. 1d. Fusobacterium nucleatum subsp. polymorphum (Knorr 1922) Dzink, Sheenan and Socransky 1990, 77VP (“Fusobacterium polymorphum” Knorr 1922) po.ly.mor¢phum. N.L. neut. adj. polymorphum (from Gr. adj. polumorphos -on) multiform, polymorphic. Type strain: ATCC 10953, CCUG 9126, DSM 20482, JCM 12990, NCTC 10562. Sequence accession no. (16S rRNA gene): AF287812. 1e. Fusobacterium nucleatum subsp. vincentii (Knorr 1922) Dzink, Sheenan and Socransky 1990, 77VP (“Fusobacterium plauti-vincentii” Knorr 1922) vin.cen¢.ti.i. N.L. masc. gen. n. vincentii of Vincent, referring to Henri Vincent who studied the organism originally isolated from Vincent’s angina and necrotizing ulcerative gingivitis. Type strain: ATCC 49256, CCUG 37843, CIP 104988, DSM 19507, JCM 11023.

Sequence accession no. (16S rRNA gene): AJ006964, AM887529. 2. Fusobacterium canifelinum Conrads, Citron, Mutters, Jang and Goldstein 2004b, 1909VP (Effective publication: Conrads, Citron, Mutters, Jang and Goldstein 2004a, 412.) ca. ni.felí num. L. gen. pl. n. canum of dogs; L. neut. adj. felinum of or belonging to a cat; N.L. neut. adj. canifelinum of dogs and cats. Strains grown on supplemented Brucella blood-agar for 2 d; colonies are convex, 1–2 mm in diameter with a slightly lobate margin, white, opaque with a granular internal appearance. Cells are slender Gram-stain-negative rods with pointed ends. Glucose and fructose are fermented weakly with a terminal pH 5.7–6.0. End product analysis of PY-glucose by gas-liquid chromatography reveals acetic and butyric acids. Threonine, but not lactate, is converted to propionate. The isolates produce indole, fail to grow in bile, do not hydrolyze esculin, and do not produce acid from lactose, maltose, mannose, raffinose, and sucrose. On agar dilution sensitivity tests, all isolates tested were susceptible to penicillin G, and metronidazole. All strains were resistant (MIC  >4  mg/ml) to levofloxacin, moxifloxacin, gemifloxacin, and other fluoroquinolones. Fusobacterium canifelinum can be distinguished phenotypically from other species of the genus Fusobacterium by the resistance to fluoroquinolones. Source: a purulent dog-bite wound in a human patient. DNA G+C content (mol%): 26–28 (Tm). Type strain: RMA 1036, ATCC BAA-689, CCUG 49733, DSM 15542. Sequence accession no. (16S rRNA gene): AY162221. 3. Fusobacterium equinum Dorsch, Love and Bailey 2001, 1962VP e.quin¢um. L neut. adj. equinum of horses. On sheep-blood agar after 2 d, colonies are circular with an entire undulate margin, convex to umbonate, 1–2 mm in diameter, and creamish in color. Gram staining of single colonies on primary plates reveals pleomorphic Gram-stainnegative rods (coccobacilli to rods, with rounded ends, and curved rods often with irregular staining), but on subculture, coccobacilli and short rods predominate. The terminal pH in glucose liquid medium ranges from pH 6.8–7.0. On GLC analysis, acetic, propionic, and butyric acids are produced and lactate and threonine are converted to propionate. The isolates produce indole and grow in bile and are nonmotile and fail to hydrolyze esculin and starch. The isolates do not ferment esculin, fructose, glucose, lactose, maltose, mannose, starch, and sucrose. They do not hemagglutinate chicken erythrocytes. They are lipase and lecithinase positive. On broth disc sensitivity tests, all isolates are sensitive to penicillin G, amoxycillin, chloramphenicol, doxycycline, and metronidazole; all strains except VPB 4076 and VPB 4014 are sensitive to erythromycin. Source: pus obtained from a discharged sinus associated with the pirulent para-oral lesion in a horse. DNA G+C content (mol%): 29–31 (Tm). Type strain: JCM 11174, NCTC 13176, VPB 4027. Sequence accession no. (16S rRNA gene): AJ295750.

Genus I. Fusobacterium

4. Fusobacterium gonidiaformans (Tunnicliff and Jackson 1925) Moore and Holdeman 1970, 45AL [Bacillus gonidiaformans Tunnicliff and Jackson 1925, 430; Sphaerophorus gonidiaformans (Tunnicliff and Jackson 1925) Prévot 1938, 299] go.ni.di.a.for¢mans. Gr. n. gone offspring, seed; N.L. n. gonidium gonidium; N.L. pl. n. gonidia gonidia; L. part. adj. formans forming; N.L. part. adj. gonidiaformans gonidia forming. Cells from glucose broth cultures are pleomorphic and vacuolated, 0.4–0.7 × 0.7–3.0 mm, often with degenerate filaments or long strands. The spheroid or gonidial forms implied by the name of this organism are seen most often in old cultures or in media that are not highly reduced. Surface colonies on horse blood agar plates are punctiform to 1 mm in diameter, circular entire, low convex, trans­lucent, and smooth. Glucose broth cultures are turbid with smooth sediment and a final pH of 5.6–6.2. Produces DNase (Porschen and Sonntag, 1974). No phosphatase is detected (Porschen and Spaulding, 1974). Hippurate is hydrolyzed. Other characteristics of the species are given in Table 148. Source: the intestinal and urogenital tracts of humans, various types of human infections and from a lamb with pneumonia. DNA G+C content (mol%): 33 (Tm) (Gharbia and Shah, 1990). Type strain: ATCC 25563, CCUG 16790. Sequence accession no. (16S rRNA gene): X55410. 5. Fusobacterium mortiferum (Harris 1901) Moore and Holdeman 1970, 45AL [Bacillus mortiferus Harris 1901, 546; Sphaerophorus mortiferus (sic) (Harris 1901) Prévot 1938, 299] mor.ti¢fer.um. L. neut. adj. mortiferum death-bringing, deathbearing. Cells from glucose broth cultures are 0.8–1.0 × 1.5–10 mm occurring singly and in pairs and short chains. Cells stain irregularly and may be extremely pleomorphic. Surface colonies on horse blood agar are 1–2 mm in diameter, circular with entire, diffuse, or slightly scalloped edge; convex; translucent; smooth. Glucose broth cultures are uniformly turbid with smooth or semiviscous sediment. No superoxide dismutase is detected (Gregory et  al., 1978). Lysine decarboxylase is not produced (Werner, 1974). Produces DNase and phosphatase (Porschen and Sonntag, 1974; Porschen and Spaulding, 1974). Heptose and KDO are present in the lipopolysaccharide (Hofstad, 1974). Fusobacterium mortiferum is the only member of the genus Fusobacterium to have a mixture of mesolanthionine and diaminopimelic acids at an equimolar ration in its cell wall. Some strains are susceptible to cephalothin, cefazolin, cefoxitin, and lincomycin (Finegold, 1977). Resistant to 3 mg/ml erythromycin; susceptible to 12 mg/ml chloramphenicol, 2 U/ml penicillin G, and 6 mg/mltetracycline (broth disk method). Source: blood and various human clinical specimens, intestinal tract and feces; and one from irradiated mice. DNA G+C content (mol%): 26–28 (Tm). Type strain: ATCC 25557, CCUG 14475. Sequence accession no. (16S rRNA gene): AJ867032.

755

6. Fusobacterium naviforme ( Jungano 1909) Moore and Holdeman 1970, 45AL [Bacillus naviformis Jungano 1909, 123; Ristella naviformis ( Jungano 1909) Prévot 1938, 291] na.vi.for¢me. L. n. navis ship; L. neut. suff. forme in shape of; N.L. neut. adj. naviforme in the shape of a ship. Cells from glucose broth cultures are 4–10 mm that are often concave. Cells in old cultures have a beaded appearance. Surface colonies are punctiform to 2.0 mm in diameter, circular entire, low convex, gray-white, translucent with mottled appearance when viewed by obliquely transmitted light. Glucose broth cultures are lightly turbid with smooth to clumpy sediment and final pH of 5.5–6.4. Produces DNase (Porschen and Sonntag, 1974). No phosphatase is detected (Porschen and Spaulding, 1974). Heptose and KDO are present in the lipopolysaccharide (Hofstad, 1974). All strains produce indole from glucose-peptone medium (Gharbia and H.N. Shah, 1989) The cell walls contain meso-lanthionine as the major diaminopimelic acid. Some strains are resistant to 3 mg/ml erythromycin. Susceptible to 12  mg/ml chloramphenicol, 1.6 mg/ml clindamycin, 2 U/ml penicillin G, and 6 mg/ml tetracycline. Source: the large intestine of a laboratory rat. Other strains have been isolated from the human gingival sulcus, from various human clinical specimens, and the bovine rumen. DNA G+C content (mol%): 32–33 (Tm). Type strain: ATCC 25832, CCUG 50052, NCTC 13121. Sequence accession no. (16S rRNA gene): not available. 7. Fusobacterium necrogenes (Weinberg, Nativelle and Prévot 1937) Moore and Holdeman 1970, 45AL [Bacillus necrogenes Weinberg, Nativelle and Prévot 1937, 681; Spherophorus necrogenes (sic) (Weinberg, Nativelle and Prévot 1937) Prévot 1938, 298] ne.cro¢ge.nes. Gr. n. necros the dead; N.L. suff. -genes (from Gr. v. gennaô to produce) producing; N.L. adj. necrogenes dead-producing, here necrosis-producing. This description is based on our study of the type and three phenotypically similar strains. Cells from 1-d-old glucose broth cultures are extremely pleomorphic with coccoid cells about 0.3–0.8 mm and thin filamentous forms 0.2–0.8 mm in diameter and up to 20 mm in length. Cells in older cultures are somewhat more uniform, irregularly staining rods 0.7–0.8 × 1.5–4.0 mm. Surface colonies on horse blood agar are minute to 0.5 mm in diameter, circular, flat to low convex, entire, translucent, white, smooth, and shiny, and colonies are surrounded by zones of b-hemolysis. Glucose broth cultures are moderately turbid with pH of 5.7–6.0 Glutamine decarboxylase negative (Terada et al., 1976). Heptose and KDO present in the lipopolysaccharide (Hofstad, 1974). Source: isolated by Kawamura (1926) from necrotic abscess of a chicken. Barnes strain EB/D/1/4a was isolated from cecal contents of a duck (Barnes and Impey, 1968). Other strains have been isolated from human feces. DNA G+C content (mol%): 27–28 (Tm). Type strain: ATCC 25556, CCUG 4949, NCTC 10723. Sequence accession no. (16S rRNA gene): AJ867034. 8. Fusobacterium necrophorum (Flügge 1886) Moore and Holdeman 1969, 12AL [Bacillus necrophorus Flügge 1886, 273;

756

Family I. Fusobacteriaceae

Fusiformis necrophorus (Flügge) Topley and Wilson 1929, 299; Sphaerophorus necrophorus (Flügge) Prévot 1938, 298] ne.cro¢pho.rum. Gr. n. necros the dead; Gr. v. phoreô to bear; N.L. neut. adj. necrophorum dead producing, here necrosis producing. Cells in glucose broth cultures are 0.5–0.7 mm in diameter with swellings up to 1.8 mm. The ends of the cells may be round or tapered. Cell length ranges from coccoid bodies to filaments over 1.00 mm. Filamentous forms with granular inclusions are more common in broth, while bacilli are more common in older cultures and growth on agar. Surface colonies on blood agar are 1–2 mm in diameter; circular with scalloped to erose edges, convex to umbonate, often with bumpy, ridged, or uneven surface; translucent to opaque, often with mosaic internal structure when viewed by transmitted light. Most strains produce either a- or b-hemolysis on rabbit blood agar. In general, the b-hemolytic strains are lipase positive (on egg yolk agar), and the a-hemolytic or nonhemolytic strains are lipase negative. No lecithinase is produced. Glucose broth cultures have a smooth, flocculent, granular, or stringy sediment and usually are turbid. The final pH of fructose and glucose cultures is 5.6–6.3. A few strains produce a pH of 5.8–5.9 in maltose medium. Human rabbit and guinea pig red blood cells are agglutinated; bovine and ovine red blood cells are not (Simon, 1975). Dextran is not hydrolyzed (Holbrook and McMillan., 1977). No superoxide dismutase (Gregory et  al., 1978) or lysine decarboxylase (Werner, 1974) is detected. Produce DNase (Porschen and Sonntag, 1974). No phosphatase is detected (Porschen and Spaulding, 1974). Heptose and KDO are present in the lipopolysaccharide (Hofstad, 1974). Source: the natural cavities of humans and other animals and from clinical specimens (necrotic lesions, abscesses, and blood) of humans and other animals particularly liver abscesses and foot rot of cattle. For a review of natural and experimental pathogenicity, see Prévot et  al. (1967) and Langworth (1977). Source: bovine liver abscess; Fievez strain 2358. DNA G+C content (mol%): 31–34 (Tm) [chromatographic separation (Sebald, 1962)]. Type strain: ATCC 25286, CCUG 9994, CIP 104559, JCM 3718, VPI 2891. Sequence accession no. (16S rRNA gene): AJ867039. Further comments: two subspecies have been described among isolates of Fusobacterium necrophorum. These correspond to Biovar A and B. 8a. Fusobacterium necrophorum subsp. necrophorum (Flügge 1886) Shinjo, Fujisawa and Mitsuoka 1991, 396VP (Fusobacterium necrophorum Moore and Holdeman 1969, 12) ne.cro¢pho.rum. Gr. n. necros the dead; Gr. v. phoreô to bear; N.L. neut.adj. necrophorum dead producing, here necrosis producing. Previously known as Fusobacterium necrophorum biovar A. The subspecies name Fusobacterium necrophorum subsp. necrophorum (Flügge, 1886) Moore and Holdeman (1969) is automatically created by the valid publication of Fusobacterium necrophorum subsp. funduliforme (ex Hallé (1898) Shinjo et al. (1991) [Rule 40d (formerly Rule 46)] and was previously known as biovar B.

Type strain: ATCC 25286, CCUG 9994, CIP 104559, JCM 3718, VPI 2891. Sequence accession no. (16S rRNA gene): AJ867039. 8b. Fusobacterium necrophorum subsp. funduliforme (ex Hallé 1898) Shinjo, Fujisawa and Mitsuoka 1991, 396VP (“Sphaerophorus funduliformis” Hallé 1898) fun.du.li.for¢me. L. n. fundulus sausage; L. neut. suff. forme in shape of; N.L. neut. adj. funduliforme sausage-shaped. Type strain: Fn524, ATCC 51357, CCUG 42162, CIP 104859, DSM 19678, JCM 3724. Sequence accession no. (16S rRNA gene): AM905356. 9. Fusobacterium perfoetens (Tissier 1905) Moore and Holdeman 1973, 72AL [Coccobacillus perfoetens Tissier 1905, 110; Ristella perfoetens (Tissier 1905) Prévot 1938, 291; Sphaerophorus perfoetens (Tissier, 1905) Sebald 1962, 149] per.foe¢tens. L. prep. per very; L. part. adj. foetens stinking; N.L. part. adj. perfoetens very stinking. Description is from Prévot et al. (1967), Weinberg et al. (1937), van Assche and Wilssens (1977). Cells from glucose cultures are 0.6–0.8 × 0.8–1.0 mm, oval, never elongated, occurring singly, in pairs, in chains of no more than three cells, or in masses. No flagella or capsule. Colonies in deep agar (2 d) are 1 mm in diameter and lenticular. Surface colonies on blood agar are 1–2 mm in diameter, circular with an entire edge, convex to raised, grayish white, translucent, smooth, and nonhemolytic on horse blood. Colonies of some strains are slightly umbonate with diffuse edges and a slightly mottled or granular appearance. Growth in glucose broth is rapid. Cultures are turbid with a fine to ropy sediment and a pH of 5.6. Gas and a fetid odor are produced. Galactose is weakly fermented (Van Assche and Wilssens, 1977). Growth is enhanced in media containing fructose, glucose, mannose, sucrose, and trehalose. The pH of cultures in these media ranges from 5.6 to 5.95. Produces CO2 and NH3. Major amounts of lactic acid produced from PYG cultures (Van Assche and Wilssens, 1977) no lactic acid detected in PYG cultures of the type strain. Inhibited by 0.001% polymyxin. Resistant to 0.001% brilliant green. Optimum temperature is 37°C; good growth occurs at 45°C, poor growth at 25–30°C. Survives up to 24 h of exposure to air. Source: isolated by Tissier in 1900 from an infant with diarrhea and in 1905 from nursing infants. Strain CC1 isolated in 1947 by Prévot from the cecum of a horse and studied by Sebald (1962) has been lost. Van Assche and Wilssens (1977) studied 6 isolates from the feces of a 2-week-old pig, one of which has been designated the neotype strain. DNA G+C content (mol%): 28–30 (Tm) (Sebald, 1962; Van Assche and Wilssens, 1977). Type strain: ATCC 29250. Sequence accession no. (16S rRNA gene): M58684. 10. Fusobacterium peridonticum Slots, Potts and Mashimo 1984, 270VP (Effective publication: Slots, Potts and Mashimo 1983, 963.) pe.ri.o¢don.ti.cum. Gr. prep. peri around; Gr. n. odous, ontos tooth; L. neut. suff. -icum suffix used with the sense of pertaining to; N.L. neutr. adj. periodonticum pertaining to periodonte.

Genus I. Fusobacterium

Description is from Slots et al. (1982). Obligately anaerobic, nonmotile, nonsporeforming, Gram-stain-negative rod. Mean cell size on blood agar is 0.5–1.0 × 4.0–7.0 mm, but filaments longer than 100 mm are often present. Cells had pointed to slightly rounded ends and generally occurred singly or in pairs lying end to end. After anerobic incubation for 2–3 d on blood agar, the colonies measured from 2.0 to 3.0 mm in diameter, were circular, convex, entire or slightly scalloped at the edge, slightly rough, and granular and opaque. Glucose broths were turbid, with flocculent or stringy sediment. Indole is produced, hydrogen sulfide formed from cysteine, litmus milk reduced, hippurate hydrolyzed, and fructose, galactose, and glucose fermented. Growth is inhibited by 1 mg/ml chloramphenicol, penicillin G, and tetracycline and was unaffected by 5 mg/ml erthryomycin and vancomycin. Addition of 2% oxgall inhibited the growth. DNA G+C content (mol%): 28 (Tm). Type strain: EK1-15, ATCC 33693, CCUG 14345, JCM 12991. Sequence accession no. (16S rRNA gene): X55405. 11. Fusobacterium russii (Hauduroy, Ehringer, Urbain, Guillot and Magrou 1937) Moore and Holdeman 1970, 45AL (Bacteroides russii Hauduroy, Ehringer, Urbain, Guillot and Magrou 1937, 73) rus¢si.i. N.L. masc. gen. n. russii of Russ, named after V. Russ, the bacteriologist who first cultured this organism. Cells in glucose broth are 1.5–4.0 mm in length and may form thin filaments 10–15 mm in length. Surface colonies on horse blood agar are 0.5–1 mm in diameter, circular, smooth, shiny, entire, convex, and translucent. The type strain is b-hemolytic on horse blood agar. Glucose-peptone broth cultures are turbid, often with stringy sediment and have a final pH of 5.9–6.1. Produces DNase and phosphatase (Porschen and Sonntag, 1974; Porschen and Spaulding, 1974). Heptose and KDO are present in the lipopolysaccharide (Hofstad, 1974). The major dibasic amino acid is mesolanthionine. Strains do not ferment any carbohydrates nor produce lactate threonine, but phosphatase activities were reported (Table 148). Susceptible to 12 mg/ml chloramphenicol, 1.6 mg/ml clindamycin, and 2 U/ml penicillin G. Some strains are resistant to 6 mg/ml tetracycline. Source: isolated by Russ (1905) from perianal abscess. Also isolated from infections of cats, including actinomycosis of cats and from human and animal feces. DNA G+C content (mol%): 31(Tm). Type strain: ATCC 25533. Sequence accession no. (16S rRNA gene): X55409. 12. Fusobacterium simiae Slots and Potts 1982, 193VP sim¢i.ae. L. fem. n. simia monkey; L. gen. n. simiae of the monkey. Cellular morphology resembles that of Fusobacterium nucleatum (q.v., species 1). Fructose and glucose are fermented (pH of 5.5–5.6). Twenty-seven other substrates tested are not fermented. Indole and lipase are produced; hippurate is hydrolyzed. Grows in media containing 2% oxgall. From glucose, butyrate is the major fermentation product; major amounts of acetate and small amounts or

757

propionate, lactate, and succinate also are produced. Neither hydrogen nor gas is detected. Lactate and threonine are converted to propionate. The type strain has 48% DNA homology with the type and one other strain of Fusobacterium nucleatum and 9% homology with the type strain of Fusobacterium necrophorum. Source: the mouth of the stump-tailed macaque (Macaca arctoides). DNA G+C content (mol%): 27–28 (Tm). Type strain: 7511 R2-13, ATCC 33568, CCUG 16798 (Slots and Potts 7511 R2–13). Sequence accession no. (16S rRNA gene): X55407. 13. Fusobacterium ulcerans Adriaans and Shah 1988, 447VP ul¢ce. rans. L. part. adj. ulcerans making sore, causing to ulcerate, referring to the source of isolation. Gram-stain-negative, nonsporeforming, obligately anaerobic, rod-shaped 0.5–4.5 mm long. Most strains consist of long cells with pointed ends. Surface colonies on anaerobic blood agar plates are 2–3 mm in diameter, circular, entire, domed to low convex, cream colored, and nonhemolytic. A second morphological type consists of rod-shaped orga­ nisms that are 0.8–4.5 × 0.2 mm and have a large round swelling (diameter 0.5 mm) in the center of the cell. Surface colonies on blood agar plates are 1–2 mm in diameter, circular, entire, and low convex. They are translucent or white in color, butyrous, and nonhemolytic. The optimal tempera­ ture for growth is 37°C with colonies appearing in 36–48 h. Culture in brain heart infusion broth produce generalized turbidity after incubation for 3–4 d. Fermentation of peptone-yeast-glucose broth produces large amounts of butyric acid and small to moderate amounts of acetic, propionic, lactic, and succinic acids. All strains convert threonine to propionic acid. Both colonial types produce gas when grown in deep culture. No H2S is detected. Acid is produced from glucose, and some strains ferment glucose and mannose. No acid is produced from fructose, lactose, sucrose, maltose, arabinose, raffinose, or rhamnose. Indole, catalase, lecithinase, urease, lipase, and oxidase are not produced. Esculin and starch are not hydrolyzed. Nitrate is reduced by all strains. All strains are susceptible to penicillin and phosphamycin and are resistant to rifampin. DNA G+C content (mol%): 29.2–29.5 (Tm). Type strain: ATCC 49185, CCUG 50053, NCTC 12111. Sequence accession no. (16S rRNA gene): AJ867037. 14. Fusobacterium varium (Eggerth and Gagnon 1933) Moore and Holdeman 1969, 12AL [Bacteroides varius Eggerth and Gagnon 1933, 409; Sphaerophorus varius (Eggerth and Gagnon 1933) Prévot 1938, 299] va¢ri.um. L. neut. adj. varium diverse, different, various. Cells from glucose broth cultures are pleomorphic, coccoid and rod-shaped, and stain unevenly. Cells are 0.3–0.7 × 0.7–2.0 mm and occur singly and in pairs. Surface colonies on blood agar are punctiform to 1 mm in diameter, circular with entire edges, flat to low convex, translucent, usually with gray-white centers, and colorless edges. Glucose broth cultures are turbid with smooth sediment and a final pH of 5.3–5.7. Dextran is not hydrolyzed (Holbrook and

758

Family I. Fusobacteriaceae

McMillan., 1977). Produces lysine decarboxylase (Werner, 1974). Produces DNase (Porschen and Sonntag, 1974). No phosphate detected (Porschen and Spaulding, 1974). Heptose and KDO are present in the lipopolysaccharide (Hofstad, 1974). A temperate lysogenic bacteriophage is harbored by the reference ATCC 27725 which causes a lytic response in other strains of Fusobacterium varium including the reference strain ATCC 8501. The phage is activated by incubation at

45°C and UV exposure (Gharbia, personal observation) Source: human feces, purulent infections of humans (upper respiratory tract, surgical wounds, peritonitis), cecal contents of mice, intestinal contents of Blatta orientalis (roach), posterior intestinal tract of Recticulitermes lucifugus (termite) and vaginal swab of chinchilla. DNA G+C content (mol%): 29 (Tm) (Sebald, 1962). Type strain: ATCC 8501, CCUG 4858, NCTC 10560. Sequence accession no. (16S rRNA gene): AJ867036.

Genus II. Cetobacterium Foster, Ross, Naylor, Collins, Ramos, Fernández-Garayzábal and Reid 1996, 362VP (Effective publication: Foster, Ross, Naylor, Collins, Ramos, Fernández-Garayzábal and Reid 1995, 206.) Kirstin J. Edwards, Julie M. J. Logan and Saheer E. Gharbia Ce.to.bac.te¢ri.um. Gr. n. kêtos whale; L. neut. n. bacterium a rod; N.L. neut. n. Cetobacterium a bacterium found in association with whales.

Short pleomorphic, nonsporeforming, rod-shaped cells. Central swelling and filaments may be present. Gram-stainnegative. Nonmotile. Microaerotolerant. Catalase negative. Fermentative. Acetic acid is the major end product from peptones or carbohydrates; butyric, propionic, lactic, and succinic acids may or may not be formed in small amounts. Indole produced and ONPG hydrolyzed. Alkaline phosphatase, and acid phosphatase positive. Produces small to moderate amounts of phosphohydrolase. May or may not produce a- or b-galactosidase and a-glucosidase. Urease may or may not be produced. Lecithinase and lipase negative. Gelatin is not hydrolyzed. Nitrate may or may not be reduced to nitrite. Resistant to 20% bile. Resistant to vancomycin. Sensitive to kanamycin and colistin sulfate discs. Susceptible to cefoxitin, clindamycin, imipenem, and metronidazole. Isolated from mammalian intestinal tract and oral cavity. DNA G+C content (mol%): 29–31. Type species: Cetobacterium ceti Foster, Ross, Naylor, Collins, Ramos, Fernández-Garayzábal and Reid 1996, 362VP (Effective publication: Foster, Ross, Naylor, Collins, Ramos, FernándezGarayzábal and Reid 1995, 206.).

Further descriptive information Cetobacterium was first isolated from the intestinal contents of a porpoise and from a mouth lesion of a minke whale (Foster

et  al., 1995). Cetobacterium somerae has subsequently been isolated from human feces (Finegold et  al., 2003a). Colonies are gray, waxy, circular with scalloped to erose edges, slightlyraised, smooth, dull, and opaque with a diameter of 2–4 mm. Weak hemolysis was observed with both sheep and horse blood. No growth was observed for Cetobacterium ceti following subculture in an atmos­phere of 10% CO2 or in air, whereas Cetobacterium somerae grows in 2% but not 6% oxygen (Finegold et al., 2003a).

Differentiation of the genus Cetobacterium from other genera Cetobacterium differs from Fusobacterium species in producing acetic and propionic acids, whereas members of the genus Fusobacterium produce butyric acid (Foster et al., 1995). Sequencing the 16S rRNA gene demonstrates the highest sequence similarities with Fusobacterium (91–94%) and 92% similarity with Propionigenium modestum. Significant sequence similarity was also observed with Leptotrichia and Sebedella (86%) (Foster et al., 1995). A large amount of acetic acid with lesser amounts of propionic, lactic, and succinic acids permits differentiation from Propionigenium modestum which does not ferment carbohydrates, but produces large amounts propionic and lesser amounts of acetic acid only from succinate and other substrates (Schink and Pfennig, 1982) A positive indole reaction allows further separation from Propionigenium modestum.

List of species of the genus Cetobacterium 1. Cetobacterium ceti Foster, Ross, Naylor, Collins, Ramos, Fernández-Garayzábal and Reid 1996, 362VP (Effective publication: Foster, Ross, Naylor, Collins, Ramos, FernándezGarayzábal and Reid 1995, 206.) ce.ti. L. gen. n. ceti of a whale. Description is from Foster et  al. (1995). Surface colonies on blood agar are 2–4 mm in diameter after 48 h at 37°C, gray, waxy, circular, with scalloped to erose edges, slightly raised, smooth, dull, opaque, and weakly hemolytic on sheep and horse blood. No growth at 25°C or 45°C. ­Catalase-negative. Indole, ONPG and phosphatase positive. Lecithinase, lipase, DNase, nitrate, urea, esculin, gelatin, and starch negative. Resistant to 20% bile. Resistant

to vancomycin. Sensitive to colistin sulfate and kanamycin. Sensitivity to penicillin varies. The major volatile fatty acids produced are acetic, propionic, lactic, and succinic. Butyric acid is not produced. DNA G+C content (mol%): 29 (Tm). Type strain: M-3333, ATCC 700028, NCIMB 703026. Sequence accession no. (16S rRNA gene): X78419. 2. Cetobacterium somerae Finegold, Vaisanen, Molitoris, Tomzynski, Song, Liu, Collins and Lawson 2003b, 1219VP (Effective publication: Finegold, Vaisanen, Molitoris, Tomzynski, Song, Liu, Collins and Lawson 2003a, 180.) so¢me.rae. N.L. gen. fem. n. somerae of Somer, to honor ­Hannele Jousimies-Somer, a contemporary Finish

Genus III. Ilyobacter

­ icro­biologist, in recognition of her important contribum tions to anaerobic microbiology. Description is from Finegold et  al. (2003a). Rod-shaped. Gram-stain-negative. Microaerotolerant. After 48 h incubation anaerobically at 37°C on brucella blood agar, colonies are 2–3 mm in diameter, smooth, circular, entire, and gray in color. Colonies do not fluoresce under UV light. Catalase negative. Indole positive after 48 h incubation. Acetic acid is the major end product in peptone yeast broth; small amounts of propionic and butyric acids may be formed; a trace of succinic acid was produced by all strains. Nitrate is reduced to nitrite. Resistant to 20% ox bile. Esculin may or may not be hydrolyzed. Gelatin is not hydrolyzed. By traditional tests, ONPG is hydrolyzed, but

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lecithinase, lipase, and b-lactamase are not produced. Urease may or may not be detected. Using the API ZYM system, a- and b-galactosidase and alkaline phosphatase are produced; phosphohydrolase is produced in lesser amounts, and a-glucosidase may or may not be produced. Susceptible to kanamycin, colistin sulfate, cefoxitin, clindamycin, imipenem, and metronidazole. Resistant to ampicillin, penicillin G, ramoplanin, trimethoprim/ sulfamethoxazole, and vancomycin. Predominant long-chain cellular fatty acids are C14:0, C16:0, and C16:1 w9c. Source: human fecal material. DNA G+C content (mol%): 31 (HPLC). Type strain: WAL 14325, ATCC BAA-474, CCUG 46254. Sequence accession no. (16S rRNA gene): AJ438155.

Genus III. Ilyobacter Stieb and Schink 1985, 375VP (Effective publication: Stieb and Schink 1984, 145.) Bernhard Schink, Peter H. Janssen and Andreas Brune I.ly.o.bac¢ter. Gr. fem. n. ilys mud; N.L. masc. n. bacter rod; N.L. masc. n. Ilyobacter a mud-inhabiting rod.

Strictly anaerobic chemoorganotrophic bacteria with fermentative metabolism, nonphotosynthetic, inorganic electron acceptors not used. Nonsporeforming. Chemoorganotrophic, fermentative type of metabolism. Media containing a reductant are necessary for growth. Catalase negative. Isolated from anoxic environments. DNA G+C content (mol%): 31.7–36.7. Type species: Ilyobacter polytropus Stieb and Schink 1985, 375VP (Effective publication: Stieb and Schink 1984, 145.).

Further descriptive information The genus Ilyobacter consists so far of four species, Ilyobacter polytropus, Ilyobacter delafieldii, Ilyobacter insuetus, and Ilyobacter tartaricus. The genus was created to house Gram-stain-negative, obligately anaerobic, nonsporeforming bacteria that do not contain cytochromes and use unusual substrates for growth. Its members differ from those of most other genera of strictly anaerobic bacteria by their unusual patterns of substrate utilization and product formation. Fermentation products include acetate, butyrate, and (on some substrates) also formate and ethanol. Malate and fumarate are fermented to acetate, formate, and propionate. The DNA base ratio of Ilyobacter species ranges from 32 to 36 mol% G+C, and so is clearly lower that that of any Bacteroides, Selenomonas, or Pelobacter species. With the exception of Ilyobacter delafieldii (see below), all Ilyobacter species are short to coccoid rods, often in pairs or short chains. Ilyobacter polytropus was enriched and isolated from marine sediment with 3-hydroxybutyrate, which was fermented to acetate and butyrate. Glycerol was fermented to 1,3-propanediol and 3-hydroxypropionate. Acetate and formate were the only products of pyruvate or citrate fermentation. Glucose and fructose were fermented to acetate, formate, and ethanol. Malate and fumarate were fermented to acetate, formate, and propionate. Ilyobacter tartaricus was enriched and isolated from marine sediment with l-tartrate as sole source of carbon and energy. Tartrate, citrate, pyruvate, and oxaloacetate are fermented to acetate, formate, and CO2. In addition, ethanol is formed from fructose and glucose. Ilyobacter insuetus was isolated from marine sediment with quinic acid (1,3,4,5-tetrahydroxy-cyclohexane-1-carboxylic acid, sodium salt) as the sole source of carbon and energy. This

bacterium is restricted to the fermentation of hydroaromatic substrates. Of more than 30 different substrates tested, only quinic acid and shikimic acid (3,4,5-trihydroxy-1-cyclohexene1-carboxylic acid) are utilized. Neither sugars, alcohols, other carboxylic acids, amino acids, nor aromatic compounds are fermented. Thus, this species represents an extreme case of specialization in substrate utilization. Ilyobacter delafieldii ( Janssen and Harfoot., 1991) was enriched and isolated from estuarine sediment with crotonate as substrate. It ferments crotonate, 3-hydroxybutyrate, lactate, pyruvate, and poly-b-hydroxybutyrate to acetate, propionate, butyrate, CO2, and H2. Poly-b-hydroxybutyrate is hydrolyzed outside the cell without cell contact, by a PHB depolymerase that is excreted into the growth medium ( Janssen and Harfoot, 1990). So far, this bacterium is unique in its capacity to degrade extracellular PHB anaerobically (Schink et al., 1992). The taxonomic status of Ilyobacter delafieldii is unclear. Since it stains Gram-negative and resembles Ilyobacter polytropus in many of its metabolic capacities, it was originally assigned to the genus Ilyobacter. However, sequence analysis of its 16S rRNA gene later revealed that it should be grouped within the genus Clostridium ( Janssen, unpublished), even though spore formation could not be demonstrated ( Janssen and Harfoot, 1990). The cellwall architecture of Ilyobacter delafieldii strain 10cr1 ( Janssen and Harfoot, 1990) is not typical of Gram-stain-negative bacteria but resembles that of a Gram-stain-positive bacterium with a complex cell-wall structure. For this reason, we do not include Ilyobacter delafieldii any further in this genus. Ilyobacter tartaricus has generated major interest because of its Na+-translocating ATP-synthase system. Tartrate and oxaloacetate are metabolized via pyruvate; the oxaloacetate decarboxylase is a Na+-translocating, membrane-bound enzyme. The Na+-gradient established this way is used for ATP-synthesis via a membrane-bound Na+-translocating ATP-synthase enzyme, and contributes to the overall energy balance of the cell. Together with a similar enzyme in Propionigenium modestum (Hilpert et al., 1984), this Na+-ATPase has become one of the model systems to study the architecture of this type of F1F0-ATPases and especially the linkage between Na+-ion transport and ATP synthesis ­(Neumann et al., 1998). The three-dimensional structure of this Na+-ATPase was recently resolved in detail (Meier et al., 2005).

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Family I. Fusobacteriaceae

The study of Na+-translocating ATPases has also contri­buted significantly to a better understanding of proton transport in the more common H+-translocating F1F0-ATPases (Gemperli et al., 2003). It appears that anoxic marine sediments are the typical habitats of these bacteria. At least with Ilyobacter tartaricus, the energy metabolism is based on sodium ions as coupling ions in energy conservation.

Enrichment and isolation procedures A strictly anoxic, sulfide-reduced mineral medium with 10 mM of either 3-hydroxybutyrate, shikimate, or l-tartrate as the sole organic carbon and energy source, and incubation at 27–30°C has proven to be highly selective for the enrichment of Ilyobacter polytropus, Ilyobacter insuetus, or Ilyobacter tartaricus, respectively. The carbonate-buffered standard medium used for enrichment and isolation has been described in detail (Schink and Pfennig, 1982; Widdel and Pfennig, 1981). After two to three transfers, the bacteria can be isolated in anoxic agar deep dilution series (Pfennig, 1978) or in roll tubes (Balch et al., 1979). Streaking on Petri dishes in an anoxic glove box has not yet been tried with these bacteria.

Maintenance procedures Cultures are maintained either by repeated transfer at intervals of 2–3 months or by freezing in liquid nitrogen using techniques common for strictly anaerobic bacteria.

Differentiation of the genus Ilyobacter from other genera The three species remaining in the genus Ilyobacter differ from most other strictly anaerobic bacteria in their unusual patterns of substrate utilization and product formation and their low G+C content. With the exception of the genus Propionigenium, they are clearly separated from all other genera by their 16S rRNA gene sequences. Comparative 16S rRNA gene sequence analysis places the members of the genera Propionigenium and Ilyobacter (with the exception of Ilyobacter delafieldii) into the Fusobacteria phylum (Brune et al., 2002). Both genera form a distinct cluster, clearly separated from the Sebaldella–Streptobacillus–Leptotrichia lineage and the Fusobacterium branch. While the 16S rRNA gene sequences did not allow resolving the branching order within the Ilyobacter– Propionigenium cluster, the 23S rRNA gene sequences supported a monophyletic status at least for the genus Ilyobacter (Brune et al., 2002). Although the metabolic properties of Propionigenium and Ilyobacter species are sufficiently different to justify maintenance of two separate genera, the situation is unsatisfying and asks for a future taxonomic revision of this group.

Further reading Dimroth, P., C. von Ballmoos and T. Meier. 2006. Catalytic and mecha­nical cycles in F-ATP synthases. Fourth in the Cycles Review Series. EMBO Rep. 7: 276–282. Brune, A. and B. Schink. 1992. Anaerobic degradation of hydroaromatic compounds by newly isolated fermenting bacteria. Arch. Microbiol. 158: 320–327.

List of species of the genus Ilyobacter 1. Ilyobacter polytropus Stieb and Schink 1985, 375VP (Effective publication: Stieb and Schink 1984, 145.)

in.su.e¢tus. L. masc. part. adj. insuetus unusual, extraordinary, referring to the organism¢s metabolism.

po.ly¢tro.pus. N.L. masc. adj. polytropus (from Gr. masc. adj. polytropos) turning many ways, versatile, referring to metabolic versatility.

Rod-shaped to coccoid cells, 0.8–1.0 mm in diameter and 1.0–1.5 mm long, with rounded ends. Nonmotile, Gramstain-negative, nonsporeforming. Strictly anaerobic chemoorganotroph. Quinic acid and shikimic acid utilized for growth and fermented to acetate, propionate, butyrate, H2, and CO2. No growth with sugars (cellobiose, fructose, glucose, erythrose, lactose, ribose, xylose), alcohols (meso-erythritol, ethanol, glycerol, mannitol), carboxylic acids (citrate, crotonate, fumarate, glycolate, 2-hydroxybutyrate, 3-hydroxybutyrate, 4-hydroxybutyrate, lactate, malate, 2-oxobutyrate, pyruvate, sorbate, tartrate), amino acids (alanine, aspartate, glycine, threonine), or aromatic compounds (gallate, phloroglucinol, protocatechuate, resorcinol, 3,4,5-trimethoxybenzoate, 3,4,5-trimethoxycinnamate). Sulfate, sulfur, thiosulfate, nitrate, and ferric iron not reduced. Strict anaerobe. No catalase activity; no super­ oxide dismutase activity; no cytochromes. Growth requires mineral media with a reductant and at least 0.7% sodium chloride. Selective enrichment in NaCl-containing mineral media with quinic acid as the sole source of carbon and energy. pH Range: 6.0–9.0, optimum at 7.0–8.0. Temperature range: 15–40°C, optimum growth temperature 30°C. Habitats: anoxic marine sediment. DNA G+C content (mol%): 35.7 ± 1.0 (HPLC). Type strain: VenChi2, ATCC BAA-291, DSM 6831. Sequence accession no. (16S rRNA gene): AJ307980.

Rod-shaped cells, 0.7 × 1.5–3.0 mm in size with rounded ends, single or in pairs. Nonmotile, Gram-stain-negative, nonsporeforming. Strictly anaerobic chemoorganotroph. 3-Hydroxybutyrate and crotonate fermented to acetate and butyrate. Glycerol fermented to 1,3-propanediol and 3-hydroxypropionate. Malate and fumarate fermented to acetate, formate, and propionate. Glucose and fructose fermented to acetate, formate, and ethanol. No other organic acids, sugars, or alcohols metabolized. Sulfate, sulfur, thiosulfate, and nitrate not reduced. Growth occurs in mineral media with a reductant. Indole not formed; gelatin and urea not hydrolyzed. No catalase activity. No cytochromes detectable. Growth requires mineral media with a reductant and at least 1% sodium chloride. Selective enrichment in NaCl-containing mineral media with 3-hydroxybutyrate as substrate. pH Range: 6.5–8.5, optimum at 7.0–7.5. Temperature range: 10–35°C, optimum growth temperature 30°C. Habitats: anoxic marine or brackish water sediment. DNA G+C content (mol%): 32.2 ± 0.5 (Tm). Type strain: CuHbu1, ATCC 51220, DSM 2926. Sequence accession no. (16S rRNA gene): AJ307981. 2. Ilyobacter insuetus Brune, Evers, Kalm, Ludwig and Schink 2002, 431VP

Genus IV. Propionigenium

3. Ilyobacter tartaricus Schink 1985, 375VP (Effective publication: Schink 1984, 413.) tar.ta¢ri.cus. N.L. n. acidum tartaricum tartaric acid; N.L. masc. adj. tartaricus referring to tartaric acid as isolation substrate. Rod-shaped cells, 1.0–1.2 × 1.2–2.5 mm in size, often in chains. Surrounded by slime capsules, nonmotile, Gramstain-negative, nonsporeforming. Strictly anaerobic chemoorganotroph. Growth on l-­ tartrate, citrate, pyruvate, oxaloacetate, glucose, fructose, raffinose, glycerol. Fermentation products include acetate, formate, and ethanol. No growth on formate, acetate, ­lactate, methanol, ethanol, ethylene glycol, 2,3-butanediol, glycerate, malate, fumarate, glyoxylate, glycolate, mannose,

761

maltose, lactose, sucrose, cellobiose, sorbose, rhamnose, trehalose, xylose, arabinose, peptone, yeast extract. Sulfate, sulfur, thiosulfate or nitrate not reduced. Indole not formed; gelatin and urea not hydrolyzed. No catalase activity, no cytochromes. Growth requires mineral media with a reductant and at least 1% sodium chloride. Selective enrichment from marine sediments with l-­ tartrate as sole carbon and energy source. pH Range: 5.5–8.0, optimum at 6.5–7.2. Temperature range: 10–40°C, optimum growth temperature 32°C. ­Habitats: anoxic marine sediment. DNA G+C content (mol%): 33.1 ± 1.0 (Tm). Type strain: GraTa2, ATCC 35898, DSM 2382. Sequence accession no. (16S rRNA gene): AJ307982.

Genus IV. Propionigenium Schink and Pfennig 1983, 896VP (Effective publication: Schink and Pfennig 1982, 215.) Bernhard Schink and Peter H. Janssen Pro.pi.o.ni.ge¢ni.um. N.L. n. acidum propionicum propionic acid; L. v. genere to make, produce; N.L. neut. n. Propionigenium propionic acid maker.

Strictly anaerobic chemoorganotrophic bacteria with fermen­ tative metabolism, nonphotosynthetic, inorganic electron acceptors not used. Nonsporeforming. Chemoorganotrophic, fermentative type of metabolism, preferentially using dicarboxylic acids as substrates. Media containing a reductant are necessary for growth. Catalase-negative. Isolated from anoxic marine or freshwater sediments. DNA G+C content (mol%): 32.9–41. Type species: Propionigenium modestum Schink and Pfennig 1983, 896VP (Effective publication: Schink and Pfennig 1982, 215.).

It has to be assumed that anoxic marine sediments are the typical habitats of these bacteria. Their energy metabolism is based on sodium ions as coupling ions in energy conservation. With this ability, they are well adapted to a marine environment. Several marine bacteria have been found to use sodium ions as energy couplers in various functions, e.g., respiration. Propionigenium maris-like bacteria were isolated also from burrows of bromophenol-producing marine infauna, where they apparently are involved in reductive debromination of bromophenols (Watson et al., 2000). They probably use organic excretions of the infauna as electron donors for this reductive reaction.

Further descriptive information

Enrichment and isolation procedures

The genus Propionigenium consists so far of two species, Propionigenium modestum and Propionigenium maris. Propionigenium modestum comprises four strains of physiologically and morphologically similar isolates from various sources (Schink and Pfennig, 1982). This genus was created to house strictly anaerobic bacteria that are able to grow by decarboxylation of succinate to propionate. Pure cultures could be obtained only with enrichment cultures from marine sources; freshwater enrichments grew much slower, and pure cultures were finally isolated when the sodium chloride concentration of the medium was increased to 100–150 mM. A further species, Propionigenium maris, was created later to comprise bacteria similar to Propionigenium modestum but which are metabolically much more versatile and are able to ferment, carbohydrates, amino acids, and other organic acids in addition to C4 dicarboxylic acids ( Janssen and Liesack, 1995). Propionigenium modestum was originally isolated from a black, anoxic, marine sediment sample taken from the Canal Grande in Venice, Italy (Schink and Pfennig, 1982). Similar strains were isolated later from many other marine habitats. Enrichments from freshwater sediments sometimes produced cells of similar morphology to Propionigenium modestum, and these could be cultivated only in media with increased (100–150 mM) sodium chloride concentrations. Also, Propionigenium maris was isolated from marine sediments ( Janssen and Liesack, 1995).

A strictly anoxic, sulfide-reduced mineral medium with 20 mM succinate as the sole organic carbon and energy source, and incubation at 27–30°C has proven to be highly selective for the enrichment of Propionigenium modestum if marine sediment samples of about 5-ml volume are used as the inoculum. The carbonate-buffered standard medium used for enrichment and isolation has been described in detail (Schink and Pfennig, 1982; Widdel and Pfennig, 1981). After two to three transfers in liquid medium, gas should no longer be formed by the enrichment cultures, and a dominant population of short, coccoid rods should be established. These bacteria can be isolated in anoxic agar deep dilution series (Pfennig, 1978) or in roll tubes (Balch et al., 1979). Streaking Petri dishes in an anoxic glove box has not yet been tried with these bacteria. Preparation of pure cultures requires two subsequent dilution series; purity should be checked after growth in selective mineral medium and in complex medium. Propionigenium maris requires yeast extract (0.1% w/v) for growth in pure culture. Although it is not recommended to add yeast extract in the liquid enrichment cultures, it is needed in the purification step.

Maintenance procedures Cultures are maintained either by repeated transfer at intervals of 2–3 months or by freezing in liquid nitrogen using techniques

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Family I. Fusobacteriaceae

common for strictly anaerobic bacteria. No information exists about survival upon lyophilization.

Differentiation of the genus Propionigenium from other genera In phase-contrast microscopy, cells of Propionigenium modestum appear as short, coccoid rods with a diameter of 0.5–0.6 mm and a length of 0.5–2.0 mm, often in short chains. They are Gramstain-negative and nonsporeforming. Propionigenium maris also forms coccoid to ovoid cells or short rod-like cells similar in size to Propionigenium modestum. Both Propionigenium species are strictly anaerobic and do not tolerate increased oxygen tensions. They are specialists for the utilization of C4-dicarboxylic acids. Whereas Propionigenium modestum is restricted to use of only few such compounds,

Propionigenium maris uses also other substrates such as sugars and amino acids, and depends on yeast extract as a medium additive. No cytochromes have been detected, which is consistent with the absence of electron transport phosphorylation. Early 16S rRNA gene sequence analyses revealed that Propionigenium maris and Propionigenium modestum are closely related and form a distinct lineage within a phylogenetically coherent group characterized by Fusobacterium nucleatum and other Fusobacterium species, together with Clostridium rectum, Leptotrichia buccalis, and Sebaldella termitidis (Both et al., 1991; Janssen and Liesack, 1995). This group has been elevated to the rank of phylum, Fusobacteria. In addition to Propionigenium, this phylum embraces the genera Fusobacterium, Ilyobacter, Leptotrichia, Sebaldella, Streptobacillus, and Sneathia (Garrity et  al., 2002).

List of species of the genus Propionigenium 1. Propionigenium modestum Schink and Pfennig 1983, 896VP (Effective publication: Schink and Pfennig 1982, 215.) mo.de¢stum. L. neut. adj. modestum modest, referring to an extremely modest type of metabolism. Rod-shaped to coccoid cells, 0.5–0.6 in diameter × 0.5–2.0 mm long, with rounded ends, single, in pairs, or in chains. Nonmotile, Gram-stain-negative, nonsporeforming. Strictly anaerobic chemoorganotroph. Succinate, fumarate, malate, aspartate, oxaloacetate, and pyruvate utilized for growth and fermented to propionate (acetate), and CO2. No other organic acids and no sugars or alcohols metabolized. Sulfate, sulfur, thiosulfate, and nitrate not reduced. Indole not formed; gelatin and urea not hydrolyzed. No catalase activity. Growth requires mineral media with a reductant and at least 1% sodium chloride. Selective enrichment in NaClcontaining mineral media with succinate as substrate. pH range 6.5–8.4, optimum at 7.1–7.7. Temperature range 15–40°C, optimum 33°C. No cytochromes detectable. Habitats: anoxic marine or brackish water sediment. DNA G+C content (mol%): 33.9 ± 1.0 (Tm). Type strain: GraSucc2, ATCC 35614, DSM 2376. Sequence accession no. (16S rRNA gene): X54275. 2. Propionigenium maris Janssen and Liesack 1996, 362VP emend. Watson, Matsui, Leaphart, Wiegel, Rainey and Lovell 2000, 1040 (Effective publication: Janssen and Liesack 1995, 33.) ma¢ris. L. neut. n. mare the sea; L. gen. n. maris of the sea, referring to the tidal mat flats from which this organism was isolated.

References Adriaans, B. and H. Shah. 1988. Fusobacterium ulcerans sp. nov. from tropical ulcers. Int. J. Syst. Bacteriol. 38: 447–448. Bachrach, G., S.K. Haake, A. Glick, R. Hazan, R. Naor, R.N. Andersen and P.E. Kolenbrander. 2004. Characterization of the novel Fusobacterium nucleatum plasmid pKH9 and evidence of an addiction system. Appl. Environ. Microbiol. 70 : 6957–6962. Baird-Parker, A.C. 1960. The classification of Fusobacteria from the human mouth. J. Gen. Microbiol. 22: 458–469. Bakken, V., S. Aaro, T. Hofstad and E.N. Vasstrand. 1989a. Outer membrane proteins as major antigens of Fusobacterium nucleatum. FEMS Microbiol. Immunol. 1: 473–483.

Coccoid to oval short rods with rounded ends, 1.0 mm in diameter × 1.2–2.5 mm long; under some culture conditions up to 50 mm long. Gram-stain-negative, nonsporeforming. Strictly anaerobic chemoorganotroph. Fermentative metabolism, external electron acceptors not used, however, bromophenols can be reductively dehalogenated. No cytochromes formed. In the presence of yeast extract, several carbohydrates and amino and organic acids are fermented. These substrates include succinate, fumarate, pyruvate, citrate, 3-hydroxybutyrate, glucose, fructose, maltose, aspartate, lysine, threonine, glutamate, and cysteine. Typical products of fermentation are propionate, acetate, and formate, depending on the substrate. Carbo-­ hydrates are fermented to formate, acetate, ethanol, and ­lactate. 3-Hydroxbutyrate is fermented to acetate and butyrate. Hydrogen is produced from carbohydrates and yeast extract, ammonia from amino acids, and sulfide from cysteine. Sulfate, sulfur, thiosulfate, and nitrate not reduced. Indole formed from l-tryptophan. Esculin and urea not hydrolyzed. No catalase activity. Growth in salt-water media with at least 5 and up to 55 g NaCl per liter. Anoxic conditions required for growth. pH range 5.3–8.8, optimum at 6.9–7.7. Temperature range 15–40°C, optimum 34–37°C. Habitat: anoxic marine or brackish water sediment. DNA G+C content (mol%): 40.0 ± 1.0 (Tm) Type strain: 10succ1, DSM 9537. Sequence accession no. (16S rRNA gene): X84049. Bakken, V., S. Aaro and H.B. Jensen. 1989b. Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum. J. Gen. Microbiol. 135: 3253–3262. Balch, W.E., G.E. Fox, L.J. Magrum, C.R. Woese and R.S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43: 260–296. Barnes, E.M. and C.S. Impey. 1968. Anaerobic gram negative ­nonsporing bacteria from the caeca of poultry. J. Appl. Bacteriol. 31: 530–541. Bolstad, A.I. and H.B. Jensen. 1993. Polymerase chain reaction­amplified nonradioactive probes for identification of Fusobacterium nucleatum. J. Clin. Microbiol. 31: 528–532.

Genus IV. Propionigenium Bolstad, A.I., J. Tommassen and H.B. Jensen. 1994. Sequence variability of the 40-kDa outer membrane proteins of Fusobacterium nucleatum strains and a model for the topology of the proteins. Mol. Gen. Genet. 244: 104–110. Both, B., G. Kaim, J. Wolters, K.H. Schleifer, E. Stackebrandt and W. Ludwig. 1991. Propionigenium modestum: a separate line of descent within the eubacteria. FEMS Microbiol. Lett. 62: 53–58. Bouma, C.L., J. Reizer, A. Reizer, S.A. Robrish and J. Thompson. 1997. 6-phospho-a-d-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases. J. Bacteriol. 179 : 4129–4137. Brazier, J.S., D.M. Citron and E.J. Goldstein. 1991. A selective medium for Fusobacterium spp. J. Appl. Bacteriol. 71: 343–346. Brown, R., H.G. Lough and I.R. Poxton. 1997. Phenotypic characteristics and lipopolysaccharides of human and animal isolates of Fusobacterium necrophorum. J. Med. Microbiol. 46: 873–878. Brune, A., S. Evers, G. Kaim, W. Ludwig and B. Schink. 2002. Ilyobacter insuetus sp. nov., a fermentative bacterium specialized in the degradation of hydroaromatic compounds. Int. J. Syst. Evol. Microbiol. 52: 429–432. Carlsson, J., J.T. Larsen and M.B. Edlund. 1994. Utilization of glutathione (l-g-glutamyl-l-cysteinylglycine) by Fusobacterium nucleatum subspecies nucleatum. Oral Microbiol. Immunol. 9: 297–300. Citron, D.M. 2002. Update on the taxonomy and clinical aspects of the genus Fusobacterium. Clin. Infect. Dis. 35: S22–27. Claesson, R., M.B. Edlund, S. Persson and J. Carlsson. 1990. Production of volatile sulfur compounds by various Fusobacterium species. Oral Microbiol. Immunol. 5: 137–142. Claros, M.C., Y. Papke, N. Kleinkauf, D. Adler, D. M. Citron, S. ­Hunt-Gerardo, Th. Montag, E.J.C. Goldstein and A.C. Rodloff. 1999. Characteristics of Fusobacterium ulcerans, a new and unusual species compared with Fusobacterium varium and Fusobacterium ­mortiferum. Anaerobe 5: 137–140. Conrads, G., M.C. Claros, D.M. Citron, K.L. Tyrrell, V. Merriam and E.J. Goldstein. 2002. 16S-23S rDNA internal transcribed spacer sequences for analysis of the phylogenetic relationships among species of the genus Fusobacterium. Int. J. Syst. Evol. Microbiol. 52: 493–499. Conrads, G., D.M. Citron, R. Mutters, S. Jang and E.J.C. Goldstein. 2004a. Fusobacterium canifelinum sp. nov., from the oral cavity of cats and dogs. Syst. Appl. Microbiol. 27: 407–413. Conrads, G., D.M. Citron, R. Mutters, S. Jang and E.J.C Goldstein. 2004b. In Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 100. Int. J. Syst. Evol. Microbiol. 54: 1909–1910. Conrads, G., D.M. Citron and E.J. Goldstein. 2005. Genetic determinant of intrinsic quinolone resistance in Fusobacterium canifelinum. Antimicrob. Agents Chemother. 49: 434–437. Dahlen, G., H. Nygren and H.A. Hannsson. 1978. Immunoelectron microscopic localization of lipopolysaccharides in the cell wall of Bacteroides oralis and Fusobacterium nucleatum. Infect. Immun. 19 : 265–271. Dahlen, G. and I. Mattsby-Baltzer. 1983. Lipid A in anaerobic bacteria. Infect. Immun. 39 : 466–468. Dorsch, M., D.N. Love and G.D. Bailey. 2001. Fusobacterium equinum sp. nov., from the oral cavity of horses. Int. J. Syst. Evol. Microbiol. 51: 1959–1963. Downes, J., A. King, J. Hardie and I. Phillips. 1999. Evaluation of the Rapid ID 32A system for identification of anaerobic Gram-negative bacilli, excluding the Bacteroides fragilis group. Clin. Microbiol. Infect. 5: 319–326. Dzink, J.L., M.T. Sheenan and S.S. Socransky. 1990. Proposal of three subspecies of Fusobacterium nucleatum Knorr 1922: Fusobacterium nucleatum subsp. nucleatum subsp. nov., comb. nov., Fusobacterium nucleatum subsp. polymorphum subsp. nov., nom. rev., comb. nov., and Fusobacterium nucleatum subsp. vincentii subsp. nov., nom. rev., comb. nov. Int. J. Syst. Bacteriol. 40 : 74–78.

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Genus IV. Propionigenium Pianotti, R., S. Lachette and S. Dills. 1986. Desulfuration of cysteine and methionine by Fusobacterium nucleatum. J. Dent. Res. 65: 913–917. Pikis, A., S. Immel, S.A. Robrish and J. Thompson. 2002. Metabolism of sucrose and its five isomers by Fusobacterium mortiferum. Microbiology 148: 843–852. Porschen, R.K. and S. Sonntag. 1974. Extracellular deoxyribonuclease production by anaerobic bacteria. Appl. Microbiol. 27: 1031–1033. Porschen, R.K. and E.H. Spaulding. 1974. Phosphatase activity of anaerobic organisms. Appl. Microbiol. 27: 744–747. Prévot, A.R. 1938. Etudes de systematique bacterienne. III. Invalidite du genre Bacteroides Castellani et Chalmers demembrement et reclassification. Ann. Inst. Pasteur 20 : 285–307. Prévot, A.R., A. Turpin and P. Kaiser. 1967. Les bactéries anaérobies. Dunod, Paris. Roberts, M.C. and J. Lansciardi. 1990. Transferable TetM in Fusobacterium nucleatum. Antimicrob. Agents Chemother. 34: 1836–1838. Robrish, S.A., C. Oliver and J. Thompson. 1987. Amino acid-dependent transport of sugars by Fusobacterium nucleatum ATCC 10953. J. Bacteriol. 169 : 3891–3897. Robrish, S.A., C. Oliver and J. Thompson. 1991. Sugar metabolism by fusobacteria: regulation of transport, phosphorylation, and polymer formation by Fusobacterium mortiferum ATCC 25557. Infect. Immun. 59 : 4547–4554. Rogers, A.H., N.J. Gully, A.L. Pfennig and P.S. Zilm. 1992. The breakdown and utilization of peptides by strains of Fusobacterium nucleatum. Oral Microbiol. Immunol. 7: 299–303. Russ, V.R. 1905. ber ein Inflenzabacillenahnliches anaerobes. Stabchen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg., I Abt. Orig. 39 : 357. Schink, B. and N. Pfennig. 1982. Propionigenium modestum gen.nov. sp. nov. a new strictly anaerobic, non-sporing bacterium growing on succinate. Arch. Microbiol. 133: 209–216. Schink, B. and N. Pfennig. 1983. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 12. Int. J. Syst. Bacteriol. 33: 896–897. Schink, B. 1984. Fermentation of tartrate enantiomers by anaerobic bacteria, and description of two new species of strict anaerobes, Ruminococcus pasteurii and Ilyobacter tartaricus. Arch. Microbiol. 139: 409–414. Schink, B. 1985. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 18. Int. J. Syst. Bacteriol. 35: 375–376. Schink, B., P.H. Janssen and J. Frings. 1992. Microbial degradation of natural and of new synthetic polymers. FEMS Microbiol. Rev. 9: 311–316. Sebald, M. 1962. Étude sur les bactéries anaérobies gram-négatives asporulées. Thèses de l’Université Paris, Imprimerie Barnéoud S.A., Laval, France. Shinjo, T., T. Fujisawa and T. Mitsuoka. 1991. Proposal of two subspecies of Fusobacterium necrophorum (Flugge) Moore and Holdeman: Fusobacteriumnecrophorum subsp. necrophorum subsp. nov., nom. rev (ex Flugge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Halle 1898). Int. J. Syst. Bacteriol. 41: 395–397. Simon, P.C. 1975. A simple method for rapid identification of Sphaerophorus necrophorus isolates. Can. J. Comp. Med. 39: 349–353. Slots, J. and T.V. Potts. 1982. Fusobacterium simiae, a new species from monkey dental plaque. Int. J. Syst. Bacteriol. 32: 191–194. Slots, J., T.V. Potts and P.A. Mashimo. 1983. Fusobacterium periodonticum, a new species from the human oral cavity. J. Dent. Res. 62: 960–963. Slots, J., T.V. Potts and P.A. Mashimo. 1984. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 14. Int. J. Syst. Bacteriol. 34: 349–353. Socransky, S.S., A.D. Haffajee, M.A. Cugini, C. Smith and R.L. Kent, Jr. 1998. Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25: 134–144.

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Spaulding, E.H. and L.F. Rettger. 1937. Fusobacterium genus I. Biochemical and serological classification. J. Bacteriol. 34: 535–548. Stieb, M. and B. Schink. 1984. A new 3-hydroxybutyrate fermenting anaerobe, Ilyobacter polytropus, gen. nov., sp. nov., possessing various fermentation pathways. Arch. Microbiol. 140: 139–146. Stieb, M. and B. Schink. 1985. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 18. Int. J. Syst. Bacteriol. 35: 375–376. Sunde, P.T., L. Tronstad, E.R. Eribe, P.O. Lind and I. Olsen. 2000. Assessment of periradicular microbiota by DNA–DNA hybridization. Endod. Dent. Traumatol. 16: 191–196. Sutter, V.L., P.T. Sugihara and S.M. Finegold. 1971. Rifampin-blood-agar as a selective medium for the isolation of certain anaerobic bacteria. Appl. Microbiol. 22: 777–780. Tadepalli, S., G.C. Stewart, T.G. Nagaraja and S.K. Narayanan. 2008. Leukotoxin operon and differential expressions of the leukotoxin gene in bovine Fusobacterium necrophorum subspecies. Anaerobe 14: 13–18. Takahashi, N. and T. Sato. 2002. Dipeptide utilization by the periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum. Oral Microbiol. Immunol. 17: 50–54. Tanner, A., M.F. Maiden, B.J. Paster and F.E. Dewhirst. 1994. The impact of 16S ribosomal RNA-based phylogeny on the taxonomy of oral bacteria. Periodontol. 2000 5: 26–51. Terada, A., K. Uchida and T. Mitsuoka. 1976. Die Bacteroidaceenflora in den faeces von schweinen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. Orig. 234: 362–370. Thompson, J., S.A. Robrish, C.L. Bouma, D.I. Freedberg and J.E. Folk. 1997. Phospho-b-glucosidase from Fusobacterium mortiferum: purification, cloning, and inactivation by 6-phosphoglucono-delta-lactone. J. Bacteriol. 179: 1636–1645. Tissier, H. 1905. Répartition des microbes dans l’intestin du nourrisson. Ann. Inst. Pasteur (Paris) 19 : 109–123. Topley, W.W.C. and G.S. Wilson. 1929. The Principles of Bacteriology and Immunity, vol. 1. Edward Arnold, London. Tunnicliff, R. and L. Jackson. 1925. Bacillus gonidiaformans (n. sp.) - an hitherto undescribed organism. J. Infect. Dis. 36: 430–438. Turner, K., L. Lindqvist and C.E. Nord. 1985. Purification and properties of a novel b-lactamase from Fusobacterium nucleatum. Antimicrob. Agents Chemother. 27: 943–947. Van Assche, P.F. and A.T. Wilssens. 1977. Fusobacterium perfoetens (Tissier) Moore and Holdeman 1973: description and proposed neotype strain. Int. J. Syst. Bacteriol. 27: 1–5. Vasstrand, E.N. 1981. Lysozyme digestion and chemical characterization of the peptidoglycan of Fusobacterium nucleatum Fev 1. Infect. Immun. 33: 75–82. Vasstrand, E.N., H.B. Jensen, T. Miron and T. Hofstad. 1982. Composition of peptidoglycans in Bacteroidaceae: determination and distribution of lanthionine. Infect. Immun. 36: 114–122. Veillon, A. and A. Zuber. 1898. Recherches sur quelques microbes strictement anaérobies et leur rôle en pathologie. Arch. Med. Exp. 10 : 517–545. Watson, J., G.Y. Matsui, A. Leaphart, J. Wiegel, F.A. Rainey and C.R. Lovell. 2000. Reductively debrominating strains of Propionigenium maris from burrows of bromophenol-producing marine infauna. Int. J. Syst. Evol. Microbiol. 50: 1035–1042. Weinberg, M., R. Nativelle and A.R. Prévot. 1937. Les microbes anaérobies. Masson et Cie, Paris. Werner, H. 1974. Demonstration of lysine decarboxylase activity in the obligately anaerobic bacterium Sphaerophorus varius. Zentrabl. Bakteriol. Parasitendk. Infektionskr. Hyg. I. Abt. Orig. A 226: 364–368. Widdel, F. and N. Pfennig. 1981. Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. 1. Isolation of new sulfatereducing bacteria enriched with acetate from saline environments: description of Desulfobacter postgatei gen. nov., sp. nov. Arch. Microbiol. 129: 395–400.

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Family II. Leptotrichiaceae

Family II. Leptotrichiaceae fam. nov. James T. Staley and William B. Whitman Lep.to.tri.chi.a.ce¢a.e. N.L. fem. n. Leptotrichia type genus of the family; suff. -aceae ending to denote a family; N.L. fem. pl. n. Leptotrichiaceae the Leptotrichia family. The family Leptotrichiaceae is described in part on the basis of phylogenetic analyses of the 16S rRNA gene sequences of its members. Facultative to obligately anaerobic organisms that stain as Gram-negative rods. All described species are nonmotile and fermentative. Ferment carbohydrates to produce various organic acids including lactic, acetic, formic or succinic depen­ding on the substrate and species. Some species are

fastidious and require serum or blood for growth. Some species have been isolated from human clinical specimens and are pathogenic to humans. Some species occur in the human oral cavity and others in the hindgut of termites. Comprises the genera Leptotrichia, Sneathia, Streptobacillus, and Sebaldella. Type genus: Leptotrichia Trevisan 1879, 138AL.

Genus I. Leptotrichia Trevisan 1879, 138AL Kirstin J. Edwards and Saheer E. Gharbia Lep.to.tri.chi¢a. Gr. adj. leptos fine, small; Gr. fem. n. thrix, thricos hair; N.L. fem. n. Leptotrichia fine hair.

Straight or slightly curved rods, 0.5–3.0 × 5–15 mm, with one or both ends pointed or rounded. Frequently arranged in pairs, separate filaments, or chains, often with flattened ends. No club formation or branching. Nonmotile. Gramstain-negative. Anaerobic on first isolation; many strains subsequently grow aerobically in the presence of CO2. Optimum temperature 35–37°C. Good growth occurs at pH 7.0–7.4. Chemoorganotrophic. Metabolize carbohydrates with formation of acid without gas. The major product of glucose fermentation is lactic acid. Acetic and succinic acids may be produced in trace amounts. Hydrogen sulfide and indole are not produced. Nitrate is not reduced. The primary habitat is the oral cavity of humans, though also found in the female periurethral region and present in the oral cavity of some animals. DNA G+C content (mol%): 25–29.7. Type species: Leptotrichia buccalis (Robin 1853) Trevisan 1879, 147AL (Leptothrix buccalis Robin 1853, 345).

Further descriptive information Colonies of Leptotrichia grown anaerobically on blood agar or tryptone-yeast extract medium are distinctive. After 2–3 d of incubation they are smooth, colorless, 2–3 mm in dia­ meter, convex and with a convoluted surface. The colonies are sometimes raised with a filamentous edge, particularly after incubation for 24 h or less (Hamilton and Zahler, 1957; Kasai, 1961). The colonies are nonhemolytic and nonadherent to the medium. Pleomorphism in colony morphology may be seen (Kasai, 1961). An anaerobic atmosphere with 5–10% CO2 is essential for good growth of Leptotrichia on solid medium. Many strains become aerotolerant upon transfer. Leptotrichia is highly saccharolastic. Several mono- and disaccharides are fermented with the production of d- and l-lactic acid alone, or accompanied by trace amounts of acetic and succinic acids. CO2 is not produced. A few strains hydrolyze starch within 2–4 d. Pyruvate is fermented with production of CO2 and acetic and formic acids ( Jackins and Barker, 1951). Within the Leptotrichia there is significant heterogeneity in enzymic/ biochemical reactions (Eribe et al., 2002) and in cellular fatty acid content (Eribe et al., 2002; Hofstad and Jantzen, 1982). Significant variation between Leptotrichia is also observed in

SDS-PAGE profiles of whole-cell proteins and RAPD patterns of DNA (Eribe and Olsen, 2002). The primary habitat is the human oral cavity, where they are typically found in plaque (Eribe et  al., 2004; Hofstad, 1984), but they have also been isolated from the normal flora of the periurethral region of healthy girls and the genitalia of women (Evaldson et  al., 1980; Moore et  al., 1976; Söderberg et  al., 1979) and occasionally been recovered from blood, mainly in immunocompromised patients with neutropenia and from endocarditis (Eribe et  al., 2004; Hammann et  al., 1993; Messiaen et al., 1996; Patel et al., 1999; Reig et al., 1985; Tee et al., 2001; Vernelen et al., 1996; Weinberger et al., 1991). Leptotrichia buccalis is normally considered to play a role in tooth decay and periodontal disease (Hofstad, 1984; Krywolap and Page, 1977) especially in immunocompromised patients. Though it is not solely dependent on tooth eruption since the organism has been isolated from the mouth of pre-dentate infants (McCarthy et al., 1965). It has also been isolated from animals which have been fed commercial pellets (Schwartz et al., 1995).

Differentiation of the genus Leptotrichia from other genera The first full description of Leptotrichia buccalis described the organism as a Gram-negative bacterium related to Fusobacterium nucleatum (Böe and Thjotta, 1944; Thjötta et al., 1939). Hamilton and Zahler (1957), Gilmour et al. (1961), and Kasai (1965) concluded that Leptotrichia buccalis was a Gram-positive organism related to Lactobacillus. But an electron microscopy study and the isolation of a potent endotoxin from the organism definitely established that the organism is a Gramnegative bacterium (Hofstad and Selvig, 1969). Its ability to produce lactic acid as the only major acid from glucose fermentation distinguishes it from other closely related genera such as Fusobacterium, and analysis of 16S rRNA sequences clearly distinguishes Leptotrichia from other related genera (Figure 122). Until 1995, there was only one species defined, Leptotrichia buccalis. The closely related species “Leptotrichia sanguinegens” (Hanff et  al., 1995b), also known as “Leptotrichia microbii” (Hanff et  al., 1995a) was recently transferred to the genus Sneathia as Sneathia sanguinegens (Collins et al., 2001). The recently defined species, Leptotrichia trevisanii (Tee et al., 2001), Leptotrichia goodfellowii, Leptotrichia ­hofstadii,

767

Genus I. Leptotrichia Leptotrichia shahii (AY029806)

60.8 68.7

Leptotrichia wadei (AY029802) Leptotrichia trevisanii (AF206305)

38.1 100.0

Leptotrichia hofstadii (AY029803) Leptotrichia buccalis (L37788)

98.1

Leptotrichia goodfellowii (AY029807)

59.8

Sebaldella termitidis (M58678) Streptobacillus moniliformis (Z35305)

99.2

Sneathia sanguinegens (L37789) Fusobacterium nucleatum (AJ133496) Figure 122.  Phylogenetic tree of full-length 16S rRNA gene sequences of the genus Leptotrichia and related species. GenBank accession numbers

are given after species names. The tree was constructed using the neighbor-joining method, and the numbers at branching points are bootstrap percentages.

Leptotrichia shahii, and Leptotrichia wadei (Eribe et  al., 2004) have been added. The main characteristics of all species are described in Table 150. A further species has been described and named “Leptotrichia amnionii” (Shukla et al., 2002), how-

ever, 16S rRNA sequencing suggests that “Leptotrichia amnionii” would be better assigned to the genus Sneathia (Eribe et al., 2004), and a full description of the strain has not been validated.

List of species of the genus Leptotrichia 1. Leptotrichia buccalis (Robin 1853) Trevisan 1879, 147AL (Leptothrix buccalis Robin 1853, 345) buc.ca¢lis. L. n. bucca the mouth; L. fem. suff. -alis suffix denoting pertaining to; N.L. fem. adj. buccalis buccal, pertaining to the mouth. Straight or slightly curved rods, 0.8–1.5 × 5–15 mm, with one or both ends pointed or rounded. Frequently arranged in pairs, chains, or separate filaments. No club formation or branching. Nonmotile. Gram-stain-negative, often with Gram-stain-positive granules distributed evenly along the long axis. May be Gram-stain-positive in very young cultures. Anaerobic on first isolation; many strains subsequently grow aerobically in the presence of CO2. Optimum growth temperature, 35–37°C; little or no growth occurs at 25°C. Good growth occurs at pH 7.0–7.4. Chemoorgano­ trophic. Metabolize carbohydrates with formation of acid without gas. The major product of glucose fermentation is lactic acid. Acetic and succinic acids may be produced in trace amounts. Catalase, hydrogen sulfide, and indole are not produced. Nitrate is not reduced. The main habitat is the oral cavity of humans, though also found in the female periurethral region, and can be present in the oral cavity of animals fed with commercial pellets. DNA G+C content (mol%): 25 (Tm). Type strain: ATCC 14201, CCUG 34316, CIP 105792, DSM 1135, JCM 12969, NCTC 10249. Sequence accession no. (16S rRNA gene): L37788. 2. Leptotrichia goodfellowii Eribe, Paster, Caugant, Dewhirst, Stomberg, Lacy and Olsen 2004, 589VP

good.fel¢low.i.i. N.L. masc. gen. n. goodfellowii of Goodfellow, named in honor of Michael Goodfellow, for his contributions to microbial systematics. The description is from Eribe et al. (2004). After 2–6 d of anaerobic incubation at 37°C, colonies on Columbia or BHI agar plates supplemented with 5% human blood, hemin, and menadione are 0.8–2.0 mm in diameter, speckled, convex, irregular, pink in the periphery, and grayish light brown in the rest of the colony. They have a glistening surface, are opaque, dry, and b-hemolytic. Catalase positive and esculin weakly positive. Oxidase and indole are not produced. Colonies grow best anaerobically and sparsely aerobically. Growth occurs at 37°C but not at 25 or 42°C; optimal temperature for growth is 37°C. Gram-stain-negative, nonsporeforming, nonmotile rods. Cells are arranged in pairs, some slightly curved, others in chains joined by flattened ends. Arginine dihydrolase, b-galactosidase, b -glucosidase, N-acetyl-b-glucosaminidase, alkaline phosphatase, arginine arylamidase, leucine arylamidase, and histidine arylamidase are produced. Mannose is fermented. Isolated from human blood. DNA G+C content (mol%): 25 (HPLC). Type strain: LB 57, CCUG 32286, CIP 107915. Sequence accession no. (16S rRNA gene): AY029807. 3. Leptotrichia hofstadii Eribe, Paster, Caugant, Dewhirst, Stomberg, Lacy and Olsen 2004, 589VP hof.stad¢i.i. N.L. masc gen. n. hofstadii of Hofstad, named in honor of Tor Hofstad, for his contributions to Leptotrichia taxonomy.

768

Family II. Leptotrichiaceae

L. hofstadii

L. shahii

L. trevisanii

L. wadei

Growth at: 25°C 42°C Hemolysis of human blood Production of: N-Acetyl-b-glucosaminidase Alkaline phosphatase a-Arabinosidase Arginine arylamidase Arginine dihydrolase a-Galactosidase b-Galactosidase b-Galactosidase-6-phosphate a-Glucosidase b-Glucosidase Histidine arylamidase Leucine arylamidase Tyrosine arylamidase Fermentation of: Mannose Raffinose Saturated fatty acids: C14:0 C16:0 Unsaturated straight-chain fatty acids: C18:1c11/t 9/t 6 or unknown with ECL of 17.834 Hydroxy fatty acids: C14:0 3-OH or C15:0 DMA Catalase Esculin

L. goodfellowii

Characteristic

L. buccalis

Table 150.  Characteristics differentiating species of the genus Leptotrichia a

+ + −

− − +

− − +

+ − −

nt nt nt

− − +

− + − − − + − + + + − − +

+ + − + + − + − − + + + −

− + − − − − − + + + − − −

− − + − − − − − + − − − −

+ + − + nt − − nt + + nt − nt

− − − − − − − − + + − − −

+ +

− −

+ −

− −

nt nt

− −

10 39

14 41

6 48

9 32

nt nt

7 45

42

28

27

36

nt

24

7 − +

9 + +

9 + +

5 + +

nt + nt

8 + +

a Symbols: +, >85% positive; d, different strains give different reactions (16–84% positive);−, 0–15% positive; w, weak reaction; nt, not tested; ECL, Equivalent chainlength; DMA, dimethyl acetal.

The description is from Eribe et al. (2004). After 2–6 d of anaerobic incubation at 37°C, colonies on Columbia or BHI agar plates supplemented with 5% human blood, hemin, and menadione are 0.5–1.8 mm in diameter. They have a glistening and granular surface, are opaque, dry, and b-hemolytic. Older colonies can be up to 4.0–6.5 mm, ­circular, convex, entire (some are irregular and lobate), and grayish in color with a dark central spot. Catalase positive and esculin weakly positive. Oxidase and indole are not ­produced. Colonies grow best anaerobically and sparsely aerobically. Growth occurs at 37°C but not at 25 or 42°C; optimal temperature is 37°C. Gram-stain-negative, nonsporeforming, nonmotile rods. Cells are arranged in pairs, some slightly curved, others in chains joined by flattened ends. b-Galactosidase6-phosphate, a-glucosidase, b-glucosidase, and alkaline phos­ phatase are produced. Mannose is fermented. Source: saliva of a healthy person. DNA G+C content (mol%): 25 (HPLC). Type strain: LB 23, CCUG 47504, CIP 107917. Sequence accession no. (16S rRNA gene): AY029803.

4. Leptotrichia shahii Eribe, Paster, Caugant, Dewhirst, Stom­ berg, Lacy and Olsen 2004, 589VP sha¢hi.i. N.L. masc. gen. n. shahii of Shah, named in honor of Haroun N. Shah, a Trinidad-born microbiologist, for his contributions to microbiology. The description is from Eribe et al. (2004). After 2–6 days of anaerobic incubation at 37°C, colonies on Columbia or BHI agar plates supplemented with 5% human blood, hemin, and menadione are 1.0–1.5 mm in diameter, very filamentous to rhizoid or convoluted, pale-speckled, and grayish in color, with a dark central spot in old colonies. These are opaque, semi-dry in consistency, and nonhemolytic. Catalase-positive and esculin weakly positive. Oxidase and indole are not produced. Colonies grow best anaerobically and sparsely aerobically. Growth occurs at 25 and 37°C but not at 42°C; optimal temperature is 37°C. Gram-stain-negative, nonsporeforming, nonmotile rods. Cells are arranged in pairs, some slightly curved, others in chains joined by flattened ends. a-Glucosidase and a-arabinosidase are produced. Source: a patient with gingivitis. DNA G+C content (mol%): 25 (HPLC).

Genus II. Sebaldella

Type strain: LB 37, CCUG 47503, CIP 107916, VPI N06A-34. Sequence accession no. (16S rRNA gene): AY029806. 5. Leptotrichia trevisanii Tee, Midolo, Janssen, Kerr and DyallSmith 2002, 686VP (Effective publication: Tee, Midolo, Janssen, Kerr and Dyall-Smith 2001,768.) tre.vi.sa¢ni.i. N.L. masc. gen. n. trevisanii of V. Trevisan, who proposed the genus Leptotrichia in 1879. The description is from Tee et al. (2001).The organism is characterized by long, fusiform, nonmotile, Gram-stainnegative bacilli that are 0.8–0.9 × 6–13 mm with tapered ends. Many cells are slightly curved. Cells are arranged in pairs and chains and, where they join, the ends are flattened. Isolates are nonsporeforming, catalase positive, indole negative, and oxidase negative. They are negative for urease, negative for hydrolysis of the p-NP-sugars a-darabinoside, a-d-galactoside, and a-l-fucoside, and are unable to hydrolyze O-NP-b-d-galactoside. They are positive for hydrolysis of p-NP derivatives of a-d-glucoside, b-dglucoside, N-acetyl-b-d-glucosamine, and p-NP-phosphate. The organism is unable to hydrolyze the naphthylamide derivatives of leucyl-glycine, glycine, proline, serine, and pyrrollidone, but is able to hydrolyze arginine and phenylalanine derivatives. Leptotrichia trevisanii grows both anaerobically and under 5% CO2 (in air) on subsequent culture on solid media. It ferments glucose to produce lactic acid as the major organic end product. On horse blood agar plates, colonies appeared smooth, grayish in color, low convex, and erose edged; they showed a dark central spot with

769

t­ ransillumination and grew to about 2 mm diameter after 5 d of incubation. Older colonies showed a slightly convoluted surface. DNA G+C content (mol%): 29.7 (HPLC). Type strain: “Wee Tee” 1999, ATCC 700907, DSM 22070. Sequence accession no. (16S rRNA gene): AF206305. 6. Leptotrichia wadei Eribe, Paster, Caugant, Dewhirst, Stomberg, Lacy and Olsen 2004, 591VP wade¢i. N.L. masc. gen. n. wadei of Wade, named in honor of William G. Wade, for his contributions to microbiology. The description is from Eribe et al. (2004). After 2–6 d of anaerobic incubation at 37°C, colonies on Columbia or BHI agar plates supplemented with 5% human blood, hemin, and menadione are 0.5–3.0 mm in diameter, convex, sparsely filamentous to irregular, and grayish brown in color, with a dark central spot in old colonies. The surface appearance is glistening and smooth with a rough edge. Colonies are opaque, dry in consistency, and b-hemolytic. Esculin and catalase are positive. Oxidase and indole are not produced. Growth occurs best anaerobically and sparsely aerobically at 37°C but not at 25 or 42°C. Gram-stain-negative, nonsporeforming, nonmotile rods. Cells are arranged in pairs, some slightly curved, others in chains joined by flattened ends. a-Glucosidase and b-glucosidase are produced. Source: saliva of a healthy person. DNA G+C content (mol%): 25 (HPLC). Type strain: LB 16, CCUG 47505, CIP 107918. Sequence accession no. (16S rRNA gene): AY029802.

Genus II. Sebaldella Collins and Shah 1986, 349VP Saheer E. Gharbia and Kirstin J. Edwards Se.bal.del¢la. N.L. dim. ending -ella; N.L. fem. dim. n. Sebaldella named after the French microbiologist Madeleine Sebald, who first described the organism.

Rods. Nonsporeforming. Nonmotile. Gram-stain-negative. Anaerobic. Acid produced from glucose and some other sugars. The major end products of glucose fermentation are acetic and lactic acids; formic acid may also be produced. Hexose monophosphate shunt enzymes, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase are absent. Glutamate dehydrogenase and malate dehydrogenase are absent. Nonhydroxylated and 3-hydroxylated long-chain fatty acids are present. The fatty acids are primarily of the straight-chain saturated and monounsaturated types. Menaquinones are absent. DNA G+C content (mol%): 32–36. Type species: Sebaldella termitidis (Sebald 1962) Collins and Shah 1986, 349VP (Sphaerphorus siccus var. termitidis Sebald 1962, 124; Bacteroides termitidis Holdeman and Moore 1970, 33).

Enrichment and isolation procedures Surface colonies are 1–2 mm in diameter, circular, and transparent to opaque. Colonies in deep agar are lenticular and nonpigmented.

Differentiation of the genus Sebaldella from other genera Sebaldella differs from Bacteroides fragilis and related species by exhibiting a lower G+C content (32–36 mol%), by producing acetic and lactic acids as major end products of glucose fermentation, and by the absence of glutamate and malate dehydrogenases (Collins and Shah, 1986). Sebaldella also differs from the Bacteroides fragilis group in lipid composition. The long chain fatty acids of Bacteroides are predominantly of the straight-chain saturated, anteiso- and iso-methyl branched chain types, with monosaturated acids either absent or present in only trace amounts. In contrast, Sebaldella primarily synthesizes acids of the straight chain saturated and monosaturated types and methyl branched acids are absent (Miyagawa et al., 1979; Shah and Collins, 1983). Sebaldella and Bacteroides also differ in isoprenoid quinone composition, with Bacteroides possessing menaquinones and Sebaldella lacking respiratory quinones (Collins and Jones, 1981; Shah and Collins, 1980). Based on 16S rRNA studies, Sebaldella is phylogentically distinct from Bacteroides and all other described eubacterial phyla (Paster et al., 1985).

770

Family II. Leptotrichiaceae

List of species of the genus Sebaldella 1. Sebaldella termitidis (Sebald 1962) Collins and Shah 1986, 349VP (Sphaerphorus siccus var. termitidis Sebald 1962, 124; Bacteroides termitidis Holdeman and Moore 1970, 33.) ter.mi¢ti.dis. L. n. termes, -itis wood-eating worm; N.L. fem. adj. termitidis pertaining to the termite. The description is from Collins and Shah (1986). The Gram-stain-negative, obligately anaerobic, nonmotile, rodshaped cells are 0.3–0.5 × 2–12 mm with central swellings and occur singly, in pairs, and in filaments. Surface colonies are 1–2 mm in diameter, circular, and transparent to opaque. Colonies in deep agar are lenticular and nonpigmented. Acetic and lactic acids are the major end products of glucose metabolism; formic acid may also be produced. Acid is produced from glucose, fructose, maltose, mannitol, mannose, rhamnose, sucrose, trehalose, and xylose. Acid is not produced from arabinose, melazitose, or starch; low levels of

acid may be produced from lactose (delayed reaction). Most strains produce H2S. Gelatin is not liquefied; coagulated proteins are not attacked. Urease, chitinase, and indole are not produced. Nitrate is not reduced. Uric acid is degraded to CO2, acetate, and ammonia. Malate dehydrogenase and glutamate dehdrogenase are not produced. Nonhydroxylated and 3-hydroxylated long-chain fatty acids are present. The fatty acids are of the straight-chain saturated and monounsaturated types, with hexadecanoic and octadecenoic acids predominating. Menaquinones are not produced. Isolated from posterior intestinal contents of termites, where these organisms are part of the predominant bacterial flora. DNA G+C content (mol%): 32–36 (Bd). Type strain: ATCC 33386, NCTC 11300. Sequence accession no. (16S rRNA gene): M58678.

Genus III. Sneathia Collins, Hoyles, Törnqvist, von Essen and Falsen 2002, 687VP (Effective publication: Collins, Hoyles, Törnqvist, von Essen and Falsen 2001, 360.) Julie M.J. Logan, Kirstin J. Edwards and Saheer E. Gharbia Sneath¢i.a. N.L. fem. n. Sneathia named after the British microbiologist Peter H.A. Sneath, in recognition of his outstanding contributions to microbial systematics.

Gram-stain-negative, asporogenous, rod-shaped bacteria; nonmotile. Cells may display pleomorphism and filaments may be observed. Anaerobic, although some strains may show poor growth in CO2. Fermentative metabolism. Acid but no gas is produced from glucose. Acid is not produced from ribose or maltose. Lactic acid, formic acid, and minor amounts of acetic acid are the end products of glucose metabolism; succinic acid may be produced. Fastidious; require serum or blood for growth. Optimum temperature for growth 35–37°C. Catalase and oxidase negative. Esculin and hippurate are hydrolyzed but starch is not. b-Glucuronidase is produced. Indole is not produced. Voges-Proskauer negative. Nitrate is not reduced to nitrite. DNA G+C content (mol%): 22–25. Type species: Sneathia sanguinegens Collins, Hoyles, Törnqvist, von Essen and Falsen 2002, 687 (Effective publication: Collins, Hoyles, Törnqvist, von Essen and Falsen 2001, 360.).

Further descriptive information Originally, Hanff et  al. (1995b) reported the isolation from blood cultures of an unusual Gram-stain-negative anaerobic rod-shaped organism from four obstetric patients with postpartum fever, two neonates, and a 100-year-old woman. This fastidious, serum-requiring bacterium was considered by Hanff et al. (1995b) to be a member of the genus Leptotrichia and was

­ esignated “Leptotrichia sanguinegens”. The species was, howd ever, not validly published, and no type strain was designated. In 2001, Collins et al. isolated three strains from amniotic fluid and blood from non-obstetric patients that resembled the bacterium described by Hanff et al. (1995b). However, based on both phenotypic and phylogenetic evidence, Collins et al. concluded that these isolates corresponded to “Leptotrichia sanguinegens” and proposed the new genus and species Sneathia sanguinegens.

Differentiation of the genus Sneathia from other genera Sneathia sanguinegens can be readily identified in the clinical laboratory on the basis of its cellular morphology and fastidious growth requirements combined with its API Rapid ID32A and ZYM biochemical profiles. In particular, using these systems it can be readily distinguished from Streptobacillus moniliformis by its positive b-glucuronidase reaction and by failing to produce chymotrypsin and proline arylamidase. Similarly, Sneathia sanguinegens can be easily distinguished from Leptotrichia buccalis in requiring serum or blood for growth, by producing b-glucuronidase, and by its negative a-glucosidase and b-glucosidase reactions. On the basis of 16S rDNA sequencing, Sneathia isolates demonstrated high sequence similarity of >99.8% and are distinct from their closest named relatives such as Streptobacillus moniliformis, Sebaldella termiditis, and Leptotrichia buccalis (Collins et al., 2001).

List of species of the genus Sneathia 1. Sneathia sanguinegens Collins, Hoyles, Törnqvist, von Essen and Falsen 2002, 687VP (Effective publication: Collins, Hoyles, Törnqvist, von Essen and Falsen 2001, 360.) san.gui.ne¢gens. L. n. sanguis, -inis blood; L. part adj. egens needing; N.L part. adj sanguinegens needing blood; because the organism requires blood or serum.

Description is from Collins et  al. (2001). Gram-stainnegative anaerobic or facultatively anaerobic, nonsporeforming, nonmotile, rod-shaped cells. Colonies on chocolate or blood agar are pin-point and convex after 72 h. Fastidious, requiring blood or serum for growth. Catalase and oxidase negative. Lactic acid, formic acid, and

Genus IV. Streptobacillus

minor amounts of acetic acid are the end products of ­glucose metabolism; succinic acid may be produced. Acid but no gas is produced from glucose. Acid may or may not be produced from mannose and raffinose. Acid is not ­produced from l-arabinose, d-arabitol, cyclodextrin, ­glycogen, lactose, mannitol, maltose, melebiose, melezitose, methyl-b-d-glucopyranoside, pullulan, d-ribose, sorbitol, sucrose, tagatose, or trehalose. Alkaline phosphatase, acid phosphatase, arginine arylamidase, phosphoamidase, and b-glucuronidase are detected. Arginine dihydrolase, alanine arylamidase, alanine phenylalanine proline arylamidase, a-arabinosidase, chymotrypsin, cystine arylamidase, a-frucosidase, a-­galactosidase, b-galactosidase, b-galactosidase-6-phosphate, a-glucosidase, b-­glucosidase, glutamic acid decarboxylase, glycyl tryptophan arylamidase,

771

­ yroglutamic acid arylamidase, leucine glycin arylamidase, p lipase C14, a-mannosidase, b-mannosidase, N-acetyl-b-­ glucosaminidase, proline arylamidase, trypsin, valine arylamidase, and urease are not detected. Activity may or may not be detected for ester lipase C8, esterase C4, glutamyl glutamic acid arylamidase, glycine arylamidase, histidine arylamidase, phenyl alanine arylamidase, leucine arylamidase, serine arylamidase, and tyrosine arylamidase. Indole-negative and Voges–Proskauer-negative. Esculin and hippurate are hydrolyzed but starch is not. Nitrate is not reduced to nitrite. Habitat is not known. Source: human clinical specimens (blood, amniotic fluid). DNA G+C content (mol%): 22–25 (HPLC). Type strain: CCUG 41628, CIP 106906. Sequence accession no. (16S rRNA gene): AJ344093.

Genus IV. Streptobacillus Levaditi, Nicolau and Poincloux 1925, 1188AL Saheer E. Gharbia and Kirstin J. Edwards Strep.to.ba.cil¢lus. Gr.adj. streptos twisted, curved; L. masc. n. bacillus a small rod; N.L. masc. n. Streptobacillus a twisted or curved small rod.

Rods with rounded or pointed ends. Occur singly or form long, wavy chains. Nonsporeforming. Nonmotile. Gram-stainnegative. Conversion to l-phase or transitional-phase variant may occur spontaneously during cultivation. Capable of growth anaerobically or aerobically. Ferments glucose to produce acid but not gas. Optimum temperature 35–37°C. Catalase and oxidase negative. Indole not produced. Nitrate not reduced to nitrite. Require serum, ascitic fluid, or blood for growth. Isolated from the throat and nasopharynx of wild and laboratory rats. Causes rat-bite fever in man. DNA G+C content (mol%): 24–26. Type species: Streptobacillus moniliformis Levaditi, Nicolau and Poincloux 1925, 1188AL.

Further descriptive information The sole species forms small (1–2 mm), smooth, convex, grayish, nonhemolytic colonies on 5% horse blood Columbia agar after 72 h of incubation in an atmosphere of carbon dioxide at an optimum temperature of 35–37°C. Direct examinations reveal pleomorphic, Gram-stain-negative filamentous rods with lateral bulbous swellings. In serum-supplemented liquid media, bacterial growth shows a typical cottonball like appearance. The organisms exist in two variant types, the “normal” bacillary form and the inducible or spontaneously occurring l-form that exhibits the typical “fried-egg” colony morphology (Wittler and Cary, 1974). The latter is regarded as an apathogenic Streptobacillus moniliformis variant (Freundt, 1956). Streptobacillus moniliformis can be distinguished from other biochemically related bacteria by its negative reactions for catalase, oxidase, indole production and reduction of nitrate to nitrite (Savage, 1984). Streptobacillus moniliformis is a pathogen for humans that was first isolated as a cause of rat-bite fever by Hugo Schottmüller in 1914. He named the organism Streptothrix muris ratti. Clinical pictures of the disease are similar despite two different modes of transmission. Oral uptake of Streptobacillus moniliformis via contaminated food leads to a disease known as Haverhill fever, named after the place (Haverhill, MA, USA) where the first

well-documented epidemic was observed (Place and Sutton, 1934). Affected individuals had consumed unpasteurized milk or milk products to which rats had access. In another well-documented epidemic (Chelmsford, UK), boarding school pupils became infected after using water from a spring in the vicinity of which rats were observed (McEvoy et al., 1987). As in Haverhill, Streptobacillus moniliformis was not isolated from captured rats. However, in both cases, epidemiological data suggested it was most probable that foodstuffs or water contaminated by rats were responsible for the epidemics. Rats are also the source of the second type of human streptobacillosis, the so-called rat-bite fever. (Another form of rat-bite fever called sodoku is caused by Spirillum minus which is not the subject of this review.) Streptobacillus moniliformis can be transmitted by rat bite, but recent reports suggest that not only the rat bite, but also simple contact with (pet) rats (Clausen, 1987; Rygg and Bruun, 1992), may result in rat-bite fever. The disease is characterized by an acute onset with chills, vomiting, malaise, headache, irregularly relapsing fever, erythematous rash (especially of the extremities), and arthralgia. Untreated, it often leads to a severe septic polyarthritis and lymphadenopathy. If untreated, mortality is estimated to be about 13% (Roughgarden, 1965; Simon and Wilson, 1986). Complications of streptobacillary rat-bite fever are endocarditis (Rey et al., 1987; Rupp, 1992), pericarditis (Carbeck et al., 1967) brain abscess (Oeding and Pedersen, 1950), amnionitis (Faro et  al., 1980), septicemia (Brown and Nunemaker, 1942; Dellamonica et  al., 1979; Renaut et al., 1982; Rygg and Bruun, 1992), interstitial pneumonia, prostatitis, and pancreatitis (Delannoy et al., 1991). So far as is known, the rat is the natural reservoir of Streptobacillus moniliformis and therefore plays the dominant role in harboring and transmitting the infectious agent. Most probably the microorganism is a member of the commensal flora of the upper respiratory tract. Hence, the main isolation sites in healthy rats are the nasopharynx (Strangeways, 1933), larynx, upper trachea (Peagle et  al., 1976), and the middle ear (Koopman et al., 1991). Although of only low pathogenicity for

772

Family II. Leptotrichiaceae

the rat, Streptobacillus moniliformis may act as a secondary invader (Weisbroth, 1979) in conjunction with presumptive pathogens such as Pasteurella pneumotropica, Mycoplasma pulmonis causing otitis media (Olson and McCune, 1968; Wullenweber et  al., 1992), conjunctivitis (Young and Hill, 1974), bronchopneumonia (Bell and Elmes, 1969), and chronic pneumonia (Gay et al., 1972).

Taxonomic comments This organism has been given various names over the years. They are found in original research papers as well as in ­textbooks on pathogenic bacteria. The list of references is: Streptothrix muris ratti (Schottmüller, 1914), Nocardia muris (de Mello and Pais, 1918), Actinomyces muris ratti (Lieske, 1921; Schottmüller, 1914), Haverhillia multiformis (Parker and Hudson, 1926), ­Actinomyces muris (de Mello and Pais, 1918; Topley and Wilson, 1936), Asterococcus muris (de Mello and Pais, 1918; Heilman, 1941), Proactinomyces muris (de Mello and Pais, 1918; Krasil’nikov, 1941), Haverhillia moniliformis (Levaditi et  al., 1925; Prévot, 1948), Actinobacillus muris (de Mello and Pais, 1918; Wilson and Miles, 1955).

Differentiation of the genus Streptobacillus from other genera Streptobacillus can be differentiated from genera which are found in the same habitat, including Cardiobacterium, Actinobacillus, and Haemophilus, on the basis of its serum requirement, flocculent growth in broth, small butyrous colony on agar, characteristic microscopic appearance, absence of catalase and oxidase activity, and failure to reduce nitrate to nitrite or produce indole. Related genera will be positive for one or more of the catalase, oxidase, nitrate reduction or indole production characteristics while Streptobacillus is negative for all (Lapage, 1974; Midgley et al., 1970). Gas-liquid chromatography analysis of the fatty acid pattern shows palmitic, stearic, oleic, and linoleic acid as major components (Edwards and Finch, 1986). On the basis of numerical analysis of SDS-PAGE protein profiles, seven subgroups were identified among 31 Streptobacillus moniliformis cultures representing 22 different strains of human, murine, and avian origin isolated in Europe, USA, and Australia. As these groups show a close similarity, it was concluded that Streptobacillus moniliformis is a very homologous species (Costas and Owen, 1987).

List of species of the genus Streptobacillus 1. Streptobacillus moniliformis Levaditi, Nicolau and Poincloux 1925, 1188AL mo.ni.li.for¢mis. L. n. monile necklace; L. masc. suff. formis in the shape of; N.L. masc. adj. moniliformis necklaceshaped.

Reference Bell, D.P. and P.C. Elmes. 1969. Effects of certain organisms associated with chronic respiratory disease on SPF and conventional rats. J. Med. Microbiol. 2: 511–519. Böe, J. and T. Thjotta. 1944. The position of Fusobacterium and Leptotrichia in the bacteriological system. Acta Pathol. Microbiol. Scand. 21: 441–450. Brown, T.M. and J.C. Nunemaker. 1942. Rat-bite fever. a review of the American cases with reevaluation of etiology; report of cases. Bull. Johns Hopkins Hosp. 70 : 201–236. Carbeck, R.B., J.F. Murphy and E.M. Britt. 1967. Streptobacillary ­rat-bite fever with massive pericardial effusion. J.A.M.A. 201: 133–134. Clausen, C. 1987. Septic arthritis due to Streptobacillus moniliformis. Clin. Microbiol. Newsl. 9: 123–124. Collins, M.D. and D. Jones. 1981. Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implication. Microbiol. Rev. 45: 316–354. Collins, M.D. and H.N. Shah. 1986. Reclassification of Bacteroides ­termitidis Sebald (Holdeman and Moore) in a new genus Sebaldella termitidis, as Sebaldella termitidis comb. nov. Int. J. Syst. Bacteriol. 36: 349–350. Collins, M.D., L. Hoyles, E. Törnqvist, R. von Essen and E. Falsen. 2001. Characterization of some strains from human clinical sources which resemble “Leptotrichia sanguinegens”: description of Sneathia sanguinegens sp. nov., gen. nov. Syst. Appl. Microbiol. 24: 358–361. Collins, M.D., L. Hoyles, E. Törnqvist, R. von Essen and E. Falsen. 2002. In Validation of publication of new names and new combinations previously effectively published outside the IJSEM. Validation List no. 85. Int. J. Syst. Evol. Microbiol. 52: 685–690.

The description and characteristics are as described for the genus. DNA G+C content (mol%): 24–26 (Tm). Type strain: ATCC 14647, CCUG 2469, CCUG 13453, DSM 12112, NCTC 10651. Sequence accession no. (16S rRNA gene): Z35305.

Costas, M. and R.J. Owen. 1987. Numerical analysis of electrophoretic protein patterns of Streptobacillus moniliformis strains from human, murine and avian infections. J. Med. Microbiol. 23: 303–311. de Mello, F. and A.S.A. Pais. 1918. Um caso de nocardiose pulmonar simulando a tísica. Arq. Hig. Pat. Exot. Lisboa 6: 133–206. Delannoy, D., P. Savinel, M.H. Balquet, J.P. Canonne, J. Amourette and P.Y. Bugnon. 1991. Manifestations digestives et pulmonaires rélévant une septicémie à Streptobacillus moniliformis: présentation atypique d’ une pathologie rare et méconnue. La Revue de Médicine Interne. 3: 5158. Dellamonica, P., E. Delbeke, D. Giraud and G. Illy. 1979. Septicémies à Streptobacillus moniliformis: à propos d’ un cas-revue de la littérature. Méd. Mal. Infect. 9 : 226–229. Edwards, R. and R.G. Finch. 1986. Characterisation and antibiotic susceptibilities of Streptobacillus moniliformis. J. Med. Microbiol. 21: 39–42. Eribe, E.R., B.J. Paster, D.A. Caugant, F.E. Dewhirst, V.K. Stromberg, G.H. Lacy and I. Olsen. 2004. Genetic diversity of Leptotrichia and description of Leptotrichia goodfellowii sp. nov., Leptotrichia hofstadii sp. nov., Leptotrichia shahii sp. nov. and Leptotrichia wadei sp. nov. Int. J. Syst. Evol. Microbiol. 54: 583–592. Eribe, E.R.K., T. Hofstad and I. Olsen. 2002. Enzymatic/biochemical and cellular fatty acid analyses of Leptotrichia isolates. Microb. Ecol. Health Dis. 14: 137–148. Eribe, E.R.K. and I. Olsen. 2002. SDS-PAGE of whole-cell proteins and random amplified polymorphic DNA (RAPD) analyses of Leptotrichia isolates. Microb. Ecol. Health Dis. 14: 193–202. Evaldson, G., G. Carlstrom, A. Lagrelius, A.S. Malmborg and C.E. Nord. 1980. Microbiological findings in pregnant women with

Genus IV. Streptobacillus ­ remature rupture of the membranes. Med. Microbiol. Immunol. p 168: 283–297. Faro, S., C. Walker and R.L. Pierson. 1980. Amnionitis with intact ­amniotic membranes involving Streptobacillus moniliformis. Obstet. Gynecol. 55: 9S-11S. Freundt, E.A. 1956. Experimental investigations into the pathogenicity of the l-phase variant of Streptobacillus moniliformis. Acta Pathol. Microbiol. Scand. 38: 248–256. Gay, F.W., M.E. Maguire and A. Baskerville. 1972. Etiology of chronic pneumonia in rats and a study of the experimental disease in mice. Infect. Immun. 6: 83–91. Gilmour, M.N., J.A.H. Howell and B.G. Bibby. 1961. The classification of organisms termed Leptotrichia (Leptrotrix) buccalis. I. Review of the literature and proposed separation into Leptotrichia buccalis Trevisan 1879 and Bacterionema gen. nov. B. matruchotii (Mendel 1919) comb. nov. Bacteriol. Rev. 25: 131–141. Hamilton, R.D. and S.A. Zahler. 1957. A study of Leptotrichia buccalis. J. Bacteriol. 73: 386–393. Hanff, P.A., J.A. Rosol-Donoghue, C.A. Spiegel, K.A. Wilson and L.A. Moore 1995a, posting date. Sneathia sanguinegens. NCBI Taxonomy Browser. [Online.] Hanff, P.A., J.A. Rosol-Donoghue, C.A. Spiegel, K.H. Wilson and L.H. Moore. 1995b. Leptotrichia sanguinegens sp. nov., a new agent of postpartum and neonatal bacteremia. Clin. Infect. Dis. 20 Suppl. 2: S237–239. Hammann, R., A. Iwand, J. Brachmann, K. Keller and A. Werner. 1993. Endocarditis caused by a Leptotrichia buccalis-like bacterium in a patient with a prosthetic aortic valve. Eur. J. Clin. Microbiol. Infect. Dis. 12: 280–282. Heilman, F.R. 1941. A study of Asterococcus muris (Streptobacillus ­moniliformis). II. Cultivation and biochemical activities. J. Infect. Dis. 69 : 45–51. Hofstad, T. and K.A. Selvig. 1969. Ultrastructure of Leptotrichia buccalis. J. Gen. Microbiol. 56: 23–26. Hofstad, T. and E. Jantzen. 1982. Fatty acids of Leptotrichia buccalis: ­taxonomic implications. J. Gen. Microbiol. 128: 151–153. Hofstad, T. 1984. Genus III. Leptotrichia. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg). Williams & Wilkins, Baltimore, pp. 637–641. Holdeman, L.V. and W.E.C. Moore (editors). 1970. Bacteroides, Outline of Clinical Methods in Anaerobic Bacteriology, 2nd revn. Virginia Polytechnic Institute Anaerobe Laboratory, Blacksburg, VA. Jackins, H.C. and H.A. Barker. 1951. Fermentative processes of the ­fusiform bacteria. J. Bacteriol. 61: 101–114. Kasai, G.J. 1961. A study of Leptotrichia buccalis. I. Morphology and ­preliminary observations. J. Dent. Res. 40: 800–811. Kasai, G.J. 1965. A study of Leptotrichia buccalis. II. Biochemical and physiological observations. J. Dent. Res. 44: 1015–1022. Koopman, J.P., M.E. van den Brink, P.P.C.A. Vennix, W. Kuypers, R. Boot and R.H. Bakker. 1991. Isolation of Streptobacillus moniliformis from the middle ear of rats. Lab. Anim. 25: 35–39. Krasil’nikov. 1941. Proactinomyces. In Guide to the Actinomycetes. Izd. Akad. Nauk., U.S.S.R, Moskau, p. 76. Krywolap, G.N. and L.R. Page. 1977. Oral Fusobacterium, Leptotrichia and Bacterionema: II. Pathogenicity: a review of the literature. J. Baltimore Coll. Dent. Surg. 32: 26–32. Lapage, S.P. 1974. Genus Cardiobacterium Slotnick and Daugherty. In Bergey’s Manual of Determinative Bacteriology, 8th edn (edited by Buchanan and Gibbons). Williams & Wilkins, Baltimore, pp. 377–378. Levaditi, C., S. Nicolau and P. Poincloux. 1925. Sur le role étiologique de Streptobacillus moniliformis (nov. spec.) dans l’erythème polymorph aigu septicémique. C. R. Hebd. Séances Acad. Sci. (Paris) 180: 1188–1190. Lieske, R. 1921. Morphologie und Biologie der Strahlenpilze ­(Actinomyceten). Borntraeger Bros., Leipzig. McCarthy, C., M.L. Snyder and R.B. Parker. 1965. The indigenous oral flora of man. I. The newborn to the 1-year-old infant. Arch. Oral Biol. 10 : 61–70.

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McEvoy, M.B., N.D. Noah and R. Pilsworth. 1987. Outbreak of fever caused by Streptobacillus moniliformis. Lancet. 2: 1361–1363. Messiaen, T., C. Lefebvre and A. Geubel. 1996. Hepatic abscess likely related to Leptotrichia buccalis in an immunocompetent patient. Liver 16: 342–343. Midgley, J., S.P. LaPage, B.A. Jenkins, G.I. Barrow, M.E. Roberts and A.G. Buck. 1970. Cardiobacterium hominis endocarditis. J. Med. Microbiol. 3: 91–98. Miyagawa, E., R. Azuma and T. Suto. 1979. Cellular fatty acid composition in Gram-negative obligately anaerobic rods. J. Gen. Microbiol. 25: 41–51. Moore, W.E.C., J.L. Johnson and L.V. Holdeman. 1976. Emendation of Bacteroidaceae and Butyrivibrio and descriptions of Desulfomonas gen. nov. and ten new species in genera Desulfomonas, Butyrivibrio, Eubacterium, Clostridium, and Ruminococcus. Int. J. Syst. Bacteriol. 26: 238–252. Neumann, S., U. Matthey, G. Kaim, and P. Dimroth. 1998. Purification and properties of the F1F0 ATPase of Ilyobacter tartaricus, a sodium ion pump. J. Bacteriol. 180: 3312–3316. Oeding, P. and H. Pedersen. 1950. Streptothrix muris ratti (Streptobacillus moniliformis) isolated from a brain abscess. Acta Pathol. Microbiol. Scand. 27: 436–442. Olson, L.D. and E.L. McCune. 1968. Histopathology of chronic otitis media in the rat. Lab. Anim. Care 18: 478–485. Parker, J.F. and N.P. Hudson. 1926. The etiology of Haverhill fever ­(erythema arthriticum epidemicum). Am. J. Pathol. 2: 375–379. Paster, B.J., W. Ludwig, W.G. Weisburg, E. Stackebrant, R.B. Hespel, C.M. Hahn, H. Reichenback, K.O. Stetter and C.R. Woese. 1985. A phylogenic grouping of the bacteroides, cytophagas, and certain flavobacteria. Syst. Appl. Microbiol. 6: 34–42. Patel, J.B., J. Clarridge, M.S. Schuster, M. Waddington, J. Osborne and I. Nachamkin. 1999. Bacteremia caused by a novel isolate resembling Leptotrichia species in a neutropenic patient. J. Clin. Microbiol. 37: 2064–2067. Peagle, R.D., R. P. Tewari, W.N. Berhard and E. Peters. 1976. ­Microbial flora of the larynx, trachea and large intestine of the rat after ­long-term inhalation of 100 per cent oxygen. Anesthesiology 44: 287–290. Place, E.H. and L.E. Sutton. 1934. Erythema arthriticum epidemicum (Haverhill fever). Arch. Intern. Med. 54: 659–684. Prévot, A.R. 1948. Manuel de classification et de determination des ­bactéries anaérobies, 2nd edn. Masson et Cie, Paris. Reig, M., F. Baquero, M. Garcia-Campello and E. Loza. 1985. Leptotrichia buccalis bacteremia in neutropenic children. J. Clin. Microbiol. 22: 320–321. Renaut, J.J., C. Pecquet, C. Verlingue, H. Barriere, M. Deriennic and A.L. Courticu. 1982. Septicémie à Streptobacillus moniliformis. Nouv. Presse. Med. 11: 1143. Rey, J.L., G. Laurans, A. Pleskof, M. Guerlin, J. Orfila, C. Tribouillov, P. Bernasconi and J.P. Lesbre. 1987. Les endocardites à Streptobacillus moniliformis. A propos de deux cas. Ann. Cardiol. Angéiol. 36: 297–300. Robin, C. 1853. Histoire naturelle des végétaux parasites qui crossent sur l’homme et sur les animaux vivants. J.-B. Baillière, Paris. Roughgarden, J.W. 1965. Antimicrobial Therapy of ratbite fever. A review. Arch. Intern. Med. 116: 39–54. Rupp, M.E. 1992. Streptobacillus moniliformis endocarditis: case report and review. Clin. Infect. Dis. 14: 769–772. Rygg, M. and C.F. Bruun. 1992. Rat bite fever (Streptobacillus moniliformis) with septicemia in a child. Scand. J. Infect. Dis. 24: 535–540. Savage, N.L. 1984. Genus Streptobacillus Levaditi, Nicolau and Poincloux 1925. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg). Williams & Wilkins, Baltimore. Schottmüller, H. 1914. Zur Atiologie und Klinik der Bisskrankheit ­(Ratten-, Katzen-, Eichhornchen-Bisskrankheit). Dermatol. ­Wochenschr. Erganzungsh. 58: 77. Schwartz, D.N., B. Schable, F.C. Tenover and R.A. Miller. 1995. Leptotrichia buccalis bacteremia in patients treated in a single bone marrow transplant unit. Clin. Infect. Dis. 20 : 762–767.

774

Family II. Leptotrichiaceae

Sebald, M. 1962. Étude sur les bactéries anaérobies gram-négatives asporulées. Thèses de l’Université Paris, Imprimerie Barnéoud S.A., Laval, France. Shah, H.N. and M.D. Collins. 1980. Fatty acid and isoprenoid quinone composition in the classification of Bacteroides melaninogenicus and related taxa. J. Appl. Bacteriol. 48: 75–87. Shah, H.N. and M.D. Collins. 1983. Genus Bacteroides. A chemotaxonomical perspective. J. Appl. Bacteriol. 55: 403–416. Shukla, S.K., P.R. Meier, P.D. Mitchell, D.N. Frank and K.D. Reed. 2002. Leptotrichia amnionii sp. nov., a novel bacterium isolated from the amniotic fluid of a woman after intrauterine fetal demise. J. Clin. Microbiol. 40: 3346–3349. Simon, M.W. and H.D. Wilson. 1986. Streptobacillus moniliformis ­endocarditis. A case report. Clin. Pediatr. (Phila) 25: 110–111. Söderberg, G., A.A. Lindberg and C.E. Nord. 1979. Bacteroides fragilis in acute salpingitis. Infection 7: 226–230. Strangeways, W.I. 1933. Rats as carriers of Streptobacillus moniliformis. J. Pathol. Bacteriol. 37: 45–51. Tee, W., P. Midolo, P.H. Janssen, T. Kerr and M.L. Dyall-Smith. 2001. Bacteremia due to Leptotrichia trevisanii sp. nov. Eur. J. Clin. ­Microbiol. Infect. Dis. 20: 765–769. Tee, W., P. Midolo, P.H. Janssen, T. Kerr and M.L. Dyall-Smith. 2002. In Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 85. Int. J. Syst. Evol. Microbiol. 52: 685–690. Thjötta, T., O. Hartmann and J. Böe. 1939. A study of the Leptotrichia ­Trevisan. History, morphology, biological and serological character­

isitics. Skr. Nor. Vidensk.-Akad. Oslo I. Mat.-Naturvidensk. K1. 5: 1–199. Topley, W.W.C. and G.S. Wilson. 1936. The Principles of Bacteriology and Immunity, 2nd edn. Edward Arnold, London, p. 274. Trevisan, V. 1879. Prime linee d’introduzione allo studio dei Batterj italiani. Rendiconti dell’Istituto Lombardo di Scienze Series 2 12: 133–151. Vernelen, K., I. Mertens, J. Thomas, J. Vandeven, J. Verhaegen and L. Verbist. 1996. Bacteremia with Leptotrichia buccalis: report of a case and review of the literature. Acta Clin. Belg. 51: 265–270. Weinberger, M., T. Wu, M. Rubin, V.J. Gill and P.A. Pizzo. 1991. ­Leptotrichia buccalis bacteremia in patients with cancer: report of four cases and review. Rev. Infect. Dis. 13: 201–206. Weisbroth, S.H. 1979. Bacterial and mycotic diseases. In The ­Laboratory Rat, vol. 1, Biology and Diseases (edited by Baker, Lindsey and ­Weisbroth). Academic Press, New York, pp. 193–241. Wilson, G.S. and A.A. Miles. 1955. Topley and Wilson’s Principles of Bacteriology and Immunology, 3rd edn, vol. 1. Williams & Wilkins, Baltimore. Wittler, R.G. and S.G. Cary. 1974. Genus Streptobacillus Levaditi, ­Nicolau and Poincloux. 1925,1188. In Bergey’s Manual of Determinative ­Bacteriology, 8th ed. (edited by Buchanan and Gibbons). Williams & Wilkins, Baltimore, pp. 378–381. Wullenweber, M., C. Jonas and I. Kunstyr. 1992. Streptobacillus moniliformis isolated from otitis media of conventionally kept laboratory rats. J. Exp. Anim. Sci. 35: 49–57. Young, C. and A. Hill. 1974. Conjunctivitis in a colony of rats. Lab. Anim. 8: 301–304.

Phylum XX. Dictyoglomi phyl. nov. Bharat K. C. Patel Dic¢ty.o.glo¢mi. N.L. n. Dictyoglomus type genus of the type order of the phylum; -i ending to denote phylum; N.L. neut. pl. n. Dictyoglomi the phylum of the order Dictyoglomales.

The phylum is currently represented by a single class, order, family, and genus. The phylum forms a deep line of descent with its related phyla Thermomicrobia and Deinococcus–Thermus (Figure 123). Gram-stain-negative, strictly anaerobic, thermophilic, and chemoorganotrophic rod-shaped to filamentous

cells that form spherical balls known as rotund bodies. Members produce a range of thermostable enzymes of significance to the biotechnology industries. Type order: Dictyoglomales ord. nov.

Chloroflexi Deferribacteria 0.10 Deinococci−Thermi Cyanobacteria

Nitrospiraceae

obium es en

rys

Ch

Thermotoga

omicr

Therm

Thermodesulfobacteriaceae

Chlorobiaceae

iog

Aquificaceae

Proteobacteria

Lentisphaerae Gemmatimonas Dictyoglomi

Verrucomicrobiales Firmicutes Fibrobacter Fusobacteria

Bacteroidetes

Planctomycetes

Chlamydiales Spirochaetales

Actinobacteria Figure 123.  Phylogenetic position of phylum Dictyoglomi. The scale bar represents 10 changes per 100 nucleotides.

775

776

Family I. Dictyoglomaceae

Class I. Dictyoglomia class. nov. Bharat K. C. Patel Dic.ty.o.glo¢mi.a. N.L. n. Dictyoglomus type genus of the type order of the class; suff. -ia ending proposed by Gibbons and Murray and by Stackebrandt et al. to denote a class; N.L. neut. pl. n. Dictyoglomia the class of the order Dictyoglomales. Slender (thin) rod to filamentous cells, 5–30 × 0.35–0.45 mm, occurring singly or in pairs. Spherical balls of cells, known as rotund bodies, are commonly observed. Stain Gram-negative, and cell walls possess an outer and an inner cell wall layer. Spores are not formed; cells are nonmotile and no flagella have been observed.

Thermophilic. Neutrophilic. Chemoorganotrophic, strict anaerobe, ferments glucose. The principal fermentation end products are acetate, lactate, ethanol, CO2, and H2. Occur in natural thermal hot springs and man-made thermal environments. Type order: Dictyoglomales ord. nov.

Order I. Dictyoglomales ord. nov. Bharat K. C. Patel Dic¢ty.o.glo.ma¢les. N.L. neut. n. Dictyoglomus type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Dictyoglomales the order of Dictyoglomus. Only one order, Dictyoglomales, currently exists, therefore the description of the order is the same as the class Dictyoglomi. Type genus: Dictyoglomus Saiki, Kobayashi, Kawagoe and Beppu 1985, 253VP.

Reference Saiki, T., Y. Kobayashi, K. Kawagoe and T. Beppu. 1985. Dictyoglomus thermophilum gen. nov., sp. nov., a chemoorganotrophic, anaerobic, thermophilic bacterium. Int. J. Syst. Bacteriol. 35: 253–259.

Family I. Dictyoglomaceae fam. nov. Bharat K. C. Patel Dic¢ty.o.glo.ma¢ce.ae. N.L. neut. n. Dictyoglomus type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Dictyoglomaceae the family of Dictyoglomus. Only one family, Dictyoglomaceae, is accepted in the order ­Dictyoglomales. The description of the family is the same as for the class. Only one genus is accepted in the family Dictyoglomaceae.

Type genus: Dictyoglomus Saiki, Kobayashi, Kawagoe and Beppu 1985, 256VP.

Genus I. Dictyoglomus Saiki, Kobayashi, Kawagoe and Beppu 1985, 256VP Bharat K. C. Patel Dic¢ty.o.glo¢mus. Gr. n. dictyon net; L. neut. n. glomus ball; N.L. neut. n. Dictyoglomus net ball.

Slender rod to filamentous cells (5–30 × 0.35–0.45 mm), which occur singly or in pairs. Nonsporeforming and nonmotile; no flagella have been observed. Stain Gram-negative, and cell walls possess outer and an inner cell wall layers. Spherical balls of cells, termed rotund bodies, formed due to either cell aggregation or by cell division, are commonly observed. The walls of the cells in the rotund bodies are portioned with the rotund bodies acquiring the outer wall layer with the cells attached to the inner wall layer. Thermophilic, neutrophilic, chemoorganotrophs which are strict anaerobes. Ferment carbohydrates to acetate, lactate, ethanol, CO2, and H2. Occur in natural thermal hot springs and man-made thermal environments (Table 151). DNA G+C content (mol%): 29–34. Type species: Dictyoglomus thermophilum Saiki, Kobayashi, Kawagoe and Beppu 1985, 256VP.

Further descriptive information Dictyoglomus species can easily be identified as they all form thin filaments and rotund bodies (Figure 124) similar to that observed for Thermus species. Only three out of 95 positive enrichment cultures initiated from 370 volcanic hot spring samples yielded Dictyoglomus isolates (Patel et al., 1987). None was isolated from the 300 positive enrichment cultures initiated from non-­volcanic geothermal waters of the Great Artesian Basin of Australia (Patel, unpublished results), suggesting that, unlike other thermoanaerobes, Dictyoglomus species are restricted in nature. Members of Dictyoglomus have so far been isolated only from natural and man-made thermal habitats. To date, two species (Dictyoglomus thermophilum H-6-12T (Saiki et al., 1985) and Dictyoglomus turgidum Z-1310T) (Svetlichnii and Svetlichnaya, 1988), both of which have been isolated from volcanic hot springs,

777

Genus I. Dictyoglomus Table 151.  Characteristics differentiating members of the genus Dictyoglomus a

Characteristics References Habitat Cell morphology and size (mm) Size of rotund bodies (mm) DNA G+C content (mol%) Temperature range for growth (°C) (optimum) pH range for growth (optimum) Generation time (min) Substrates utilized: Arabinose Carboxymethylcellulose Casein hydrolysate Cellobiose Cellulose Fructose Fucose Galactose Glucose Glycerol Glycogen Inositol Lactose Maltose Mannitol Mannose Pectin Pyruvate Raffinose Rhamnose Sorbitol Starch Sucrose Xylose Enzymes produced: Amylase Cellulase Xylanase End-products from carbohydrates Resistance to vancomycin (100 mg/ml)

D. thermophiilum strain H-6-12T

D. turgidum strain Z-1310T

Dictyoglomus strain Rt46-B1

Dictyoglomus strain B1

Saiki et al. (1985)

Patel et al. (1987)

Hot spring, Kumamoto Prefecture, Japan Rods to filaments (5–20 × 0.4–0.6) 15–100

Svetlichnii and Svetlichnaya (1988) Hot spring, Kamchatka, Russia Rods to filaments (10–30 × 0.3–0.4) 15–100

Hot spring, Kuirau Park, New Zealand Rods to filaments (5–25 × 0.35–0.45) 3–20

Mathrani and Ahring (1991) Pulp mill cooling tower, Kirkniemi, Finland Rods to filaments (up to 5–20 × 0.3) 5–25

29

32.5

29.5

34

51–80 (73–78)

48–86 (72)

40–82 (70)

>50–85% positive; −, 0–15% positive; w, weak reaction; nd, not determined.

a

have been validly published. Several other strains have also been described. These include Dictyoglomus strains Z-1311 to Z-1317 and Dictyoglomus strain Rt46-B1 (formerly named Fervidothrix), Rt8N2, and Tos-80-la-d. All of these were isolated from volcanic hot springs (pH ranging from 2.8 to 9.0 and temperatures ranging from 55 to 90°C) from Japan, Italy, and Russia (Patel et  al., 1986, 1987; Plant et  al., 1987; Svetlichnii and Svetlichnaya, 1988). Dictyoglomus strain B1 was isolated from a manmade pulp mill cooling tower which had a temperature range

of 70–80°C and a pH range of 5.5–6.5 (Mathrani and Ahring, 1991, 1992). With the exception of Dictyoglomus strain B isolated from a cooling tower of a pulp mill, all other Dictyoglomus isolates are reported to be nutritionally versatile and use a wide range of carbohydrates. However, all strains studied to date are slow growers with a generation time reported to vary between 260 and 600 min depending on the substrate used. A variety of extracellular enzymes have been identified for their potential in

778

Family I. Dictyoglomaceae

hyperthermophilic microbes (Fukusumi et  al., 1988; Horinouchi et al., 1988; Janecek, 1998; Jeon et al., 1997; Kobayashi et al., 1988). Studies on other enzymes such as mannases (Gibbs et al., 1999) and phosphofructokinase (Ding et al., 1999, 2000) have also been reported. Nielsen et al. (2007) provided evidence that the bioaugmentation of xylanase-producing Dictyoglomus species and cellulase/xylanase-producing Caldicellusiruptor species in cattle manure, led to a 93% increase in methane production. Their physiological processes in pure and/or mixed cultures are not well understood but if this significant gap in our knowledge were to be bridged, then we may be able to significantly improve the microbial energy-yielding processes. At the time of writing, the genomes of Dictyoglomus thermophilum and Caldicellulosiruptor saccharolyticus are being sequenced, and it will be interesting to await the analysis of the data in a bid to unravel the mysteries of their metabolic pathways.

Enrichment and isolation procedures

Figure 124.  Large spherical rotund bodies of Dictyoglomus thermophilum (strain H-6-12T). (top) Phase-contrast microphotograph; bar = 10 mm. (bottom) Thin section of cells showing that they are connected by the outer cell wall layer to form the rotund bodies; bar = 0.5 mm.

biotechnology. A number of reports have shown that xylanases from Dictyoglomus strains have the potential to pretreat wood pulp in paper manufacturing processing and achieve comparable levels of whiteness with much less bleach (Kenealy and Jeffries, 2003; Morris et al., 1998; Ratto et al., 1994). This has led to marked improvements in methods for the production of xylanases, both by conventional culture methods (Adamsen et al., 1995) and by gene recombinant techniques (Gibbs et al., 1995; Te’o et al., 2000). The structure of a xylanase has also been elucidated (Sunna and Bergquist, 2003) and metagenomic approaches have been used to clone and study Dictyoglomus-like xylanase genes directly from natural volcanic thermal samples. Amylases from Dictyglomus are reported to have a high degree of homology to amylases from a number of hyperthermophilic members of domain Archaea. This has stimulated an interest in the origin, evolution, and functioning of amylolytic enzymes in thermophilic and

Members of the genus Dictyoglomus can be enriched and isolated on complex anaerobic media containing yeast extract and/or ­peptones and a variety of fermentable polysaccharides. All media are prepared and dispensed anaerobically using Hungate techniques in an atmosphere of 100% nitrogen. Dictyoglomus thermophilum are grown in a medium containing per liter (pH 7.2): 1.5 g KH2PO4, 4.2 g Na2HPO4·12H2O, 0.5 g NH4Cl, 0.38 g MgCl2·6H2O, 0.05 g CaCl2, 0.039 g Fe(NH4)2(SO4)2·6H2O, 1.0 g Na2CO3, 2.0 mg resazurin, 1.0 g l-cysteine. HCl, 10.0 ml Trace Metal Solution (from a ×100 stock containing per liter 290.0 mg CoCl2·6H2O, 240.0 mg Na2MoO4·2H2O, 17.0 mg Na2SeO3, 200.0 mg MnCl2· 4H2O, 280.0 mg ZnSO4·7H2O), 2.0 g yeast extract, and 2.0 g ­peptone) and 10.0 ml filter-sterilized Wolfe’s vitamin solution (from a ×100 stock solution containing 2.0 mg biotin, 2.0 mg folic acid, 10.0 mg pyridoxine hydrochloride, 5.0 mg thiamine HCl, 5.0 mg riboflavin, 5.0 mg nicotinic acid, 5.0 mg calcium D-(+)pantothenate, 0.1 mg vitamin B12, 5.0 mg p-aminobenzoic acid, and 5.0 mg thioctic acid), added aseptically to the sterile medium with soluble starch (5 g/l) as the fermentable substrate. Dictyoglomus turgidum and Dictyoglomus strain B are cultured on a complex medium containing xylan as the fermentable substrate, whereas Dictyoglomus strain Rt46-B1 and a number of other volcanic hot spring isolates are enriched on the complex medium Trypticase Yeast Extract Medium (TYE). TYEG contains glucose as the fermentable substrate (Patel et al., 1985a, b). Enrichments are usually initiated by adding sediment and/ or water samples into anaerobic medium followed by incubation for up to 7 d at temperatures 70–80°C. In general, the use of glucose should be avoided if the enrichments are to be incubated for more than a few days at temperatures higher than 70°C as furfurals, a toxic by-product are produced under these conditions. Polysaccharides such as xylan and starch are preferable, as they do not form furfurals.

Maintenance procedures Dictyoglomus strains grown in Trypticase Yeast Extract Starch (TYES) or Trypticase Yeast Extract Glucose (TYEG) Medium remain viable for at least up to 3 months on the bench at room temperature and can be successfully subcultured. For long-term storage, mid-exponential-phase grown cultures are preserved in a 50:50 glycerol:TYEG medium to which 0.1 ml of iron sulfide (Brock and Od’ea, 1977) is added, and cultures are stored at −20 or −80°C.

Genus I. Dictyoglomus

Differentiation of the genus Dictyoglomus from other genera Though members of Dictyoglomus are thermophilic (optimum growth temperature >70°C), strictly anaerobic, carbohydratefermenting bacteria, they can be differentiated from all other thermoanaerobes due to their unique slender filamentousshaped cells and their phylogenetic position in the tree of life. Dictyoglomus strains possess a number of phenotypic traits, such as the production of rotund bodies, that are in common with certain members of the order Thermotogales and Thermales. Members of Dictyoglomus and of the order Thermotogales can be readily differentiated from Thermales as the former contains large amounts of C16 and C18 fatty acids whereas the latter contain C15 iso and C17 iso in their cell envelope phospholipid fatty acids (Patel et al., 1991). There are numerous phenotypic features which differentiate members of the Thermotogales order and Dictyoglomus species, e.g., the presence of terminal spheroids or blebs (ballooning of the ends of the cell) in members of the order Thermotogales but not in Dictyoglomus species. Morphologically, members of Dictyoglomus species are slender filamentous cells. Additionally, members of Thermotogales appear to be restricted to volcanic springs and oilfields with low salinity, but this is not the case with Dictyoglomus species. Furthermore, Dictyoglomus strains are slow growers (e.g., 4.3–10 h generation

779

times in batch cultures in a complex glucose medium), but members of the order Thermotogales are fast growers (1.5 h generation time in the same medium).

Taxonomic comments Early 16S rRNA gene based phylogeny placed Dictyoglomus thermophilum H-6-12T and Dictyoglomus strain Rt46-B1 as a deep line of descent in the vicinity of the phylum Thermotogales (Love et  al., 1993). The high concentrations of saturated phospholipid fatty acids in the cell membranes is another trait that is shared between members of Dictyoglomus and the phylum Thermotogales (Patel et  al., 1991). However, the phylogenetic relationship was not confidently predicted by bootstrap analysis, and the inclusion of different sequences changed the tree topology suggesting that the placement of members of Dictyglomus as a deep line of descent was tenuous. The re-evaluation of the phylogeny, based on the selection of new sequences that have been added to the 16S rRNA database, suggests that the members of Dictyoglomus do form a separate line of descent, but that it is closer to the members of the phylum Firmicutes (Figure 1).

Acknowledgements This research was supported by the Australian Research Council and by Griffith University Grants Scheme.

List of species of the genus Dictyoglomus 1. Dictyoglomus thermophilum Saiki, Kobayashi, Kawagoe and Beppu 1985, 256VP ther.mo¢phil.um Gr. n. thermê heat; N.L. neut. adj. philum (from Gr. neut. adj. philon) loving; N.L. neut. adj. thermophilum heat-loving. Rods to filaments measuring 5–20 × 0.4–0.6 mm that occur singly, in pairs, and in bundles. Several to several dozen cells form large spherical bodies (50–100 mm in diameter) known as rotund bodies (Figure 124). Stain Gram-negative. Nonsporeforming, the cells are nonmotile, and flagella are not observed. Pigment is not formed. Strict fermentative anaerobe which grows optimally at 73°C (temperature growth range of >45 and 5.4 and 90% of the sequences in the phylum. These environmental clones comprise several unique order- to family-level lineages (Figure 127, Table 152). The two cultivated orders plus three uncultivated lineages were termed subgroups L1–L5 by Cho et  al. (2004b); however, to account for the paraphyly of subgroup L4, accommodate new sequences, and avoid confusion, we use the term subphylum to denote well-supported deep monophyletic groups of three or more environmental clone sequences. This term has no taxonomic value, therefore the taxonomy of these subphyla should be ­re-evaluated and formally addressed as new organisms are ­isolated and characterized. The two cultivated genera belong to two well-supported clades, formally designated the orders Lentisphaerales and Victivallales. The order Lentisphaerales occupies a deep phylogenetic position within the Lentisphaerae and is comprised of Lentisphaera along with two other 16S rRNA gene sequences from marine environments. This clade is well supported and phylogenetically distant from other members of the phylum. The order Victivallales is comprised of Victivallis, indigenous to the human colon, along with 16S rRNA gene sequences from a turkey cecum (Scupham, 2007), dugong feces, termite gut contents, and anaerobic digesters fed with waste from a wine distillery or municipal sludge (Chouari et  al., 2005; ­Delbes et al., 2000; Gordon et al., 1997). The order Victivallales groups with subphylum 3 (formerly subgroup L2 (Cho et al., 2004b), which is represented by 16S rRNA gene sequences from the ­cattle rumen, an anaerobic wine distillery waste digester ­(Gordon et al., 1997), and freshwater lake and river sediments (Crump and Hobbie, 2005; Nercessian et al., 2005). Subphylum 4 (formerly subgroup L3 (Cho et  al., 2004b) and subphylum 5 are each comprised of sequences from mucosal secretions of marine invertebrates or deep-sea marine sediments (Alain et al., 2002; Li et al., 1999; Zhu et al., 2008). Subphyla 6 and 7 form a well-supported clade. Subphylum 6 16S rRNA gene sequences have been recovered from a ­variety

785

786

Phylum XXII. Lentisphaerae turkey cecum clone CFT114A6, DQ456068 Dugong feces clone dgC−74, AB218348 anaerobic digester clone AA08, AF275917 anaerobic digester clone vadinHB65, U81755 Victivallis vadensis, AY049713 Evry municipal wastewater clone 055H08_P_DI_P58, CR933072 Evry municipal wastewater clone 062G10_P_SD_P93, CR933035 termite gut clone BCf1−10, AB062771 cattle rumen clone F23−D04, AB185535 rumen clone RFN44, AB009200 rumen clone RFN4, AB009195 anaerobic digester clone vadinBE97, U81707 Lake Washington sediment clone pLW−89, DQ067008 Parker River clone PRD18C08, AY948016 marine worm isolate Dex80−43, AJ431234 mucus secretion clone P. palm C/A 24, AJ441226 deep sea sediment clone BD2−3, AB015533 marine sponge isolate PRPR22, DQ903989 cold seep sediment clone JT75−104, AB189358 marine clone Arctic95B−14, AF355055 marine clone Arctic95B−10, AY028222 mucus secretion clone P. palm C 41, AJ441225 wastewater treatment plant clone 030B08_P_DI_P15, CR933087 freshwater pond clone MVP−94, DQ676364 Lake Cadagno clone 362, AJ536842 Pearl River Estuary sediment clone MidBa45, EF999382 Zodletone Spring sediment clone Zplanct33, EF602494 gorilla feces clone Z20, DQ353908 Kings Bay sediment clone SS1_B_08_35, EU050937 hypersaline mat clone MAT−CR−H4−C09, EU245220 hypersaline mat clone MAT−CR−P6−B02, EU246270 hypersaline mat clone MAT−CR−M1−F05, EU245440 hypersaline mat clone MAT−CR−M3−E03, EU245595 hypersaline mat clone MAT−CR−M3−A05, EU245555 Mono Lake clone ML635J−58, AF507900 marine worm isolate Dex80−64, AJ431235 freshwater clone LiUU−11−182, AY509522 Kings Bay sediment clone SS1_B_02_17, EU050935 hypersaline mat clone MAT−CR−M6−B01, EU245769 Lentisphaera araneosa HTCC2160, AY390429 Lentisphaera araneosa HTCC2155, AY390428 Kings Bay sediment clone SS1_B_03_46, EU050936 Lentisphaera sp. clone EC115, DQ889896 Chlamydiae

Victivallales

Subphylum 3

Subphylum 4 Subphylum 5

Subphylum 6

Subphylum 7

Lentisphaerales

0.10

Figure 127.  Maximum likelihood tree with heuristic correction produced by using ARB (Ludwig et al., 2004). Solid squares represent nodes

supported by maximum likelihood, parsimony, and distance (neighbor-joining, Kimura 2-parameter correction). Open nodes were supported by two of the three methods. Parsimony and neighbor joining trees were made with 1000 bootstraps and heuristic corrections. Alignments were unmasked. The dataset was purged of sequences suggested to be anomalous by Mallard (Ashelford et al., 2006).

Table 152.  Habitat distribution of the orders of Lentisphaera including major uncultivated groups

Freshwater Sediment Order Lentisphaerales Order Victivallales Subphylum 3, uncultivated Subphylum 4, uncultivated Subphylum 5, uncultivated Subphylum 6, uncultivated Subphylum 7, uncultivated

Marine

Water

Sediment

Water

Invertebrate mucous

• ○ • ○

○

○ ○

• •

Vertebrate digestive tract

Anaerobic digesters

• •

• ○

•

Symbols: •, strains isolated and/or 16S rRNA gene clones from several different cultivation-independent studies of habitat type; from a cultivation-independent study of habitat type. No symbol, little or no evidence of colonization of the habitat.

of anaerobic habitats, including municipal sewage (Chouari et  al., 2005), anoxic freshwater sediments (Briee et  al., 2007; Elshahed et al., 2007), and feces of a wild gorilla (Frey et al., 2006). Subphylum 7 is composed of clone sequences from aquatic habitats varying in salinity, including a basal sequence from a freshwater lake (Eiler and Bertilsson, 2004), a marine

Hypersaline

○,

more than one 16S rRNA gene clone

clone from the marine tube worm Alvinella pompejana, and ­several derived sequences isolated from hypersaline environments (Humayoun et  al., 2003; Isenbarger et  al., 2008). The branching pattern within subphylum 7 suggests a freshwater ancestor with later niche invasion into saline and hypersaline environments.

Class I. Lentisphaeria

References Alain, K., M. Olagnon, D. Desbruyeres, A. Page, G. Barbier, S.K. Juniper, J. Quellerou and M.A. Cambon-Bonavita. 2002. Phylogenetic characterization of the bacterial assemblage associated with mucous secretions of the hydrothermal vent polychaete Paralvinella palmiformis. FEMS Microbiol. Ecol. 42: 463–476. Ashelford, K.E., N.A. Chuzhanova, J.C. Fry, A.J. Jones and A.J. ­Weightman. 2006. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Appl. Environ. Microbiol. 72: 5734–5741. Briee, C., D. Moreira and P. Lopez-Garcia. 2007. Archaeal and bacterial community composition of sediment and plankton from a suboxic freshwater pond. Res. Microbiol. 158: 213–227. Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004a. In ­Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 98. Int. J. Syst. Evol. Microbiol. 54: 1005–1006. Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004b. ­Lentisphaera araneosa gen. nov., sp. nov., a transparent exopolymer producing marine bacterium, and the description of a novel bacterial phylum, Lentisphaerae. Environ. Microbiol. 6: 611–621. Chouari, R., D. Le Paslier, C. Dauga, P. Daegelen, J. Weissenbach and A. Sghir. 2005. Novel major bacterial candidate division within a municipal anaerobic sludge digester. Appl. Environ. Microbiol. 71: 2145–2153. Crump, B.C. and J.E. Hobbie. 2005. Synchrony and seasonality in ­bacterioplankton communities of two temperate rivers. Limnol. Oceanogr. 50: 1718–1729. Delbes, C., R. Moletta and J.J. Godon. 2000. Monitoring of activity dynamics of an anaerobic digester bacterial community using 16S rRNA polymerase chain reaction – single-strand conformation ­polymorphism analysis. Environ. Microbiol. 2: 506–515. Eiler, A. and S. Bertilsson. 2004. Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes. Environ. Microbiol. 6: 1228–1243. Elshahed, M.S., N.H. Youssef, Q. Luo, F.Z. Najar, B.A. Roe, T.M. Sisk, S.I. Buhring, K.U. Hinrichs and L.R. Krumholz. 2007. Phylogenetic and metabolic diversity of Planctomycetes from anaerobic, sulfide- and sulfur-rich Zodletone Spring, Oklahoma. Appl. Environ. Microbiol. 73: 4707–4716. Frey, J.C., J.M. Rothman, A.N. Pell, J.B. Nizeyi, M.R. Cranfield and E.R. Angert. 2006. Fecal bacterial diversity in a wild gorilla. Appl. Environ. Microbiol. 72: 3788–3792. Gordon, J.J., E. Zumstein, P. Dabert, F. Habouzit and R. Moletta. 1997. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl. Environ. Microbiol. 63: 2802–2813. Humayoun, S.B., N. Bano and J.T. Hollibaugh. 2003. Depth distribution of microbial diversity in Mono Lake, a meromictic soda lake in California. Appl. Environ. Microbiol. 69: 1030–1042.

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Isenbarger, T.A., M. Finney, C. Rios-Velazquez, J. Handelsman and G. Ruvkun. 2008. Miniprimer PCR, a new lens for viewing the microbial world. Appl. Environ. Microbiol. 74: 840–849. Janssen, P.H., P.S. Yates, B.E. Grinton, P.M. Taylor and M. Sait. 2002. Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl. Environ. Microbiol. 68: 2391–2396. Lee, S.Y., J. Bollinger, D. Bezdicek and A. Ogram. 1996. Estimation of the abundance of an uncultured soil bacterial strain by a ­competitive quantitative PCR method. Appl. Environ. Microbiol. 62: 3787–3793. Li, L., C. Kato and K. Horikoshi. 1999. Bacterial diversity in deep-sea sediments from different depths. Biodivers. Conserv. 8: 659–677. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Forster, I. Brettske, S. Gerber, A.W. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. Konig, T. Liss, R. Lussmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N. Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode and K.H. Schleifer. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32: 1363–1371. Nercessian, O., E. Noyes, M.G. Kalyuzhnaya, M.E. Lidstrom and L. Chistoserdova. 2005. Bacterial populations active in metabolism of C1 compounds in the sediment of Lake Washington, a freshwater lake. Appl. Environ. Microbiol. 71: 6885–6899. Noll, M., D. Matthies, P. Frenzel, M. Derakshani and W. Liesack. 2005. Succession of bacterial community structure and diversity in a paddy soil oxygen gradient. Environ. Microbiol. 7: 382–395. Rappe, M.S. and S.J. Giovannoni. 2003. The uncultured microbial majority. Annu. Rev. Microbiol. 57: 369–394. Sangwan, P., X. Chen, P. Hugenholtz and P.H. Janssen. 2004. Chthoniobacter flavus gen. nov., sp. nov., the first pure-culture representative of subdivision two, Spartobacteria classis nov., of the phylum Verrucomicrobia. Appl. Environ. Microbiol. 70: 5875–5881. Sangwan, P., S. Kovac, K.E. Davis, M. Sait and P.H. Janssen. 2005. Detection and cultivation of soil Verrucomicrobia. Appl. Environ. Microbiol. 71: 8402–8410. Scupham, A.J. 2007. Succession in the intestinal microbiota of preadolescent turkeys. FEMS Microbiol. Ecol. 60: 136–147. Wagner, M. and M. Horn. 2006. The Planctomycetes, Verrucomicrobia, ­Chlamydiae and sister phyla comprise a superphylum with biotechnological and medical relevance. Curr. Opin. Biotechnol. 17: 241–249. Zhu, P., Q. Li and G. Wang. 2008. Unique microbial signatures of the alien Hawaiian marine sponge Suberites zeteki. Microb. Ecol. 55: 406–414. Zoetendal, E.G., C.M. Plugge, A.D. Akkermans and W.M. de Vos. 2003. Victivallis vadensis gen. nov., sp. nov., a sugar-fermenting anaerobe from human faeces. Int. J. Syst. Evol. Microbiol. 53: 211–215.

Class I. Lentisphaeria class. nov. Jang-Cheon

Cho, Muriel Derrien and Brian P. Hedlund

Len.ti.spha.e¢ria. N.L. fem. n. Lentisphaera type genus of the type order; N.L. neut. pl. n. Lentisphaeria class of the order Lentisphaerales. Lentisphaeria the class of bacteria having 16S rRNA gene sequences related to those of the members of the Lentisphaerales. The class Lentisphaeria encompasses bacteria and related 16S rRNA gene sequences mainly from marine habitats, freshwater habitats, anaerobic digesters, and vertebrate feces. It contains the orders Lentisphaerales and Victivallales in the phylum Lentisphaerae and is currently represented only by the genera Lentisphaera and

Victivallis. The delineation of the class is primarily determined based on phylogenetic information of 16S rRNA sequences. Type order: Lentisphaerales Cho, Vergin, Morris and Giovannoni 2004a, 1005VP (Effective publication: Cho, Vergin, Morris and Giovannoni 2004b, 617.).

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Family I. Lentisphaeraceae

References Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004a. In Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 98. Int. J. Syst. Evol. Microbiol. 54: 1005–1006.

Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004b. ­Lentisphaera araneosa gen. nov., sp. nov., a transparent exopolymer producing marine bacterium, and the description of a novel ­bacterial phylum, Lentisphaerae. Environ. Microbiol. 6: 611–621.

Order I. Lentisphaerales Cho, Vergin, Morris and Giovannoni 2004a, 1005VP (Effective publication: Cho, Vergin, Morris and Giovannoni 2004b, 617.) Jang-Cheon Cho and Brian P. Hedlund Len.ti.spha.e.ra¢les. N.L. fem. n. Lentisphaera type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Lentisphaerales the order of the genus Lentisphaera The order Lentisphaerales encompasses bacteria and related 16S rRNA gene sequences (detected by cultivation-independent methods and retrieved from pelagic marine habitats) within the phylum Lentisphaerae. The order comprises two strains of the genus Lentisphaera. The delineation of the order is primarily determined based on phylogenetic information of 16S rRNA sequences. Type genus: Lentisphaera Cho, Vergin, Morris and ­Giovannoni 2004a, 1005VP (Effective publication: Cho, Vergin, Morris and Giovannoni 2004b, 618.).

References Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004a. In ­Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 98. Int. J. Syst. Evol. Microbiol. 54: 1005–1006. Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004b. ­Lentisphaera araneosa gen. nov., sp. nov., a transparent exopolymer producing marine bacterium, and the description of a novel bacterial phylum, Lentisphaerae. Environ. Microbiol. 6: 611–621.

Family I. Lentisphaeraceae fam. nov. Jang-Cheon Cho and Brian P. Hedlund Len.ti.spha.e.ra.ce¢ae. N.L. fem. n. Lentisphaera type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Lentisphaeraceae the family of the genus Lentisphaera. The family Lentisphaeraceae encompasses bacteria and related 16S rRNA gene sequences (detected by cultivation-independent methods and retrieved from pelagic marine habitats) within the phylum Lentisphaerae. The family comprises two strains of the genus Lentisphaera. The delineation of the family is primarily

determined based on phylogenetic information of 16S rRNA sequences. Type genus: Lentisphaera Cho, Vergin, Morris and ­Giovannoni 2004a, 1005VP (Effective publication: Cho, Vergin, Morris and Giovannoni 2004b, 618.).

Genus I. Lentisphaera Cho, Vergin, Morris and Giovannoni 2004a, 1005VP (Effective publication: Cho, Vergin, Morris and Giovannoni 2004b, 618.) Jang-Cheon Cho and Stephen J. Giovannoni Len.ti.spha.e¢ra. L. adj. lentus sticky; L. fem. n. sphaera sphere; N.L. fem. n. Lentisphaera a sticky sphere.

Cells are spherical (coccus). Gram-stain negative. Gram-­negative cell structure. Cells have thin layer of extracellular slime and buds or appendages around the cells. Nonmotile. Strictly aerobic, chemoheterotrophic, and facultatively oligotrophic bacteria. Do not produce pigments. Require NaCl for growth. Do not utilize amino acids as sole carbon sources. Produce transparent exopolymers. The major cellular fatty acids are C16:1 w9c (50.8%) and C14:0 (25.9%). The genome size is 6.02 Mb, based on full-genome sequencing. Phylogenetically, the genus belongs to the order Lentisphaerales in the phylum Lentisphaerae. DNA G+C content (mol%): 48–49. Type species: Lentisphaera araneosa Cho, Vergin, Morris and Giovannoni 2004a, 1005VP (Effective publication: Cho, Vergin, Morris and Giovannoni 2004b, 618.).

Further descriptive information The genus Lentisphaera currently contains only one species with the type strain, Lentisphaera araneosa HTCC2155T. Phylogenetic analyses based on almost complete 16S rRNA gene sequences of strains HTCC2155T and HTCC2160 show that the genus forms a distinct clade together with Victivallis vadensis and several environmental sequences of the candidate phylum VadinBE97. The phylogenetic clade containing the genera Lentisphaera and Victivallis is clearly separated from the nearest phylum, Verrucomicrobia (Figure 128). Based on this phylogenetic analysis, the genus Lentisphaera is classified as the type genus in the order Lentisphaerales of the phylum Lentisphaerae. The comparative analyses of 16S rRNA gene sequence show that Lentisphaera araneosa has

Genus I. Lentisphaera

only 65.1–82.1% similarity with the sequences of representatives of all other phyla in the domain Bacteria. Cells are nonmotile, spherical forms with 0.8 mm in diameter, dividing by binary fission. Cells have a Gram-stain-negative cell structure. Flagella, endospore, and intracellular granules are not found. Colonies are 0.6–1.1 mm in diameter, milkish, uniformly circular, convex, and opaque, with smooth surfaces and entire margins, after incubation on marine agar 2216 at 20°C for 3 weeks. The genus Lentisphaera is an obligately aerobic, NaClrequiring, and psychrotolerant marine chemoheterotroph. The temperature range, pH range, and salinity range for growth are 4–25°C (optimum at 16–20°C), pH 7.0–9.0 (optimum at pH 8.0), and 0.75–15% (w/v) of NaCl (optimum at 3.0%), respectively. b-Galactosidase activity is positive. Catalase, oxidase, denitrification, indole production, glucose acidification, arginine dihydrolase, urease, and hydrolysis of esculin and gelatin are negative. Cells utilize the following carbon sources (0.02%) as sole carbon sources; d-glucose, d-galactose, d-fructose, b-lactose, d-trehalose d-cellobiose, d-maltose, d-mannose, d-glucosamine, d-mannitol, d-sorbitol, pyruvic acid, succinic acid, and d-malic acid. However, the cells do not utilize amino acids as sole carbon sources. The most important physiological property of the genus Lentisphaera is transparent exopolymer (TEP) production in oligotrophic conditions. The genus produces TEP in artificial seawater medium (ASW; 30 g of NaCl, 1.0 g of MgCl2·6H2O, 4.0 g of Na2SO4, 0.7 g of KCl, 0.15 g of CaCl2·2H2O, 0.5 g of NH4Cl, 0.2 g of NaHCO3, 0.1 g of KBr, 0.27 g of KH2PO4, 0.04 g

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of SrCl2·6H2O, 0.025 g of H3BO3, 0.001 g of NaF, 10 ml of Tris– Cl [pH 8.0]), and 1 ml of SN trace metal solution (Waterbury et  al., 1986) per 1 l deionized water) and LNHM medium (0.2  mm-­filtered and autoclaved seawater supplemented with 1.0 mM NH4Cl and 0.1 mM KH2PO4) amended with 1× mixed carbons (MC; LNHM plus 1× MC); 1× MC was composed of 0.001% (w/v) of each of the following carbon compounds: d-glucose, d-ribose, succinic acid, pyruvic acid, glycerol, N-acetyl d-­glucosamine, and 0.002% (v/v) of ethanol. The culture medium increases in viscosity at the end of exponential growth phase and reaches a maximum in stationary phase. The TEP stains bright to deep blue with the dye Alcian Blue. The composition of the TEP is rhamnose (62.2%), galactose (14.2%), mannose (12.2%), and glucose (11.4%). Lentisphaera araneosa was isolated from the surface seawater of the Pacific Ocean; thus its major habit is considered to be marine environments. The members of the phylum Lentisphaerae including cultured and uncultured members seem to inhabit mainly marine environments and animal (or human)related anaerobic terrestrial environments. Marine habitats include seawaters of the Arctic Ocean, the Pacific Ocean, deep-sea sediments, and mucous of the hydrothermal vent polychaete Paralvinella palmiformis. Bulk nucleic acid hybridization values determined by using Lentisphaera araneosa-specific probe (5¢-TTAGCAAGTAAGGATATGGGT-3¢) from the Pacific Ocean and the oligotrophic Atlantic seawater samples indicate that Lentisphaera araneosa is a common marine bacterium which accounts for less than 1% of total bacterial 16S rRNA.

Figure 128.  Maximum-likelihood 16S rRNA phylogenetic tree showing the phylogenetic positions of the genus Lentisphaera and the phylum Lentisphaerae. Bootstrap proportions over 70% from both neighbor-joining (above nodes) and maximum parsimony (below nodes) are shown. The closed circles and open circles at each node in the Lentisphaerae indicate recovered nodes in all three treeing methods and recovered nodes in two treeing methods respectively. Scale bar, 0.1 substitutions per nucleotide position.

790

Family I. Lentisphaeraceae

Enrichment and isolation procedures The original liquid cultures of Lentisphaera araneosa were ­cultivated from the Oregon coast of the Pacific Ocean surface by a dilution-to-extinction method, using LNHM amended with 1× MC after incubation at 16°C for 21 d. The liquid cultures were spread on LNHM plus 1× MC agar medium and colonies were isolated after incubation for 20 d at 16°C. The colonies were transferred on marine agar 2216 and stored as glycerol suspension in liquid nitrogen. Currently there is no special method to enrich Lentisphaera from the mixed marine microbial community. Because the growth rate of Lentisphaera araneosa is much slower than that of standard colony forming marine bacteria, use of an oligotrophic medium such as LNHM plus 1× MC medium or 1/10 marine R2A is recommended rather than Zobell’s medium or marine agar 2216 for the isolation of Lentisphaera araneosa from the complex marine microbial community. Recommended incubation temperature to isolate the genus is 16°C.

Maintenance procedures Frozen stocks as a glycerol suspension (10–30%) stored in liquid nitrogen or at −70°C are routinely used to start a new culture. Either colonies scraped from the surface of agar medium or broth cultures can be used for preparing glycerol stocks. Neither marine broth 2216 nor 1/10 marine R2A broth is suitable for maintaining or preserving the cultures. Incubating the cultures in LNHM plus 1× MC medium or ASW plus 10× MC is recommended for the maintenance and storage of the cultures. Working cultures can be maintained on marine agar 2216 at 16°C for 2 months. Working cultures (slants) can be maintained at 4°C for 3 months.

Procedures for testing special characters Colonies of Lentisphaera araneosa can be visualized as small rounded colonies after 10 d of incubation. To check for culture

purity, DAPI stained cells are viewed by epifluorescent microscopy. When Lentisphaera araneosa is cultured in ASW plus 10× MC medium, the viscosity of the medium can be ­easily checked by swirling the culture flasks and measuring ­viscosity. Lentisphaera araneosa can be specifically identified by using Lentisphaera araneosaspecific PCR primers HTCC2155-195F (5¢-AAGGTTACGCTTA GGGATGA-3¢) and HTCC2155-1345R (5¢-GTAGCTGATGCCCATTTACT-3¢), and a standard PCR ­protocol using an annealing temperature of 55°C.

Differentiation of the genus Lentisphaera from other related genera Phylogenetically, the most closely related genus is Victivallis within the order Victivallales in the phylum Lentisphaera. The genus Lentisphaera is differentiated from the genus Victivallis by 16S rDNA sequence similarity (84.4%), habitat (seawater vs human), oxygen requirement (obligately aerobic vs obligately anaerobic), mode of glucose utilization (oxidation vs fermen­ tation), DNA G+C composition (48.3 mol% vs 59.2 mol%), and optimum growth temperature (20°C vs. 37°C).

Taxonomic comments The genus Lentisphaera is the type genus of the order Lentisphaerales, which encompasses bacteria retrieved from marine habitats, within the phylum Lentisphaerae. The phylum Lentisphaerae was named after the genus Lentisphaera, although Victivallis is the first validly published genus in the phylum. Because the category phylum is not covered by the Rules of the Bacteriological Code, Lentisphaerae as nomenclature of the phylum is legitimate. As the phylum contains some cultured-but-­undescribed species and many uncultured organisms, culturing endeavors and more formal description of novel taxa in the phylum would be very helpful to elucidate the taxonomy of the phylum ­Lentisphaerae.

List of species of the genus Lentisphaera 1. Lentisphaera araneosa Cho, Vergin, Morris and ­Giovannoni 2004a, 1005VP (Effective publication: Cho, Vergin, ­Morris and Giovannoni 2004b, 618.) a.ra.ne.o¢sa. L. fem. adj. ­araneosa similar to cobwebs, pertaining to the morphology of ­transparent exopolymer particles produced by the species.

References Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004a. In Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 98. Int. J. Syst. Evol. Microbiol. 54: 1005–1006. Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004b. ­Lentisphaera araneosa gen. nov., sp. nov., a transparent exopolymer

The characteristics of the species are as described for the genus. DNA G+C content (mol%): 48–49 (HPLC). Type strain: HTCC2155, ATCC BAA-859, KCTC 12141. Sequence accession no. (16S rRNA gene): AY390428.

producing marine bacterium, and the description of a novel ­bacterial phylum, Lentisphaerae. Environ. Microbiol. 6: 611–621. Waterbury, J.B., S.W. Watson, F.W. Valois and D.G. Franks. 1986. ­Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. In Canadian Bulletin Fisheries and Aquatic Sciences, vol. 214 (edited by Platt and Li). Department of Fisheries and Oceans, Ottawa, pp. 71–120.

Genus I. Victivallis

791

Order II. Victivallales Cho, Vergin, Morris and Giovannoni 2004a, 1005VP (Effective publication: Cho, Vergin, Morris and Giovannoni 2004b, 618.) Muriel Derrien, Jang-Cheon Cho and Brian P. Hedlund Vic.ti.val.lal¢les. N.L. fem. n. Victivallis type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Victivallales the order of the genus Victivallis The order comprises the genus Victivallis and uncultured 16S rRNA genes from mainly anaerobic environments that branch within the phylum Lentisphaerae. Clones have been retrieved from the intestinal contents of humans, dugongs, turkeys, and termites, as well as anaerobic digesters and wastewater. The delineation of the order is primarily based on phylogenetic information from 16S rRNA sequences. Type genus: Victivallis Zoetendal, Plugge, Akkermans and de Vos 2003, 214VP (Effective publication: Cho, Vergin, Morris and Giovannoni 2004b, 618.).

References Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004a. In ­Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 98. Int. J. Syst. Evol. Microbiol. 54: 1005–1006. Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004b. ­Lentisphaera araneosa gen. nov., sp. nov., a transparent exopolymer producing marine bacterium, and the description of a novel bacterial phylum, Lentisphaerae. Environ. Microbiol. 6: 611–621. Zoetendal, E.G., C.M. Plugge, A.D. Akkermans and W.M. de Vos. 2003. Victivallis vadensis gen. nov., sp. nov., a sugar-fermenting anaerobe from human faeces. Int. J. Syst. Evol. Microbiol. 53: 211–215.

Family I. Victivallaceae fam. nov. Muriel Derrien, Jang-Cheon Cho and Brian P. Hedlund Vic.ti.val.la.ce¢ae. N.L. fem. n. Victivallis type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Victivallaceae the family of the genus Victivallis The family comprises the genus Victivallis and uncultured 16S rRNA genes from mainly anaerobic environments that branch within the phylum Lentisphaerae. Clones have been retrieved from the intestinal contents of humans, dugongs, turkeys, and termites, as well as anaerobic digesters and wastewater. The

delineation of the family is primarily based on phylogenetic information from 16S rRNA sequences. Type genus: Victivallis Zoetendal, Plugge, Akkermans and de Vos 2003, 214VP.

Genus I. Victivallis Zoetendal, Plugge, Akkermans and de Vos 2003, 214VP Muriel Derrien, Caroline M. Plugge, Willem M. de Vos and Erwin G. Zoetendal Vic.ti.val¢lis. L. masc. n. victus food; L. fem. n. vallis valley; N.L. fem. n. victivallis food valley which refers to the Wageningen “Food Valley”. This is an area in The Netherlands in which food science is a major research topic.

Coccoid cells, occurring singly and in pairs. The diameters of the cells vary between 0.5 and 1.3 mm. Gram-negative cell structure. Nonmotile. Strictly anaerobic. Victivallis does not grow on standard agar plates, but beige, shiny, lens-shaped colonies are formed on agar plates with 0.75% (w/v) agar after 10 d of incubation. Cells grow optimally at 37°C and pH 6.5. Growth of Victivallis is chemoorganotrophic and restricted to a variety of sugars. Acetate, ethanol, H2 and bicarbonate are the main fermentation products from glucose. 16S rRNA gene sequence analysis indicated that Victivallis belongs to the recently described order Victivallales of the phylum Lentisphaerae. The closest cultured relative is Lentisphaera araneosa (84.4% 16S rRNA gene similarity). DNA G+C content (mol%): 59.2 (HPLC). Type species: Victivallis vadensis Zoetendal, Plugge, ­Akkermans and de Vos 2003, 214VP.

Further descriptive information Light microscopic and transmission electronic microscopic (TEM) analyses revealed that Victivallis vadensis is a coccoid cell

with Gram-negative cell structure (Figure 129). The cells are variable in diameter (0.5–1.3 mm) and surrounded by haloes, which consist of an extracellular slime layer. In addition, many intracellular electron-dense structures are observed (Figure 129). Victivallis vadensis was initially isolated in a bicarbonate­buffered anaerobic mineral salts medium (for details see Plugge (2005) supplemented with 10 mM cellobiose and 0.7% (v/v) clarified sterile rumen fluid. However, rumen fluid is not required for growth. In this basal medium Victivallis can be cultivated using cellobiose, fructose, galactose, glucose, lactose, lactulose, maltose, maltotriose, mannitol, melibiose, myo­inositol, raftilose, rhamnose, ribose, sucrose and xylose as sole carbon and energy source. All sugars are utilized fermentatively. Besides growth on the above-mentioned defined media, Victivallis grows well on Wilkens–Chalgren broth (Oxoid; 16 g/l) and the medium described by Kamlage et al. (1999) (designated KA medium) with minor modifications [no hemin, bacteriological peptone (Oxoid) instead of tryptic peptone from meat], both supplemented with 0.7% clarified sterile rumen fluid. Optimal growth conditions include strict anoxic conditions, pH 6.5,

792

Family I. Victivallaceae

contaminants. Triplicate incubations in soft agar (0.75%) with ­subsequent transfer of single colonies into liquid medium will result in the isolation of Victivallis. Growth in liquid cultures will take 1 d; formation of colonies on soft agar will take approximately 10 d.

Maintenance procedures Victivallis can be stored in basal medium containing cellobiose or any other sugar for several months at 4°C under anaerobic conditions. For longer periods up to several years, cultures can be maintained in basal medium containing 25% glycerol and 0.1% of a titanium-citrate solution (100 mM) at −80°C. Victivallis can also be freeze-dried.

Procedures for testing special characters

Figure 129.  Transmission electronic micrograph of the type strain, Victivallis vadensis. Scale bar, approximately 0.5 mm.

temperature of 37°C and addition of 0.2% yeast extract. Under these conditions, the doubling time may reach 0.5 h. Victivallis can grow syntrophically with Methanospirillum hungatei JF-1T (DSM 864) in co-culture; glucose is converted exclusively to acetate and methane. Little is known about the habitat of Victivallis since it has only been isolated from a single fecal sample. Denaturating gradient gel electrophoretic analysis (DGGE) of 16S rRNA genes indicated that it is not a dominant member from this fecal sample. Two related uncultured Victivallales clones which share only 94% 16S rRNA sequence similarity with Victivallis vadensis were detected in an anaerobic digester that was fed with wastewater containing plant material (Godon et al., 1997). This indicates that Victivallis may also be present in these types of habitats.

Enrichment and isolation procedures Victivallis can be enriched and eventually isolated from human feces in liquid anaerobic basal medium, with cellobiose as sole carbon and energy source, by repeated transfers in serial dilution. For a detailed protocol concerning the preparation of basal medium see Plugge (2005). Incubations are performed at 37°C under anaerobic conditions under a gas phase of 182 kPa (1.8 atm) N2/CO2 (80:20, v/v). The antibiotics streptomycin and polymyxin B may be used to inhibit growth of potential

Victivallis cells are characterized as coccoid cells with Gramnegative cell structure varying in size between 0.5 and 1.3 mm, and surrounded by a slime layer. This slime layer increases the viscosity of TE or 1× PBS when cells are washed with these solutions for DNA isolation. A special characteristic of Victivallis is its inability to grow on standard agar plates.

Differentiation of the genus Victivallis from other genera Phylogenetic analysis of 16S rRNA genes revealed that ­Victivallis belongs to the order Victivallales of the phylum Lentisphaerae (Figure 130). The closest cultured relative of the type strain ­Victivallis vadensis is Lentisphaera araneosa, another isolate from the phylum Lentisphaerae, although it only shares 84.4% ­similarity at the16S rRNA gene level (Cho et al., 2004). While Victivallis and Lentisphaera both have limited metabolic capacities, the two genera differ by: habitat (human intestine vs sea water); oxygen requirement (strict anaerobe vs strict ­aerobe), glucose utilization (fermentation vs oxidation), DNA G+C ­composition (59.2 vs 48.3 mol%), and optimum growth temperature (37°C vs 20°C).

Taxonomic comments To date, Victivallis vadensis is the only isolate representing the genus Victivallis and order Victivallales. This limits our ability to comprehensively describe this group at the order, genus and species level. Fortunately, culture-independent data shows the detection of bacteria that are more closely related (94% 16S rRNA gene sequence similarity). This suggests that additional species of Victivallis or of a closely related genus are waiting to be isolated.

Differentiation of the species of the genus Victivallis Only one species is described, the type species Victivallis vadensis.

List of the species of the genus Victivallis 1. Victivallis vadensis Zoetendal, Plugge, Akkermans and de Vos 2003, 214VP va.den¢sis. N.L. fem. adj. vadensis of or belonging to Vada, referring to Wageningen. Victivallis vadensis refers to the Wageningen “Food Valley”. Cells are coccoid-shaped, 0.5–1.3 mm, nonmotile, with Gram-stain-negative cell structure and surrounded by an extracellular slime layer. Cells occur singly or in pairs. In pure culture, the cells can grow on a variety of sugars, only

under strictly anaerobic conditions. Glucose is converted to acetate, ethanol, H2 and bicarbonate. Growth on solid agar media is possible below 0.75% (w/v) agar. Cells grow optimally on bicarbonate-buffered mineral salts medium with cellobiose at 37°C and pH 6.5. Source: human feces. DNA G+C content (mol%): 59.2 (HPLC). Type strain: Cello, DSM 14823, ATCC BAA-548. Sequence accession no. (16S rRNA gene): AY049713.

Genus I. Victivallis

793

Figure 130.  Phylogenetic neighbor-joining dendogram showing the relationships of Victivallis vadensis with

­representatives of the phyla Verrucomicrobia and Lentisphaerae. Bar = 0.1% sequence difference.

References Cho, J.C., K.L. Vergin, R.M. Morris and S.J. Giovannoni. 2004. ­Lentisphaera araneosa gen. nov., sp. nov., a transparent exopolymer producing marine bacterium, and the description of a novel bacterial phylum, Lentisphaerae. Environ. Microbiol. 6: 611–621. Godon, J.J., E. Zumstein, P. Dabert, F. Habouzit and R. Moletta. 1997. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl. Environ. Microbiol. 63: 2802–2813.

Kamlage, B., L. Hartmann, B. Gruhl and M. Blaut. 1999. Intestinal microorganisms do not supply associated gnotobiotic rats with ­conjugated linoleic acid. J. Nutr. 129: 2212–2217. Plugge, C.M. 2005. Anoxic media design, preparation, and considerations. In Methods in Enzymology, vol. 397. Academic Press, New York, NY, pp. 3–16. Zoetendal, E.G., C.M. Plugge, A.D. Akkermans and W.M. de Vos. 2003. Victivallis vadensis gen. nov., sp. nov., a sugar-fermenting anaerobe from human faeces. Int. J. Syst. Evol. Microbiol. 53: 211–215.

Phylum XXIII. Verrucomicrobia phyl. nov. Brian P. Hedlund Ver.ru¢co.mi.cro¢bi.a. N.L. fem. pl. n. Verrucomicobiales type order of the phylum; N.L. neut. pl. n. Verrucomicrobia the Verrucomicrobiales phylum.

The phylum Verrucomicrobia is defined by phylogenetic analysis of 16S rRNA gene sequences of cultured strains and environmental clone sequences retrieved from a wide variety of environments. All cultivated members of the phylum stain Gram-negative; many have intracellular compartments bounded by internal membranes. Menaquinones are the dominant respiratory quinones; ubiquinones have not been detected. Most members are chemoheterotrophs, preferring carbohydrates including complex natural polysaccharides. Recently isolated members are thermoacidophilic methylotrophs. Type order: Verrucomicrobiales Ward-Rainey, Rainey, Schlesner and Stackebrandt 1996, 625VP (Effective publication: WardRainey, Rainey, Schlesner and Stackebrandt 1995, 3249.) emend. Yoon, Matsuo, Adachi, Nozawa, Matsuda, Kasai and Yokota 2008, 1002.

Further descriptive information To date, there are seven class-level groups in the phylum including three classes that have been formally defined, Verrucomicrobiae, Spartobacteria, and Opitutae. Two additional subphyla groups include recently cultivated organisms that have not yet been formally defined. Lastly, two subphyla groups are represented solely by 16S rRNA gene sequences from cultivation-independent studies (Figure 131). Each class-level group exhibits distinct habitat preferences that are not yet fully elucidated (Table 153). Classes Verrucomicrobiae, Spartobacteria, and Opitutae are each described in their own class chapters. Here, traits of the four remaining class-level groups are described. Subphylum 3 is known mostly from 16S rRNA gene sequences from cultivation-independent studies of soils and marine environments. Recently, six strains were isolated from soils by prolonged incubation on gellan gum-solidified medium with xylan as the sole carbon and energy source, including Ellin 514, Ellin 516, and Ellin 518 (Sangwan et al., 2005; Figure 132). The genus name “Pedosphaera” will be proposed to encompass these Gram-stain-negative, saccharolytic cocci (Sangwan and Janssen, pers. Comm.). Ellin 514 has been shown to have a compartmentalized cell including a membrane-enclosed compartment containing a condensed nucleoid and ribosomes (termed the pirellulosome) and a ribosome-free compartment between the pirellulosome and the cytoplasmic membrane (termed the paryphoplasm) (Lee et al., 2009). This cellular architecture has also been described in Verrucomicrobium, Prosthecobacter, and Chthoni­ obacter species (Lee et al., 2009). The phylogenetic structure of subphylum 3 includes two primary lineages (Figure 132). The lineage including “Pedosphaera” is comprised almost entirely of 16S rRNA genes recovered from soil habitats (Cruz-Martinez

et al., 2009; Janssen, 2006; Sangwan et al., 2005) where it is typically the second most abundant class-level group of Verruco­ microbia in clone libraries at 0–4.7% of total 16S rRNA gene clones ( Janssen, 2006). The second lineage is comprised of 16S rRNA genes recovered from marine habitats, particularly in the deep sea (Lau and Armbrust, 2006; Penn et al., 2006). Subphylum 6 has been predominantly recovered from methane-emitting mud volcanoes showing evidence of methane consumption (Alain et  al., 2006; Niemann et  al., 2006). Subsequently, three separate groups reported isolation of methanotrophic Verrucomicrobia from thermoacidic mud volcanoes, provisionally named “Methylacidiphilum infernorum” (Dunfield et  al., 2007), “Methylacidiphilum kamchatkensis” (Islam et  al., 2008), and “Acidimethylosilex fumarolicum” (Pol et  al., 2007). “Methylacidiphilum infernorum” and “Methylacidiphilum kamchat­ kensis” were isolated from Hell’s Gate, New Zealand, and Uzon Caldera, Kamchatka, Russia, respectively. These two isolates have similar pH growth optima at pH 3.5 and ranges from pH 0.8 to 5.8. The temperature ranges for growth were 40–65°C with optima at 55°C. However, only “Methylacidiphilum kamchat­ kensis” appears to be able to fix atmospheric N2. “Acidimethylo­ silex fumarolicum” was isolated from the Pozzouli Solfatara, Italy with a pH range of 1.0–6.0 (optimum 2.5) at 60°C. All three isolates have three divergent operons encoding the components of the particulate (membrane-bound) methane monooxygenase (pMMO) and possess intracellular membrane structures described as tubular or polyhedral which are likely to increase membrane surface area for pMMO in the absence of stacked membrane structures typical of methylotrophic Proteobacteria. Analysis of pyrosequencing data from “Acidimethylosilex fumaroli­ cum” (Pol et al., 2007) and a complete genome from “Methyla­ cidiphilum infernorum” (Hou et al., 2008) show simple, modified methanotrophic pathways with many homologs from methylotrophic Proteobacteria. The genome size of “Methylacidiphilum infernorum” is much smaller than Proteobacteria methylotrophs at 2.3 Mb; it was suggested that genes for C1 metabolism were obtained by horizontal gene transfer from Proteobacteria. Subphylum 5 is known only by cultivation-independent studies (Figure 133). Two major clades exist. One is found primarily in anaerobic sediments such as those showing anaerobic ammonia oxidation activity (Freitag and Prosser, 2003), contaminated aquifers (Dojka et al., 1998), geothermal mats (both marine and freshwater) (Hirayama et al., 2007), RNA from freshwater sediments (Nercessian et al., 2005), and hypersaline terminal-basin lakes (Humayoun et al., 2003). The second clade is also found in anaerobic habitats such as those listed above, however, this clade has also been detected in digestive tracts of vertebrates

795

796

Phylum XXIII. Verrucomicrobia  Class Verrucomicrobiae Class “Spartobacteria” subphylum 3, uncultivated subphylum 6, uncultivated Class Opitutae subphylum5, uncultivated

subphylum 7, uncultivated Phylum Lentisphaerae 0.10 Figure 131.  Phylogeny of Verrucomicrobia calculated with maximum likelihood methods with heuristic correc-

tion showing nearly complete 16S rRNA gene sequences present in version 91 of the Silva database (Pruesse et  al., 2007). All class-level groups were included and wedged to show the relationship between existing classes and class-level groups or “subphyla” not yet formally described. Subphylum 3 includes strains recently isolated by Janssen’s group but not yet formally described. The genus name “Pedosphaera” will be proposed (Sangwan et al., 2005). Recent thermoacidophilic methylotrophs provisionally named “Methylacidiphilum infernorum” (Dunfield et al., 2007), “Methylacidiphilum kamchatkensis” (Islam et al., 2008), “Acidimethylosilex fumarolicum” (Pol et al., 2007), and related 16S rRNA gene sequences belong to subphylum 6. The phylogeny was produced by using ARB (Ludwig et al., 2004) using Escherichia coli 16S rRNA gene nucleotides 110–1274. Alignments were unmasked.

Table 153.  Habitat distribution of the classes of Verrucomicrobia including uncultivated class-level groups from studies published prior to June 2008a,b

Habitat Soil Sediment: Fresh Marine Water: Fresh Marine Symbionts or commensals: Vertebrate Invertebrate Geothermal

Class Verrucomicrobiae

Class Opitutae

Class Spartobacteria

Class 3, uncultivatedc

○

●

●

●

○ ● ● ● ● ●

○ ○ ●

○

● ○

●

Class 5, uncultivated

Class 6, uncultivatedc

Class 7, uncultivated

○

○

● ●

●

○ ○

○ ○

● ○ ○

○

○ ●

○ ○

a Symbols: ●, multiple strains isolated from different samples and/or >25% of 16S rRNA gene clones in the class from cultivation-independent studies come from habitat type; ○, single strain isolated or multiple strains from the same sample and/or 0.5% NaCl Presence of C18:2 fatty acids High content of C18:1w9c DNA G+C content (mol%)

Ovoid or ellipsoidal +

Ellipsoidal

Cell shape

Blastopirellula

Schlesneria

Characteristic

Table 176.  Characteristics differentiating Schlesneria from other aerobic budding bacteria lacking peptidoglycana

+ v − − + − + 51–58

−

+

Ovoid or spherical

Planctomyces

− + − − + − + 53–57

−

+

Ovoid or ellipsoidal

Rhodopirellula

− − + − − + + 57–60

−

−

Spherical

Singulisphaera

912 Family I. Planctomycetaceae

Genus VIII. Singulisphaera

913

List of species of the genus Schlesneria 1. Schlesneria paludicola Kulichevskaya, Ivanova, Belova, Baulina, Bodelier, Rijpstra, Sinninghe Damsté, Zavarzin and Dedysh 2007, 2686VP pa.lu.di.co¢la. L. n. palus, -udis a marsh, bog; L. suff. cola (from L. n. incola) inhabitant, dweller; N.L. n. paludicola a bog-dweller. Description as for the genus with the following additional information. Colonies are circular, smooth, raised, opaque, uncolored, 1–3 mm in diameter. Carbon sources (0.05%, w/v) include glucose, N-acetylglucosamine, cellobiose, maltose, sucrose, trehalose, fucose, and salicin. Capable of hydrolyzing fucoidan, laminarin, esculin, pectin, chondroitin sulfate, pullulan, gelatin, and xylan. Nitrogen sources (0.05%, w/v)

are ammonia, nitrate, N-acetylglucosamine, Bacto peptone, Bacto Yeast Extract, alanine, aspartate, glutamine, and threonine. Growth factors are not required, but yeast extract slightly increases the growth rate. Members of this species are resistant to ampicillin, streptomycin, chloramphenicol, lincomycin, and novobiocin, but are sensitive to neomycin, kanamycin, and gentamicin. Optimal growth occurs at pH 5.0–6.2 and at temperatures 15 – 26°C. NaCl inhibits growth at concentrations above 0.5% (w/v). DNA G+C content (mol%): 54.4–56.5 (Tm). Type strain: MPL7, ATCC BAA-1393, VKM 2452. Sequence accession no. (16S rRNA gene): AM162407.

Genus VIII. Singulisphaera Kulichevskaya, Ivanova, Baulina, Bodelier, Sinninghe Damsté and Dedysh 2008, 1191VP Svetlana N. Dedysh and Irina S. Kulichevskaya Sin.gu.li.spha.e′ra. L. adj. singuli single, separate; L. fem. n. sphaera sphere; N.L. fem. n. Singulisphaera a single spherical cell.

Spherical cells, up to 3–4 mm in diameter, occurring singly, in pairs, or in shapeless aggregates. Crateriform structures are scattered over the whole cell surface. Encapsulated. Immobile. Reproduce by budding. Stalk-like structures are absent. Attach to surfaces by means of amorphous holdfast material. Gram-stain-negative. Chemoorganotrophic aerobes. Capable of growth in microaerobic conditions. Colonies are opaque and nonpigmented. Various carbohydrates or N-acetylglucosamine are the preferred substrates. No growth factors are required. Possess hydrolytic capabilities. Organic nitrogen compounds, ammonium, and some amino acids serve as nitrogen sources. Nitrate is not utilized. Catalase, oxidase, and urease positive. Dissimilatory nitrate reduction is negative. Mesophilic and moderately acidophilic bacteria growing in the pH range of 4.2–7.5, with an optimum at pH 5.0–6.2. Sensitive to NaCl. This genus is a member of the phylum Planctomycetes, order Planctomycetales, family Planctomycetaceae. Acidic wetlands are the main habitat. DNA G+C content (mol%): 57.8–59.9. Type species: Singulisphaera acidiphila Kulichevskaya, Ivanova, Baulina, Bodelier, Sinninghe Damsté and Dedysh 2008, 1191VP.

Further descriptive information A single species, Singulisphaera acidiphila, has been described based on characterization of four independent isolates (Kulichevskaya et  al., 2008). Mature cells of this species vary in size from 1.6 to 2.6 mm (Figure 188), but some cells in old cultures are up to 3–4 mm in diameter (Figure 188). No stable cell chains or filaments are present in cultures of Singulisphaera acidiphila. Examination of old cultures grown on agar plates shows the presence of an amorphous holdfast substance excreted from the cell poles (Figure 188). Thin sections show the pattern of cell compartmentalization typical for Isosphaera-like planctomycetes (Figure 188) (Fuerst, 2005; Giovannoni et  al., 1987b; Lindsay et al., 2001). The cell wall consists of two electron-dense layers separated by an electron-transparent layer; the total wall thickness is about 12 nm. Crateriform structures with a diameter of 25 nm are scattered over the whole cell surface. An enlarged view of the cross-section of one of these pit-like structures shows

that it represents a cell-wall invagination, which is connected to underlying paryphoplasm (Figure 188). The intracytoplasmic membrane compartmentalizes the cell to produce irregularly shaped membrane-bounded regions. The paryphoplasm in cells of Singulisphaera acidiphila is invaginated in a way to form a central region and numerous peripheral regions which are filled with an electron-dense substance. Nucleoid and electron-dense ribosome-like particles are located in the central part of the cell. On agar media, Singulisphaera acidiphila forms raised, opaque, uncolored, circular colonies with an entire edge and a smooth surface. One-month-old colonies are 1–4 mm in diameter. Members of the genus Singulisphaera are obligately aerobic chemoheterotrophs, however, they grow well in microaerobic conditions. Most sugars and N-acetylglucosamine are the preferred growth substrates, but good growth occurs also on media with various biopolymers. Organic acids are either not utilized or poorly utilized by some of the strains. Representatives of Singulisphaera acidiphila are moderately acidophilic (pH range of 4.2–7.5) and mesophilic (temperature range of 4–33°C). High sensitivity to NaCl reflects adaption of these bacteria to dilute environments such as ombrotrophic peat bogs. Growth inhibition of 50–80% occurs in the presence of NaCl in the medium at concentrations of 0.2–0.5% (w/v), and NaCl at concentrations above 0.5% (w/v) completely inhibits growth. Cells of Singulisphaera acidiphila contain menaquinone-6 (MK-6) as the predominant isoprenoid quinone. The major fatty acids are C16:0 (23.9–36.6%), C18:1 w9c (23.5–46.7%), and C18:2 w6,12c (13.8–25.6%); the latter is genus-characteristic. The neutral lipids are dominated by an n-C31:9 hydrocarbon; squalene, diplopterol, and 3-methyl-diplopterol are also present.

Enrichment and isolation procedures Enrichment strategy and the medium used for isolation procedure (M31) are the same as for members of the genus Schlesneria (above). The colonies appearing on the plates are subjected to examination by phase microscopy for the presence of spherical cells that occur singly or in shapeless aggregates.

914

Family I. Planctomycetaceae

FIGURE 188.  (a) Phase-contrast micrographs of cells of Singulisphaera acidiphila grown in liquid culture and (b) on agar medium; black arrows show amorphous holdfast substance excreted by the cells grown on agar medium. (c) Electron micrograph of an ultrathin section of a cell of Singulisphaera acidiphila; CW, cell wall; ICM, intracytoplasmic membrane; P, paryphoplasm; R, ribosome-like particles; N, nucleoid. Black arrow indicates pit-like invagination of a cell wall [enlarged view is shown in (d)]. Bars = 10 mm [(a) and (b)] and 0.2 mm [(c) and (d)]. [(c) and (d), Printed with permission of Olga I. Baulina.]

Maintenance procedures Strains can be maintained on agar medium M31 by subculturing once in 2–3 months. Alternatively, they can be kept in liquid medium M31 with 10% glycerol at −70°C or stored by lyophilization.

Differentiation of the genus Singulisphaera from other genera Table 176 gives characteristics of Singulisphaera that differentiate it from other aerobic budding bacteria that lack peptidoglycan. Spherical cell shape makes Singulisphaera different from Schlesneria, Planctomyces, Pirellula, Blastopirellula, and Rhodopirellula. Absence of cell filaments, pigmentation, and gliding motility differentiate Singulisphaera from Isosphaera. High sensitivity to NaCl distinguishes it from Pirellula, Blastopirellula, and ­Rhodopirellula.

The ability to grow at pH below 5.0 distinguishes it from all other currently known planctomycetes with the only exception being Schlesneria. Finally, the presence of C18:2 fatty acids is a genus-characteristic feature of Singulisphaera that is unique among other planctomycetes.

Taxonomic comments Comparative sequence analysis of the 16S rRNA gene shows that Singulisphaera acidiphila is a member of the order Planctomycetales and belongs to a phylogenetic lineage defined by the genus Isosphaera. The 16S rRNA gene sequence identity between the Isosphaera pallida (Giovannoni et al., 1987b) and representatives of the Singulisphaera acidiphila is about 90%. Four taxonomically described strains of Singulisphaera acidiphila were obtained from acidic Sphagnum-dominated boreal wetlands of northern Russia.

Genus VIII. Singulisphaera

915

List of species of the genus Singulisphaera 1. Singulisphaera acidiphila Kulichevskaya, Ivanova, Baulina, Bodelier, Sinninghe Damsté and Dedysh 2008, 1191VP a.ci.di¢phi.la. N.L. n. acidum acid from L. adj. acidus sour; N.L. adj. philus -a -um (from Gr. adj. philos -ê -on), friend, loving; N.L. fem. adj. acidiphila acid-loving. Description as for the genus with the following additional information. Carbon sources (0.05%, w/v) include glucose, fructose, galactose, lactose, cellobiose, maltose, mannose, melibiose, rhamnose, ribose, trehalose, saccharose, xylose, leucrose, N-acetylglucosamine, and salicin. Ability to utilize fucose, lactate, and pyruvate is variable. Capable of hydrolyzing laminarin, pectin, chondroitin sulfate, esculin, gelatin, pullulan, lichenan, and xylan. Shows the following enzyme activities: alkaline and acid phosphatase, esterase, esterase lipase, leucine arylamidase, cystine arylamidase, valine

References Anagnostidis, K. and R. Rathsack-Künzenbach. 1967. Isocystis pallida Blaualge oder hefeartiger Pilz. 29: 191–198. Bauer, M., T. Lombardot, H. Teeling, N.L. Ward, R.I. Amann and F.O. Glöckner. 2004. Archaea-like genes for C1-transfer enzymes in Planctomycetes: phylogenetic implications of their unexpected presence in this phylum. J. Mol. Evol. 59 : 571–586. Bauld, J. and J.T. Staley. 1976. Planctomyces maris sp. nov., marine isolate of Planctomyces blastocaulis group of budding bacteria. J. Gen. Microbiol. 97: 45–55. Bauld, J. and J.T. Staley. 1980. Planctomyces maris sp. nov., nom. rev. Int. J. Syst. Bacteriol. 30 : 657–657. Bomar, D., S. Giovannoni and E. Stackebrandt. 1988. A unique type of eubacterial 5S rRNA in members of the order Planctomycetales. J. Mol. Evol. 27: 121–125. Burdett, I.D. and R.G. Murray. 1974. Septum formation in Escherichia coli: characterization of septal structure and the effects of antibiotics on cell division. J. Bacteriol. 119 : 303–324. Butler, M.K., J. Wang, R.I. Webb and J.A. Fuerst. 2002. Molecular and ultrastructural confirmation of classification of ATCC 35122 as a strain of Pirellula staleyi. Int. J. Syst. Evol. Microbiol. 52 : 1663–1667. Butler, M.K. and J.A. Fuerst. 2004. Comparative analysis of ribonuclease P RNA of the Planctomycetes. Int. J. Syst. Evol. Microbiol. 54 : 1333–1344. Castenholz, R.W. 1988. Culturing methods (cyanobacteria). In Cyanobacteria: Methods in Enzymology, vol. 167 (edited by Packer and Glazer), pp. 68–93. Chistoserdova, L., C. Jenkins, M.G. Kalyuzhnaya, C.J. Marx, A. Lapidus, J.A. Vorholt, J.T. Staley and M.E. Lidstrom. 2004. The enigmatic planctomycetes may hold a key to the origins of methanogenesis and methylotrophy. Mol. Biol. Evol. 21 : 1234–1241. Chouari, R., D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach and A. Sghir. 2003. Molecular evidence for novel planctomycete diversity in a municipal wastewater treatment plant. Appl. Environ. Microbiol. 69 : 7354–7363. Claus, H., H.H. Martin, C.A. Jantos and H. Konig. 2000. A search for betalactamase in chlamydiae, mycoplasmas, planctomycetes, and cyanelles: bacteria and bacterial descendants at different phylogenetic positions and stages of cell wall development. Microbiol. Res. 155 : 1–6. Cohen-Bazire, G., W.R. Sistrom and R.Y. Stanier. 1957. Kinetic studies of pigment synthesis by non-sulfur purple bacteria. J. Cell Phys. 49  : 25–68. Dahlberg, C., M. Bergstrom and M. Hermansson. 1998. In situ detection of high levels of horizontal plasmid transfer in marine bacterial communities. Appl. Environ. Microbiol. 64 : 2670–2675.

arylamidase, phosphohydrolase, N-acetyl-b-glucosaminidase, and b-galactosidase. Nitrogen sources (0.05%, w/v) are ammonia, N-acetylglucosamine, Bacto Peptone, Bacto Yeast Extract, alanine, aspartate, arginine, glutamine, threonine, tryptophan, and glycine. Some strains can also utilize asparagine, isoleucine, lysine, phenylalanine, proline, and valine. Nitrate or nitrite are not utilized. Vitamins are not required. Resistant to ampicillin, streptomycin, chloramphenicol, lincomycin, kanamycin, and novobiocin, but sensitive to neomycin and gentamicin. Optimal growth occurs at pH 5.0–6.2 and at temperatures 20–26°C. NaCl inhibits growth at concentrations above 0.5% (w/v). DNA G+C content (mol%): 57.8–59.9 (Tm). Type strain: MOB10, ATCC BAA-1392, DSM 18658, VKM B-2454. Sequence accession no. (16S rRNA gene): AM850678.

Dedysh, S.N., T.A. Pankratov, S.E. Belova, I.S. Kulichevskaya and W. Liesack. 2006. Phylogenetic analysis and in situ identification of bacteria community composition in an acidic Sphagnum peat bog. Appl. Environ. Microbiol. 72 : 2110–2117. DeLong, E.F., D.G. Franks and A.I. Alldredge. 1993. Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages. Limnol. Oceanogr. 38 : 924–934. Euzéby, J.P. and T. Kudo. 2001. Corrigenda to the Validation Lists. Int. J. Syst. Evol. Microbiol. 51 : 1933–1938. Famurewa, O., H.G. Sonntag and P. Hirsch. 1983. Avirulence of 27 bacteria that are budding, prosthecate, or both. Int. J. Syst. Bacteriol. 33 : 565–572. Fott, B. and J. Komarek. 1960. Das Phytoplankton der Teiche im Teschner Schlesien. Preslia 32 : 113–141. Franzmann, P.D. and V.B.D. Skerman. 1981. Agitococcus lubricus gen. nov. sp. nov., a lipolytic, twitching coccus from fresh water. Int. J. Syst. Bacteriol. 31 : 177–183. Franzmann, P.D. and V.B. Skerman. 1984. Gemmata obscuriglobus, a new genus and species of the budding bacteria. Antonie van Leeuwenhoek 50 : 261–268. Franzmann, P.D. and V.B. Skerman. 1985. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 18. Int. J. Syst. Bacteriol. 35 : 375–376. Fuerst, J.A., S.K. Sambhi, J.L. Paynter, J.A. Hawkins and J.G. Atherton. 1991. Isolation of a bacterium resembling Pirellula species from primary tissue culture of the giant tiger prawn (Penaeus monodon). Appl. Environ. Microbiol. 57 : 3127–3134. Fuerst, J.A. and R.I. Webb. 1991. Membrane-bounded nucleoid in the eubacterium Gemmata obscuriglobus. Proc. Natl. Acad. Sci. U. S. A. 88 : 8184–8188. Fuerst, J.A., H.G. Gwilliam, M. Lindsay, A. Lichanska, C. Belcher, J.E. Vickers and P. Hugenholtz. 1997. Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon. Appl. Environ. Microbiol. 63 : 254–262. Fuerst, J.A. 2005. Intracellular compartmentation in Planctomycetes. Annu. Rev. Microbiol. 59  : 299–328. Gade, D., H. Schlesner, F.O. Glöckner, R. Amann, S. Pfeiffer and M. Thomm. 2004. Identification of Planctomycetes with order-, genus-, and strain-specific 16S rRNA-targeted probes. Microb. Ecol. 47 : 243–251. Gade, D., J. Gobom and R. Rabus. 2005a. Proteomic analysis of carbohydrate catabolism and regulation in the marine bacterium Rhodopirellula baltica. Proteomics 5 : 3672–3683.

916

Family I. Planctomycetaceae

Gade, D., T. Stührmann, R. Reinhardt and R. Rabus. 2005b. Growth phase dependent regulation of protein composition in Rhodopirellula baltica. Environ. Microbiol. 7 : 1074–1084. Gade, D., D. Theiss, D. Lange, E. Mirgorodskaya, T. Lombardot, F.O. Glockner, M. Kube, R. Reinhardt, R. Amann, H. Lehrach, R. Rabus and J. Gobom. 2005c. Towards the proteome of the marine bacterium Rhodopirellula baltica: mapping the soluble proteins. Proteomics 5 : 3654–3671. Gebers, R., U. Wehmeyer, T. Roggentin, H. Schlesner, J. Kölbel-Boelke and P. Hirsch. 1985. Deoxyribonucleic acid base compositions and nucleotide distributions of 65 strains of budding bacteria. Int. J. Syst. Bacteriol. 35 : 260–269. Geitler, L. 1963. Die angebliche Cyanophyceae Isosphaera pallida is ein hefeartiger Pilz. Arch. Mikrobiol. 46 : 238–242. Gimesi, N. 1924. Hydrobiologiai tanulmanyok (Hydrobiologische Studien). I. Planctomyces bekefii Gim. nov. gen. et sp. (in Hungarian, with German transl.). Kiadja a Magyar Ciszterci Rend, Budapest, pp. 1–8. Giovannoni, S.J., W. Godchaux, 3rd, E. Schabtach and R.W. Castenholz. 1987a. Cell wall and lipid composition of Isosphaera pallida, a budding Eubacterium from hot springs. J. Bacteriol. 169  : 2702–2707. Giovannoni, S.J., E. Schabtach and R.W. Castenholz. 1987b. Isosphaera pallida, gen. and comb. nov., a gliding, budding Eubacterium from hot springs. Arch. Microbiol. 147 : 276–284. Giovannoni, S.J., E. Shabtach and R.W. Castenholz. 1995. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 54. Int. J. Syst. Bacteriol. 45 : 619–620. Gliesche, C. 1980. Isolierung und Charakterisierung von Bakteriophagen knospender Bakterien (Isolation and characterization of bacteriophages of budding bacteria). Diploma thesis, ChristianAlbrechts-Universitaet, Kiel. Glöckner, F.O., B.M. Fuchs and R. Amann. 1999. Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization. Appl. Environ. Microbiol. 65 : 3721–3726. Glöckner, F.O., M. Kube, M. Bauer, H. Teeling, T. Lombardot, W. Ludwig, D. Gade, A. Beck, K. Borzym, K. Heitmann, R. Rabus, H. Schlesner, R. Amann and R. Reinhardt. 2003. Complete genome sequence of the marine planctomycete Pirellula sp. strain 1. Proc. Natl. Acad. Sci. U. S. A. 100 : 8298–8303. Granberg, K. 1969. Kasviplanktonin merkityksesta vesilaitoksen raakvaveden tarkkailussa. Limnol. Foren. I. Finland Limnol. Symp. 1968 : 34–43. Griepenburg, U., N. Ward-Rainey, S. Mohamed, H. Schlesner, H. Marxsen, F.A. Rainey, E. Stackebrandt and G. Auling. 1999. Phylogenetic diversity, polyamine pattern and DNA base composition of members of the order Planctomycetales. Int. J. Syst. Bacteriol. 49  : 689–696. Henrici, A.T. and D.E. Johnson. 1935. Studies of freshwater bacteria: II. Stalked bacteria, a new order of Schizomycetes. J. Bacteriol. 30 : 61–93. Heynig, H. 1961. Zur Kenntnis des Planktons mitteldeutscher Gewaesser. Arch. Protistenkd. 105 : 407–416. Hindák, F. 2001. Thermal micro-organisms from a hot spring on the coast of Lake Bogoria, Kenya. Nova Hedwigia, Beiheft 123 : 77–93. Hirsch, P. 1972. Two identical genera of budding and stalked bacteria: Planctomyces Gimesi 1924 and Blastocaulis Henrici and Johnson 1935. Int. J. Syst. Bacteriol. 22 : 107–111. Hirsch, P., M. Müller and H. Schlesner. 1977. New aquatic budding and prosthecate bacteria and their taxonomic position. In Symposium on Aquatic Microbiology, Lancaster, UK. Academic Press, London, pp. 107–133. Hirsch, P. and M. Müller. 1985. Planctomyces limnophilus sp. nov., a stalked and budding bacterium from freshwater. Syst. Appl. Microbiol. 6 : 276–280. Hirsch, P. and M. Müller. 1986. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 20. Int. J. Syst. Bacteriol. 36 : 354–356. Hortobágyi, T. 1965. Neue Planctomyces Arten. Tot. Koezlem. 52 : 111–119.

Hortobágyi, T. 1968. Planctomyces from Vietnam. Acta Phytopathol. Acad. Sci. Hung 3 : 271–273. Hortobágyi, T. 1980. Aquatic bacteria and fungi in Danube River and in the water producing systems of the Budapest Waterworks. Acta Microbiol. Acad. Sci. Hung. 27 : 259–268. Hortobágyi, T. and L. Hajdú. 1984. A critical survey of Planctomyces research. Acta Bot. Hung. 30 : 3–9. Jenkins, C. and J.A. Fuerst. 2001. Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu. J. Mol. Evol. 52 : 405–418. Kerger, B.D., C.A. Mancuso, P.D. Nichols, D.C. White, T. Langworthy, M. Sittig, H. Schlesner and P. Hirsch. 1988. The budding bacteria, Pirellula and Planctomyces, with atypical 16S rRNA and absence of peptidoglycan, show eubacterial phospholipids and uniquely high proportions of long chain beta-hydroxy fatty acids in the lipopolysaccharide lipid A. Arch. Microbiol. 149  : 255–260. Kodaka, H., A.Y. Armfield, G.L. Lombard and V.R. Dowell, Jr. 1982. Practical procedure for demonstrating bacterial flagella. J. Clin. Microbiol. 16 : 948–952. Kölbel-Boelke, J., R. Gebers and P. Hirsch. 1985. Genome size determination for 33 strains of budding bacteria. Int. J. Syst. Bacteriol. 35 : 270–273. Komárek, J. and K. Anagnostidis. 2005. Cyanoprokaryota 2. In Oscillatoriales. Elsevier/Spektrum, Munich, pp. 336–339. König, H., H. Schlesner and P. Hirsch. 1984. Cell wall studies on budding bacteria of the Planctomyces - Pasteuria group and on a Prosthecomicrobium sp. Arch. Microbiol. 138 : 200–205. Krasil’nikov, N.A. 1949. Guide to the bacteria and actinomycetes. In Akad. Nauk. S.S.S.R. Moscow, pp. 1–830. Kristiansen, J. 1971. On Planctomyces bekefii and its occurrence in Danish lakes and ponds. Bot. Tidsskr. 66 : 293–392. Kulichevskaya, I.S., T.A. Pankratov and S.N. Dedysh. 2006. Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation approaches. Microbiology (En. transl. from Mikrobiologiya) 75 : 329–335. Kulichevskaya, I.S., A.O. Ivanova, S.E. Belova, O.I. Baulina, P.L.E. Bodelier, W.I.C. Rijpstra, J.S. Sinninghe Damsté, G.A. Zavarzin and S.N. Dedysh. 2007. Schlesneria paludicola gen. nov., sp. nov., the first acidophilic member of the order Planctomycetales, from Sphagnum-dominated boreal wetlands. Int. J. Syst. Evol. Microbiol. 57 : 2680–2687. Kulichevskaya, I.S., A.O. Ivanova, O.I. Baulina, P.L.E. Bodelier, J.S. Sinninghe Damsté and S.N. Dedysh. 2008. Singulisphaera acidiphila gen. nov., sp. nov., a non-filamentous, Isosphaera-like planctomycete from acidic northern wetlands. Int. J. Syst. Evol. Microbiol. 58 : 1186–1193. Kustu, S., E. Santero, J. Keener, D. Popham and D. Weiss. 1989. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol. Rev. 53 : 367–376. Leary, B.A., N. Ward-Rainey and T.R. Hoover. 1998. Cloning and characterization of Planctomyces limnophilus rpoN : complementation of a Salmonella typhimurium rpoN mutant strain. Gene 221 : 151–157. Liesack, W., H. König, H. Schlesner and P. Hirsch. 1986. Chemical composition of the peptidoglycan-free cell envelopes of budding bacteria of the Pirella/Planctomyces group. Arch. Microbiol. 145 : 361–366. Liesack, W. and E. Stackebrandt. 1989. Evidence for unlinked rrn operons in the planctomycete Pirellula marina. J. Bacteriol. 171 : 5025–5030. Lindsay, M., R.I. Webb, H.M. Hosmer and J.A. Fuerst. 1995. Effects of fixative and buffer on morphology and ultrastrcuture of a freshwater planctomycete, Gemmata obscuriglobus. J. Microbiol. Methods 21 : 45–54. Lindsay, M.R., R.I. Webb and J.A. Fuerst. 1997. Pirellulosomes: a new type of membrane-bounded cell compartment in planctomycete bacteria of the genus Pirellula. Microbiology 143 : 739–748.

Genus VIII. Singulisphaera Lindsay, M.R., R.I. Webb, M. Strous, M.S. Jetten, M.K. Butler, R.J. Forde and J.A. Fuerst. 2001. Cell compartmentalisation in Planctomycetes: novel types of structural organisation for the bacterial cell. Arch. Microbiol. 175 : 413–429. Llobet-Brossa, E., R. Rossello-Mora and R. Amann. 1998. Microbial community composition of wadden sea sediments as revealed by fluorescence in situ hybridization. Appl. Environ. Microbiol. 64 : 2691–2696. Lyman, J. and R.H. Fleming. 1940. Composition of sea water. J. Mar. Res. 3 : 134–146. Majewski, D.M. 1985. Molekularbiologische Charakterisierung von Bakteriophagen knospender Bakterien (Molecular biological characterisation of bacteriophages of budding bacteria). Diploma thesis, Christian-Albrechts-Universitaet, Kiel. Menke, M.A.O.H., W. Liesack and E. Stackebrandt. 1991. Ribotyping of 16S and 23S rRNA genes and organization of rrn operonsin ­members of the bacterial genera Gemmata, Planctomyces, Thermotoga, Thermus and Verrucomicrobium. Arch. Microbiol. 155 : 263–271. Neef, A., R. Amann, H. Schlesner and K.H. Schleifer. 1998. Monitoring a widespread bacterial group: in situ detection of Planctomycetes with 16S rRNA-targeted probes. Microbiology 144 : 3257–3266. Olah, J. and L. Hajdu. 1973. Electron microscopic morphology of Planctomyces bekefii (sic) Gimesi. Arch. Hydrobiol. 71 : 271–275. Parra, O. 1972. Presencia del genero Planctomyces (Fungi Imperfecti Moniliales) en Chile. Bol. Soc. Arg. Bot. 14 : 282–284. Pearson, A., M. Budin and J.J. Brocks. 2003. Phylogenetic and biochemical evidence for sterol synthesis in the bacterium Gemmata obscuriglobus. Proc. Natl. Acad. Sci. U. S. A. 100 : 15352–15357. Rabus, R., D. Gade, R. Hellwig, M. Bauer, F.O. Glöckner, M. Kube, H. Schlesner, R. Reinhardt and R. Amann. 2002. Analysis of N-acetyl­ glucosamine metabolism in the marine bacterium Pirellula sp. strain 1 by a proteomic approach. Proteomics 2 : 649–655. Razumov, A.S. 1949. Gallionella kljasmensis sp. n. a bacterial component of the plankton (in Russian). Mikrobiologiya 18 : 442–446. Rönner, S., W. Liesack, J. Wolters and E. Stackebrandt. 1991. Cloning and sequencing of a large fragment of the atpD-gene of Pirellula marina : a contribution to the phylogeny of Planctomycetales. Endocytobios Cell Res. 7: 219–229. Schlesner, H. and P. Hirsch. 1984. Assignment of ATCC 27377 to Pirella gen. nov. as Pirella staleyi comb. nov. Int. J. Syst. Bacteriol. 34 : 492–495. Schlesner, H. 1986. Pirella marina sp. nov., a budding, peptidoglycanless bacterium from brackish water. Syst. Appl. Microbiol. 8 : 177–180. Schlesner, H. and E. Stackebrandt. 1986. Assignment of the genera Planctomyces and Pirella to a new family Planctomycetaceae fam. nov. and description of the order Planctomycetales ord. nov. Syst. Appl. Microbiol. 8 : 174–176. Schlesner, H. and P. Hirsch. 1987. Rejection of the genus name Pirella for pear-shaped budding bacteria and proposal to create the genus Pirellula gen. nov. Int. J. Syst. Bacteriol. 37 : 441–441. Schlesner, H. and E. Stackebrandt. 1987. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 23. Int. J. Syst. Bacteriol. 37 : 179–180. Schlesner, H. 1989. Planctomyces brasiliensis sp. nov., a halotolerant bacterium from a salt pit. Syst. Appl. Microbiol. 12 : 159–161. Schlesner, H. 1990. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 32. Int. J. Syst. Bacteriol. 40 : 105–106. Schlesner, H. 1994. The development of media suitable for the microorganisms morphologically resembling Planctomyces spp., Pirellula spp., and other Planctomycetales from various aquatic habitats using dilute media. Syst. Appl. Microbiol. 17 : 135–145. Schlesner, H., C. Rensmann, B.J. Tindall, D. Gade, R. Rabus, S. Pfeiffer and P. Hirsch. 2004. Taxonomic heterogeneity within the Planctomycetales as derived by DNA–DNA hybridization, description of Rhodopirellula baltica gen. nov., sp. nov., transfer of Pirellula marina to the

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genus Blastopirellula gen. nov. as Blastopirellula marina comb. nov. and emended description of the genus Pirellula. Int. J. Syst. Evol. Microbiol. 54 : 1567–1580. Schmidt, J.M. 1978. Isolation and ultrastructure of freshwater strains of Planctomyces. Curr. Microbiol. 1 : 65–70. Schmidt, J.M. and M.P. Starr. 1978. Morphological diversity of freshwater bacteria belonging to the Blastocaulis–Planctomyces group as observed in natural populations and enrichments. Curr. Microbiol. 1 : 325–330. Schmidt, J.M. and M.P. Starr. 1979a. Morphotype V of the Blastocaulis– Planctomyces group of budding and appendaged bacteria: Planctomyces guttaeformis Hortobágyi (sensu Hajdu). Curr. Microbiol. 2 : 195–200. Schmidt, J.M. and M.P. Starr. 1979b. Corniculate cell surface protrusions in morphotype II of the Blastocaulis–Planctomyces group of budding and appendaged bacteria. Curr. Microbiol. 3 : 187–190. Schmidt, J.M. and M.P. Starr. 1980a. Some ultrastructural features of Planctomyces bekefii, morphotype I of the Blastocaulis–Planctomyces group of budding and appendaged bacteria. Curr. Microbiol. 4 : 189–194. Schmidt, J.M. and M.P. Starr. 1980b. Current sightings, at the respective type localities and elsewhere, of Planctomyces bekefii Gimesi 1924 and Blastocaulis sphaerica Henrici and Johnson 1935. Curr. Microbiol. 4 : 183–188. Schmidt, J.M., W.P. Sharp and M.P. Starr. 1981. Manganese and iron encrustations and other features of Planctomyces crassus Hortobágyi 1965, morphotype Ib of the Blastocaulis–Planctomyces group of budding and appendaged bacteria, examined by electron microscopy and X-ray micro-analysis. Curr. Microbiol. 5 : 241–246. Schmidt, J.M. and M.P. Starr. 1981. The Blastocaulis–Planctomyces group of budding and appendaged bacteria. In The Prokaryotes: a Handbook on Habitats, Isolation, and Identification of Bacteria, vol. 1 (edited by Starr, Stolp, Trüper, Balows and Schlegel). Springer, New York, pp. 496–504. Schmidt, J.M., W.P. Sharp and M.P. Starr. 1982. Metallic-oxide encrustations of the nonprosthecate stalks of naturally occurring populations of Planctomyces bekefii. Curr. Microbiol. 7 : 389–394. Schmidt, J.M. and M.P. Starr. 1982. Ultrastructural features of budding cells in a prokaryote belonging to morphotype IV of the Blastocaulis– Planctomyces group. Curr. Microbiol. 7 : 7–11. Sittig, M. and H. Schlesner. 1993. Chemotaxonomic investigation of various prosthecate and or budding bacteria. Syst. Appl. Microbiol. 16 : 92–103. Skerman, V.B. 1968. A new type of micromanipulator and microforge. J. Gen. Microbiol. 54 : 287–297. Skuja, H. 1964. Grundzuege der Algenflora und Algenvegetation der Fjeldgegenden um Abisko in Schwedisch-Lappland. Nova Acta Reg. Soc. Sci. Upsal. Ser. IV 18 : 1–139. Stackebrandt, E., W. Ludwig, W. Schubert, F. Klink, H. Schlesner, T. Roggentin and P. Hirsch. 1984. Molecular genetic evidence for early evolutionary origin of budding peptidoglycan-less eubacteria. Nature 307 : 735–737. Stackebrandt, E., U. Wehmeyer and W. Liesack. 1986. 16S Ribosomal RNA and cell wall analysis of Gemmata obscuriglobus, a new member of the order Planctomycetales. FEMS Microbiol. Lett. 37 : 289–292. Staley, J.T. 1968. Prosthecomicrobium and Ancalomicrobium : new prosthecate freshwater bacteria. J. Bacteriol. 95 : 1921–1942. Staley, J.T. 1973. Budding bacteria of the Pasteuria–Blastobacter group. Can. J. Microbiol. 19  : 609–614. Staley, J.T., K.C. Marshall and V.B.D. Skerman. 1980. Budding and prosthecate bacteria from freshwater habitats of various trophic states. Microb. Ecol. 5 : 245–251. Staley, J.T. 1981a. The genera Prosthecomicrobium and Ancalomicrobium. In The Prokaryotes: a Handbook on Habitats, Isolation, and Identification of Bacteria (edited by Starr, Stolp, Trüper, Balows and Schlegel). Springer, New York, pp. 456–460.

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Order II. “Candidatus Brocadiales”

Staley, J.T. 1981b. The genus Pasteuria. In The Prokaryotes: a Handbook on Habitats, Isolation, and Identification of Bacteria (edited by Starr, Stolp, Trüper, Balows and Schlegel). Springer, New York, pp. 490–492. Staley, J.T., J.A. Fuerst, S. Giovannoni and H. Schlesner. 1992. The order Planctomycetales and the genera Planctomyces, Pirellula, Gemmata and Isosphaera. In The Prokaryotes: a Handbook on the Biology of Bacteria : Ecophysiology, Isolation, Identification, Applications, 2nd edn, vol. 4 (edited by Balows, Trüper, Dworkin, Harder and Schleifer). Springer, New York, pp. 3710–3731. Starr, M.P., R.M. Sayre and J.M. Schmidt. 1983. Assignment of ATCC 27377 to Planctomyces staleyi sp. nov. and conservation of Pasteuria ramosa Metchnikoff 1888 on the basis of type descriptive material: Request for an Opinion. Int. J. Syst. Bacteriol. 33 : 666–671. Starr, M.P. and J.M. Schmidt. 1984. Planctomyces stranskae (ex Wawrik 1952) sp. nov., nom. rev. and Planctomyces guttaeformis (ex Hortobágyi 1965) sp. nov., nom. rev. Int. J. Syst. Bacteriol. 34 : 470–477. Starr, M.P. and J.M. Schmidt. 1989. Genus Planctomyces Gimesi 1924. In Bergey’s Manual of Systematic Bacteriology, vol. 3 (edited by Staley, Bryant, Pfennig and Holt). Williams & Wilkins, Baltimore, pp. 1946–1958. Teiling, E. 1942. Schwedische Planktonalgen. 3. Neue oder wenig bekannte Formen. Bot. Not. 1942 : 63–68. Tekniepe, B.L., J.M. Schmidt and M.P. Starr. 1981. Life cycle of a budding and appendaged bacterium belonging to morphotype IV of the Blastocaulis–Planctomyces group. Curr. Microbiol. 5 : 1–6. Tell, G. 1975. Presencia de Planctomyces bekefii (Fungi Imperfecti, Moniliales) en la Argentina. Physis (B. Aires) 34 : 71. Van Ert, M. and J.T. Staley. 1971. Gas-vacuolated strains of Microcyclus aquaticus. J. Bacteriol. 108 : 236–240.

Wang, J., C. Jenkins, R.I. Webb and J.A. Fuerst. 2002. Isolation of Gemmatalike and Isosphaera-like planctomycete bacteria from soil and freshwater. Appl. Environ. Microbiol. 68 : 417–422. Ward-Rainey, N. 1996. Genetic diversity in members of the order Planctomycetales. PhD thesis, University of Warwick, Coventry. Ward-Rainey, N., F.A. Rainey, E.M. Wellington and E. Stackebrandt. 1996. Physical map of the genome of Planctomyces limnophilus, a representative of the phylogenetically distinct planctomycete lineage. J. Bacteriol. 178 : 1908–1913. Ward-Rainey, N., F.A. Rainey and E. Stackebrandt. 1997. The presence of a dnaK (HSP70) multigene family in members of the orders Planctomycetales and Verrucomicrobiales. J. Bacteriol. 179  : 6360–6366. Ward, N., F.A. Rainey, E. Stackebrandt and H. Schlesner. 1995. Unraveling the extent of diversity within the order Planctomycetales. Appl. Environ. Microbiol. 61 : 2270–2275. Ward, N.L., F.A. Rainey, B.P. Hedlund, J.T. Staley, W. Ludwig and E. Stackebrandt. 2000. Comparative phylogenetic analyses of members of the order Planctomycetales and the division Verrucomicrobia : 23S rRNA gene sequence analysis supports the 16S rRNA gene sequencederived phylogeny. Int. J. Syst. Evol. Microbiol. 50 : 1965–1972. Wawrik, F. 1952. Planctomyces-Studien. Sydowia Ann. Mycol. Ser. II 6 : 443–451. Wawrik, F. 1956. Neue Planktonorganismen aus Waldviertler Fischteichen. Oesterr. Bot. Z. 103 : 291–299. Weisburg, W.G., T.P. Hatch and C.R. Woese. 1986. Eubacterial origin of Chlamydiae. J. Bacteriol. 167 : 570–574. Woronichin, N.N. 1927. Materiali k agologitscheskoj flore i rastitjelnosti mineralnich istotchnikov gruppie Kaukaskich mineralnich wod. Trav. Inst. Balneol. aux Eaux Miner du Caucase 5 : 90–91. Zavarzin, G.A. 1961. Budding bacteria. Mikrobiologiya 30 : 774–791.

Order II. “Candidatus Brocadiales” ord. nov. Mike S.M. Jetten, Huub J.M. Op den Camp, J. Gijs Kuenen and Marc Strous Bro.ca.di.a¢les. N.L. fem. n. “Candidatus Brocadia” type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Brocadiales the order of “Candidatus Brocadia”. The description is the same as for the family “Candidatus Brocadiaceae”. Type genus: “Candidatus Brocadia” Strous et  al. 1999; Kuenen and Jetten 2001.

References Kuenen, J.G. and M.S.M. Jetten. 2001. Extraordinary anaerobic ammonium-oxidizing bacteria. ASM News 67: 456–463. Strous, M., J.A. Fuerst, E.H.M. Kramer, S. Logemann, G. Muyzer, K.T. van de Pas-Schoonen, R. Webb, J.G. Kuenen and M.S.M. Jetten. 1999. Missing lithotroph identified as new planctomycete. Nature 400 : 446–449.

Family I. “Candidatus Brocadiaceae” fam. nov. Mike S.M. Jetten, Huub J.M. Op den Camp, J. Gijs Kuenen and Marc Strous Bro.ca.di.a.ce¢ae. N.L. fem. n. [Candidatus] Brocadia type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Brocadiaceae the family of “Candidatus Brocadia”. Coccoid cells, generally 0.7–1.1 × 1.1–1.3 mm. Depending on the growth conditions, anammox cells occur singly or in aggregates. Phenomenologically “Gram-stain-negative”. No endospores are formed. Ultrastructure similar to that of the Planctomycetales, with typical membrane-bound riboplasm and paryphoplasm. An internal organelle (the anammoxosome), the locus of anammox metabolism, is present in the cytoplasm.

Nonmotile. Obligately anaerobic. Facultatively chemolithoautotrophic on ammonium and nitrite. Nitrite (which is converted to nitrate) is the electron donor for autotrophic CO2-fixation. Electron transport is cytochrome-based with nitrate, nitrite, Mn(IV), or Fe(III) as terminal electron acceptor. Hydrazine is produced from ammonium and hydroxylamine. Nitrate: nitrite oxidoreductase activity is present. Catalase- and hydrogen

Family I. “Candidatus Brocadiaceae”

peroxidase-positive. Media supporting an autotrophic lifestyle, containing ammonium, nitrite, and bicarbonate, are required. Optimum growth temperature for enriched species 15–40°C. Very slow growth with doubling times of 2–3 weeks. Type genus: “Candidatus Brocadia” Strous et al. 1999a; Kuenen and Jetten 2001.

Taxonomic comments The family “Candidatus Brocadiaceae” contains the bacteria responsible for anaerobic oxidation of ammonium (anammox). So far all anammox bacteria identified on the basis of 16S rRNA gene sequencing comprise a monophyletic cluster within the Planctomycetes lineage of descent ( Jetten et al., 2005b; Quan et al., 2008; Schmid et al., 2005; Woebken et al., 2008). Although knowledge on the microbiology of the anammox bacteria is steadily increasing, at this point the information is still too limited to describe the five recognized genera of the family “Candidatus Brocadiaceae” separately (Strous and Jetten, 2004). As isolation of pure cultures using traditional methods has so far not been successful, all type species have the Candidatus status (Kartal et  al., 2007b, 2008; Kuenen and Jetten, 2001; Quan et al., 2008; Schmid et al., 2001, 2003; Strous et al., 1999a; Woebken et  al., 2008). Highly purified cultures have been obtained by physical separation. The genome of one of the anammox bacteria “Candidatus Kuenenia stuttgartiensis” has been sequenced (Strous et al., 2006).

Further descriptive information Phylogeny and detection.  Comparative 16S rRNA gene sequence analysis shows more than 15% sequence difference between the anammox bacteria and cultivated members of the

Planctomycetales (Figure 189). The similarity of the five anammox genera is well below the 97% threshold value typically used to separate species. The deep monophyletic branching of the anammox bacteria has been confirmed by analysis of large datasets of concatenated ribosomal proteins and genes (Strous et  al., 2006). Since the anammox bacteria have several features in common with members of the order Planctomycetales, notably its compartmentalized ultrastructure, it is not surprising that the oligonucleotide probe S-P-Planc-0046-a-A-18 (Table 177; Neef et al., 1998) also hybridizes with all five genera of anammox bacteria. Most of the initial probes designed for anammox bacteria targeted only “Candidatus Brocadia anammoxidans” (Table 177; Schmid et al., 2005; Strous et al., 1999a). However, probe S-*-Amx-0820-a-A-22 also hybridized with “Candidatus Kuenenia stuttgartiensis”. “Candidatus Kuenenia” and “Candidatus Brocadia” can be distinguished by fluorescence in situ hybridization (FISH) microscopy using the probes S-S-Kst-0157-a-A-18 and S-S-Ban-0162-a-A-18 (Table 177; Schmid et al., 2005). Probe S-*-Kst-1275-a-A-20 (Table 177) is specific for “Candidatus Kuenenia stuttgartiensis”. The genus “Candidatus Scalindua” can be detected by probes S-GSca-1309-a-A-21, S-*-Scabr-1114-a-A-22 and S-*-BS-820-a-A-22 (Table 177). Specific probes for the genera “Candidatus Anammoxoglobus” and “Candidatus Jettenia” have recently become available (Kartal et al., 2007b; Quan et al., 2008). The 23S rRNA targeting probe L-*-Amx-1900-a-A-21 was designed to detect “Candidatus Brocadia” and “Candidatus Kuenenia” (Schmid et al., 2001). Recently the intergenic spacer and large parts of the 23S ribosomal gene of the five different anammox genera have become available to design new probes (Quan et al., 2008; Woebken et al., 2008).

Brocadia anammoxidans, AF375994

95

Brocadia fulgida, DQ459989

93

Anammoxoglobus propionicus, DQ317601

99 100 100

Jettenia asiatica, DQ301513 Kuenenia stuttgartiensis, AF375995 Scalindua wagneri, AY254882 Scalindua brodae, AY254883

100 100

Scalindua sorokinii, AY257181

Rhodopirellula baltica, RBr03 Planctomyces limnophilus, X62911 Gemmata obscuriglobus, AJ231191

69 79

919

Isosphaera pallida, AJ231195

0.02

Figure 189.  Phylogenetic affiliation of the Brocadiales to the Planctomycetales, based on 16S rRNA gene analysis.

The evolutionary history was inferred using the neighbor-joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 0.93 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al., 2004) and are expressed as the number of base substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (pairwise deletion option). There were a total of 1633 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007). Accession numbers are given after the species names.

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Family I. “Candidatus Brocadiaceae”

Table 177.  Phenotypic characteristics of members of the order “Candidatus Brocadiales”

Characteristic Gram stain Motility Catalase Oxidase Nitrate reduction Nitrite, gas N2H4 production HZO gene Anammoxosome Ladderane lipids Salt tolerance Phosphate tolerance Oxidation of organic acids: Formate Acetate Propionate Oxidation of methylamine Oxidation of hydrogen Hybridization in FISH: Probe AMX368 Probe AMX820 Probe KST1275 Probe BS820

Brocadia

Anammoxoglobus

Jettenia

Kuenenia

Scalindua

− − + + + + + + + + − −

− − + + + + + + + + nd nd

− − nd nd nd + nd + + nd nd nd

− − + + + + + + + + + +

− − + + + + + + + + + nd

+ ++ + + −

++ ++ ++ nd nd

nd nd nd nd nd

+ + + nd −

+ + − nd −

+ + − −

+ − − −

+ − − −

+ + + −

+ − − +

Symbols: +, greater than 90% of strains positive; ++, high activity; −, less than 10% positive; nd, not determined.

Cell-wall composition.  Peptidoglycan appears to be absent and electron microscopy shows a visible proteinaceous S-layer (van Niftrik et al., 2008a, b). The absence of a complete peptidoglycan biosynthesis route was confirmed by genomic analysis of Kuenenia stuttgartiensis (Strous et al., 2006). Ultrastructure.  Like the cells of other planctomycetes, anammox bacteria possess the shared planctomycete cell plan involving a single-membrane-bounded compartment containing the nucleoid and ribosomes, as well as a paryphoplasm region surrounding the outer rim of the cell (Fuerst, 2005). Within the riboplasm, however, members of the family “Candidatus Brocadiaceae” possess another single membranebounded compartment, the anammoxosome, unique to these bacteria. The anammoxosome harbors at least one specialized enzyme, a hydrazine/hydroxylamine oxidoreductase functional in the mechanism of anaerobic ammonium oxidation (Lindsay et al., 2001; Schalk et al., 2000; Shimamura et al., 2007, 2008; van Niftrik et  al., 2008a, b). The anammoxosome is wrapped in a single-membrane envelope possessing unique cyclobutanecontaining ladderane lipids which may confer relatively high density and reduced permeability to the anammoxosome membrane. Ladderane lipids, fatty acids, and sterols.  The main components in the membrane lipids of anammox bacteria were shown to be so-called “ladderane lipids” (Damste et  al., 2002, 2005). These lipids are comprised of three-to-five linearly concatenated cyclobutane moieties with cis ring junctions, which occur as fatty acids, fatty alcohols, alkyl glycerol monoethers, dialkyl glycerol diethers, and alkyl acyl glycerol ether/esters (Damste et  al., 2005). The internal membrane surrounding the anammoxosome organelle is nearly exclusively composed of ladderane

lipids as a barrier against diffusion of protons and intermediates (van Niftrik et  al., 2004). The most abundant fatty acids of anammox bacteria are 14-methylpentadecanoic acid (C16), 10-methylhexadecanoic acid, and 9,14-dimethylpentadecanoic acid. In addition, the anammox bacteria also contain squalene, and a number of hopanoids (hop-22[29]-ene, diplopterol, 17b,21b(H)-bis-homohopanoic acid, 17b,21b(H)-32-hydroxytrishomohopanoic acid, 22,29,30-trisnor-21-oxo-hopane; Damste et al., 2004). Recently the intact phospholipids of anammox bacteria containing phosphoethanolamine and phosphocholine headgroups have been investigated and compared to each other (Boumann et al., 2006; Rattray, 2008). Nutrition and growth conditions.  A medium sustaining a chemolithoautotrophic lifestyle on ammonium, nitrite, and carbon dioxide in the absence of oxygen is required for growth of anammox bacteria ( Jetten et al., 2005a; van de Graaf et al., 1995, 1996). Furthermore, a continuous flow reactor set up with very efficient biomass retention is required to maintain optimal conditions for (exponential) growth (Strous et al., 1998, 2002; van der Star, 2008a). A sequencing fed-batch reactor has been proven to be very suitable for the enrichment and maintenance of a growing anammox culture. Recently, also a membrane reactor was described to produce high quality single cells (van der Star, 2008b). The basic mineral medium contains (in g/l): KHCO3, 1.25; NaH2PO4, 0.05; CaCl2·2H2O, 0.3; MgSO4·7H2O, 0.2; FeSO4, 0.00625; EDTA, 0.00625; trace element solution, 1.25 ml/l. The trace element solution contains (g/l): EDTA, 15; ZnSO4·7H2O, 0.43; CoCl2·6H2O, 0.24; MnCl2·4H2O, 1.0; CuSO4·H2O, 0.25; NaMoO4·2H2O, 0.22; NiCl2·6H2O, 0.19; NaSeO4·10H2O, 0.21; H3BO4, 0.014; NaWO4·2H2O, 0.050. For the enrichment of

Family I. “Candidatus Brocadiaceae”

marine “Candidatus Scalindua” species an appropriate seawater medium should be composed (Kartal et al., 2006; Nakajima et al., 2008; van de Vossenberg, 2008; Windey et al., 2005). The genus “Candidatus Anammoxoglobus” and “Candidatus Brocadia fulgida” obtain a competitive advantage when using propionate and acetate, as an additional energy source, respectively. Thus propionate or acetate can be added to the ammonium-nitrite enrichment medium in a C/N ratio of 1/6 (Guven et al., 2005; Kartal et al., 2007b, 2008). The medium concentrations of NaNO2, (NH4)2SO4, and NaNO3 are initially set to 5, 5, and 10 mM each. Suitable starting material for the enrichment of anammox bacteria is activated sludge from a treatment plant with a long sludge age, or anoxic sediments which receive a continuous supply of ammonium and nitrate (Dapena-Mora et al., 2004; Fujii et al., 2002; Kartal et al., 2007b, 2008; Schmid et al., 2000, 2003; Toh et al., 2002; van de Vossenberg, 2008; van der Star et al., 2007; Zhang et al., 2007). The biomass concentration in the enrichment cultures should stay above 1 g dry wt per liter to prevent loss of activity. The inflowing ammonium and nitrite concentrations can be increased gradually (to a maximum of 45 mM each) as long as the nitrite is completely consumed. Successful enrichments often have a reddish, pink, or brownish appearance due to the high cytochrome content of the anammox bacteria. In these enrichments anammox bacteria comprise about 80–85% of the community as determined by specific oligonucleotide probes using FISH. Purified cells can be obtained via physical separation using density centrifugation (Kartal et  al., 2007a, 2008; Strous et al., 1999a, 2002). Antibiotic sensitivity.  Penicillin G can be added to the media at 0.1 mg/ml to suppress growth of undesirable contaminants (van de Graaf et al., 1995). Whether the insensitivity of anammox bacteria to Penicillin G is caused by lack of peptidoglycan or the growth in aggregates remains to be established. Ecology.  Anammox bacteria were first discovered in wastewater treatment systems and most successful enrichments have used inocula from such environments ( Jetten et  al., 2005a, b; Schmid et  al., 2000, 2003). However, it is now clear that the occurrence of anammox bacteria is practically ubiquitous. Many studies have detected the presence and activity of anammox bacteria in more than 30 natural freshwater and marine ecosystems all over the world (Francis et  al., 2007; Op den Camp et al., 2006; Penton et al., 2006; Tsushima et al., 2007). The detection and quantification of the anammox bacteria in these ecosystems was based on a combination of different techniques: enrichment cultures, nutrient profiles, 15N labeling incubations with sediments or water samples, ladderane lipid analysis, FISH microscopy, or 16S rRNA gene sequencing (Dalsgaard et al., 2005; Egli et al., 2001; Kuypers et al., 2003, 2005; Pynaert et al., 2003; Rich et al., 2008; Risgaard-Petersen et  al., 2004; Rysgaard and Glud, 2004; Schubert et  al., 2006; Tal et al., 2005; Thamdrup and Dalsgaard, 2002). As the anammox bacteria require the simultaneous presence of ammonium and nitrite for growth, they are typical interface organisms, thriving in sediments, biofilms, and stratified waterbodies. In anoxic sediments with low organic carbon content, the anammox bacteria can account for 20–79% of total N2 production. Studies in the anoxic water columns of the Black Sea and the Golfe Dulce showed that anammox bacteria were responsible

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for 20–50% of the total N2 production (Dalsgaard et al., 2003; Kuypers et al., 2003; Thamdrup et al., 2004). Anammox bacteria are also mainly responsible for nitrogen loss in the oxygen minimum zone (OMZ) that are most productive regions of the world oceans (Hamersley et al., 2007; Kuypers et al., 2005; Thamdrup et  al., 2006). The strong N-deficit in the OMZ was until now attributed to denitrification, but recent studies showed unequivocally that the anammox bacteria are responsible for the majority of nitrogen transformation in the OMZs. Based on these observations, it is likely that anammox also plays an important role in other OMZ waters and sediments of the ocean. In various ecosystems, anammox bacteria will be dependent on the activity of aerobic ammonium-oxidizing bacteria especially when the oxygen supply is limited for example in biofilms or aggregates (Schmidt et  al., 2002). In these oxygen-limited environments, the ammonium-oxidizing bacteria would oxidize ammonium to nitrite and keep the oxygen concentration low, while anammox bacteria would convert the produced nitrite and the remaining ammonium to dinitrogen gas (Strous et al., 1997; Third et  al., 2001, 2005). Such conditions have been established in many different man-made ecosystems (Schmidt et al., 2002). FISH analysis and activity measurements showed that aerobic as well as anammox bacteria were present and active, but aerobic nitrite oxidizers (i.e., Nitrobacter or Nitrospira) were not detected (Third et al., 2001, 2005). It seems likely that under these conditions anaerobic and aerobic ammonium oxidizers form a quite stable community. The cooperation of ammonium-oxidizing bacteria is not only relevant for wastewater treatment, but might play an important role in natural environments at the oxic/anoxic interface (Kindaichi et  al., 2007; Kuypers et al., 2005; Lam et al., 2007; Nielsen et al., 2005; Woebken et al., 2007). Ecophysiology.  The anammox bacteria have a relatively wellstudied ecophysiology (Strous et al., 1999b). The anammox bacteria grow very slowly. This is caused by a low maximum substrate conversion rate, rather than by a low biomass yield. The Ks values of anammox bacteria for ammonium and nitrite are below the detection level (