Microbial Fundamentals of Biotechnology [1 ed.] 3527306153, 9783527306152

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Deutsche Forschungsgemeinschaft Microbial Fundamentals of Biotechnology

Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright © 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

Deutsche Forschungsgemeinschaft

Microbial Fundamentals of Biotechnology Final report of the collaborative research centre 323, „Mikrobielle Grundlagen der Biotechnologie: Struktur, Biosynthese und Wirkung mikrobieller Stoffe“, 1986 – 1999 Edited by Volkmar Braun and Friedrich Götz Collaborative Research Centres

Deutsche Forschungsgemeinschaft Kennedyallee 40, D-53175 Bonn, Federal Republic of Germany Postal address: D-53175 Bonn Phone: ++49/228/885-1 Telefax: ++49/228/885-2777 E-Mail: (X.400): S = postmaster, P = dfg, A = d400, C = de E-Mail (Internet RFC 822): [email protected] Internet: http://www.dfg.de

This book was carefully produced. Nevertheless, editor, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Cover: Crystal structure of E.coli FhuA with bound rifamycin CGP 4832 as determined by Ferguson et al. Structure 9:707-16 (2001)

Library of Congress Card No.: applied for

A catalogue record for this book is available from the British Library.

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 WILEY-VCH Verlag GmbH, Weinheim (Federal Republic of Germany), 2001 Printed on acid-free and chlorine-free paper. All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form ± by photoprinting, microfilm, or any other means ± nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition: ProSatz Unger, Weinheim Printing: betz-druck gmbh, Darmstadt Bookbindung: J. Schåffer GmbH & Co. KG, Grçnstadt Printed in the Federal Republic of Germany

Contents

Antibiotics and Other Biologically Active Microbial Metabolites 1

1.1 1.2 1.3 1.4 1.5 1.6 2 2.1 2.2 2.3

3 3.1 3.2 3.3 3.4 3.5 3.6 3.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volkmar Braun and Friedrich Götz Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibiotic research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The unique features of microbial iron transport . . . . . . . . . . . . . . . . Transport of bacterial proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane components and membrane polarization . . . . . . . . . . . . Chemistry of microbial peptides and proteins . . . . . . . . . . . . . . . . . Summary of short-term projects of the collaborative research centre Screening for New Secondary Metabolites from Microorganisms Hans-Peter Fiedler and Hans Zähner Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening methods and novel compounds . . . . . . . . . . . . . . . . . . . . Increasing structural diversity by directed fermentations . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthesis of the Lantibiotics Epidermin and Gallidermin . . . . . Friedrich Götz and Günther Jung History of lantibiotics and lantibiotic research in Tübingen . . . . . . . Primary structure and proposed maturation of epidermin in staphylococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic organization and regulation of the epidermin genes . . . . . Isolation and characterization of genetically engineered gallidermin and epidermin analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of the epidermin immunity genes epiFEG . . . . . . . . . . . . . Inactivation and characterization of the epidermin leader peptidase EpiP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The flavoenzyme EpiD and formation of peptidyl-aminoenethiolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 3 3 6 9 10 12 14 16 16 18 41 45 47 52 52 55 56 59 66 72 74 V

Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

Contents 3.8

3.9

4 4.1 4.2 4.3 4.4 4.5

5 5.1 5.2 5.3 5.4 5.5 5.6

6

6.1 6.2 6.3 6.4

7

7.1 7.2 7.3

VI

Incorporation of d-alanine into S. aureus teichoic acids confers resistance to defensins, protegrins, and other antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fermentation of Lantibiotics Epidermin and Gallidermin . Uwe Theobald Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strains for gallidermin/epidermin production . . . . . . . . . . . . Disadvantages during gallidermin process development . . . Gallidermin – a lantibiotic and its way towards industrial production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83 86 88

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93 94 94

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95 99 100

Genetics of Nikkomycin Production in Streptomyces tendae Tü 901 Christiane Bormann Introduction: nikkomycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation of nikkomycin biosynthetic genes . . . . . . . . . . . . . . . . . . . Isolation of the nikkomycin gene cluster and expression in Streptomyces lividans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organization of the nikkomycin gene cluster . . . . . . . . . . . . . . . . . . Roles of the nik genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcriptional organization and regulation of the nik cluster . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

102

Glycosylated Antibiotics: Studies on Genes Involved in Deoxysugar Formation, Modification and Attachment, and their Use in Combinatorial Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andreas Bechthold Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cloning of the avilamycin, landomycin, urdamycin, and granaticin biosynthetic gene clusters . . . . . . . . . . . . . . . . . . . . . Organization of avilamycin, landomycin, urdamycin, and granaticin biosynthetic genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New genetically engineered natural compounds . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of the Biosynthesis of Glycopeptide Antibiotics: Basis for Creating New Structures by Combinatorial Biosynthesis . . . . . . . Stefan Pelzer and Wolfgang Wohlleben Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

102 104 108 109 110 120 121

124 124 127 128 132 136

139 139 141 149 149

Contents 8

8.1 8.2 8.3

Homologous Recombination and the Induction of the SOS-Response in Antibiotic Producing Streptomycetes Günther Muth and Wolfgang Wohlleben Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutational analysis of the S. lividans recA gene . . . . . . Regulation of RecA activity . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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151 152 156 160

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Membrane Processes 9

9.1 9.2

9.3

9.4 9.5 9.6

10 10.1 10.2 10.3 10.4

11

11.1 11.2 11.3 11.4

Regulated Transport and Signal Transfer Channels involved in Bacterial Iron Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volkmar Braun and Helmut Killmann Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Fhu proteins catalyze active transport of ferrichrome and the antibiotic albomycin across the outer membrane and the cytoplasmic membrane of E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transduction of energy from the cytoplasmic membrane into the outer membrane for the activation of FhuA as a transporter and phage receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transport of ferrichrome across the cytoplasmic membrane . . . . . . . Ferric-carboxylate transport system of Morganella morganii Volkmar Braun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transport of ferric iron ions by the Sfu system of Serratia marcescens Volkmar Braun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Iron Transport in Gram-negative and Gram-positive Bacteria . Klaus Hantke Ferric iron transport in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . Ferrous-iron transport systems (Feo) of E. coli . . . . . . . . . . . . . . Regulation of iron transport and metabolism . . . . . . . . . . . . . . . An [2Fe-2S] protein is involved in ferrioxamine B utilization . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

163 163

165

175 178 181

182 183 183

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188 194 195 198 201

Regulation of the Ferric-Citrate Transport System by a Novel Transmembrane Transcription Control . . . . . . . . . . . . . . . . . . . . . . Volkmar Braun and Sabine Enz Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transport of Fe3+ is mediated by citrate . . . . . . . . . . . . . . . . . . . . . . Transcription initiation by a signaling cascade from the cell surface into the cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Iron regulation of fecIR and fecABCDE transcription . . . . . . . . . . . .

205 205 205 207 209 VII

Contents

12 12.1 12.2 12.3 12.4 12.5 12.6

13

13.1 13.2 13.3

14

14.1 14.2 14.3 14.4 14.5 14.6

15

15.1 15.2 15.3 15.4

VIII

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

209 211

Structure, Function, Import, and Immunity of Colicins . . . . . . . . . Volkmar Braun and Helmut Pilsl Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colicin M inhibits murein biosynthesis and thus displays a unique activity among the colicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colicins 5 and 10 are taken up by a novel mechanism . . . . . . . . . . . Colicins evolved by the exchange of DNA fragments which precisely defined functional domains . . . . . . . . . . . . . . . . . . . . . . . . Pore-forming colicins are inactivated by the cognate immunity proteins shortly before the formation of the transmembrane pores . Pesticin is a muramidase which is inactivated by the immunity protein in the periplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

212

Structure, Activity, Activation, and Secretion of the Serratia marcescens Hemolysin/Cytolysin . . . . . . . . . . . . . . . . . . . . . . . Volkmar Braun and Ralf Hertle Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of the S. marcescens hemolysin (ShlA) . . . . . . Pathogenicity of S. marcescens hemolysin/cytolysin . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

213 214 215 217 218 220 220

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222 224 231 235

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Staphylococcal Lipases: Molecular Characterization and Use as an Expression and Secretion System . . . . . . . . . . . . . . . . . . . . . . . . Friedrich Götz and Ralf Rosenstein Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular organization of staphylococcal lipases . . . . . . . . . . . . . . . Biochemical characterization of staphylococcal lipases . . . . . . . . . . Role of the pro-peptide region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The use of ShyL as expression and secretion system . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Multienzyme Complex Involved in Murein Synthesis of Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Moritz von Rechenberg, Waldemar Vollmer, and Joachim-Volker Höltje The murein sacculus, a “growing” molecule . . . . . . . . . . . . Murein growth is accompanied by massive turnover . . . . . . Enlargement and division of a stress bearing structure . . . . Interaction of murein hydrolases and synthases as indicated by affinity chromatography . . . . . . . . . . . . . . . .

212

238 238 239 241 244 244 246 246

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249 252 253

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Contents 15.5 15.6 15.7 15.8

Dimerization of the bifunctional transpeptidase/transglycosylase PBP1B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reconstitution of the core particle of a murein synthesizing machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proposed structure of a hypothetical holoenzyme of murein synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recent insights in the mechanism of growth of the murein sacculus reveal novel targets for antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The Changing Path of Hopanoid Research: From Condensing Lipids to New Membrane Enzymes . . . . . . . . . . . . . . . . . . . . . . Karl Poralla 16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2 The cyclization reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3 Purification of squalene cyclases . . . . . . . . . . . . . . . . . . . . . . . . . 16.4 Properties of purified cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.5 Cloning of squalene-hopene cyclases . . . . . . . . . . . . . . . . . . . . . 16.6 Properties of SHC sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.7 The structure of squalene-hopene cyclase . . . . . . . . . . . . . . . . . 16.8 Site directed mutagenesis of squalene-hopene cyclase . . . . . . . 16.9 Hopanoid biosynthesis gene clusters . . . . . . . . . . . . . . . . . . . . . 16.10 Miscellaneous results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.11 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

255 256 256 259 260

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263 265 266 267 270 271 272 274 278 279 280 280 281

17

Genetic and Biochemical Analysis of the Biosynthesis of the Orange Carotenoid Staphyloxanthin of Staphylococcus aureus . . Friedrich Götz 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.2 Cloning of the carotenoid biosynthetic genes from S. aureus Newman in S. carnosus and E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3 Function of CrtM and CrtN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.4 Identification of carotenoids in S. carnosus (pOC21), E. coli (pUG1), and S. aureus Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.5 Identification of dehydrosqualene in E. coli (pUG1) and E. coli (UG9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.6 Squalene is very likely no substrate for CrtN, the proposed dehydrosqualene desaturase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.7 The crt operon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.8 Homology of CrtO, CrtP, and CrtQ . . . . . . . . . . . . . . . . . . . . . . . . . . 17.9 Construction of crtM mutants of S. aureus strain Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.10 sB-regulated promoter of the crt operon from S. aureus strain Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

284 284 285 286 287 287 288 288 289 289 290 IX

Contents 17.11 The carotenoid biosynthesis genes . . . . . . . . . . . . . . . . . . . . . . . . . . 17.12 Function of the pigments in S. aureus strain Newman . . . . . . . . . . . 17.13 Distribution of pigment biosynthesis genes among staphylococcal species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

290 292

Second Messenger Systems in Paramecium . . . . . . . . . . . . . . . . . . Joachim E. Schultz and Jürgen Linder Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification and characterization of cGMP and cAMP second messenger signaling systems in Paramecium . . . . . . . . . . . . . . . . . . Biochemical properties of an adenylyl cyclase . . . . . . . . . . . . . . . . . A guanylyl cyclase disguised as an adenylyl cyclase . . . . . . . . . . . . On the way to an adenylyl cyclase with an intrinsic ion conductance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Downstream of second messengers . . . . . . . . . . . . . . . . . . . . . . . . . In vivo screening of bacterial secondary metabolites . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

295

18 18.1 18.2 18.3 18.4 18.5 18.6 18.7

292 293

295 296 300 302 306 308 311 312 312

Chemical Synthesis and Structure Elucidation 19

19.1 19.2 19.3 19.4

Structure Elucidation and Chemical Synthesis of Microbial Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roderich D. Süßmuth, Jörg Metzger, and Günther Jung Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure elucidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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319 320 325 337 339

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345 345 345 348 349 349 355 364 364 365 365

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Documentation 20 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9 20.10 X

Documentation of the Collaborative Research Centre 323 List of institutes involved . . . . . . . . . . . . . . . . . . . . . . . . . . . List of supported project areas . . . . . . . . . . . . . . . . . . . . . . Promotion of members of the collaborative research centre Recruitment of new project leaders . . . . . . . . . . . . . . . . . . . Alphabetical list of members and participants . . . . . . . . . . Support of young scientists . . . . . . . . . . . . . . . . . . . . . . . . . Alphabetical list of guests . . . . . . . . . . . . . . . . . . . . . . . . . . International cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . International conferences . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Preface

This book summarizes the scientific contributions of members of the collaborative research centre 323. Tübingen is one of the very few academic places worldwide where microbes have been systematically screened for biologically active metabolites. This research started in 1964 when the first chair of microbiology was created at the University of Tübingen and was led by Hans Zähner. Research on antibiotics in Tübingen continued in the 1980s, when most pharmaceutical companies had abandoned the development of new antibiotics as more than 100 antibiotics were already available to treat seemingly all relevant microbial infections. We now know that this decision was premature. Bacterial antibiotic resistance was already emerging and continued to progress at an increasing pace, resulting in multi-resistant pathogens which now can only be controlled by newly developed antibiotics. The collaborative research centre 323 was an ideal instrument for bringing together scientists of different disciplines and defining common interests. It consisted of chairs and groups of Microbiology/Biotechnology, Microbiology/Membrane Physiology, Microbial Genetics, Pharmaceutical Chemistry, Pharmaceutical Biology, and Organic Chemistry of the University and various groups of the Max-Planck Institutes of Developmental Biology (Biochemistry Department), and Infection Biology. The members of the collaborative research centre met regularly in seminars, which led to very successful co-operations in nearly all scientific projects. Striking examples include the lantibiotic research, the identification of new siderophores in pathogenic microorganisms, the screening/isolation/and subsequent structure elucidation of new antibiotics, and the synthesis of defined substrates for iron transport analysis or hemolysin function. The extremely fruitful cooperation between microbiologists and chemists is documented by co-authorship in numerous publications and is specified in more detail in the “Research Projects”. What is the secret of success of a collaborative research centre? There are several reasons why the scientific outcome of a collaborative research centre is usually more than the sum of the individual projects. First of all, the duration of a collaborative research centre is long-term. Our collaborative research centre 323 was designed to run for 15 years, which allowed the realization of long-term XI Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

Preface future-oriented projects. Research at universities is normally hampered by short-term grants and fellowships. The collaborative research centre enabled researchers to delve into scientific topics without the persistent fear of premature termination of the project, which would render the field open for competitors to “harvest the fruit”. Secondly, successful scientific cooperation is a matter of trust, competence, and bilateral benefit. At a single university or research institution, it is not easy to find a configuration that meets these prerequisites. A foundation of trust takes time to develop and is largely dependent on individuals. The environment of our collaborative research centre 323 facilitated and stimulated scientific cooperation. The importance of the collaborative research centre for our place at the frontier of science may be best illustrated by the recruitment of the scientific staff to meet the requirements of the collaborative research centre 323. It is no exaggeration to say that through the efforts of the members of the collaborative research centre 323, fundamental and important results were achieved in a number of areas, and many colleagues worldwide were influenced and stimulated by the achievements of the collaborative research centre. We are deeply indebted to the Deutsche Forschungsgemeinschaft for continuous support and to its reviewers for dedicated evaluation of the general concept of the collaborative research centre and the individual research projects. We would also like to thank the University of Tübingen and the Ministry for Science of Baden-Württemberg for their understanding and financial support. Volkmar Braun

XII

Antibiotics and Other Biologically Active Microbial Metabolites

Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

1 Introduction Volkmar Braun* and Friedrich Götz**

Abstract

A summary of the major scientific achievements in the antibiotic, the membrane-traffic, and chemistry projects of the collaborative research centre 323 from 1986 to 1999 will be presented in the Introduction, which is followed by a more detailed description of the research projects of 1995 to 1999.

1.1 Antibiotic research

Because of the threatening spread of multi-resistant pathogenic microorganisms, the search for and development of new antibiotics (anti-infective drugs) has become more compelling than ever before. Vancomycin-resistant Staphylococcus aureus strains have already been isolated in various countries, and one can foresee that we are facing an increased morbidity and mortality due to treatment failure. Therefore, the search for new anti-infectives and lead compounds and the development of new strategies will always be important. Antibiotics are considered secondary metabolites, which, at least under laboratory conditions, do not participate in the primary metabolism essential for microbial growth. Their role in the natural environment has always been an issue in the collaborative research centre. Antibiotics inhibit microorganisms com-

* Mikrobiologie/Membranphysiologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen ** Mikrobielle Genetik, Universität Tübingen, Waldhäuser Str. 70/8, D-72076 Tübingen

3 Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

1 Introduction peting for the same ecological niche. It was proposed that secondary metabolism also forms the basis for the evolution of new metabolic pathways. Through elucidation of the mode of action of various antibiotics, a large number of distinct metabolic activities of both prokaryotic and eukaryotic organisms were identified. Because of the beneficial activities of compounds related to antibiotics, such as siderophores, which deliver iron to microorganisms, antibiotics can no longer be regarded as secondary metabolites. Antibiotics with structures similar to those of iron complexes were found to be actively transported into target cells. This finding led to the concept of increasing the efficacy of antibiotics by linking them to actively transported compounds.

1.1.1 Screening and fermentation Antibiotic research in Tübingen involved the screening of many newly isolated microorganisms for novel antibiotics and production of the antibiotics in amounts sufficient for structure elucidation and utility testing. In the course of the collaborative research centre, a state-of-the-art fermentation technology was maintained, and new assay systems, and analytical and synthetic tools were developed. Metabolite production was increased by optimizing the fermentation conditions, aided by a sophisticated on-line analysis of the products released into the culture media. An extensive antibiotic database was created to differentiate new compounds from known compounds at an early stage of the investigation. The spectrum of products usually formed by the microorganisms was modified by directed fermentation and more recently by genetic means, such as mutation and combinatorial pathway recombination. The concept was not a large-scale screening in the sense that thousands of strains and compounds were tested per day. Rather, a selective screening was employed using special growth conditions, novel test systems, and various strains, some of which synthesized a number of different secondary metabolites. The screening was also not target-oriented, and an unbiased selection procedure was used. Some test systems were established within the collaborative research centre, and others were used in cooperation with research groups outside the collaborative research centre. Potential applications were focused not only on antibacterial compounds, antifungal products and therapeutic compounds were also considered. Apart from the biotechnological purpose of searching for new microbial metabolites, the wealth of compounds found demonstrated the extremely high metabolic potential of microorganisms. For example, in the original nikkomycinproducing Streptomyces strain, 14 nikkomycin variants were determined and mutagenesis resulted in 24 additional derivatives. The metabolic diversity of Streptomycetes along with their capability of differentiation is reflected by the size of their genome, which is at the upper limit of bacterial genomes. 4

1.1 Antibiotic research

1.1.2 Marketed compounds developed under the collaborative research centre Among the four commercially most interesting compounds developed under the collaborative research centre, only gallidermin and avilamycin are antibacterial compounds; the latter is employed as a food additive. Desferrioxamine B (trade name Desferral) is used to treat iron-overload diseases, phosphinothricin (trade name BASTA) is a herbicide, and the nikkomycins are potent inhibitors of chitin synthases and thus kill fungi, insects, and acarids without toxic side effects on mammals. Nikkomycin Z is currently in the second stage of clinical trial.

1.1.3 Lantibiotics The lantibiotic era in Tübingen began in 1985 with the determination of the structure of epidermin, a peptide antibiotic isolated from Staphylococcus epidermidis. Four research teams in the collaborative research centre worked to reveal this new class of natural products with a novel biosynthesis pathway. Tübingen, where the name “lantibiotic” was coined, has been a stronghold in this research area over the years. The functions of most of the proteins and enzymes involved in biosynthesis were elucidated, although the mechanism of the key reaction, lanthionine formation, is still unknown. Since chemical synthesis of large amounts of epidermin and gallidermin is currently impossible, great efforts were spent to improve the fermentation process, the scale up, and the downstream processing.

1.1.4 Glycosyl antibiotics and regulation of biosynthesis New derivatives of the glycosylated antibiotics avilamycin, landomycin, urdamycin, and granaticin were generated. The gene clusters involved in the biosynthesis of each of the antibiotics were analyzed, and the functions of most of genes were identified. Especially those genes involved in deoxy-sugar formation, modification, and attachment were used to create novel natural products. A series of new genetically engineered natural compounds were created by inactivation or overexpression of certain genes. Balhimycin is a glycopeptide with properties similar to those of vancomycin, which is often used to combat bacterial infections when no other antibiotic is effective. Balhimycin has the same heptapeptide core as vancomycin and dif5

1 Introduction fers in the glycosylation pattern. A cloning system for the producing strain was developed, and a number of genes were identified by using DNA probes based on consensus sequences of the typical biosynthetic enzymes, such as those encoding peptide synthetases and glycosyltransferases. Most of the balhimycin biosynthesis gene cluster was sequenced, and the function of a number of genes analyzed. The door is now open for the generation of hybrid antibiotics using the combinatorial biosynthesis strategy. Antibiotic production of mycelia-forming Streptomycetes is controlled by a complex regulatory network that allows the cells to sense different growth conditions and to react to these changes by producing antibiotics. Antibiotic production of Streptomyces coelicolor was shown to be affected by cell density, nutritional limitations, nutritional shiftdown, imbalance in metabolism, and different kinds of stress. The key player of the SOS response in Streptomyces lividans, RecA, was investigated.

1.1.5 Antibiotics and transport Membrane studies were important for gaining knowledge on the entry of antibiotics into cells, resistance via permeability barriers, and active export systems, and for identifying novel targets and altering the antibiotics to exert their detrimental function. One antibiotic studied is albomycin, which is actively taken up by cells through an iron siderophore (ferrichrome) transport system. Inside the target cell, the antibiotically active portion is cleaved off the carrier, which is released from the cells. Active transport reduces the minimal inhibitory concentration to the lowest value known for an antibiotic that kills Escherichia coli. Another example is phosphinothricyl-alanyl-alanine, which is transported into cells by an oligopeptide system; the antibiotic is released from the peptide carrier by intracellular proteases to generate phosphinothricin, which then inhibits glutamine synthetase. These findings prompted research by the collaborative research centre and by pharmaceutical companies aimed at developing antimicrobial compounds that are taken up by transporters.

1.2 The unique features of microbial iron transport

It was clear from the beginning that iron transport systems must have special features not shared by transport systems of any other nutrient. Under oxic conditions, iron occurs as the completely insoluble Fe3+. Since iron is an important 6

1.2 The unique features of microbial iron transport cofactor of redox enzymes, iron shortage must be overcome by microorganisms; they handle this by synthesizing iron-complexing compounds of low molecular weight, designated siderophores (originally called sideramines, siderochromes). The antibiotic-screening group of the collaborative research centre also had a long-standing interest in microbial iron complexes because sideromycins, potent antibiotics, belong to this class of compounds. Studies of the uptake of sideromycins, the intracellular metabolism, and their mode of action were essential for understanding antibiotic activity. In addition, it was clear that the iron supply must be carefully balanced since iron overload is toxic due to iron-catalyzed radical formation, which results in the destruction of DNA, proteins, and membrane lipids. Therefore, transport of iron-loaded siderophores and sideromycins and regulation of siderophore synthesis and transport were the focus of the iron projects.

1.2.1 Iron transport through the outer membrane of E. coli and other pathogenic bacteria Novel iron transport and regulatory mechanisms were expected and also found. Transport of Fe3+-siderophores was unique in several respects. Transport across the outer membrane of Gram-negative bacteria consumes energy, which is provided by the proton-motive force of the cytoplasmic membrane. Energy transfer from the cytoplasmic membrane to the outer membrane became an important research topic. A major breakthrough was the identification of the proteins involved in energy transfer: TonB, ExbB, and ExbD (Ton system). The Ton system was extensively characterized at molecular, biochemical, and structural levels. Seven E. coli K-12 Fe3+-siderophore transport systems were identified, and those of ferrichrome and ferric citrate were studied in detail. The receptors undergo conformational changes upon substrate binding and through interaction with the energized Ton system, as supported by the analysis of the crystal structure of the FhuA transporter. Upon binding of ferrichrome to FhuA close to the cell surface, a strong structural transition occurs. The long-range conformational change takes place across most of the molecule and the width of the outer membrane. The link to antibiotics is provided by FhuA, which serves as the active transporter of two antibiotics: albomycin is structurally related to ferrichrome; rifamycin CGP 4832 is structurally unrelated to ferrichrome. Surprisingly, both occupy the same position as ferrichrome on FhuA. Iron siderophores transporters homologous to the E. coli K-12 outer membrane transporters were identified in Yersinia enterocolitica and Morganella morganii. The ferrioxamine B transport system of the highly pathogenic Y. enterocolitica O8 strain explains the occurrence of yersiniosis upon treatment of patients suffering from iron overload with Deferral (mesylate salt of desferri-ferrioxamine B). In addition, a siderophore, designated yersiniabactin, was detected in the culture supernatants of highly pathogenic strains. Yersiniabactin 7

1 Introduction was isolated in amounts sufficient for determination of its novel structure. Furthermore, the genes of the entire heme transport system of Y. enterocolitica were cloned and sequenced, and functions were assigned to the encoded proteins. This was the first characterized heme transport system. Characterization of the iron transport systems of Serratia marcescens was initiated by the finding that transcription of the hemolysin genes is iron-regulated. These studies revealed a plethora of Fe3+-siderophore transport systems, one of which transports Fe3+ across the cytoplasmic membrane without involvement of a siderophore. Other research groups later related this system to the uptake of iron delivered by human transferrin to a variety of human pathogenic bacteria.

1.2.2 Iron transport through the cytoplasmic membrane Transport of Fe3+-siderophores, heme, and Fe3+ across the cytoplasmic membrane is catalyzed by ABC transporters, which consist of a periplasmic binding protein, one or two integral membrane proteins, and a cytoplasmic ATPase. ABC transporters represent the most frequently occurring transport systems in bacteria. Regions of interaction between the periplasmic binding protein and the cytoplasmic membrane transporter were shown for the first time with FhuB/D. The same type of ferrichrome transport system was shown to occur in Bacillus subtilis, a Gram-positive bacterium, which lacks a periplasm. Here, a protein similar to the periplasmic binding protein of Gram-negative bacteria is linked by a lipid anchor of the murein lipoprotein type to the cytoplasmic membrane.

1.2.3 Iron transport regulation Fur was the first iron regulatory gene to be mapped, cloned, and sequenced. Fur functions as an oligomer and binds when loaded with Fe2+ to iron-regulated promoters and inhibits transcription. An assay for the identification of Fur-regulated promoters was developed. The ferric citrate transport system displays the particular property that it is not only repressed by iron, but is induced by ferric citrate. Ferric citrate binds to the outer membrane FecA transport protein; this binding initiates a signal that is transmitted by the FecR protein across the cytoplasmic membrane. In the cytoplasm, FecI is converted to an active sigma factor, which in turn transcribes the fecABCDE transport genes. In this dual stepwise control, first iron limitation is recognized and subsequently the transport system is synthesized only when the cognate substrate is in the culture medium. 8

1.3 Transport of bacterial proteins Under anoxic conditions, bacteria may acquire Fe2+, which is much more soluble than Fe3+ and does not require chelating agents. Feo of E. coli, the only Fe2+ transport system characterized at the molecular level, is encoded by three genes; one gene, feoB, is very likely involved in energizing the transport by nucleotide triphosphate hydrolysis. Mutants in feo were shown to be attenuated in the mouse gut.

1.2.4 Intracellular iron metabolism Very little is known about the intracellular iron metabolism in bacteria. The atypical [2Fe-2S] protein FhuF was characterized; the cysteine residues that bind the iron-sulfur center were identified by amino acid replacement studies. FhuF mutants no longer utilize ferrioxamine B as an iron source, which suggests that FhuF may be involved in iron mobilization from ferrioxamine B. The two ironregulated genes sufS and sufD play a role in utilization of ferrioxamine B as an iron source and possibly in intracellular iron metabolism. Sequence similarities of SufS to NifS suggest that SufS is involved in the formation of the iron-sulfur center of FhuF.

1.3 Transport of bacterial proteins

1.3.1 Transport of colicins and toxins The activities, import, immunity, and evolution of bacterial protein toxins were studied. The genes of eight colicins and pesticin were cloned and sequenced. Cells that synthesize the toxins are protected by immunity proteins with a high specificity for the cognate colicin. Most of the colicins are released from cells by lysis proteins that are encoded downstream of the activity and immunity genes. Colicins can be subdivided into the N-terminal translocation region, the central receptor recognition region, and the C-terminal activity and immunity regions. Comparison of amino acid sequences clearly demonstrated evolution of the pore-forming colicins by exchange of DNA fragments that encode functional domains. How and when the immunity proteins in the cytoplasmic membrane inactivate the pore-forming colicins was a major question. The transmembrane topology of the immunity proteins and the regions of interaction with the colicins in9

1 Introduction dicate that the colicins are inactivated shortly before the pores are opened. In contrast, colicin M, which inhibits murein and O-antigen biosynthesis by interfering with C55-lipid carrier regeneration, and pesticin, which degrades murein by a mechanism similar to that of lysozyme, are inactivated by their immunity proteins in the periplasm before they reach their targets. The hemolysin/cytolysin (ShlA) of Serratia marcescens is activated by a single protein (ShlB) in the outer membrane through a novel mechanism during secretion. The hemolysin is a large protein that remains in a non-hemolytic form in the periplasm of cells that synthesize no ShlB protein. The N-terminal portion of ShlA is important for activation and secretion, the central portion for binding to erythrocytes, and the C-terminus for the formation of small pores in the membrane of erythrocytes, leukocytes, and epithelial cells. ShlA represents one of the very few cases where a major phospholipid of a biomembrane also serves as a cofactor for activity. ShlB has the potential to form pores through which ShlA might be exported.

1.3.2 Transport of staphylococcal (phospho)lipases Five different staphylococcal lipase genes of S. aureus, S. epidermidis, and Staphylococcus hyicus were cloned and sequenced. All corresponding proteins are organized as pre-pro-enzymes in which the pro-region comprises between 207 and 267 amino acids. The pro-region acts as an intramolecular chaperone that facilitates translocation of the native lipase; the pro-peptide can also translocate a number of completely unrelated proteins fused to it. The pro-region protects the proteins from proteolytic degradation. The lipase pro-peptide-based expression and secretion system is used by an increasing number of groups for production of human proteins and peptides in Staphylococcus carnosus, a food-grade microorganism for which a cloning system was developed.

1.4 Membrane components and membrane polarization

1.4.1 Biosynthesis of triterpenes in bacteria There is a tremendous variety of triterpenes in the plant kingdom; a single higher plant always contains several types of triterpenes. The triterpenoic hopanoids found in a large number of Gram-positive and Gram-negative bacteria 10

1.4 Membrane components and membrane polarization show less structural variability. In some bacteria, hopanoid biosynthesis genes are present, but are not expressed under laboratory conditions; therefore, an even wider range of bacteria may synthesize hopanoids. The study of triterpene biosynthesis and the structural variation of triterpenes in nature was approached by investigating the membrane-bound squalene-hopene cyclase of Alicyclobacillus acidocaldarius, which proved to be easier to work with than that of plants. The encoding gene was cloned, sequenced, and expressed, and the gene product was purified and characterized. Comparisons of the amino acid sequence with those of other triterpene cyclases revealed a conserved 16amino acid repeat. Interestingly, the highly purified A. acidocaldarius squalenehopene cyclase forms minor products of mostly tetracyclic structure; this finding was important for a better understanding of the cyclase reaction mechanism. The studies formed the basis for the determination of the crystal structure, which, together with the crystal structures of two sesquiterpene cyclases, are the first three-dimensional structures of terpene cyclases. The squalene-hopene cyclase is a monotopic membrane-bound enzyme. Knowledge of the structure allowed site-directed mutagenesis of specific residues in the catalytic cavity. Some mutant squalene-hopene cyclases significantly increased the synthesis of tetracyclic and bicyclic byproducts; a “new” cyclase in which a leucine in the central cavity is replaced by lysine produced a bicyclic compound. Additional genes involved in hopanoid biosynthesis were detected upstream of the squalene-hopene cyclase genes of Bradyrhizobium japonicum, Zymomonas mobilis, and Methylococcus capsulatus; the first bacterial gene found to encode a squalene synthase is among them. In the aerial mycelium of Streptomyces coelicolor, a differentiation-dependent formation of hopanoids was found; hopanoids are not formed in substrate mycelium and when cultures are grown in liquid.

1.4.2 Biosynthesis of staphyloxanthin The yellow to orange colony color of S. aureus is one of the classical species criteria. The main pigment is staphyloxanthin, a C30-carotenoid that is integrated into the cytoplasmic membrane. The genes involved in the biosynthesis of staphyloxanthin were identified and analyzed. Through the creation of deletion mutants and the analysis of the intermediary compounds formed, a biosynthetic pathway was postulated. The function of staphyloxanthin is still unclear; however, the expression of its gene responds to the stress sigma factor, SigB, which suggests that staphyloxanthin is necessary for survival under certain conditions.

11

1 Introduction

1.4.3 Signal transduction by cAMP and cGMP The initial steps in signal transduction in Paramecium involving the second messengers cAMP and cGMP were characterized. Unlike in metazoans, where hormones as first messengers elicit intracellular second messenger formation, in the ciliate Paramecium abrupt changes in the cell’s membrane potential activated second messenger biosynthesis. Characterized behavioral mutants of the ciliate with defined defects in electrogenesis showed that cAMP generation depends on a K+-outward current, whereas cGMP formation is enhanced by a depolarizing Ca2+-inward current. Analysis of clones carrying genes of the respective protozoan nucleotide triphosphate cyclases demonstrated the presence of an adenylyl cyclase embedded in a protein background that strongly resembles a potassium ion channel. Most surprisingly, the guanylyl cyclase is disguised in a membrane topology identical to that of canonical mammalian adenylyl cyclases and, in addition, carries an extended N-terminus that closely resembles a P-type ATPase unit with a total of ten transmembrane-spanning helices. These findings obtained with the ciliate Paramecium open new vistas on the structural and functional evolution of nucleotide triphosphate cyclases and provide Rosetta Stone sequences to decipher novel binding/regulating partnerships.

1.5 Chemistry of microbial peptides and proteins

During the course of the collaborative research centre, innovative analytical and synthetic methods were introduced. These methods allowed high-level biochemical investigations to solve microbiological research problems in interdisciplinary co-operations. Very often the analytical and synthetic work was even decisive for the success of projects.

1.5.1 Structure determinations The major contributions of the chemistry group were the structure determinations of a large number of antibiotics, and the synthesis of precursors, which allowed the elucidation of the activities of enzymes involved in biosynthesis. The 3-D structures of gallidermin and actagardine are the basis for the model of their mode of action, which very recently became of increased interest due to the inhibitory activity on peptidoglycan biosynthesis. One of the most unusual and in12

1.5 Chemistry of microbial peptides and proteins teresting peptide structures ever found is the 43-peptide antibiotic microcin B17, elucidated by multidimensional NMR of the 13C-, 15N-labeled polypeptide containing eight oxazole and thiazole rings in its backbone. This gyrase (topoisomerase II) inhibitor is ribosomally synthesized as a precursor peptide which is post-translationally modified. To elucidate such structures at that time, innovative instrumental methods, such as greatly improved NMR methods, HPLC-ESI-MS, and the Edman sequencer coupled to an ESI-mass spectrometer, and novel chemical transformations had to be introduced. Unusual peptide structures can now be sequenced using very small amounts of samples. The number and complexity of the elucidated natural products increased considerably, e. g. lipoglycopeptides and other unusual peptides, and new nonpeptidic metabolites, such as siderophores, macrolides, polyols, lactam antibiotics, and steroidal antibiotics. Recently, the complex structure of CDA (calciumdependent peptide antibiotic), a peptide pheromone carrying a thiolactone ring, of intermediates in nikkomycin biosynthesis, and of the first linear glycopeptide precursors in the biosynthesis of the antibiotic balhymicin were elucidated.

1.5.2 Peptide chemistry, peptide libraries, and mass spectrometric analysis The continuous improvements in parallel automated synthesis of peptides, peptide mimetics, and peptide libraries contributed extraordinarily to structure activity studies in various groups of the collaborative research centre. The outstanding synthetic capabilities combined with novel achievements in library analytics by ESI-MS, HPLC-ESI-MS, ICR-MS, and Edman pool sequencing stimulated collaborations in microbiology and immunology, in and outside of Tübingen. The binding regions of the gating loop of FhuA were identified using synthetic peptides. A number of enzyme activities (e. g. oxidative decarboxylase in the epidermin biosynthesis) and binding domains of proteins to the cell wall (e. g. autolysin) could only be studied with the aid of synthetic peptides and peptide libraries. Furthermore, many new proteins were characterized by LCMS and Edman sequencing, circular dichroism, peptide mapping, and antipeptide antibodies, such as the novel antifungal protein from Streptomyces. The recent introduction of a high-resolution, Fourier-transform, ion cyclotron resonance mass spectrometer will provide powerful technologies for further fruitful research between microbiologists and organic chemists.

13

1 Introduction

1.6 Summary of short-term projects of the collaborative research centre

The collaborative research centre was designed to run for 15 years. During this period, the details of the scientific program changed; however, the basic concept was maintained. Detailed descriptions of the results are contained in the research reports of the collaborative research centre from 1986–87, 1988–1990, 1990–1993, and 1993–1995. This book contains the detailed reports of 1995–1999. The following paragraphs describe the contributions made in short-term projects by the groups of the listed project leaders. Karl-Dieter Entian. Major contributions to the cloning and sequencing of the epidermin and the pep5 lantibiotic biosynthesis gene clusters were made. Heterologous proteins and peptides in yeast were synthesized. Bernd Hamprecht. Intercellular communication in the human nerve system was studied. A method was developed and successfully applied to the quantitative determination of adenosine binding to neural adenosine receptors, which paved the way for the isolation of adenosine receptors. The activities of glycogen phosphorylase, creatine kinase, and sorbitol dehydrogenase were determined in an attempt to analyze metabolic processes regulated by cyclic AMP in astroglia-enriched primary cultures. The group was further involved in the study of the role carnosine plays in the brain, taurine transport, and the mode of action of bradykinin. The neuronal cell cultures were used to analyze the activities of products isolated in the microbial screening programs. Thomas F. Meyer. Secretion of the IgA protease by Neisseria gonorrhoeae was investigated, and a novel mechanism was discovered. The mode of action of the translocating b-domain in the outer membrane was studied, and the domain was fused with heterologous proteins that became exposed at the cell surface. The OmpT protease was identified as the enzyme that degrades the fused proteins, which results in decreased yields. The formation of disulfide bonds by oxidation in the periplasm was shown to prevent secretion by locking fusion proteins in a secretion-incompetent conformation. The b-domain is therefore suitable for exposing antigens at the cell surface with the aim to produce antibodies and to stimulate the human immune system. Johannes Pohlner. The a-domain of the IgA protease of N. gonorrhoeae was studied since it contains a sequence of basic amino acids found in proteins that enter the nucleus of eukaryotic cells. The group also studied the post-translational processing of the IgA protease polypeptide to form the protease proper, which is released into the culture medium along with the a-domain and the bdomain that reside in the outer membrane. Rainer Haas. The VacA cytotoxin of Helicobacter pylori was characterized. The growth conditions for the production of the toxin were established, the vacA structural gene was cloned and sequenced, the occurrence of vacA in var14

1.6 Summary of short-term projects of the collaborative research centre ious Helicobacter strains was determined, vacA mutants were isolated and characterized, and the secretion mechanism of VacA was studied. In addition, tools for the genetic analysis of Helicobacter were developed. Susanne Klumpp. The protein phosphatases type 1, 2A, and 2C of Paramecium were studied. The genes of the phosphatases 1 and 2C were cloned and sequenced. The three phosphatases were purified to electrophoretic homogeneity, and functions were ascribed to protein domains. As part of a collaboration, the biochemistry of sensory transduction in Paramecium was studied.

15

2 Screening for New Secondary Metabolites from Microorganisms Hans-Peter Fiedler* and Hans Zähner

2.1 Introduction

Originally screening for secondary metabolites was focused on antibacterial compounds. Later on the screening was extended to antifungal, antiviral and antitumor activity and today it has expanded to human medicine, animal health, and plant protection. The initial idea, using only natural products produced by microorganisms has been replaced by the search for novel lead structures, accompanied by the development of novel targets in all application fields. Still, the most prominent source for novel leads is found in nature and especially in the secondary metabolism of microorganisms [1]. The new lead compounds can be used for derivatisation programs or as platform for chemical synthesis. Therefore, the screening for novel secondary metabolites received an increased interest in the last 15 years. More than hundred new test systems are described till today for applications in pharmaceutical and agricultural fields [2]. These in vitro-assays are mostly based on key enzymes or receptors and differ from classical antibiotic assays by the following aspects: . Proteases in microbial samples or extracts lead to false positive results by degradation of assay enzymes or protein receptors. . Numerous assays are sensitive to metabolites from the intermediary metabolism, which are found in variable concentrations in all microbial cultures. . The assays are sensitive to infections, osmotic conditions, and changes in the pH value. . The assays are selective for a distinct mode of action within a cascade and do not take account to the whole cascade.

* Mikrobiologisches Institut, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen

16 Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

2.1 Introduction . Not all new developed assays are suitable for automated robot screening. . Quite a number of assays are sensitive to already known compounds. . None of the target directed assays is suitable to detect chemical diversity in microbial cultures or extracts. Within the collaborative research centre 323 we developed screening strategies which aimed not only on classical antibacterial activity, but also on antifungal activity, on activities which are involved in differentiation processes and on detection of novel siderophores. However, our main screening strategy is based on physico-chemical methods to detect a maximal number of novel secondary metabolites in freshly isolated Actinomycete strains. The so-called “chemical screening” which is based on thin-layer chromatography and staining reagents was first introduced by Hamao Umezawa [3] and continued a few years later by Satoshi Omura, who detected staurosporin by this assay which his most prominent compound found by chemical screening [4]. Hans Zähner modified this method with respect to staining reagents, sample preparation, and variation of the cultivation conditions of the isolated strains [5, 6]. The detection of a biological activity comes only second order. A new dimension of insights into the chemical diversity of produced secondary metabolites was the coupling of high-performance liquid chromatography with computer-assisted diode array detection (HPLC-DAD) and the construction of a database of antibiotics and other natural products based on HPLC and UV-visible absorbance spectral libraries (HPLC-UV-Vis Database) by HansPeter Fiedler [7]. This efficient method allowed the identification of known compounds in raw extracts at a very early stage of investigations or permitted a classification of the compound by comparing UV-visible spectra data. The efficiency of HPLC-DAD screening technique was extended by HPLC-ESI-MS analysis in co-operation with the members of the collaborative research centre Prof. Günther Jung and Prof. Jörg Metzger. The additional information of the molecular mass permitted a more accurate search in commercially available chemical databases. The goal of our strategy was to detect a novel compound which then was isolated and broadly tested for its biological activity, considering that each secondary metabolite will have an activity. A further advantage of chemical screening was achieved by testing pure compounds in the assays. Quite a number of test systems are not compatible with microbial cultures or crude extracts and need the application of pure compounds. Nevertheless, we have not sufficient assay systems available and for many new compounds we have so far not found any biological activity despite intensive co-operation with various pharmaceutical and agrobiological companies. We expect that the “Naturstoffpool” (sponsored since 1996 by BMBF and German pharmaceutical companies) comprising a collection of compounds will be tested by the end of 1999 by a large variety of molecular robot assays. For the analysis of the structure-activity relationship of secondary metabolites we increased the number of original compounds by “directed fermentations” and feeding, by modification of precursors during the production phase, or, by “mutasynthesis” using blocked mutants. 17

2 Screening for New Secondary Metabolites from Microorganisms In the following subchapters all new secondary metabolites are described which were detected in various screening programs during 1986 and 1999 in the groups of Prof. Hans Zähner and Prof. Hans-Peter Fiedler within the collaborative research centre 323.

2.2 Screening methods and novel compounds 2.2.1 Classical screening for antimicrobial activity The classical agar plate diffusion assay for the detection of antimicrobial agents produced by Gram-positive and Gram-negative bacteria, yeasts or filamentous fungi was applied only until 1988. By this assay system we found pyridazomycin [8] and chlorotetain [9]. Both antibiotics show a selective antifungal activity. Pyridazomycin is distinguished by the unusual pyridazine ring that was not described before in microbial secondary metabolites, chlorotetain is a dipeptide containing an unusual chlorinated amino acid. A screening for growth inhibitors against Bacillus subtilis revealed a novel peptide antibiotic named aborycin [10]. The structures of the novel antifungal antibiotics are shown in Fig. 2.1.

Cl+ N H2N

N

O COO+ H3N

Cl

H

H2C

CH3

O +

Pyridazomycin

H3N

Streptomyces violaceoniger sp. griseofuscus Tü 2557

CH

CO

NH

CH

H -

COO

Chlorotetain Bacillus subtilis ATCC 6633

Trp

Val

Val

Phe HO

Ala

Tyr

Cys Ala

S S Cys

Aborycin Streptomyces griseoflavus Tü 4072

Asn

Gly

P he

Cys

Asp

S

Ser

Cys

Gly

Leu Ile

Gly

S

Gly

Figure 2.1: Microbial secondary metabolites detected by classical screening for antimicrobial activity.

18

2.2 Screening methods and novel compounds

2.2.2 Screening for antibiotics causing morphological changes of hyphae of Botrytis cinerea This assay is based on both, growth inhibition of Botrytis cinerea and morphological changes of the hyphae, a so-called “bulging effect“. By this assay substances with antifungal action in the presence of polyene antibiotics were found. Nikkomycin Z and X [11, review] were the most prominent antibiotics analysed in the group of Prof. Hans Zähner during the collaborative research centre 76. Nikkomycin Z is a potent inhibitor of chitin synthase and is non-toxic for humans. It was several years under intensive investigation as acaricide for agricultural use at BAYER AG but cancelled in 1984 because of too high costs and its too narrow application in plant protection. From 1994 till 1998 nikkomycin Z was developed as an antimycotic agent for therapy of histoplasmosis, blastomycosis and coccidoidomycosis in human medicine by Shaman Pharmaceuticals in the USA and passed to the second clinical trial. The Botrytis-assay was continued during the beginning of the collaborative research centre 323 and resulted in the detection of galbonolides [12, 13], four new members of antifungal macrolide antibiotics. Their structures are shown in Fig. 2.2. HO

OH

O CH3

R HO

H3C

O

CH2

A B

CH3

O

CH3

: R = OCH3 : R = CH3

OH

OH H3C

CH3

H3C

O O

CH2

CH3

O

CH3

OH CH3

H3CO O

H3C

O

CH2

C

CH3

O

CH3

D

Galbonolides A-D Streptomyces galbus Tü 2253

Figure 2.2:

Microbial secondary metabolites detected in the Botrytis assay.

2.2.3 Screening for novel siderophores With the exception of lactobacilli all other microorganisms are dependent on the uptake of external iron ions to supply their iron-containing enzymes. Because of the extreme water insolubility of Fe3+-ions all microorganisms have developed very efficient iron-chelating compounds and specific iron-uptake systems. Iron-chelating compounds are for example trihydroxamates, catecholes, tricarboxylates, and other compounds which are able to chelate iron. In the course of the collaborative research centre 323 the following novel chelating metabolites were isolated: 19

2 Screening for New Secondary Metabolites from Microorganisms Maduraferrin was isolated from strain Actinomadura madurae DSM 43067 after detection by an HPLC assay [14]. The complexing centres are a salicylamide moiety, a hydroxamic acid group and an acid hydrazide group. The highly hydrophilic carboxylate-type siderophores staphyloferrins A and B were isolated from Staphylococcus hyicus DSM 20459 grown under strong iron-restricted conditions [15–18]. Both compounds are strictly iron-regulated. Staphyloferrin A consists of two molecules citric acid, each linked to D-ornithine by an amino bond, whereas staphyloferrin B consists of 2,3-diaminopropionic acid, citrate, ethylenediamine and 2-ketoglutaric acid. From Bacillus sp. strain DSM 6940 was isolated besides schizokinen the new dihydroxamate siderophore schizokinen B, in which citrate is replaced by aconitate [19]. Rhizoferrin is a novel carboxylate-type siderophore which was isolated in collaboration with the groups of Prof. Winkelmann and Prof. Jung from Rhizopus microsporus and other fungi of the Mucorales [20, 21]. Rhizoferrin is similar in structure to staphyloferrin A. In case of rhizoferrin, D-ornithine is replaced by putrescin as bridge. From a highly virulent Yersinia enterocolitica strain H1852 a siderophore named yersinibactin was isolated [22]. The novel compound contains a benzene and a thiazolidine ring, as well as two thiazoline rings. It forms stable complexes with trivalent cations such as iron and gallium. While we investigated the fermentation of S,S-ethylenediamine disuccinic acid (EDDS) with Amycolatopsis orientalis strain we found out that EDDS is not an iron but a zinc chelator which opens new biotechnological applications. A novel, not ferrioxamine-type siderophore named amycolachrome was isolated that is similar in structure to the fungal ferrichrome-hexapeptides [23]. The structures of the isolated new siderophores are shown in Fig. 2.3.

2.2.4 Screening for secondary metabolites involved in differentiation processes Actinomycetes are characterised by complex differentiation processes. The search for metabolites which influence these processes is of general importance because they give insight in the tricky sequence and regulation of this dramatic event in the live cycle of these organisms. Prof. Heinz Wolf in the group of Prof. Zähner developed a screening system that allows detection of compounds that stimulate formation of aerial mycelium in Streptomycetes and he isolated hormaomycin from Streptomyces griseoflavus W384, a novel peptide-lactone antibiotic [24, 25]. Hormaomycin induces not only aerial mycelium formation but also antibiotic production in these organisms. Germicidins A and B were isolated from Streptomyces viridochromogenes NRRL B-1551 [26]. Germicidin A is the first known autoregulative inhibitor of spore germination in the genus Streptomyces. Another novel peptide, streptofactin, was isolated from the nikkomycin producer Streptomyces tendae Tü 901/8 c in the group of Prof. Fiedler. This bio20

2.2 Screening methods and novel compounds HO O HN NH H N

O

O OH N

O

H N

O

NH

O

N

COOH

H CH3

H N H

HOOC

COOH

OH

Maduraferrin

HOOC

Rhizoferrin

Actinomadura madurae DSM 43067

Cunninghamella elegans, Rhizopus microsporus

COOH

H

H N

O

HO

OH

O

O

H N

OH O

N H COOH

H N

O

NH HN

COOH O

O

HO

OH

HOOC

COOH

H2N

O

COOH

HOOC

COOH

B

A Staphyloferrins

Staphylococcus hyicus DSM 20459

O

O N H

N

CH3

CH3

OH OH

OH

COOH

OH

H N

N

N

N

CH3

COOH S

H3C

S

O

N

CH3

S

O

Schizokinen B

Yersiniabactin

Bacillus sp. DSM 6940

Yersinia enterocolitica

OH

HO N

CH2

O C

CH2

CH2

CH

C

C

C O

O

NH

O

CH2 CH

NH CH

C

CH2

O

C NH

CH3

OH

CH2 H2C

N

Figure 2.3:

CH

CH

CH2

O

O

NH

C NH

H2C C

CH

O

H2C

H3C

NH

OH

CH2

OH

CH2 HO

Amycolachrome Amycolatopsis orientalis

N H

Novel iron-chelating compounds.

21

2 Screening for New Secondary Metabolites from Microorganisms surfactant plays a structural role in aerial mycelium development of Streptomycetes and supports the erection of aerial hyphae by lowering the surface tension of water films enclosing the colonies. Mass spectrometry results and amino acid analysis revealed the peptide sequence H2N-Leu-Leu-Ala-Val-Ala-Leu-Lys-Thr and a molecular mass of 1021 Daltons, including a further valine. The missing small part with 94 Daltons of the molecule is bound to the N-terminal end of the peptide [27]. Streptofactin is the first peptide described having structurally and autoregulatory functions. The structures of the isolated secondary metabolites involved in differentiation processes are summarised in Fig. 2.4.

O HC

CH3 H3C

CH2

HC

H C

O

C

H N

C

CH3

CH

NH O C HC

C O

C

O

C

CH

C O CH CH

C

NH

O

H3C

C CH2

O2N

NO2

NH

NH CH3

CH2

N H

CH3

O

CH NH C

HO

O

N

Hormaomycin Cl

Streptomyces griseoflavus W-384

OH

OH

CH3

CH3 H3C

O

O

H3C

H3C

O

O

H3C

Germicidin A

Germicidin B

Streptomyces viridochromogenes NRRL B-1551

Figure 2.4:

22

New secondary metabolites involved in differentiation processes.

2.2 Screening methods and novel compounds

2.2.5 Chemical screening by TLC, monitoring coloured secondary metabolites Concentrated extracts of culture filtrates and mycelia of Streptomyces strains were separated on silica gel TLC. Such strains were investigated whose extracts showed coloured spots on TLC. The assay lead to the detection of various novel anthraquinone, phenazine and polyene antibiotics. Urdamycins A–F were the most prominent secondary metabolites detected by this method [28–32]. These novel angucycline antibiotics, produced by Streptomyces fradiae Tü 2717, are biologically active against Gram-positive bacteria and show a strong cytotoxic activity against stem cells of murine L1210 leukaemia. For the para-quinone metabolites cinnaquinone and di-cinnaquinone, which were isolated from Streptomyces griseoflavus ssp. thermodiastaticus Tü 2484, no biological activities have been detected so far [33, 34]. Two dark green substances, the esmeraldines A and B, were isolated from Streptomyces antibioticus 2706 [35]. They formally derive by condensation of two phenazine residues of the saphenic acid family. They don’t have any antibacterial activity, but esmeraldine B shows a cytotoxic activity against various tumor cell lines. From Streptomyces violaceus Tü 3556 the new naphthoquinone complex naphthgeranines A–D was isolated [36], from which naphthgeranines A and B show a weak antibacterial and antifungal activity, whereas A, B and C have a moderate cytocidal activity against various tumor cell lines. In addition, strain Tü 3556 produced the new naphthoquinone compounds naphtherythrins D–F. The bright-yellow polyene carboxylic acid serpentene which shows an antibacterial activity against Bacillus subtilis was isolated from Streptomyces sp. Tü 3851 [37]. Remarkable regarding the structure is the benzene ring nearly in the middle of the molecule. The structures of the isolated secondary metabolites screened by this approach are summarised in Fig. 2.5.

2.2.6 Chemical screening by TLC, monitoring fluorescent secondary metabolites Two novel metabolites were detected regarding their blue fluorescence on TLC plates by irradiation with UV light. Pyridindolol glycosides were isolated from Streptomyces parvulus Tü 2480 [38]. No biological activity could be observed of all three compounds. Depsichlorins, isolated from Streptomyces antibioticus ssp. griseorubinosus Tü 1661, represent a group of new cyclopeptide antibiotics which show biological activity against Gram-positive and Gram-negative bacteria [39, 40]. The structures of pyridindolol glycosides and depsichlorins are summarised in Fig. 2.6. 23

2 Screening for New Secondary Metabolites from Microorganisms

O

OH

CH3

Urdamycins Streptomyces fradiae Tü 2717 CH3

O

O

O

OH

CH3 O

HO

OH

O H3C

CH3 O

HO

R OH

O

O

A : R = H

O HO

E : R = SCH3 O

CH3

O

OH

CH3 O

HO O H3C

CH3

O

OH

O

O

HO

B

O HO

O

CH3

OH

O R

O

CH3

O O

OH

CH3 O

HO

OH

O H3C

CH3

O

O

HO

O

HO

OH

HO

O

C: R = H N

D: R =

O

O

CH3

CH3

O O

OH

CH3 O

HO

OH

OH

O H3C

CH3 HO

O O

OH

O

O

OH

F

HO

Figure 2.5: New secondary metabolites by chemical screening using TLC and monitoring colored compounds.

24

2.2 Screening methods and novel compounds O

O

COOH

HO

COOH

HO

O H2N

NH2

NH2 O

O

OH

HOOC

di-Cinnaquinone

O

Cinnaquinone

Streptomyces griseoflavus ssp. thermodiastaticus Tü 2486

H3C CH3 H3C

HN N

HOOC

O

CH3 H3C

R HN O

N

N

HOOC

O

N

OH

O

N

N

COOH

A

COOH

B Esmeraldins

Streptomyces antibioticus Tü 2706

a

R = C13H27

b

R =

(CH2)10

CH CH2 CH3

c

R =

(CH2)12

CH CH3

d

R = C15H31

CH3 CH3 (CH2)13

e

R =

f

R = C16H33

g

R = (CH2)14

CH2R1

CH CH3

H

CH3

HO

CH CH3 CH3

R4

O

h R = C17H33 i

R1

OH

R2

R3

R4

R2

O

R3 H CH3 CH3

A

H

H

H

H

B

OH

H

H

H

C

OH

OH

H

H

D

OH

OH

OH

H

O

R = C17H31

CH2OH OH O

E

HO

O OH

CH3 R2

C

CH3

Naphthgeranins Streptomyces violaceus Tü 3556

O

CH3 OH

O R1

O

NH CH3 HOOC

CH3

O

R1 D E F

R2

CHO H H H H OH

COOH

CH3

Naphtherythrins D-F Streptomyces violaceus Tü 3556

Serpentene Streptomyces sp. Tü 3851

Fig. 2.5 continued

25

2 Screening for New Secondary Metabolites from Microorganisms

OR1 N H H

N

Pyridindolol glucosides

OR2

Streptomyces parvulus Tü 2480

R3

R2

R1

OR3

OH

Tü 2480 F2

H

Tü 2480 F3

H

HO

OH OH

O

H OH HO

H

OH OH

O

OH HO

Tü 2480 F4

CH

HO

O

O

C

C

H

O NH

CH

H3C

CH

CH

O

C

O

C

N

OH

CH

CH

Cl O HO

CH2

Y

C

O

X

C

NH

Y

CH NH

C

N

CH3 CH2 CH3 CH3 CH3

C O

O H3C

OAc

A

H

CH3

Cl

X

OH OH

O

O O

Leu N

CH3

Depsichlorins Streptomyces antibioticus ssp. griseorubinosus Tü 1661

O OAc

B

Homo-Ile N

CH3

O OAc

C

CH3

Leu N

CH3

O OAc

D

CH3

Homo-Ile N

CH3 O

Figure 2.6: New secondary metabolites by chemical screening using TLC and monitoring fluorescent compounds.

26

2.2 Screening methods and novel compounds

2.2.7 Chemical screening by TLC and Ehrlich reagent Ehrlich reagent reacts mainly with primary amines and the products appear as red-violet zones within few seconds on the TLC. This reagent was successfully applied for detection of pyrrol-3-yl-2-propenoic acid and pyrrol-3-yl-2-propenamide, two further non-active secondary metabolites isolated from Streptomyces parvulus Tü 2480, the producer of pyridindolol glucosides [41]. The group of pyrrolams are four biosynthetically new pyrrolozidinones produced by Streptomyces olivaceus Tü 3082 [42]. They show no antibacterial and antifungal activities, but a weak herbicidal activity against wheat and rice seedlings. Pyrrolam influences the embryonic development of the fish Brachydanio rerio. Obsurolides A2 and A3 produced by Streptomyces viridochromogenes Tü 2580 represent a novel class of phosphodiesterase inhibitors [43]; they have no growth inhibiting potency against bacteria, yeasts and filamentous fungi. Two new phenylpentadienamides were detected in Streptomyces sp. Tü 3946 by orange spots on the TLC stained with Ehrlich reagent, 5-(4-aminophenyl)penta-2,4-dienamide and N2-[5-(4-aminophenyl)penta-2,4-dienoyl]-L-glutamine [44]. Both secondary metabolites show no antibacterial and antifungal activities. The structures of the novel secondary metabolites are summarised in Fig. 2.7.

2.2.8 Chemical screening by TLC and blue tetrazolium staining reagent Blue tetrazolium is a relatively specific derivatisation reagent for steroids and reducing compounds. Blue or violet coloured zones are formed on a light background on the TLC sheet. From Streptomyces aurantiogriseus Tü 3149 a compound was isolated which revealed a yellow-orange colour by staining with blue tetrazolium. Because of its stimulation of aerial mycelium and spore formation of Streptomyces glaucescens, the compound was named differolid [45]. No growth inhibiting activity against bacteria, yeasts and filamentous fungi was observed. A further blue tetrazolium positive compound, (2S,3R,4R,6R)-2,3,4-trihydroxy-6-methylcyclohexanone, was isolated from Streptomyces phaeochromogenes ssp. venezuelae Tü 3154 and Streptomyces albus Tü 3226 [46]. The compound shows no biological activity to bacteria and fungi. A new member of natural compounds having a thiotetronic acid structure was isolated from Streptomyces olivaceus Tü 3010 [47]. The secondary metabolite (2S)-4-ethyl-2,5-dihydro-3-hydroxy-2-[(1E)-2-methyl-1,3-butadienyl]-5-oxo2-thienylacetamide shows antibacterial activity especially against Streptomyces strains. From Streptomyces griseoflavus Tü 2880 the bright yellow colabomycins A–C were isolated which represent new members of the manumycin group [48, 49]. They react with blue tetrazolium as brown spots, with vanillin-sulphuric 27

2 Screening for New Secondary Metabolites from Microorganisms H

COR

R

N

N H

O

Pyrrol-3-yl-2-propenoic acid: R = OH Pyrrol-3-yl-2-propenamide: R = NH2

N O

Pyrrolam

A : R = OH B : R = OCH 3

Streptomyces parvulus Tü 2480

C : R = O

CH O CH2 CH3 CH3

Streptomyces olivaceus Tü 3082 O O

5-(4-Aminophenyl)penta-2,4-dienamide

N H

R

CH3

H2N O

HO

Obscurolides A2:: R = CHO A3:: R = CH2OH Streptomyces viridochromogenes Tü 2580

NH2

N2-(5-(4-aminophenyl)penta-2,4-dienoyl)-L-glutamine H2N O COOH NH

Streptomyces sp. Tü 3946

Figure 2.7: agent.

CONH2

New secondary metabolites by chemical screening using TLC and Ehrlich re-

acid as dark violet and with molybdatophosphoric acid as black spots, indicating their reducing character. The main compound, colabomycin A, is active against Gram-positive bacteria and shows a cytotoxic activity against stem cells of murine L1210 leukaemia. In collaboration with Hoechst AG and Prof. Fiedler, seven musacin compounds were detected in extracts of Streptomyces griseoviridis FH-S 1832 on TLC plates. The compounds were detected by blue tetrazolium chloride (showing a blue-violet colour), by anisaldehyde, orcinol, and Ehrlich’s reagent, respectively. The determination of their structure revealed that six of the seven compounds were new [50]; musacin C shows an anthelmintic activity against Caenorhabditis elegans and Trichostrongylus colubriformis. The structures of the isolated secondary metabolites are summarised in Fig. 2.8.

28

2.2 Screening methods and novel compounds O

H

O

H3C

H

OH

O O

O

OH OH

Differolid

(2S,3R,4R,6R)-2,3,4-trihydroxy6-methylcyclohexanone

Streptomyces aurantiogriseus Tü 3149

Streptomyces phaechromogenes ssp. venezuelae Tü 3154

O H N

H O

O

CH2

H OH

CH3 CH3 OH

O C

CH2

H2N

S CH2 CH3 O

Colabomycin A Thiotetronic acid Tü 3010

Streptomyces griseoflavus Tü 2880

O

Streptomyces olivaceus Tü 3010

NH HO

O

OH

A

OH

HO

O

CH3 O

OH

OH

O

OH

B

O

CH3

O O

OH

OH OH HO

O

CH3

C O O

D

OH

OCH3

H3C O

O

O

O

OH HO

F

H3C OH

Musacins Streptomyces griseoviridis FH-S 1832

Figure 2.8: New secondary metabolites by chemical screening using TLC and blue tetrazolium staining reagent.

29

2 Screening for New Secondary Metabolites from Microorganisms

2.2.9 Chemical screening by TLC and anisaldehyde and orcinol reagent With anisaldehyde-sulphuric acid reagent sugars, steroids, and terpenes can be detected. After heating the stained TLC sheets, a great variety of coloured spots from violet, blue, grey to green were formed on a weakly ochre coloured background. The same strain, Streptomyces griseoviridis FH-S 1832, that showed blue violet musacin spots on the TLC plate when sprayed with blue tetrazolium chloride, showed another pattern of spots with altered Rf values and colour, when the plate was sprayed with anisaldehyde and orcinol reagent, respectively. Besides cineromycin B, three new members of the cineromycin group of macrolide antibiotics were isolated [50]. The cineromycins showed weak activity against Gram-positive bacteria; no further biological activities have yet been observed. The structures of the new cineromycins are summarised in Fig. 2.9. O

HO

HO

O

CH3

OH

OH

CH3 OH CH3

CH3

H3C

O

Dehydrocineromycin B

H

H3C

CH3 CH3

H3C

H

HOH2C

H3C

O

H3C

O

Oxycineromycin B

O

O

2,3-Dihydrocineromycin B

Streptomyces griseoviridis FH-S 1832

Figure 2.9: New secondary metabolites by chemical screening using TLC and anisaldehyde or orcinol staining reagent.

2.2.10 Chemical screening by TLC and vanillin-sulphuric acid staining reagent Vanillin-sulphuric acid reacts relatively specific with higher alcohols, phenols and steroids. Coloured zones are produced on a pale background on the TLC sheet. In the mycelium of the colabomycin producing strain Streptomyces griseoflavus Tü 2880, a further compound, called 2880-II, was detected by vanillin-sulphuric acid staining reagent, resulting in a dark brown spot on the TLC sheet. The compound is related to ferulic acid and shows no antibacterial and antifungal activity [51]. (3S,5R,6E,8E)-Deca-6,8-diene-1,3,5-triol and (3S,6E,8E)-1,3-dihydroxydeca6,8-diene-5-one were isolated from the (3S,8E)-1,3-dihydroxydec-8-en-5-one 30

2.2 Screening methods and novel compounds OH

OH

OCH3

OH OH

H3C

(3S,5R,5E,8E)-Deca-6,8-diene-1,3,5-triol

O

O

NH

HO

O

OH OH

H3C

(3S,6E,8E)-1,3-Dihydroxydeca-6,8-diene-5-one 2880-II Streptomyces griseoflavus Tü 2880

Streptomyces fimbriatus Tü 2335

Figure 2.10: New secondary metabolites by chemical screening using TLC and vanillinsulphuric acid staining reagent.

producer Streptomyces fimbriatus Tü 2335. All compounds are inactive against bacteria and fungi [52]. The structures of the isolated secondary metabolites are summarised in Fig. 2.10.

2.2.11 Screening for new secondary metabolites by polystyrene resin fermentation Addition of the non-polar polystyrene resins Amberlite XAD-16 or XAD-1180 to growing cultures of microorganisms, preferably at the end of the growth phase, enables the absorption of unstable intermediate products or stimulates the producing organism to an altered metabolite pattern. The naphthgeranine and naphtherythrine producer Streptomyces violaceus Tü 3556 (see Section 2.2.5) synthesised the novel series of naphtherythrins A–C, when Amberlite XAD-1180 was added to growing cultures after 36 hours of incubation [53]. The main compounds, naphtherythrins A and B show a biological activity against Gram-positive bacteria and a weak activity against fungi. Under the same conditions Streptomyces exfoliates Tü 1424 produced a group of three new naphthoquinone antibiotics named exfoliamycins [54, 55]. They inhibit growth of Gram-positive bacteria, whereas Gram-negative bacteria and fungi are not sensitive against these antibiotics. The structures of secondary metabolites produced during polystyrene resin fermentations are summarised in Fig. 2.11.

31

2 Screening for New Secondary Metabolites from Microorganisms H3C

CH3

CH3

H3C

OH

O

O

OH

O

O OH

O

O

O

HOOC

O

N H O

HOOC

O

A : R = CH3 B: R=H

O

O

N R

O

CH2OH

O

C Naphtherythrins A-C Streptomyces violaceus Tü 3556

Exfoliamycines Streptomyces exfoliatus Tü 1424 CH3

CH3 HO OH

HO

O

O H3C

OH OH O

CH2OH

OH

O

O

O

CH2OH

H3C

OR O

Exfoliamycin R=H 3-O-Methylexfoliamycin R = CH3

O

Anhydroexfoliamycin

Figure 2.11: New secondary metabolites by screening using polystyrene resin fermentation.

2.2.12 Screening for new secondary metabolites by HPLC and photoconductivity detection Photoconductivity detection has a complete different detection window than UV-Vis spectroscopy and offers the detection of new secondary metabolites in culture filtrates and extracts of microorganisms by HPLC analysis. Streptomyces antibioticus Tü 99, who is known as a producer of chlorothricin, juglomycins A and B, ketomycin, nikkomycins Z and J, as well as nocardamine, was reinvestigated using a HPLC photoconductivity screening system. With this method we could detect four new butenolides [56]. The compounds, which are summarised in Fig. 2.12, show a weak antibiotic activity against Pseudomonas aeruginosa and also a weak inhibition of the chitinase from Serratia marcescens.

32

2.2 Screening methods and novel compounds OH

OH

O

H3C

O

H3C O

H3C H3C

HO

H

Tü 99-1 OH

Tü 99-2 OH

O

H3C

O

H3C H3C H

O

H3C H3C

HO

O

H3C H3C H3C

Tü 99-3

O

Tü 99-4 Tü 99 Butenolides

Streptomyces antibioticus Tü 99

Figure 2.12:

New secondary metabolites by HPLC-photoconductivity screening.

2.2.13 Screening for new secondary metabolites by HPLC and diode-array detection This method represents a modification of the classical chemical screening procedure. TLC and staining reagents were replaced by the more efficient reversedphase HPLC technique coupled with computerised diode-array detection (HPLC-DAD). Commercially available antibiotics and secondary metabolites from our institute pool were analysed with identical standardised HPLC conditions as culture filtrates and raw extracts from freshly isolated Actinomycete strains. Retention times and UV-visible absorbance spectra of references and biological samples were stored in libraries of a HPLC-UV-Vis-Database [7]. Till today more than 600 secondary metabolites, mostly antibiotics, are stored in the database. The technique was first used for detection of minor congeners and for characterisation of blocked mutants and intermediate products of biosynthetic pathways, and since 1990 as screening method for identification of new secondary metabolites in freshly isolated strains. All new secondary metabolites resulting from this screening strategy are summarised in Fig. 2.13. In raw extracts from the elloramycin producer Streptomyces olivaceus Tü 2353 five minor congeners, elloramycins B–F, were detected by HPLC-DAD and determined in structure [57, 58]. All elloramycins are strongly active against Streptomyces strains. As expected, the less methylated elloramycin B shows the best activity against Gram-positive bacteria. The new tetracenomycins B3 and D3 were detected in a blocked mutant of the elloramycin producer, Streptomyces olivaceus Tü 2353-R [59]. The main 33

2 Screening for New Secondary Metabolites from Microorganisms R2 O

O

O

OH

CH3

O OCH3

H3CO

O OH O

OH

R1

CH3

O

OCH3 OR3 O

CH3 O

O

OH

CH3

OR4

R1

R2

R3

R4

B

H

H

H

CH3

C

H

CH3

CH3

H

D F

H

CH3

H

CH3

OH

CH3

CH3

CH3

O OCH3

H3CO

Elloramycins B-F

O

Streptomyces olivaceus Tü 2353

OH O

O

CH3

O

E

OCH3 OCH3

OCH3

O OH

RO

COOH OH

O

OH

CH3

B3 R = CH3 D3 R = H Tetracenomycins B3 and D3 H3C

Streptomyces olivaceus Tü 2353-R

O

H3C

N

OCH3

N

N

N

COOH

COOH

6-Acetylphenazine-1carboxylic acid

H3C N

Saphenic acid methyl ether

O

R O

A

R = 12-methyltridecanoic acid

B

R = tetradecanoic acid

D

R = 12-methyltetradecanoic acid

E

R = 14-methylpentadecanoic acid

G

R = hexadecanoic acid

I

R = 14-methylhexadecanoic acid

J

R = unsaturated C18 acid

K

R = 16- methylheptadecanoic acid

N COOH

Saphenyl fatty acid esters

Streptomyces antibioticus Tü 2706

Figure 2.13:

34

New secondary metabolites by HPLC-diode-array screening.

2.2 Screening methods and novel compounds O

O CHO HN

A =

HN

B = O

N

HN

O

O

N

R2

R6 O

R3

O

C

R1

CH CH CH C NH CH

R4 N

O

NH2

OH

R5 OH OH

O CHO HN

A =

HN

B = O

N

O

N

R2 CH3

O

C

O

CH CH CH C NH CH N

R1 O

NH2

OH

OH OH

O

CHO HN

A =

HN

B = O

N

O

N

OH O HO

C

O

CH2 CH C NH CH

R1 O

NH2

Nikkomycins Streptomyces tendae Tü 901

NH

C =

OH OH

Fig. 2.13 continued

35

2 Screening for New Secondary Metabolites from Microorganisms R1

R2

O HN

Sz

H

O

N

CHO HN

Sx O

O

N R2

O

Soz

R1

OH

H

HN O

O C

OH

O

OH

OH N

CHO HN

Sox O

OH

Nikkomycins

N

Streptomyces tendae Tü 901

O

O

O

O

R

R

N H

N H

CH3

A1 : R = COOH A4 : R = CH2OCH3

HO

CH3

HO

B2 : R = CHO B3 : R = CH2OH B4 : R = CH2OCH3 O

COOR2 O

OH

R1

N H

OHC

N H

CH3 HO

C : R1 = CHO; R2 = H C2 methyl ester: R1 = CHO; R2 = CH3

D2

Obscurolides Streptomyces viridochromogenes Tü 2580

OH

O CH3

Juglomycin Z

O HO

Streptomyces tendae Tü 901

Fig. 2.13 continued

36

H COOH

CH3 O

2.2 Screening methods and novel compounds CH2OH O

3

O

HO

CH2OH

CH2OH

5

O

OH

CH3

N H

O

OH

1-(3-Indolyl)-2,3-dihydroxypropan-1-one

Naphthgeranine F Streptomyces violaceus Tü 3556

Streptomyces violaceus Tü 3556

N

H3C C

N

H H N C

O

CH3 O CH3

O C

N C H H

HOCH2

HO C O

H C

C N CH

S

O H C C

C

C H

CH CH3 O H3C CH3

N

O C

C H

OH

SCH3 CH2OH

CH2 N

CH3

CH3 CH

N

C

CH3 O

H C

H N C

CH3

C H

N H

O C

N

O N

Echinoserin Streptomyces tendae Tü 4031

O

NH2

O

OH

O

NH2

O

O

O

O

O H

OR

O O

COOH

H3C

Dioxolid A R = H Dioxolid B R = COCH3

NH2

Dioxolid D

H

OR

OH

Dehydrodioxolid A R = H Dehydrodioxolid B R = COCH3

para-Hydroxybenzamide Streptomyces tendae Tü 4042

Streptomyces tendae Tü 4042

OH

CH3

CH3

CH3

COOH

O O

OH

OH

O

OCH3

O

1-Hydroxy-4-methoxy-2-naphthoic acid Streptosporangium cinnabarinum ATCC 31213

Spirofungin OH

CH3

CH3

Streptomyces violaceusniger Tü 4113

Fig. 2.13 continued

37

2 Screening for New Secondary Metabolites from Microorganisms OH

OH

H3C

OH

OH HO

10 5

HO

CH3

20

15

O OR2

CH3

O

1

O H3C

O 1'

R1

O 2'

3'

OH

NH

A

35

OH

NH

OH

H NH2+

CH3

NH

D

NH2

45

CH3 O

R1

40

NH2

45

C

30

CH3

H O

OH

O

NH2

45

25

R2

Kanchanamycins Streptomyces olivaceus Tü 4018

O O

H3C OH

HO

(E)-4-oxonon-2-enoic acid

HO O

Streptomyces olivaceus Tü 4018

O

2E,4Z,7Z-decatrienoic acid

2E,4Z-decadienoic acid

Streptomyces viridochromogenes Tü 6105

CH3

O OH O

CH3

O

H3C

HO H3C

OH O

O

O

OH O

CH3

O

OH O

CH3

OH O

O

CH3

O

O O O

H3C

O HO

H3C

CH3

O O

CH3

OH

Tigloside Amycolatopsis sp.

Fig. 2.13 continued

38

O

CH3

2.2 Screening methods and novel compounds Simocyclinones

CH3

O

O

Streptomyces antibioticus TuÈ 6040

HO

Simocyclinones A

O

A1: R = H A2: R = OH OH

OH

R

O

O R2 = H R2 = H R2 = COCH3

CH3

HO

Simocyclinones B B1: R1 = H B3: R1 = OH B4: R1 = OH

OH

R2

H3C

O

O

O

OH

HO OH

OH

R1

Simocyclinones C C2: R1 = OH C3: R1 = H C4: R1 = OH

R2 = H R2 = COCH3 R2 = COCH3

HO R2

OH

CH3

O

O H3C

O

O

O

OH

O O OH

OH

R1

O R3 O

O

OH

O

O

CH3

HO R2

HN OH

H3C

O

O

O

OH

O O OH

OH

R1

O

Simocyclinones D D2: D3: D4: D6: D7: D8:

R1 = OH R1 = H R1 = OH R1 = OH R1 = H R1 = OH

R2 = H R2 = COCH3 R2 = COCH3 R2 = H R2 = COCH3 R2 = COCH3

R3 = H R3 = H R3 = H R3 = Cl R3 = Cl R3 = Cl

O O

O

OH

CH3 CH3

OH

O

OH

Kyanomycin

HN

Nonomuria sp. NN22303

O O P O C16-18H33-37

O

OH OH

O O

C13-16H27-33

Fig. 2.13 continued

O

39

2 Screening for New Secondary Metabolites from Microorganisms compound B3 is antibiotically inactive against Gram-positive and Gram-negative bacteria, but D3 shows a moderate activity against Bacillus subtilis and Arthrobacter aurescens. The importance of the new compounds is based in their role as key intermediates and in the elucidation of the biosynthetic pathway of elloramycins and tetracenomycins. Seven phenazine compounds were isolated from Streptomyces antibioticus Tü 2706. Besides saphenamycin, saphenic acid and tubermycin B, three new phenazines were detected by HPLC-DAD, 6-acetylphenazine-1-carboxylic acid, saphenic acid methyl ether and a group of eight saphenyl fatty acid esters [60]. A great success of HPLC-DAD screening was the identification of new nikkomycin compounds in mutants of Streptomyces tendae Tü 901. Twenty new nikkomycins were detected by this method [61–67] allowing intensive studies on structure activity relationships [68] and getting new insights in the biosynthetic pathway of nikkomycins. A further new compound from the juglomycin family, juglomycin Z, was detected in the nikkomycin producing strain Streptomyces tendae Tü 901, when the organism was grown under modified nutrition conditions [69]. The naphthoquinone antibiotic shows biological activity against Gram-positive and Gram-negative bacteria and against yeasts. A series of eight new obscurolides was detected besides the main compounds A2 and A3 in Streptomyces viridochromogenes Tü 2580 [43, 70]. B4 is the most active obscurolide in the phosphodiesterase assay. All obscurolides revealed no growth inhibiting potency against bacteria, yeasts and filamentous fungi. In the naphthgeranine producing strain Streptomyces violaceus Tü 3556 besides tubermycin B and 1-phenazinecarboxylate, a new minor congener naphthgeranine F, as well as 1-(3-indolyl)-2,3-dihydroxypropan-1-one, which was not previously described as a natural product, were detected [71]. Naphthgeranine F showed a similar antibacterial activity against Gram-positive bacteria as naphthgeranine C, the main compound in fermentations of strain Tü 3556, whereas 1-(3-indolyl)-2,3-dihydroxypropan-1-one shows no biological activity. A new member of the quinoxaline group antibiotics, echinoserine, was detected in strain Streptomyces tendae Tü 4031 [72]. The new compound is a noncyclic form of echinomycin, but is not a biosynthetic precursor. Echinoserine is less antibiotically active than echinomycin. Dioxolides, a novel class of secondary metabolites, were detected in the culture filtrate of Streptomyces tendae Tü 4042 [73]. Besides dioxolides, which consist of an unusual substituted dioxolane ring, para-hydroxybenzamide was detected, which was not yet described as a natural product. All compounds show no biological activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi. The kanchanamycins, a group of novel 36-membered polyol macrolide antibiotics were detected in Streptomyces olivaceus Tü 4018 [74, 75]. The compounds show antibacterial and antifungal activities, and are especially effective against Pseudomonas. In the same strain the fatty acid (E)-4-oxonon-2-enoic acid was detected, isolated and determined in structure [76]. This new second40

2.3 Increasing structural diversity by directed fermentations ary metabolite shows an antibacterial activity against various Gram-positive and Gram-negative bacteria, especially against Staphylococcus aureus, but not against yeasts and other fungi with the exception of Paecilomyces variotii. Streptosporangium cinnabarinum ATCC 31213 was characterised as a producer of the known antibiotics 43,334 and 43,596, but also as a producer of the new naphthalene compound 1-hydroxy-4-methoxy-2-naphthoic acid, which shows herbicidal activity in the Lemna minor assay [77]. In the culture filtrates and extracts of Streptomyces violaceusniger Tü 4113 the new secondary metabolite spirofungin was detected, a compound having a polyketide-spiroketal structure that shows various antifungal activities, particularly against yeasts [78]. Tigloside, a new tigloylated tetrasaccharide was detected in Amycolatopsis sp. NN0 21702 [79]. This secondary metabolite shows unusual structural elements, which have never before been isolated from Actinomycete strains. Until now, no biological activity could be observed. Two fatty acids, (2E,4Z)-decadienoic acid and (2E,4Z,7Z)-decatrienoic acid, the latter one being described for the first time as a natural product, were detected in the culture filtrate of Streptomyces viridochromogenes Tü 6105 [80]. Both metabolites show strong herbicidal activities against Lemna minor and Lepidium sativum. Simocyclinones, a novel group of angucyclinones, were detected during the fermentation of Streptomyces antibioticus Tü 6040 [81–84]. They are novel natural “hybrid” polyketide antibiotics and consist of an unusual angucyclinone ring with a tetraene side chain and a coumarin ring. Simocyclinones can be subdivided in A-, B-, C- and D-series regarding structures and UV-visible spectra. Compounds of the D-series are distinguished by an activity against Gram-positive bacteria and against several tumor cell lines. Kyanomycin, a blue-coloured secondary metabolite, was detected in the mycelium extract of Nonomuria sp. by HPLC-DAD and HPLC-ESI-MS screening and determined as an unusual anthracycline-phosphatidylethanolamine hybrid that shows weak antibacterial activity [85].

2.3 Increasing structural diversity by directed fermentations 2.3.1 Biomodification of saphenamycin and esmeraldine Streptomyces antibioticus Tü 2706 produces the antibacterial and antitumor active saphenamycin, a phenazine compound, and the cytotoxic active esmeraldine B (see Fig. 2.5). Esmeraldine is the condensation product of one molecule saphenic acid and one molecule saphenamycin. As saphenamycin is a methylsalicylic acid ester of saphenic acid, esmeraldine B shows the same methylsalicyclic acid side-chain than saphenamycin. It was of interest to investigate whether strain Tü 2706 modifies this side chain when derivatives of methylsalicylic acid 41

2 Screening for New Secondary Metabolites from Microorganisms were fed during directed fermentations, and if modified saphenamycins and esmeraldins show an altered antibacterial and antitumor spectrum. Biomodification was achieved by feeding acetylsalicylic acid, 3-methylsalicylic acid, 4-methylsalicylic acid, 5-fluorosalicylic acid, 5-chlorosalicylic acid and 5-bromosalicylic acid, resulting in six new saphenamycin and six new esmeraldine compounds. No incorporation into the molecules was achieved by feeding 5-iodosalicylic acid, 5-bromo-4-hydroxysalicylic acid, 3-hydroxysalicylic acid, 3-methoxysalicylic acid, 3,5-dinitrosalicyclic acid, 4-aminosalicylic acid, 5sulfosalicylic acid, and thiosalicylic acid [86]. The structural modifications are summarised in Fig. 2.14. All saphenamycin derivatives show an antibacterial activity, however, only 4-methylsaphenamycin is as active as saphenamycin. The cytotoxic activity towards an urinary bladder carcinoma cell line was lower in case of the derivatives than with saphenamycin. In comparison to esmeraldine B, 4-methyl- and 5-fluoro-esmeraldine B show an altered antibacterial spectrum and an increased antibacterial activity. 3-Methylesmeraldine B shows a higher cytotoxic activity against the tested tumor cell line than the original esmeraldine B [86].

2.3.2 Biomodification of ferrioxamines Streptomyces olivaceus Tü 2718 is naturally overproducing the iron-chelating compound desferrioxamine E, also known as the antibiotically active nocardamine. Optimisation of the fermentation conditions yielded in amounts of more than 10 g per litre desferrioxamine E. The siderophore consists of three mole succinic acid and three mole L-lysine, forming a trihydroxamate ring. Desferrioxamine E shows the strongest iron(III)-chelating complexing constant related to desferrioxamines A–I. The specificity for the incorporation of diamines containing two to six carbon atoms was investigated by feeding the following diamines: 1,2-diamine ethane, 1,3-diamine propane, 1,4-diamine butane (putrescine), 1,5-diamine pentane (cadaverine), 1,6-diamine hexane, 1,7-diamine heptane, 1,8-diamine octane, 1,5-diamine ethylether, S-2-aminoethyl cysteine, and N-glycin-1,2-ethylenediamine. The incorporation was monitored by HPLC-DAD that allowed the detection of all modified ferrioxamines [87]. Diamines with a space of more than four carbon atoms between the amino groups were not incorporated into the molecule, such as diamines with more than six carbon atoms. All other diamines were incorporated and led to the isolation and identification of 13 new ferrioxamines besides ferrioxamine D2 and E [88], and were determined in their differences of iron-complexation [89]. The new desferrioxamine structures are shown in Fig. 2.15.

42

2.3 Increasing structural diversity by directed fermentations R2 R3

R1 H3C

O R4

N

O

OR5

N COOH

R2 R1 CH3 H3C

HN HOOC

N

N

R3

O R4 O

OR5

N COOH

Figure 2.14: Biomodifications of saphenamycin and esmeraldine B by directed fermentations with Streptomyces antibioticus Tü 2706.

43

2 Screening for New Secondary Metabolites from Microorganisms O NH

C

R3

(CH2)2 N

C OH

O

C (CH2)2 C

O

HO

O

R4 N R1 NH

N

R2

NH

O C (CH2)2 C O

Figure 2.15: Desferrioxamine structures produced by directed fermentations with strain Streptomyces olivaceus Tü 2717.

44

Acknowledgments

2.3.3 Biomodification of rhizoferrins Although rhizoferrin represents a fairly simple molecule that consists of two molecules of citric acid linked to 1,4-diaminobutane through two amide bonds (see Fig. 2.3), it may have potential application in biotechnology due to its appreciable metal-binding properties and the ability to be easily degraded by various microorganisms. The specificity of the biosynthetic enzymes of the rhizoferrin producing fungus Cunninghamella elegans was investigated by modifying both, the chain length of the diamine and the citric acid part of the molecule [21]. Variations in chain length of the diamine backbone were very well tolerated. Branching of functionalization in b-position to the amino group of the diamine compounds was also accepted. However, it was not possible to introduce aamino acids. Therefore, the biosynthesis of rhizoferrin must be different to the biosynthesis of staphyloferrin A, produced by staphylococci. Neither by feeding of D-ornithine nor by application of inhibitors of ornithine decarboxylase, with and without simultaneous addition of D-ornithine, it was possible to detect even trace amounts of staphyloferrin A in Cunninghamella elegans. A higher degree of enzyme specificity was involved in the formation of the activated citryl species since analogues of citric acid are more difficult to be incorporated into rhizoferrin analogues than diamines. The structures are summarised in Fig. 2.16 (series A are structures modified in the diamine part, series B are structures modified in the citric acid part). All derivatives obtained by directed fermentations showed similar iron-chelating properties.

Acknowledgments Following scientists from the “Mikrobiologie/Antibiotika” group contributed to the success in search for novel secondary metabolites: Post-docs: J. Bielecki, C. Bormann, Z. Chen, H. Decker, H. Drautz, U. Fauth, M. Harder, T. Hörner, J. Müller, A. Plaga, O. Potterat, T. Schüz, and U. Theobald. Doctoral students: M. Alverado-Kirigin, N. Andres, S. Blum, M. Brandl, D. Braun, K. Burkhardt, I. Cebulla, H.-J. Cullmann, H. Haag, U. Hartjen, H. Hoff, W. Huhn, C. Isselhorst-Scharr, O. Jung,W. Katzer,W. Kuhn, J. Meiwes, F. Petersen, C. Pfefferle, U. Pfefferle, P. Reuschenbach, M. Richter, J. Schimana, P. Schneider, U. Schneider, A. Seiffert, J. Stümpfel, M. Tschierske, B. Wahl, and F. Walz. The excellent collaboration with the groups of Walter Keller-Schierlein (ETH Zürich), Axel Zeeck and Jürgen Rohr (Universität Göttingen), Günther Jung and Jörg W. Metzger (Universität Tübingen), Wilfried König (Universität Hamburg), Urs Séquin (Universität Basel), and Gerhard Bringmann (Universität Würzburg), which were involved in structure elucidation of isolated secondary metabolites, is gratefully acknowledged. 45

2 Screening for New Secondary Metabolites from Microorganisms A-Series

B-Series

H N

O

H N

COO-

O

O

COO-

HO

C

C

-

C

OOC

OH

H

-

COO-

H N

H N

COO-

O

OH

H

-

H N

H N

O

COO-

O

H C

-

OOC -

OOC

COO-

C

C

OOC

CH3

-

COO-

H N CH3

O

C

OOC

OOC

OOC

2-Methylhomorhizoferrin

H N

COOC CH3

-

COO-

-

H N

O

HO -

OH

C

OOC

Monomethylmonodesoxyrhizoferrin

Oxahomorhizoferrin

C

O

HO -

COO-

OOC

H N

H N

H N

O

HO -

OH

COO-

O

Didesoxyrhizoferrin

C

O

H N

COO-

OOC

Norrhizoferrin

O

OOC

COOC

OOC

COO-

-

H N

O

C

-

C

OOC

Monodesoxyrhizoferrin

HO

C

O

HO -

COO-

OOC

Homorhizoferrin

O

H N

H N

COO-

H3C -

O

C

OOC -

OOC

Dimethyldidesoxyrhizoferrin

O H N

O

H N

COOC OH COO-

O

HO C

-

OOC -

OOC

2-Oxorhizoferrin

Figure 2.16: Rhizoferrin structures produced by directed fermentations with Cunninghamella elegans.

46

References

References

1. Bérdy, J. (1995) Are actinomycetes exhausted as a source of secondary metabolites? Biotekhnologiya 8, 13–34. 2. Hill, D. C., Wrigley, S. K., and Nisbet, L. J. (1998) Novel screen methodologies for identification of new microbial metabolites with pharmaceutical activity. Adv. Biochem. Engin. Biotechnol. 59, 73–121. 3. Umezawa, S., Tsuchiya, T., Tatsuta, K., Horiuchi, Y., Usui, T., Umezawa, H., Hamada, M., and Yagi, A. (1970) A new antibiotic, dienomycin. I. Screening method, isolation and chemical studies. J. Antibiot. 23, 20–27. 4. Omura, S., Iwai, Y., Hirano, A., Nakagawa, A., Awaya, J., Tsuchiya, H., Takahashi, Y., and Masuma, R. (1977) A new alkaloid AM-2282 of Streptomyces origin, taxonomy, fermentation, isolation and preliminary characterization. J. Antibiot. 30, 275–282. 5. Drautz, H. and Zähner, H. (1981) A new method of screening for secondary metabolites in Streptomycetes. In: Actinomycetes, Zbl. Bakt. Suppl., Vol. 11 (Schaal, K. P. and Pulverer, G., eds), Gustav Fischer Verlag, Stuttgart, pp. 515–519. 6. Zähner, H. and Drautz, H. (1985) Methods in the search for new secondary metabolites from microorganisms – a comparison. In: VIII International Symposium on Medicinal Chemistry, Proceedings Vol 2 (Dahlbohm, R. and Nilsson, J. L. G., eds.), Swedish Pharmaceutical Press, Stockholm, pp. 49–68. 7. Fiedler, H.-P. (1993) Screening for secondary metabolites by HPLC and UV-visible absorbance spectral libraries. Nat. Prod. Lett. 2, 119–128. 8. Grote, R., Chen, Y., Zeeck, A., Chen, Z., and Zähner, H. (1988) Pyridazomycin, a new antifungal antibiotic produced by Streptomyces violaceoniger. J. Antibiot. 41, 595–601. 9. Rapp, C., Jung, G., Katzer, W., and Loeffler, W. (1988) Chlorotetain aus Bacillus subtilis, ein antifungisches Dipeptid mit einer ungewöhnlichen chlorierten Aminosäure. Angew. Chem. 100, 1801–1802. 10. Potterat, O., Stephan, H., Metzger, J. W., Gnau, V., Zähner, H., and Jung, G. (1994) Aborycin – a tricyclic 21-peptide antibiotic isolated from Streptomyces griseoflavus. Liebigs Ann. Chem. 1994, 741–743. 11. Fiedler, H.-P., Schüz, T., and Decker, H. (1993) An overview of nikkomycins: history, biochemistry, and applications. In: Cutaneous Antifungal Agents: Compounds in Clinical Practice and Development (Rippon, J. W. and Fromtling, R. A., eds), Marcel Dekker, New York, pp. 325–352. 12. Fauth, U., Zähner, H., Mühlenfeld, A., and Achenbach, H. (1986) Galbonolides A and B – two non-glycosidic antifungal macrolides. J. Antibiot. 39, 1760–1764. 13. Achenbach, H., Mühlenfeld, A., Fauth, U., and Zähner H. (1988) The galbonolids. Novel, powerful antifungal macrolides from Streptomyces galbus ssp. eurothermus. Ann. New York Acad. Sci. 544, 128–140. 14. Keller-Schierlein, W., Hagman, L., Zähner, H., and Huhn, W. (1988) Maduraferrin, ein neuartiger Siderophor aus Actinomadura madurae. Helv. Chim. Acta 71, 1528–1540. 15. Meiwes, J., Fiedler, H.-P., Haag, H., Zähner, H., Konetschny-Rapp, S., and Jung, G. (1990) Isolation and characterization of staphyloferrin A, a compound with siderophore activity from Staphylococcus hyicus DSM 20459. FEMS Microbiol. Lett. 67, 201–206. 16. Konetschny-Rapp, S., Jung, G., Meiwes, J., and Zähner, H. (1990) Staphyloferrin A: a structurally new siderophore from staphylococci. Eur. J. Biochem. 191, 65–74. 17. Drechsel, H., Freund, S., Nicholson, G., Haag, H., Jung, O., Zähner, H., and Jung, G. (1993) Purification and chemical characterization of staphyloferrin B, a hydrophilic siderophore from staphylococci. BioMetals 6, 185–191.

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2 Screening for New Secondary Metabolites from Microorganisms 18. Haag, H., Fiedler, H.-P., Meiwes, J., Drechsel, H., Jung, G., and Zähner, H. (1994) Isolation and biological characterization of staphyloferrin B, a compound with siderophore activity from staphylococci. FEMS Microbiol. Lett. 115, 125–130. 19. Seiffert, A. and Zähner, H. (1992) Siderophore production in alkalophilic bacilli. In: Dechema Biotechnology Conferences, Vol. 5 (Kreysa, G. and Driesel, A. J., eds.), VCH, Weinheim, pp. 725–728. 20. Drechsel, H., Metzger, J., Freund, S., Jung, G., Boelart, J. R., and Winkelmann, G. (1991) Rhizoferrin – a novel siderophore from the fungus Rhizopus mircrosporus var. rhizopodiformis. BioMetals 4, 238–243. 21. Drechsel, H., Tschierske, M., Thieken, A., Jung, G., Zähner, H., and Winkelmann, G. (1995) The carboxylate type siderophore rhizoferrin and its analogs produced by directed fermentation. J. Industr. Microbiol. 14, 105–112. 22. Drechsel, H., Stephan, H., Lotz, R., Haag, H., Zähner, H., Hantke, K., and Jung, G. (1995) Structure elucidation of yersiniabactin, a siderophore from highly virulent Yersinia strains. Liebigs Ann. 1995, 1727–1733. 23. Cebulla. I. (1995) Gewinnung komplexbildender Substanzen mittels Amycolatopsis orientalis. Doctoral Thesis, Universität Tübingen. 24. Andres, N., Wolf, H., Zähner, H., Rössner, E., and Zeeck, A. (1989) Hormaomycin, ein neues Peptid-lacton mit morphogener Aktivität auf Streptomyceten. Helv. Chim. Acta 72, 426–437. 25. Andres, N., Wolf, H., and Zähner, H. (1990) Hormaomycin, a new peptide lactone antibiotic effective in inducing cytodifferentiation and antibiotic biosynthesis in some Streptomyces species. Z. Naturforsch. 45 c, 851–855. 26. Petersen, F., Zähner, H., Metzger, J. W., Freund, S., and Hummel, R.-P. (1993) Germicidin, an autoregulative germination inhibitor of Streptomyces viridochromogenes NRRL B-1551. J. Antibiot. 46, 1126–1138. 27. Richter, M., Willey, J. M., Süssmuth, R., Jung, G., and Fiedler, H.-P. (1998) Streptofactin, a novel biosurfactant with aerial mycelium inducing acitivity from Streptomyces tendae Tü 901/8 c. FEMS Microbiol. Lett. 163, 165–171. 28. Drautz, H., Zähner, H., Rohr, J., and Zeeck, A. (1986) Urdamycins, new angucycline antibiotics from Streptomyces fradiae. I. Isolation, characterization and biological properties. J. Antibiot. 39, 1657–1669. 29. Zeeck, A., Rohr, J., Sheldrick, G. M., Jones, P. G., and Paulus, E. F. (1986). Structure of the new antibiotic and cytotoxic indicator substance, urdamycin A. J. Chem. Res. (Synopsis) 1986, 104–105. 30. Rohr, J. and Zeeck, A. (1987) Urdamycins, new angucycline antibiotics from Streptomyces fradiae. II. Structural studies of urdamycins B to F. J. Antibiot. 40, 459–467. 31. Rohr, J., Zeeck, A., and Floss, H. G. (1988) Urdamycins, new angucycline antibiotics from Streptomyces fradiae. II. The structures of urdamycins C and D. J. Antibiot. 41, 126–129. 32. Rohr, J., Beale, J. M., and Floss, H. (1989) Urdamycins, new angucycline antibiotics from Streptomyces fradiae. IV. Biosynthetic studies of urdamycins A–D. J. Antibiot. 42, 1151–1157. 33. Korff, U. (1988) Strukturaufklärung, Synthese und Derivatisierung eines Pigments aus dem Stamm Tü 2484. Doctoral Thesis, Universität Tübingen. 34. Alvarado-Kirigin, M. (1990) Monomeres und dimeres Cinnachinon aus Streptomyces griseoflavus (Tü 2484). Doctoral Thesis, Universität Tübingen. 35. Keller-Schierlein, W., Geiger, A., Zähner, H., and Brandl, M. (1988) Die Esmeraldine A und B, tief grüne Farbstoffe aus Streptomyces antibioticus, Stamm TÜ 2706. Helv. Chim. Acta 71, 2058–2070. 36. Wessels, P., Göhrt, A., Zeeck, A., Drautz, H., and Zähner, H. (1991) Naphthgeranines, new naphthoquinone antibiotics from Streptomyces sp. J. Antibiot. 44, 1013–1018. 37. Ritzau, M., Drautz, H., Zähner, H., and Zeeck, A. (1993) Serpentene, a novel polyene carboxylic acid from Streptomyces. Liebigs Ann. Chem. 1993, 433–435.

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References 38. Hagmann, L., Keller-Schierlein, W., Wahl, B., and Zähner, H. (1988) Pyridindolol glucosides from Streptomyces parvulus. J. Antibiot. 41, 289–295. 39. Gröne, C. (1991) Strukturaufklärung der Depsichlorine: Neue Depsipeptid-Antibiotika aus Streptomyces Tü 1661. Doctoral Thesis, Universität Göttingen. 40. Cullmann, H. J. (1994) Depsichlorine und andere Metabolite aus Streptomyces antibioticus Tü 1661. Doctoral Thesis, Universität Tübingen. 41. Keller-Schierlein, W., Müller, A., Hagmann, L., Schneider, U., and Zähner, H. (1985) (E)-3-(1H-Pyrrol-3-yl)-2-propensäure und (E)-3-(1H-Pyrrol-3-yl)-2-propensäureamid aus Streptomyces parvulus. Helv. Chim. Acta 68, 559–562. 42. Grote, R., Zeeck, A., Stümpfel, J., and Zähner, H. (1990) Pyrrolams, new pyrrolizidinones produced by Streptomyces olivaceus. Liebigs Ann. Chem. 1990, 525–530. 43. Hoff, H., Drautz, H., Fiedler, H.-P., Zähner, H., Schultz, J. E., Keller-Schierlein, W., Philipps, S., Ritzau, M., and Zeeck, A. (1992) Obscurolides, a novel class of phosphodiesterase inhibitors from Streptomyces. I. Production, isolation, structural elucidation and biological activity of obscurolides A1 to A4. J. Antibiot. 45, 1096–1107. 44. Potterat, O., Zähner, H., Metzger, J. W., and Freund, S. (1994) 5-Phenylpentadienoicacid derivatives from Streptomyces sp. Helv. Chim. Acta 77, 569–574. 45. Keller-Schierlein,W., Bahnmüller, U., Dobler, M., Bielecki, J., Stümpfel, J., and Zähner, H. (1986) Isolierung und Strukturaufklärung von Differolid. Helv. Chim. Acta 69, 1833–1836. 46. Müller, A., Keller-Schierlein, W., Bielecki, J., Rak, G., Stümpfel, J., and Zähner, H. (1988) (2S,3R,4R,6R)-2,3,4-Trihydroxy-6-methylcyclohexanon aus zwei ActinomycetenStämmen. Helv. Chim. Acta 69, 1829–1832. 47. Rapp, C., Jung, G., Isselhorst-Scharr, C., and Zähner, H. (1988) A new member of the class of antibiotics with thiotetronic acid structure isolated from Streptomyces olivaceus TÜ 3010. Liebigs Ann. Chem. 1988, 1043–1047. 48. Grote, R., Zeeck, A., Drautz, H., and Zähner, H. (1988) Colabomycins, new antibiotics of the manumycin group from Streptomyces griseoflavus. I. Isolation, characterization and biological properties. J. Antibiot. 41, 1178–1185. 49. Grote, R., Zeeck, A., and Beale Jr., J. M. (1988) Colabomycins, new antibiotics of the manumycin group from Streptomyces griseoflavus. II. Structure of colabomycin A. J. Antibiot. 41, 1186–1195. 50. Burkhardt, K., Fiedler, H.-P., Grabley, S., Thiericke, R., and Zeeck, A. (1996) New cineromycins and musacins obtained by metabolite pattern analysis of Streptomyces griseoviridis (FH-S 1832). I. Taxonomy, fermentation, isolation and biological activity. J. Antibiot. 49, 432–437. 51. Grote, R., Zeeck, A., Drautz, H., and Zähner, H. (1988) 2880-II, a metabolite related to ferulic acid from Streptomyces griseoflavus. J. Antibiot. 41, 1275–1276. 52. Niederer, D., Séquin, U., Drautz, H., and Zähner, H. (1990) (3S,5R,6E,8E)-Deca-6,8dien-1,3,5-triol, ein neues Stoffwechselprodukt von Streptomyces fimbriatus (Millard et Burr 1926). Helv. Chim. Acta 73, 2129–2135. 53. Wessels, P. (1990) Strukturaufklärung neuer Naphthochinon-Antibiotika aus Streptomyces Tü 3556 und Beiträge zum chemischen Screening mit extrem thermophilen Archaebakterien. Doctoral Thesis, Universität Göttingen. 54. Potterat, O., Zähner, H., Volkmann, C., and Zeeck, A. (1993) Exfoliamycin and related metabolites, new naphthoquinone antibiotics from Streptomyces exfoliatus. J. Antibiot. 46, 346–349. 55. Volkmann, C., Zeeck, A., Potterat, O., Zähner, H., Bohnen, F.-M., and Herbst-Irmer, R. (1995) The structures of the exfoliamycins. J. Antibiot. 48, 431–432. 56. Braun, D., Pauli, N., Séquin, U., and Zähner, H. (1995) New butenolides from the photoconductivity screening of Streptomyces antibioticus (Waksman and Woodruff). Waksman and Henrici 1948. FEMS Microbiol. Lett. 126, 37–42. 57. Fiedler, H.-P., Rohr, A., and Zeeck, A. (1986) Elloramycins B, C, D, E and F: minor congeners of the elloramycin producer Streptomyces olivaceus. J. Antibiot. 39, 856–859.

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2 Screening for New Secondary Metabolites from Microorganisms 58. Fiedler, H.-P. (1986) Identification of new elloramycins, anthracycline-like antibiotics, in biological cultures by high-performance liquid chromatography and diode array detection. J. Chromatogr. 361, 432–436. 59. Rohr, J., Eick, S., Zeeck, A., Reuschenbach, P., Zähner, H., and Fiedler, H.-P. (1988) Tetracenomycins B3 and D3, key intermediates of the elloramycin and tetracenomycin C biosynthesis. J. Antibiot. 41, 1066–1073. 60. Geiger, A., Keller-Schierlein, W., Brandl, M., and Zähner, H. (1988) Phenazines from Streptomyces antibioticus strain TÜ 2706. J. Antibiot. 41, 1542–1551. 61. König, W. A., Hahn, H., Rathmann, R., Hass, W., Keckeisen, A., Hagenmaier, H., Bormann, C., Dehler, W., Kurth, R., and Zähner, H. (1996) Drei neue Aminosäuren aus dem Nikkomycin-Komplex – Strukturaufklärung und Synthese. Liebigs Ann. Chem. 1986, 407–421. 62. Decker, H., Bormann, C., Fiedler, H.-P, Zähner, H., Heitsch, H., and König, W. A. (1989) Isolation of new nikkomycins from Streptomyces tendae. J. Antibiot. 42, 230– 235. 63. Heitsch, H., König, W. A., Decker, H., Bormann, C., Fiedler, H.-P., and Zähner, H. (1989) Structure of the new nikkomycins pseudo-Z and pseudo-J. J. Antibiot. 42, 711–717. 64. Bormann, C., Mattern, S., Schrempf, H., Fiedler, H.-P., and Zähner, H. (1989) Isolation of Streptomyces tendae mutants with an altered nikkomycin spectrum. J. Antibiot. 42, 913–918. 65. Decker, H., Walz, F., Bormann, C., Zähner, H., and Fiedler, H.-P. (1990) Nikkomycins Wz and Wx, new chitin synthetase inhibitors from Streptomyces tendae. J. Antibiot. 43, 43–48. 66. Decker, H., Pfefferle, U., Bormann, C., Zähner, H., Fiedler, H.-P., van Pée, K.-H., Rieck, M., and König, W. A. (1991) Enzymatic bromination of nikkomycin Z. J. Antibiot. 44, 626–634. 67. Schüz, T. C., Fiedler, H.-P., Zähner, H., Rieck, M., and König, W A. (1992) Nikkomycins Sz, Sx, Soz and Sox, new intermediates associated to the nikkomycin biosynthesis. J. Antibiot. 45, 199–206. 68. Decker, H., Zähner, H., Heitsch, H., König, W. A., and Fiedler, H.-P. (1991) Structureactivity relationships of the nikkomycins. J. Gen. Microbiol. 137, 1805–1813. 69. Fiedler, H.-P., Kulik, A., Schüz, T. C., Volkmann, C., and Zeeck, A. (1994) Juglomycin Z, a new naphthoquinone antibiotic from Streptomyces tendae. J. Antibiot. 47, 1166– 1122. 70. Ritzau, M., Philipps, S., Zeeck, A., Hoff, H., and Zähner, H. (1993) Obscurolides, a novel class of phosphodiesterase inhibitors from Streptomyces. II. Minor components belonging to the obscurolide B to D series. J. Antibiot. 46, 1625–1628. 71. Volkmann, C., Hartjen, U., Zeeck, A., and Fiedler, H.-P. (1995) Naphthgeranine F, a minor congener of the naphthgeranine group produced by Streptomyces violaceus. J. Antibiot. 48, 522–524. 72. Blum, S., Fiedler, H.-P., Groth, I., Kempter, C., Stephan, H., Nicholson, G., Metzger, J. W., and Jung, G. (1995) Echinoserine, a new member of the quinoxaline group, produced by Streptomyces tendae. J. Antibiot. 48, 619–625. 73. Blum, S., Groth, I., Rohr, J., and Fiedler, H.-P. (1996) Dioxolides, novel secondary metabolites from Streptomyces tendae. J. Basic Microbiol. 36, 19–25. 74. Fiedler, H.-P., Nega, M., Pfefferle, C., Groth, I., Kempter, C., Stephan, H., and Metzger, J. W. (1996) Kanchanamycins, new polyol macrolide antibiotics produced by Streptomyces olivaceus Tü 4018. I. Taxonomy, fermentation, isolation and biological activities. J. Antibiot. 49, 101–107. 75. Stephan, H., Kempter, C., Metzger, J. W., Jung, G., Potterat, O., Pfefferle, C., and Fiedler, H.-P. (1996) Kanchanamycins, new polyol macrolide antibiotics produced by Streptomyces olivaceus Tü 4018. II. Structure elucidation. J. Antibiot. 49, 109–113.

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References 76. Pfefferle, C., Kempter, C., Metzger, J. W., and Fiedler, H.-P. (1996) (E)-4-oxonon-2enoic acid, an antibiotically active fatty acid produced by Streptomyces olivaceus Tü 4018. J. Antibiot. 49, 135–137. 77. Pfefferle, C., Breinholt, J., Gürtler, H., and Fiedler, H.-P. (1997) 1-Hydroxy-4-methoxy2-naphthoic acid, a herbicidal compound produced by Streptosporangium cinnabarinum ATCC 31213. J. Antibiot. 50, 1067–1068. 78. Höltzel, A., Kempter, C., Metzger, J. W., Jung, G., Groth, I., Fritz, T., and Fiedler, H.P. (1998) Spirofungin, a new antifungal antibiotic from Streptomyces violaceusniger Tü 4113. J. Antibiot. 51, 487–495. 79. Breinholt, J., Kulik, A., Gürtler, H., and Fiedler, H.-P. (1998) Tigloside: a new tigloylated tetrasaccharide from Amycolatopsis sp. Acta Chem. Scand. 52, 1239–1242. 80. Maier, A., Müller, J., Schneider, P., Fiedler, H.-P., Groth, I., Tayman, F. S. K., Teltschik, F., Günther, C., and Bringmann, G. (1999) (2E,4Z)-Decadienoic acid and (2E,4Z,7Z)decatrienoic acid, two herbicidal metabolites from Streptomyces viridochromogenes Tü 6105. Pesticide Sci. 55, 733–739. 81. Schimana, J., Fiedler, H.-P., Groth, I., Süßmuth, R., Beil, W., Walker, M., and Zeeck, A. (2000) Simocyclinones, novel cytostatic angucyclinone antibiotics produced by Streptomyces antibioticus Tü 6040. I. Taxonomy, fermentation, isolation and biological activities. J. Antibiot. 53, 779–787 82. Walker, M., Zeeck, A., Schimana, J., and Fiedler, H.-P. (2000) Simocyclinones, novel cytostatic angucyclinone antibiotics produced by Streptomyces antibioticus Tü 6040. II. Structure determination and biosynthesis. J. Antibiot., in press. 83. Walker, M., Schimana, J., Süßmuth, R., Beil, W., Fiedler, H.-P., and Zeeck, A. (2000) New simocyclinones of the A-, B-, C- and D-series, novel angucyclinone antibiotics from Streptomyces antibioticus Tü 6040. J. Antibiot., in press. 84. Schimana, J., Walker, M., Zeeck, A., and Fiedler, H.-P. (2000) Simocyclinones: diversity of metabolites is dependent on fermentation conditions. J. Ind. Microbiol. Biotechnol., in press. 85. Pfefferle, C., Breinholt, J., Olsen, C. E., Kroppenstedt, R. M., Gürtler, H., and Fiedler, H.-P. (2000) Kyanomycin, a complex of unusual anthracycline-phospholipid hybrid from Nonomuria species. J. Nat. Prod. 63, 295–298. 86. Kuhn, W. (1993) Untersuchungen zur Produktion der Esmeraldine und der Physiologie von Streptomyces antibioticus. Doctoral Thesis, Universität Tübingen. 87. Fiedler, H.-P., Meiwes, J., Werner, I., Konetschny-Rapp, S., and Jung, G. (1990) Identification of new ferrioxamines by HPLC and diode array detection. J. Chromatogr. 513, 255–262. 88. Meiwes, J., Fiedler, H.-P., Zähner, H., Konetschny-Rapp, S., and Jung, G. (1990) Production of desferrioxamine E and new analogues by directed fermentation and feeding fermentation. Appl. Microbiol. Biotechnol. 32, 505–510. 89. Konetschny-Rapp, S., Jung, G., Raymond, K. N., Meiwes, J., and Zähner, H. (1992) Solution thermodynamics of the ferric complexes of new desferrioxamine siderophores obtained by directed fermentations. J. Am. Chem. Soc. 114, 2224–2230.

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3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin Friedrich Götz* and Günther Jung**

3.1 History of lantibiotics and lantibiotic research in Tübingen

The first lantibiotic structure elucidated was that of nisin [23], and nisin was probably the first lantibiotic identified [65]. Nisin is produced by the cheese starter culture organism Lactococcus lactis subsp. lactis and has an antimicrobial effect on a broad variety of Gram-positive bacteria [23]. The pioneering research of E. Gross and coworkers demonstrated that the peptide antibiotics nisin and subtilin, the latter produced by Bacillus subtilis, actually contain lanthionine (Lan) and 3-methyllanthionine (MeLan) as well as (Dha) and (Dhb), confirming previous hypotheses [6, 21, 22]. The lantibiotic era in Tübingen began in 1985 with the publication of the peptide sequence of epidermin isolated from Staphylococcus epidermidis. With the elucidation of the structure of epidermin, it became clear that it is a heterodet tetracyclic 22-amino acid (2164 Da), amide peptide [1] that contains one residue each of Dhb and MeLan and two residues of Lan. The fourth cyclic structure results from the novel C-terminal mono-carboxy, di-amino acid, AviCys. It was not clear at that time how these amino acid structures arose and how the complex structures of nisin and epidermin are synthesized. Are they non-ribosomally synthesized, as are gramidicin and valinomycin, which are typically synthesized by large multi-enzyme complexes in the cell and for which no structural genes exists [27, 33], or are they synthesized ribosomally as a precursor peptide, which is subsequently posttranslationally modified? In 1987, when Friedrich Götz took over the chair in Mikrobielle Genetik in Tübingen, he and his coworkers started to unravel the biosynthetic principles of

* Mikrobielle Genetik, Universität Tübingen, Waldhäuser Str. 70/8, D-72076 Tübingen ** Institut für Organische Chemie, Universität Tübingen, Auf der Morgenstelle 18, D-72076 Tübingen

52 Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

3.1 History of lantibiotics and lantibiotic research in Tübingen epidermin. In co-operation with Karl-Dieter Entian’s group, a gene corresponding to the epidermin amino acid sequence was identified on a 54-kb plasmid of the epidermin-producing strain [72]. These pioneering results brought lantibiotic research a big step forward. For the first time it was shown that a lantibiotic is ribosomally synthesized, and clues on the organization of epidermin as a precursor protein and the processing and post-translational modification steps were found. In the following years, the principles of the genetic organization and biosynthesis first found with epidermin were subsequently also found with other lantibiotics – all other lantibiotics studied later were also shown to be ribosomally synthesized. To date, antibiotics have been found in many Gram-positive genera, such as Bacillus, Lactococcus, Pediococcus, Staphylococcus, Streptococcus, and Streptomyces. Lantibiotics have not been found in a Gram-negative species. Subsequent studies of lantibiotics have revealed that they are post-translationally modified at specific positions to give rise to the large number of modified amino acids found in these peptides. In addition, the peptides are produced with a leader peptide, which is removed during maturation, and are transported by specific transport-related proteins out of the cell. Still other lantibiotic-specific proteins are involved in the genetic regulation of biosynthesis and generation of the specific producer-cell self-protection mechanism(s) frequently observed. The name “lantibiotics” was coined in Tübingen and refers to the rapidly expanding group of antibiotic-like peptides that contain the non-protein amino acids lanthionine and 3-methyllanthionine [72]. The discussion whether epidermin and nisin should be regarded as antibiotics or as bacteriocins was intense. The small size and compact structure of the compounds and the wide range of antibacterial activity against most Gram-positive and some Gram-negative bacteria favors consideration as antibiotics – bacteriocins are usually larger proteins with a narrow range of activity, such as colicins. On the other hand, typical amino-acid-derived antibiotics, such as penicillin and gramicidin, are non-ribosomally synthesized. With the knowledge available today, it is clear that lantibiotics fulfill criteria of both, antibiotics and bacteriocins and cannot be placed in one or the other category. The name lantibiotic belies the full extent and complexity of this class of bacterial peptides. A strong and very fruitful collaboration was developed with Hans-Georg Sahl (Bonn) and his coworkers. Since 1983, they had been studying the mode of action of Pep5 and nisin, and later also of gallidermin and other lantibiotics. Earlier studies suggested that nisin could interfere with the biosynthesis of the bacterial cell wall [46, 64]. This appears to be indeed true, as recent studies show [13]. However, the binding to cell wall precursors does not explain the observed bactericidal effect of lantibiotics. Type A lantibiotics form non-specific transmembrane pores in an energy-dependent fashion and allow efflux of preaccumulated intracellular components [66, 69, 73]. In 1984, Friedrich Götz, then at the Technical University in München, isolated an antibiotic compound from a Staphylococcus gallinarum strain, which belongs to a species previously described by Devriese et al. [17]. Preliminary 53

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin studies showed that this antibiotic compound exerts a rather broad activity against Gram-positive bacteria. However, it was not until he moved to Tübingen that the compound was isolated in the group of Hans Zähner and the structure was elucidated in the group of Günther Jung [28]. The compound was named gallidermin after the species name of the producing staphylococcus. Gallidermin is a structural analogue of epidermin, but is slightly more active than epidermin. Today, after long and laborious work to optimize the fermentation and thus increase gallidermin production approximately hundred-fold [29], S. gallinarum Tü 3928 is used for the biotechnological production of gallidermin by the groups of Peter Fiedler (fermentation) and Rolf Werner (downstream processing; Boehringer Ingelheim, Biberach). To say that in the years following 1986, Tübingen became a stronghold in lantibiotic research, by setting the pace of the research, inspiring many other groups, and initiating quite a number of national and international co-operations, is no exaggeration. Three international workshops on lantibiotics have already taken place. Günther Jung (Tübingen) initiated together with HansGeorg Sahl (Bonn) the first lantibiotic workshop, held in April 1991 in Bad Honnef. The contributions were published under the title “Nisin and novel lantibiotics” with Jung and Sahl as editors (Leiden: Escom). The second workshop on “Lantibiotics: a unique group of antibiotic peptides” was organized by Ruud Konigs and Cees Hilbers (Nijmegen, The Netherlands) and held in Arnhem in November 1994. The contributions were published in Antonie van Leeuwenhoek, vol. 69 in 1996. The third workshop on “Lantibiotics and related modified antibiotic peptides” was organized by Friedrich Götz, R. Jack, Günther Jung, and Hans-Georg Sahl and was held in Blaubeuren in April 1998. The ever present international interest in the lantibiotic subject is best documented by the increasing number of applicants and participants from workshop to workshop. A number of potential applications have been found for the mature lantibiotics, including the use as an anti-infective in medical and veterinary areas, in food, beverage and cosmetic preservation, and as regulators of both, human immune function and blood pressure [26, 50, 62, 67]. Lantibiotics represent new lead structures, but their chemical synthesis is too complicated and costly; therefore, the only way to produce large enough quantities for marketing is through biotechnology. In the following sections, more detailed information on lantibiotic research is presented. Since this is a final report on all achievements obtained in the framework of the collaborative research centre 323, we will focus on the results obtained by the groups in Tübingen. However, the picture would be incomplete if major achievements of other groups were not included. The topics on novel structures, mechanism(s) of biosynthesis, genetic organization and regulation, biological activities and mode of actions as well as potential applications for this fascinating, novel class of bacterial-derived, biologically active peptides will be addressed.

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3.2 Primary structure and proposed maturation of epidermin in staphylococci

3.2 Primary structure and proposed maturation of epidermin in staphylococci

The nucleotide sequence of the epidermin structural gene, epiA, revealed that epidermin is part of a pre-peptide that consists of a 30-amino-acid N-terminal leader peptide and a 22-amino-acid C-terminal propeptide [72]. A comparison of the chemical structure of epidermin [2] with the peptide sequence deduced from epiA indicates that this propeptide undergoes several modifications (Fig. 3.1) before it is transported out of the cell. At each pair of positions where the mature epidermin contains a lanthionine bridge, the precursor peptide contains one serine or threonine and one cysteine. Lanthionine is proposed to be formed in a two-step process involving dehydration of serine and threonine and the subsequent addition of a cysteine thiol group [10, 24].

Figure 3.1: Posttranslational modifications of the epidermin pre-peptide. (A) Model of lanthionine synthesis: dehydration of serine (a) is followed by a nucleophilic addition reaction of a thiol group, thereby forming a thioether bridge (b); the formation of meso-lanthionine from threonine is analogous. (B) Biosynthesis of epidermin: dehydration (a), nucleophilic addition (b), oxidative decarboxylation (c), and removal of the leader peptide (d). Abu, aminobutyric acid; Dhb, dehydrobutyrine.

55

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin

3.3 Genetic organization and regulation of the epidermin genes

3.3.1 Organization and function of epidermin genes The gene cluster for biosynthesis of the lantibiotic epidermin is composed of eleven genes in five transcription units. The genes are involved in 1) synthesis of the epidermin precursor peptide, 2) modification of the precursor peptide, 3) secretion of epidermin, 4) cleavage of the leader peptide, 5) immunity of the producer to epidermin, and 6) regulation of the gene cluster. Four of the transcription units are activated by the DNA-binding regulator EpiQ. The factors or conditions leading to activation of the EpiQ protein or its expression remain unclear. The gene cluster for epidermin biosynthesis, located on plasmid pTü32 of S. epidermidis Tü 3298 [72], contains genes for all activities proposed to be involved in the biosynthetic pathway (Fig. 3.2). Like all lantibiotics, epidermin is ribosomally synthesized and the gene cluster accordingly contains the structural gene epiA, which encodes the precursor peptide [70, 72]. In the same operon, the genes for modification reactions leading to the formation of thioether bridges (epiB and epiC) [4, 41, 58] and to oxidative decarboxylation of the C-terminus (epiD) [42, 43] are encoded. Between epiA and epiB, a weak terminatorlike structure reduces the expression of the subsequent genes [56, 70] whose products are required only in catalytic amounts. In a second transcriptional unit, the extracellular leader peptidase EpiP [20] and the regulator protein EpiQ [56] are encoded. Upstream of epiA, the individually expressed genes epiT and epiH are located [60]. The deduced product of epiT shares sequence similarity with an ABC-type transporter that is involved in secretion of certain peptides and proteins [75]. The coding sequence

Figure 3.2: Organization of the epidermin gene cluster encoded on plasmid pTü32. Epidermin genes are shown as white arrows; flanking genes with putative functions in plasmid replication (rep) and maintenance (resolvase) are indicated in gray. Gene functions: epiA, structural gene; epiB and epiC, dehydration and lanthionine formation; epiD, flavoprotein (oxidative decarboxylation); epiQ, activator; epiP, pro-epidermin processing protease; epiH and epiT, translocation (export) of pro-epidermin; epiFEG, epidermin immunity. Arrows indicate promoters that are activated by EpiQ. The epiT gene is incomplete due to a deletion and frame-shift mutation, but its function can be complemented by the intact gallidermin gene, gdmT.

56

3.3 Genetic organization and regulation of the epidermin genes is disrupted by two frame-shift deletions, and it is very questionable whether epiT has a function in epidermin biosynthesis. Transcriptional reporter gene fusions have demonstrated, however, that epiT is expressed (see below). The homologous gene gdmT from the gene cluster of the closely related lantibiotic gallidermin, has an intact sequence; it mediates an increase of epidermin production in the heterologous host Staphylococcus carnosus [60] and thereby substantiates the proposed capacity of GdmT to secrete epidermin or gallidermin. The gene adjacent to gdmT – gdmH – is also necessary for increased epidermin production; GdmH may be an accessory factor for secretion. EpiH and GdmH are hydrophobic proteins without conspicuous similarities to other proteins; they furthermore exert a limited level of immunity to epidermin [57]. Upstream of epiH, three cotranscribed genes, epiF, epiE, and epiG, are encoded. They mediate resistance (immunity) to epidermin, and their products very likely constitute the subunits of an ABC transporter that expels the harmful epidermin molecules from the cytoplasmic membrane [54, 57]. The epidermin genes are flanked by genes which apparently encode factors for replication and stability of plasmid pTü32 (Fig. 3.2). The res gene product has a high level of identity to plasmid resolvases, whose function is the resolution of cointegrates [58]. The open reading frame upstream of epiP shares significant similarity with plasmid replication genes from various Gram-positive bacteria (M. Hille and A. Peschel, unpublished). The epidermin gene cluster thus seems to be complete; however, several chromosomally encoded genes are also very likely involved in epidermin biosynthesis.

3.3.2 EpiQ is an unusual transcriptional regulator EpiQ shares similarities with DNA-binding proteins of the response regulator family [70] and binds to the epiABCD promoter region to activate expression [56]. Unlike conventional response regulators, EpiQ does not contain the conserved aspartic acid residue that is phosphorylated by a corresponding sensor kinase to activate the regulator [76]. Since a corresponding kinase gene is absent from the epidermin gene cluster, it is very questionable whether EpiQ is phosphorylated at all. Several other lantibiotic gene clusters encode a conventional kinase and regulator pair [75]; therefore, the regulator gene epiQ is unusual. The twocomponent regulatory systems of nisin and subtilin activate the biosynthetic genes in response to the extracellular concentration of the respective lantibiotic [36]. Nisin and subtilin thus act as bacteriocin-like antimicrobial peptides and as quorum-sensing peptide pheromones that control their own expression. Synthesis of epidermin seems to be regulated differently. Regulating agents or conditions have not yet been identified, but EpiQ seems to be active under all laboratory conditions tested so far (A. Peschel, unpublished).

57

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin

3.3.3 Regulation of epidermin genes

Lipase act ivit y ( U/ g cell dry wt .)

Trans-activation experiments with transcriptional reporter gene fusions have demonstrated that EpiQ controls most of the transcriptional units in the epidermin gene cluster; the promoters of the epiFEG, epiH, epiT, and epiABCD gene clusters are activated [56, 58, 60] (Fig. 3.3). All these promoter regions have a palindromic sequence motif (agAaAATTAC – 6 bp – GTAATTtTct) located immediately upstream of the only weakly conserved –35 regions. The motif upstream of the epiABCD promoter is the EpiQ binding site [56]. The EpiQ operator is also present in the gallidermin gene clusters at the same positions, and the promoters are also sensitive to EpiQ. - E p iQ

1000

+ E pi Q 100

10

1

Pep i FE G

Pep i H

Pep i T

Pep i AB C D

Pep i PQ

Figure 3.3: Promoter activities of various epidermin genes. Extracellular lipase activities of S. carnosus strains carrying a promoter test plasmid containing the lipase gene as a reporter under the control of the indicated epidermin gene promoters (Pepi). Lipase expression was determined in the presence and absence of the activator gene, epiQ, encoded on a second plasmid.

The only transcriptional unit not controlled by EpiQ is the epiPQ operon (Fig. 3.2); EpiQ therefore exerts no autoregulation. The epidermin gene cluster contains additional promoters within the operon structures that are not controlled by EpiQ, e. g. in front of epiC, epiD, and epiQ. The epiD promoter has a particularly high activity (Peschel et al., unpublished). The regulation of the epidermin genes remains elusive since no activating agents have been found. Recent literature illustrates the importance of global regulatory relays in Gram-positive bacteria [31]. Quorum-sensing systems control the production of antimicrobial substances in B. subtilis, and it is conceivable that a similar, yet unknown system controls the epidermin genes. A further possibility is the involvement of special sigma factors that couple epidermin production to certain growth phases or environmental conditions. In this respect, it is interesting to note that epidermin production occurs mainly during the exponential growth phase and is obviously switched off in the stationary phase [54]. Since EpiQ con58

3.4 Isolation and characterization of genetically engineered gallidermin trols most of the epidermin genes, the promoters controlling its own expression are likely targets for a chromosomally encoded regulatory system. Chromosomally encoded proteins may also be involved in secretion of epidermin, thereby substituting for the defective epiT, and may be involved in the modification reactions.

3.4 Isolation and characterization of genetically engineered gallidermin and epidermin analogues

Gallidermin (Gdm) and epidermin (Epi) are highly similar. To study the substrate specificity of the modifying enzymes and to find variants of the lantibiotics with altered or new biological activities, we exchanged certain amino acids of the gallidermin and epidermin structural genes, gdmA and epiA, respectively, by sitespecific mutagenesis [53]. No epidermin/gallidermin analogues are found in the supernatant when 1) the hydroxyamino acids involved in thioether amino acid formation are substituted by non-hydroxyamino acids (S3N and S19A); 2) cysteine residues involved in thioether bridging are deleted (C21, C22 and C22); or 3) a ring amino acid is substituted by an amino acid with a completely different character (G10E and Y20G). Production is greatly decreased when serine residues involved in thioether amino acid formation are exchanged by threonine residues (S16T, S19T). A number of conservative exchanges at positions 6, 12, or 14 of the gallidermin backbone are tolerated and lead to analogues with altered biological properties, such as an enhanced antimicrobial activity (L6V) or a remarkable resistance to proteolytic degradation (A12L and Dhb14P). The T14S substitution leads to the simultaneous production of two gallidermin species formed by an incomplete posttranslational modification (dehydration) of the S14 residue. The fully modified Dhb14Dha analogue has antimicrobial activity similar to gallidermin, whereas the Dhb14S analogue is less active. Both peptides are more sensitive to tryptic cleavage than gallidermin. The construction and characterization of the various analogues are described in more detail in the following sections.

3.4.1 Characterization of two Epi – mutants and development of a host-vector system for expression of wild type and mutated gdmA and epiA genes We isolated a series of Epi – mutants of S. epidermidis Tü 3298 by ethyl-methanesulfonate(EMS) mutagenesis; two mutants (EMS5 and 6) carry mutations in the epiA region [4]. Both mutants can be complemented to an Epi+ phenotype by 59

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin transformation with a plasmid carrying epiA, which indicates that other epi biosynthetic genes are functional. Single point mutations in epiA result in an S3N substitution (ring A) in EMS5 and a G10E substitution (ring B) in EMS6 (Fig. 3.1). Analytical HPLC analyses indicated that no epidermin analogue or precursor peptide is present in the culture supernatants of EMS5 and EMS6 [53]. We chose the mutant S. epidermidis EMS6 as an expression host for the synthesis of epidermin and gallidermin analogues. To test this mutant for its ability to synthesize the heterologous gallidermin, a 1.6-kb SalI/EcoRI subfragment (encoding gdmA and a part of gdmB and of gdmT) of the 4.8-kb EcoRI chromosomal fragment of S. gallinarum Tü 3928 [71] was cloned into the polylinker region of the staphylococcal plasmid pT181mcs. S. carnosus TM300 was transformed with the resultant plasmid, pTgdmA [53]. After isolation, the plasmid was transferred into S. epidermidis EMS6 by electroporation. This indirect transformation method is necessary because S. epidermidis Tü 3298 and derivative strains are poorly transformed with ligation products; only ccc plasmids are electroporated efficiently. EMS6 transformants carrying plasmid pTgdmA formed halos on plates containing Micrococcus luteus, and analyses revealed that only fully modified gallidermin is produced.

3.4.2 Characterization of gallidermin and epidermin analogues generated by site-directed mutagenesis The gallidermin and epidermin analogues were isolated from the culture supernatant of various S. epidermidis EMS6 clones, purified, and analyzed by analytical HPLC, electrospray mass spectrometry (ES-MS), and continuous Edman sequencing. MIC values and tryptic sensitivity were determined for all gallidermin analogues, except Gdm L6V/S16T and epidermin S19T because these peptides were only produced in trace amounts. The results are summarized in Table 3.1 [53].

3.4.2.1 Mutations in ring A Since Gdm (L6) is more active than Epi (I6) against various Gram-positive bacteria (23), we generated two more analogues of gallidermin with mutations at position 6: L6V and L6G. The production and modification of these two analogues are not impaired. The L6G analogue is less active than gallidermin, regardless of the indicator strain employed. The L6V analogue is twice as active as gallidermin against Micrococcus luteus and Corynebacterium glutamicum, and just as active against Arthrobacter cristallopoietes and B. subtilis. Antimicrobial activity against S. aureus is reduced fivefold (Table 3.1). The L6G analogue is almost as sensitive to trypsin as gallidermin, whereas the L6V analogue is more resistant. 60

3.4 Isolation and characterization of genetically engineered gallidermin Table 3.1: Antimicrobial activities of gallidermin and its analogues against various Grampositive indicator strains [53]. Gdm + derivatives

Minimal inhibitory concentration (lg/ml)a Micrococcus Arthrobacter luteus cristallopoietes

Corynebacterium glutamicum

Bacillus subtilis

Staphyloc. aureus Cowan I

Gdm Gdm Dhb14Dha Gdm Dhb14S Gdm Dhb14A Gdm Dhb14P Gdm A12L Gdm L6G Gdm L6V

0.004 0.004 0.008 0.004 0.12 0.15 0.008 0.002

0.065 0.065 0.13 0.065 0.52 0.13 0.13 0.032

3.0 3.0 15.0 >20.0 >20.0 10.0 8.0 3.0

4.0 5.0 25.0 30.0 >30.0 12.0 30.0 20.0

a

0.005 0.005 0.01 0.02 0.08 0.02 0.04 0.005

MIC values obtained for M. luteus, A. cristallopoietes, and C. glutamicum are located within the nanomolar range (0.002–0.52 mg/ml; *1–240 nM), whereas those observed for B. subtilis and S. aureus are located within the micromolar range (3.0 to 630 mg/ml; 1.4 to 614 mM).

3.4.2.2 Mutations in ring C and D To determine the importance of the C-terminal bicyclic structure (intertwined rings C and D) for epidermin/gallidermin biosynthesis, we exchanged various amino acids in this region. Using the gdmA-L6V gene, we created the double mutant Gdm L6V/S16T. The production of this analogue is only 8% of that observed for Gdm L6V. Molecular mass determinations of Gdm L6V/S16T revealed that the T16 residue is dehydrated. However, it was not possible to verify the formation of the 3-methyllanthionine in ring C by ES-MS. Gdm L6V/S16T has antimicrobial activity; however, the MIC values were not determined because of the very low production of this analogue. The S19T codon of EpiA was replaced in order to create a posttranslationally formed S-(2-aminovinyl)-2-methyl-d-cysteine residue as found in mersacidin. This analogue has antimicrobial activity and is also produced in very low amounts. The production is only 0.4% of that observed for the gallidermin-producing EMS6 (pTgdmA) clone, which produces approximately 12 mg Gdm/l. ES-MS analysis revealed an average molecular mass of 2177 Da, indicating that T19 is dehydrated and that the C-terminus is decarboxylated. It was also not possible to verify the formation of the S-(2-aminovinyl)-2-methyl-d-cysteine residue by ES-MS; however, thioether bridge formation is very likely because of the observed chemical instability of the enethiol structure [42, 43]. To prevent formation of the S-(2-aminovinyl)-d-cysteine residue in ring D, we generated an S19A (pTepiA-S19A) substitution and a C22 deletion (pTepiADC22). These mutations could possibly lead to the formation of alternative thioether bridges. However, the corresponding EMS6 clones form no halos on 61

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin plates of M. luteus, and no epidermin analogue or precursor form was detected by analytical HPLC. In addition no antimicrobial activity and no extracellular product were detected when the last two C residues (pTepiA-DC21, C22) were deleted. An amino acid substitution not directly involved in thioether bridge formation of ring D was created by replacing the bulky Y20 residue with a G (pCUgdmA-Y20G). The corresponding EMS6 clone has no antimicrobial activity, and no gallidermin analogue or precursor peptide was detected by analytical HPLC.

3.4.2.3 Mutations in the flexible middle region (A12 to G15) With the A12L substitution, a more bulky residue was introduced in the flexible middle region. The production of this analogue is comparable to that of gallidermin; however, antimicrobial activity against most of the test strains is reduced threefold (Table 3.1). A striking feature of this analogue is its high resistance to tryptic cleavage. Further amino acid alterations mainly focused on the 2,3-unsaturated Dhb14 residue to investigate whether the reactive C=C double bond is important for biological activity, as suggested for the Dha5 residue of subtilin and nisin [38, 48], or whether this residue plays a role in stabilizing a distinct structure required for pore-forming activity, as reported for the P residue in the channelforming peptides alamethicin and melittin [78]. With the T14S substitution, two analogues were produced in nearly equimolar ratios, as judged from the respective peak areas. By ES-MS and continuous Edman degradation, the two peptides were identified as gallidermin analogues possessing either a dehydrated (Dha14) or an unmodified S14 residue (Fig. 3.1A, B). Antimicrobial activity of Gdm Dhb14Dha is similar to that of gallidermin, whereas the Dhb14S analogue is less active, especially against B. subtilis and S. aureus (Table 3.1). Both analogues were more sensitive to tryptic cleavage than gallidermin; the Dhb14S analogue was one of the most sensitive analogues, being completely cleaved after 30 min. To remove the reactive C=C double bond, a Dhb14A analogue was created. Slightly more of this analogue is produced as compared to gallidermin. Antimicrobial activity of Gdm Dhb14A is similar to that of gallidermin against M. luteus and C. glutamicum and approximately seven-fold less against B. subtilis and S. aureus (Table 3.1). Like Dhb14S, the Dhb14A analogue is more sensitive to tryptic cleavage and is also fully degraded within 30 min. The Dhb14P substitution was expected to cause the strongest conformational change of the middle region. Production of this analogue is comparable to that of gallidermin. The antimicrobial activity is generally reduced 8- to 30-fold (Table 3.1). This analogue has a pronounced resistance to tryptic cleavage.

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3.4 Isolation and characterization of genetically engineered gallidermin

3.4.3 Overview of the characteristics of gallidermin and epidermin analogues We demonstrated that although epidermin and gallidermin are produced by two different staphylococcal species, gallidermin can be synthesized successfully using the heterologous S. epidermidis EMS6 as expression host and the cloned gdmA gene. The gdmA promoter is controlled by the transcriptional activator EpiQ with an efficiency similar to the epiA promoter [60], and the enzymes involved in epidermin biosynthesis (including maturation and secretion) function efficiently with the gdmA gene product. Mutagenesis of amino acid positions that are directly involved in thioether amino acid formation result in the loss of or a large decrease in production. Loss of production (i. e. no HPLC-detectable extracellular product) is observed with the analogues containing the S19A substitution, the deletion of the C22 residue (ring D, Fig. 3.1), and the S3N substitution (ring A). Thus, it can be hypothesized that biosynthesis and/or secretion of epidermin and gallidermin are severely impaired when formation of only one of the four thioether bridges is prevented. There is also no production observed with the analogues containing the G10E (ring B) and the Y20G (ring D) substitutions, which suggests that these mutations interfere with thioether bridge formation even though the substituted residues are not directly involved. The replacement of the G10 residue for a bulky and negatively charged E residue may impose steric hindrance on thioether bridge formation at ring B. 2D-NMR studies of gallidermin [18] revealed that ring B, which is identical to ring B of nisin and subtilin, adopts a typical b-turn type II conformation. This specific conformation would be disturbed by a G10E exchange and, as a consequence, thioether bridge formation might be affected, regardless whether this reaction is enzyme-catalyzed or occurs spontaneously in a Michael-additionlike reaction [77]. Recent investigations of the substrate specificity of EpiD [43] indicate that a precursor molecule possessing a G residue instead of the Y20 residue is not a substrate of EpiD. This result suggests that at least oxidative decarboxylation of the last C residue of the mutant Y20G precursor peptide is prevented. For all mutations that lead to the loss of production, further investigations will determine whether only secretion or whether also other stages of biosynthesis are blocked. It also cannot entirely be ruled out that partially modified precursor peptides are rapidly degraded by the EMS6 host. The only thioether amino acid mutations that result in the production of antimicrobially active substances are the S19T (ring D) and the S16T (ring C) substitutions. The extremely low production of Epi S19T and Gdm L6V/S16T could be explained by assuming that dehydration of T16 and T19 is inefficient and that only fully modified (dehydrated and subsequently thioether-bridged) precursor peptides are efficiently secreted. The simultaneous production of Gdm Dhb14S and Gdm Dhb14Dha is an indication of different efficiencies of Sand T-dehydration. For the applied use of peptide antibiotics, an advantageous trait would be resistance to proteolytic degradation. Epidermin and gallidermin already natu63

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin rally have a rather high resistance. Among various peptidases tested (pronase, trypsin, pepsin, thermolysin, collagenase, and carboxypeptidases A and B), only pronase and trypsin cleave epidermin [2]. Proteolytic degradation by the other peptidases is sterically hindered by the thioether bridges. In one study, purified gallidermin and analogues were tested for their sensitivity to tryptic cleavage in order to screen for analogues with an increased resistance to proteolytic degradation and, as a consequence, a prolonged period of action. All gallidermin analogues are cleaved by trypsin only in the central part of the molecule between K13 and the residue at position 14, which is in agreement with earlier reports on gallidermin [28] and epidermin [2]. Tryptic cleavage at the second putative tryptic cleavage site, the K4-F5 bond (ring A), is sterically hindered, at least under the conditions employed. 2D-NMR studies of the gallidermin molecule [18, 19] revealed that the rigid rings A/B and C/D are connected by a flexible middle region (A12 to G15). The conformation (e. g. flexibility of the peptide backbone) of this region appears to be important for antimicrobial activity and resistance to tryptic cleavage. Gallidermin analogues with a supposed decreased flexibility of the middle region, such as Gdm Dhb14P and Gdm A12L, are highly resistant to tryptic cleavage, whereas their antimicrobial activity against various Gram-positive indicator bacteria is greatly reduced (Table 3.1). According to modeling studies of the gallidermin/trypsin interaction [26], the gallidermin molecule must adopt a bent structure in order to fit into the catalytic cleft of the trypsin molecule. This bent structure is supported by the flexibility of the peptide backbone within the middle region. Thus, the high resistance to tryptic cleavage observed for Gdm Dhb14P and Gdm A12L may reflect a decreased flexibility of the middle region, impeding the formation of the bent structure. A P residue following a K residue usually has a strong negative influence on trypsin action. This also may explain the high resistance to tryptic cleavage observed for Gdm Dhb14P (tryptic cleavage site: K13–P14). However, a negative influence of a preceding L residue is not known [28], which suggests that the large increase in resistance to tryptic cleavage observed for Gdm A12L (tryptic cleavage site identical to that of gallidermin) is mainly caused by a restricted flexibility of the middle region. The K13–Dhb14 bond of gallidermin is cleaved by trypsin with a strikingly lower efficiency than bonds of K to normal protein amino acids [28]. Thus, an increased substrate affinity of the trypsin molecule and an increased flexibility of the peptide backbone within the middle region, which may lead to a greater accessibility of the tryptic cleavage site, may be responsible for the greatly reduced resistance to tryptic cleavage observed for Gdm Dhb14A and Gdm Dhb14S (tryptic cleavage sites: K13–A14 and K13–S14, respectively). This effect was strikingly less enhanced for Gdm Dhb14Dha (tryptic cleavage site: K13– Dha14). Interestingly, the tryptic sensitivity of Gdm L6V and Gdm L6G differ, although these gallidermin analogues are not altered in the vicinity of the tryptic cleavage site; this observation possibly indicates a slight overall change in conformation of the L6V and the L6G analogue. 64

3.4 Isolation and characterization of genetically engineered gallidermin Of all the gallidermin derivatives altered within the middle region, only the Dhb14Dha analogue has antimicrobial activity similar to that of gallidermin (Table 3.1). This may indicate the importance of the 2,3-didehydroamino acids for maintaining a structure required for efficient pore formation. A critical role of the polypeptide backbone flexibility for biological activity has also been suggested for the channel-forming peptides alamethicin and melittin [78]. With Gdm Dhb14A, a decrease in antimicrobial activity is only observed for three of five indicator strains (Table 3.1). Thus, one can speculate that gallidermin can exert its antimicrobial activity by different mechanisms: a Dhb/Dha-dependent and a Dhb/Dha-independent mechanism. Subtilin exerts its antimicrobial activity against Bacillus by two different mechanisms. The inhibitory effect on Bacillus spore germination is dependent on the Dha5 residue and is most likely caused by a Michael-type reaction of the didehydro residue with nucleophilic membrane sulfhydryl groups [48]. The ability to lyse vegetative Bacillus cells, however, is independent of the Dha5 residue [47]. Thus, our results [53] and the results obtained previously by mutagenesis of other type A lantibiotics strongly indicate that the molecular mechanisms by which these peptides exert their antimicrobial activity may differ and that a general function of the 2,3-didehydroamino acids for biological activity is not yet known. The various test strains varied greatly in their susceptibility to gallidermin and its analogues (Table 3.1), possibly because of differences in the membrane phospholipid composition [25, 35] or different membrane potentials [34, 68]. Both factors are crucial for the formation of voltage-dependent transmembrane pores. Furthermore, the composition of the bacterial cell wall might influence the kinetics of pore formation. Cationic lantibiotics interact with polyanions (e. g. teichoic acids) of the cell wall [11] and with membrane-bound cell wall precursor molecules [64]. Binding of lantibiotics to these components may influence the kinetics of pore formation, and may also be responsible for secondary killing mechanisms observed for Pep5 and nisin, such as activation of autolytic enzymes and inhibition of murein synthesis [64]. Recently it was indeed demonstrated that an increased negatively charged cell wall leads to an increased sensitivity to gallidermin and other positively charged antimicrobial peptides [59]. Since B. subtilis and S. aureus produce a variety of exoproteases, the strikingly lower susceptibility of these test strains (Table 3.1) might be partially due to proteolytic degradation of gallidermin and its analogues. In summary, we successfully engineered gallidermin and epidermin analogues with increased antimicrobial activities and/or resistance to proteolytic degradation. Furthermore, we obtained valuable information about the structural elements required for proper biosynthesis of epidermin and gallidermin. NMR analyses of the generated analogues and black lipid membrane studies may provide further information on the structure/activity relationship of these lantibiotics and may pave the way for target-oriented peptide engineering.

65

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin

3.5 Function of the epidermin immunity genes epiFEG

Epidermin, gallidermin, Pep5, subtilin, and nisin are the best-studied members of type A group of lantibiotics, which are all synthesized by Gram-positive bacteria. Type A lantibiotics act by forming membrane-potential-dependent pores in the cytoplasmic membrane of bacteria [5, 68, 69]. Because of this activity, self-protection against the lantibiotic is of vital importance to the producing organism. Several genes responsible for producer self-protection have been found in the gene clusters of the respective systems: 1) nisI and spaI, which code for lipoproteins [32, 37]; 2) epiF, epiE and epiG [57] which are homologous to nisF, nisE, and nisG of the nisin system [74] and to spaF and spaG of the subtilin system [32], all of which code for ABC transporters; and 3) pepI, which does not share any sequence similarity to any known genes [63]. Analysis of the amino acid sequences suggests that the EpiFEG-type transporters and the lipoproteins NisI and SpaI are membrane-located; this has been experimentally shown for PepI [63]. The mechanism of action has not been elucidated for any of the gene products. The sequence similarity of the EpiFEG proteins to that of ABC transporters led to the proposal that the EpiFEG-type proteins act by transporting the lantibiotic out of the cytoplasmic membrane, thus keeping its concentration below a critical level and preventing pore formation. Two conceivable directions of transport have been discussed: import into the cell for inactivation by proteolytic cleavage and export into the surrounding medium. As shown by heterologous expression experiments in S. carnosus, the selfprotection (immunity) of the epidermin-producing strain S. epidermidis Tü 3298 against the pore-forming lantibiotic epidermin is mediated by an ABC transporter composed of the EpiF, EpiE, and EpiG proteins. We developed a sensitive assay based on HPLC analysis of the substrate gallidermin in cell supernatants to investigate the mechanism of the EpiFEG transporter. Our results indicate that the EpiFEG transporter works by expelling the lantibiotic from the cytoplasmic membrane into the surrounding medium with a high substrate specificity. Thus, the EpiFEG transporter functions according to the “hydrophobic vacuum cleaner” mechanism [12]. Furthermore, we showed that the gallidermin derivative L6G has an EpiE-dependent enhanced activity. These results will be covered in more detail in the following sections. To our knowledge, the EpiFEG transporter is the first of its kind to be investigated that has a pore-forming peptide as substrate. Future research on the EpiFEG transporter will focus on the question whether the EpiFEG transporter can substitute in S. epidermidis Tü 3298 for the non-functional EpiT transporter, whose task is to export completely modified pre-epidermin (with the leader peptide still present) out of the producer cell.

66

3.5 Function of the epidermin immunity genes epiFEG

3.5.1 The epiFEG genes confer immunity to the epidermin producer We characterized a DNA region located upstream of the structural gene epiA that mediates immunity and increased epidermin production. The sequence of a 2.6-kb DNA fragment revealed three ORFs (Fig. 3.2), epiF, E, and G, which form an operon [57]. In the cloning host S. carnosus, the three genes mediate an increased tolerance to epidermin, and the highest level of immunity (sevenfold) is achieved with S. carnosus carrying epiFEG and epiQ (Fig. 3.4). The promoter of the first gene epiF responds to the activator protein EpiQ and contains a palindromic sequence similar to the EpiQ-binding site of the epiA promoter, which is also activated by EpiQ. Inactivation of epiF, E, or G results in the complete loss of the immunity phenotype. An epidermin-sensitive S. epidermidis Tü 3298 mutant is complemented by a DNA fragment containing all three genes. When the epiFEG genes are cloned together with plasmid pTepi14, which contains the biosynthetic genes epiABCDQP, the production of epidermin is approximately fivefold higher. The deduced amino acid sequences of EpiF, E, and G are similar in sequence and proposed structure to the components of various ABC transporter systems. EpiF is a hydrophilic protein with conserved ATP-binding sites, while EpiE and G have six alternating hydrophobic regions and very likely constitute the integral membrane domains. When EpiF is overproduced in S. carnosus, it is at least partially associated with the cytoplasmic membrane.

Figure 3.4: The epiFEG genes confer immunity against gallidermin in the heterologous host S. carnosus. MIC values were determined with S. carnosus TM300 harboring all or only two of the epiFEG genes (in all possible combinations) on plasmid pRB473. Black bars represent MIC values of strains harboring in addition the positive regulator of the epidermin biosynthetic system, epiQ, on plasmid pTepiQ10.

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3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin

3.5.2 Transport mechanism In most cases, it has been assumed that ABC transporters reported to be involved in resistance to non-pore-forming antibiotics have to export the antibiotic from the cytoplasm to the surrounding medium. For an antibiotic whose target is located in the cytoplasm, this mechanism seems self-evident, whereas for a pore former whose target is the cytoplasmic membrane, import into the cell as well as export to the surrounding medium are both conceivable mechanisms for removing the antibiotic from its target (Fig. 3.5).

Figure 3.5: Two alternative models for the mechanism of the EpiFEG transporter. The model shows the EpiE and EpiG proteins situated within the cytoplasmic membrane and an EpiF dimer bound to the complex at the cytoplasmic side of the membrane. EpiF harbors the ATPase binding site, as predicted by hydropathy plots and sequence alignments (Peschel and Götz 1996). Black arrows illustrate the mechanism model 1: the export of gallidermin into the surrounding medium. Every exported molecule in model 1 is likely to be able to reintegrate into the membrane, thereby causing an incessant expulsion process. Gray arrows illustrate model 2: the import of gallidermin into the cytoplasm, where one would expect proteolytic degradation of the lantibiotic.

To investigate the direction of transport mediated by the EpiFEG transporter, it was not possible to use membrane vesicle systems because the cytoplasmic orientation of the ATPase site in the EpiF part of the transporter constitutes the only means to discriminate between right-side-out and inside-out vesicles and because the substrate gallidermin is known to cause efflux of small molecules such as ATP out of the cell. This would, unfortunately, lead to an equal 68

3.5 Function of the epidermin immunity genes epiFEG distribution of ATP in the internal and external fluid. We therefore developed an assay in which the amount of the substrate gallidermin remaining in the supernatant of whole cells incubated with gallidermin was determined. In this transporter assay, a concentration of gallidermin in the supernatant of cells expressing the epiFEG genes higher than that of a control strain would suggest that the EpiFEG transporter works by export; a lower concentration in the supernatant would suggest that the transporter works by import. All experiments were performed with S. carnosus TM300 as a host for heterologous expression of all or some of the epiFEG genes. Gallidermin was quantified by HPLC detection. Our results indicate that model 1 shown in Fig. 3.5 represents the mechanism used by the EpiFEG transporter. It is not known whether the transporter recognizes the substrate in its monomeric or oligomeric form; both possibilities are illustrated in Fig. 3.5. Optimal results were observed with rather low gallidermin concentrations, and HPLC quantification was optimized and resulted in a detection limit of about 20 ng at the very specific wavelength of 266 nm (absorption of C-terminal aminovinylcysteine in gallidermin and all derivatives). At 2 mg gallidermin/ml, the extracellular gallidermin content is about fourfold higher for the epiFEG-expressing strain than for the control strain. At higher and lower concentrations of applied gallidermin, the difference is less pronounced. To exclude the possibility that the observed difference in extracellular gallidermin concentration is caused by non-specific interaction of gallidermin with one of the EpiFEG proteins and not by the functional transporter, the epiFEGexpressing strain was compared with strains harboring the genes encoding only two of the three transporter components (epiF, epiE and epiG) in all three possible combinations. The gallidermin concentration in the assay supernatant of each of the three strains showed a similar basal level, whereas in the epiFEGexpressing strain, this concentration was about fourfold higher, demonstrating that the observed effect is not caused by non-specific interaction with one of the protein components of the EpiFEG transporter (Fig. 3.6).

3.5.2.1 Energy dependence of transport Energy dependence of the EpiFEG transporter could not be directly studied using ATPase inhibitors because destruction of the membrane potential would also result in inhibition of gallidermin activity. Instead, dependence of the EpiFEG-mediated transport on the glucose concentration was determined. Glucose was the only energy source in the incubation buffer. In the absence of glucose, transport is strongly reduced. Increases in the concentration of glucose up to 1% result in increases in the transport efficiency; at concentrations of glucose higher than 1%, transport efficiency does not increase further. These results are in accordance with the expected energy dependence of the ATP-consuming EpiFEG transporter.

69

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin

Figure 3.6: Comparison of the effect of the entire EpiFEG transporter with that of the transporter protein components. The transporter assay was carried out with S. carnosus strains harboring plasmid pTepiQ10, which contains the positive regulator of the epidermin system, and two (“FE”, “EG”, “FG”), none of (“473”), or all of (“FEG”) the epiF, epiE and epiG genes on plasmid pRB473. Gallidermin was used as substrate at a concentration of 2 mg/ml. The values of the determined extracellular gallidermin content from five samples for each strain were averaged.

3.5.2.2 Specificity of the transporter Eight gallidermin derivatives with single amino acid substitutions were used to determine the specificity of the EpiFEG transporter in the transporter assay. In addition to epidermin (gallidermin L6I), seven other derivatives originating from site-specific mutagenesis of the gallidermin structural gene gdmA [53] were investigated. Transporter efficacy was defined as the value obtained with the epiFEG-expressing strain divided by the value obtained with the control strain. This factor is in the range of 3 to 5 for each substance, with the exception of gallidermin Dhb14P and gallidermin L6G, where there is almost no detectable effect. No differences among the test strains were detected with nisin, which suggests that nisin is not a substrate of the EpiFEG transporter. The most prominent feature of gallidermin Dhb14P is that the flexibility of the so-called hinge region is strongly decreased; gallidermin L6G was the most hydrophilic derivative investigated (as concluded from reversed-phase HPLC retention). The three-step model for the mode of action of nisin and type A lantibiotics in general includes adhesion to the outer surface of the cell membrane mediated by electrostatic interaction between the cationic peptide and anionic charges on the surface of the cytoplasmic membrane, integration into the membrane with a DC or DpH present where hydrophobicity and flexibility are considered to be important, and formation of oligomeric pores [51, 52]. Thus, gallidermin Dhb14P and gallidermin L6G appear to be impaired in the integration into the mem70

3.5 Function of the epidermin immunity genes epiFEG brane, and this is presumably also the cause for their low bactericidal activity. Adhesion to the cytoplasmic membrane is not likely to be affected in any of the gallidermin derivatives tested because the charge of the molecule is not changed. Since specifically gallidermin Dhb14P and gallidermin L6G are by far the poorest substrates among all gallidermin derivatives investigated, we assume that integration of the substrate into the membrane is important for EpiFEG activity. This suggests that the substrate binding site of the EpiFEG transporter is within the membrane-spanning part of the protein complex, an assumption that is further supported by the interaction of gallidermin L6G with the internal membrane protein EpiE (see below). MIC values of gallidermin, epidermin, gallidermin derivatives, and nisin of S. carnosus (pRBepiFEG/pTepiQ10) and S. carnosus (pRB473/pTepiQ10) were also determined. No difference in the MIC values of nisin among the test strains was detected. Gallidermin, epidermin, and the gallidermin derivatives Dhb14S, Dhb14Dha, and A12L are more active against the control strain, whereas the activity of gallidermin Dhb14P is only slightly influenced by the presence of the epiFEG genes in the test strain. In contrast, the MICs of gallidermin derivatives L6V and Dhb14A are not influenced by the presence of the epiFEG genes in the strain; however, they are good substrates of the EpiFEG transporter, as shown in the transporter assay. While conditions in the transporter assay were selected to optimize EpiFEG transporter efficacy, this was not the case in the complex medium used for MIC determinations. Conditions in the complex medium seemed to suppress the interaction of EpiFEG with some derivatives.

3.5.2.3 Interaction of EpiE with gallidermin L6G Paradoxically, the activity of gallidermin L6G is clearly higher in the presence of the epiFEG genes in the test strain. Further experiments were designed to investigate this phenomenon. The MIC of gallidermin L6G of S. carnosus (pRBepiFE/pTepiQ10), S. carnosus (pRBepiEG/pTepiQ10), S. carnosus (pRBepiFG/ pTepiQ10), and the strains used before was determined (Table 3.2). The activity of gallidermin L6G is higher whenever the epiE gene is expressed in the strain; the expression of the other two genes (epiF and epiG) is not necessary for this effect. The activity of gallidermin L6G is also higher against S. carnosus (pTXepiE), in which the epiE gene is under the regulation of a xylose-inducible promoter, than against the control strain S. carnosus (pTX16). The integration of gallidermin L6G into the cytoplasmic membrane is usually impaired because of its comparatively low hydrophobicity, yet the activity of gallidermin L6G against strains that expressed the epiE gene was always relatively high (Table 3.2). As an explanation for this phenomenon, we propose a direct protein-protein interaction of gallidermin L6G with EpiE; this interaction would also suggest a participation of EpiE in substrate binding in general. Gallidermin L6G probably binds to the substrate binding site on EpiE and remains bound there, forming a nucleus to which other gallidermin L6G molecules adhere to form a pore. Without this interaction, gallidermin L6G would be too hydrophilic to form a pore. 71

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin Table 3.2: Activity of gallidermin L6G [53]. Strain

MIC a (mg/ml)

S. carnosus (pRB473/pTepiQ10) S. carnosus (pRBepiFEG/pTepiQ10) S. carnosus (pRBepiFE/pTepiQ10) S. carnosus (pRBepiEG/pTepiQ10) S. carnosus (pRBepiFG/pTepiQ10) S. carnosus pTX16 S. carnosus pTXepiE

1.0 0.1 0.1 0.1 1.0 1.0 0.2

a

MIC values of gallidermin L6G (Ottenwälder et al. 1995) of various strains were determined as described in the experimental procedures to investigate the gallidermin L6GEpiE interaction. Strains S. carnosus pTXepiE, in which epiE is under the control of a xylose-inducible promoter, and S. carnosus pTX16 as a control were grown in basic medium without glucose and with 0.5% xylose.

3.6 Inactivation and characterization of the epidermin leader peptidase EpiP

The sequences of lantibiotic leader peptides differ from Sec-dependent protein export signal sequences. Type A lantibiotics have a negatively charged, amphiphilic a-helix that is removed at the characteristic processing-site P–2–Q/R–1 ;X+1 (Fig. 3.7). In nearly all lantibiotic gene clusters, a serine protease is encoded, which is proposed to mediate precursor processing. These proteases are usually located in the cytoplasm. In contrast, NisP, the nisin leader peptidase, and EpiP contain a signal sequence, indicating that they act extracellularly. Therefore, the location of the processing of the various lantibiotics may differ [75]. The serine protease EpiP from S. epidermidis Tü 3298 catalyzes the extracellular processing of the epidermin precursor peptide, as shown in experiments where epiP in the xylose-regulated expression vector pCX15 in S. carnosus is

Figure 3.7: The processing site of modified pre-epidermin. The arrow indicates the processing site of the secreted EpiP protease.

72

3.6 Inactivation and characterization of the epidermin leader peptidase EpiP overexpressed. The cleavage of the unmodified EpiA precursor peptide to leader peptide and pro-epidermin by EpiP-containing culture filtrate of S. carnosus (pCX15epiP) was followed by reversed-phase chromatography and subsequent electrospray mass spectrometry [20]. The epidermin leader peptidase gene epiP was inactivated so that the final intermediates of epidermin biosynthesis could be isolated to characterize the mechanism of precursor processing. The isolated precursor peptides may also serve as natural substrates for EpiP in future experiments.

3.6.1 epiP gene replacement in S. epidermidis Tü 3298 To construct a plasmid for the replacement of epiP, 1 kb upstream and 1 kb downstream of the gene were amplified by PCR and sequenced. The two amplified fragments were ligated into pBT2, a temperature-sensitive shuttle vector [14], flanking an erythromycin resistance cassette. The resulting plasmid was introduced into S. epidermidis Tü 3298 by electroporation. A homologous recombination event deleted the entire epiP gene, leaving the epiP promoter intact to ensure expression of the regulator epiQ [56] downstream of epiP (Fig. 3.2); there is no terminator structure in the erythromycin resistance cassette that could inhibit transcription of epiQ. The antimicrobial activity of S. epidermidis Tü 3298DepiP mutant strains on agar plates containing the epidermin-sensitive M. luteus was strongly decreased. By transforming the mutant with an epiP-expressing plasmid, epidermin production could be reconstituted (Kies and Götz, unpublished).

3.6.2 Detection and isolation of epidermin precursor peptides Epidermin and epidermin precursor peptides were purified by reversed-phase chromatography of the supernatant from S. epidermidis Tü 3298DepiP grown in synthetic medium. A silver-stained nitrocellulose blot of an SDS-polyacrylamide gel with fractions from the reversed-phase chromatography showed peptides with an apparent molecular weight similar to that of mature epidermin as well as peptides with an apparent molecular weight similar to that of the N-terminally cleaved precursor peptides isolated from the S. epidermidis Tü 3298 wild type strain grown in defined medium. These preliminary results indicate that in S. epidermidis Tü 3298DepiP, the same or at least a similar processing at the Nterminus occurs as in the S. epidermidis Tü 3298 wild type strain. To test this hypothesis, the precursor peptides isolated from S. epidermidis Tü 3298DepiP were analyzed by mass spectrometry and Edman degradation. 73

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin From S. carnosus (pTepiABCDQ) grown in synthetic medium, completely modified epidermin precursors could be isolated that were N-terminally processed at different positions within the leader peptide, i. e. before amino acids 17, 19, or 20 (Fig. 3.7). Mature epidermin was also produced. The peptides were characterized by Edman degradation and mass spectrometry. The same peptide mixture was detected in S. epidermidis TÜ 3298 (wild type strain) when grown in synthetic medium, where EpiP shows a significant reduction in activity because of the low buffer capacity of this medium, which becomes rather acidic. The precursor peptides isolated are not antimicrobially active against the epidermin-sensitive M. luteus. Treatment with endoprotease ArgC, a protease that simulates the EpiP reaction in vitro, leads to antimicrobially active, mature epidermin, as shown by mass spectrometry and in the M. luteus bioassay. In conclusion, the serine protease EpiP catalyzes the last modification step in epidermin biosynthesis – the processing of the epidermin precursor peptide. Due to the inactivation of epiP in S. epidermidis Tü 3298DepiP, it was possible to detect the final intermediate of epidermin biosynthesis, i. e. the completely modified precursor peptide. The precursor had undergone proteolytic cleavage within the leader peptide. Similar results have been observed for the S. epidermidis Tü 3298 wild type strain in synthetic medium, where EpiP shows decreased activity, and for S. carnosus (pTepiABCDQ), which indicate a two-step processing of the epidermin precursor peptide.

3.7 The flavoenzyme EpiD and formation of peptidylaminoenethiolates

One of the goals of lantibiotic research is the analysis of the enzymatic mechanisms involved in the modification process from pro-epidermin to pre-epidermin. The key function is certainly associated with the EpiB and EpiC enzymes, which are apparently involved in dehydration of serine and threonine residues and in the thioether bridge formation. Homologous protein sequences that occur in all other lantibiotic genes are referred to as LanB and LanC. Several groups are studying the enzymatic mechanism of these two enzymes; however, to date, no clean in vitro reaction has been achieved. It was speculated that the enzymatic reaction is strictly oxygen sensitive and/or that unusual co-factors are involved. Epidermin and gallidermin modification involves a third enzyme, EpiD, whose enzymatic function was extensively studied by Thomas Kupke (see below). An epiD homologue has so far only been found in the mersacidin biosynthesis gene cluster [9], but not the nisin, pep5, or subtilisin gene clusters.

74

3.7 The flavoenzyme EpiD and formation of peptidyl-aminoenethiolates

3.7.1 Preparation of precursor peptide EpiA To investigate the enzymes involved in epidermin biosynthesis, it is necessary to produce sufficient amounts of pre-epidermin (EpiA) as a substrate and to design methods to detect EpiA. EpiA was therefore expressed in Escherichia coli using a malE-epiA fusion. The malE gene encodes the maltose-binding protein MBP. The fusion protein MBP-EpiA was expressed from pIH902-epiA and purified in one step by amylose affinity chromatography. EpiA was cleaved from MBP-EpiA by factor Xa, and the identity of purified EpiA was confirmed by ES-MS and amino acid sequencing. Upon prolonged incubation, factor Xa not only cleaves EpiA from the fusion protein, but also less efficiently cleaves EpiA internally between R–1 and I+1. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A–4-E-P-R–1- to -A–4-E-P-Q–1- by site-directed mutagenesis. Since the mutant peptide EpiA R–1Q) has properties different than EpiA (the molecular mass is 28 Da and the isoelectric point is approximately 1.5 pH units lower), it is used as a control peptide in incubation experiments. Anti-EpiA antisera were raised to detect EpiA [44].

3.7.2 Construction and purification of MBP-EpiD fusion proteins According to the nucleotide sequence, epiD encodes a 181-amino acid protein. In order to obtain more information on the function and biochemical properties of EpiD, epiD was expressed in E. coli using the maltose-binding protein (MBP) fusion system. The soluble 67-kDa MBP-EpiD fusion protein was purified in one step by amylose affinity chromatography and was used to raise polyclonal antibodies directed against EpiD. The MBP-EpiD fusion protein was yellow and identified as a flavoprotein by its absorption spectrum with maxima at 277, 378, and 449 nm (compare Fig. 3.8). The coenzyme is very tightly, but not covalently attached. It could only be removed by TCA extraction of the fusion protein and was identified by thin-layer chromatography (TLC) as FMN. S. epidermidis Tü 3298/EMS11 [4] carries a mutation within epiD, designated epiD*, which was PCR-amplified using purified pTü32 from the mutant as a template. This DNA fragment was inserted in the StuI site of the pIH902 polylinker. Plasmids were isolated from four E. coli TB1 (pIH902-epiD*) clones, and the entire epiD* region was sequenced. The epiD* start codon immediately follows the factor Xa cleavage sequence and all four isolated plasmids have a point mutation in codon 93, substituting the wild type GGT (Gly) with GAT (Asp) in epiD*. This G–A transition concurs with the use of EMS as the mutagenizing agent. MBP-EpiD* is not yellow and does not exhibit the characteristic absorption maxima of MBP-EpiD when similar concentrations of fusion proteins are used; it was necessary to dilute MBP-EpiD because of the low amount of soluble MBP75

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin

Figure 3.8:

Absorption spectrum of purified EpiD.

EpiD*. MBP-EpiD* does not bind FMN. Since MBP-EpiD* differs from MBPEpiD only in the substitution of Gly93 by Asp, Gly93 may be important for FMN binding [45].

3.7.3 Characterization of purified EpiD EpiD was expressed using the T7 RNA polymerase promoter system in E. coli and was purified to homogeneity in three column chromatography steps: DEAE-Sepharose Fast Flow, Mono-Q, and Phenylsuperose chromatography. The first 18 N-terminal amino acids of purified EpiD were determined by Edman degradation. The amino acid sequence correlates exactly with the amino acids (Met-Tyr-Gly-Lys-…) deduced from the epiD sequence [70]. Purified EpiD is yellow. The absorption spectrum exhibits maxima at 274, 382 and 453 nm, which are characteristic for flavoproteins in the oxidized state (Fig. 3.8). The liberated flavin component was analyzed by TLC and ES-MS. The mass of the flavin coenzyme was determined to be 455.5 Da (457 Da in a second experiment), which closely agrees with the theoretical value of 456.3 Da and confirms that the flavin component is FMN and not FAD (theoretical mass 785.6 Da). The average molecular mass of EpiD was determined to be 20,827 +/– 5 Da, which is in close agreement with the theoretical value of 20,825 Da calculated for the amino acid sequence derived from the nucleotide sequence, indicating that EpiD is not covalently modified.

76

3.7 The flavoenzyme EpiD and formation of peptidyl-aminoenethiolates

3.7.4 Model of oxidative decarboxylation of peptides Since flavin coenzymes are normally involved in oxidation-reduction reactions, EpiD belongs to the class of oxidoreductases. There is only one obvious reaction involved in epidermin biosynthesis that requires an oxidoreductase activity: The synthesis of S-((Z)-2-aminovinyl)-D-cysteine [1] includes the removal of two reducing equivalents from a -C–C- group to form a -C=C- group. Regarding the proposed order of the modification reactions [72], it was assumed that the four lanthionine rings are formed first, followed by the oxidation of the C-terminal lanthionine by EpiD and the subsequent decarboxylation reaction. In the course of the oxidation, EpiD-FMN becomes reduced. According to this first published hypothesis, EpiD catalyzes the final step in the modification of pre-epidermin, and its reaction product is then processed by the leader peptidase, forming mature epidermin [45]. Thioether formation is only possible if dehydrated serine and threonine residues are present, but in principle, oxidative decarboxylation could be independent of the other modification reactions. Therefore, the following alternative model of the C-terminal oxidative decarboxylation of peptides has been suggested (Fig. 3.9): in the first reaction, EpiD catalyzes the removal of two reducing equivalents from the C-terminal cysteine residue of unmodified EpiA. A double bond is formed, and FMN is reduced to FMNH2. The C-terminal carboxyl group is then removed, catalyzed either by EpiD or occurring spontaneously [42]. The occurrence of a reaction between EpiD and the unmodified precursor peptide EpiA was investigated. CO2

EpiD-FMNH2

O

EpiD-FMN

HN CH C OH

O HN C C OH

HN C

CH2

CH

CH

SH

SH

SH

H

Figure 3.9: Model of the C-terminal oxidative decarboxylation of unmodified precursor peptide EpiA and the role of flavoprotein EpiD. The C-terminal cysteine residue of the precursor peptide EpiA is shown. After oxidation and decarboxylation, the peptidyl-aminoenethiols are formed.

3.7.5 Interaction between EpiD and EpiA EpiA or EpiAR–1Q was coupled to an NHS-activated HiTrap column in order to purify the enzymes involved in epidermin biosynthesis by affinity chromatography. Binding studies were carried out with purified EpiD or extracts of induced E. coli K38 (pGP1–2, pT7–5epiD) cells. In both cases, the migration of EpiD is re77

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin tarded only under reducing conditions. The applied EpiD focuses as a sharp yellow band on the EpiA-HiTrap column. Other proteins of the cell extract are not retarded, which indicates the specificity of the interaction. Even under reducing conditions, the interaction is weak, and EpiD already elutes with the loading buffer containing no salt. In a control experiment, a column without any coupled peptide was used, and as expected, the migration of EpiD was not retarded [40, 43].

3.7.6 Reaction of EpiD with EpiA and pro-epidermin Under reducing conditions, purified EpiD reacts with unmodified precursor peptide EpiA. Several products were identified after separation of the reaction mixture by reversed-phase chromatography and ES-MS analysis. The product formed depends on the incubation time. The first product formed, product 3 (the products were designated according to increasing hydrophobicity), is more hydrophobic than the unmodified peptide EpiA and has a molecular mass of 5,579–5,585 Da (values obtained from several experiments), i. e. 45–48 Da less than EpiA. In order to determine the mass difference between EpiA and product 3 with greater accuracy, the peptides were measured together, giving a mass difference of 46.5 Da. Furthermore, product 3 has an absorbance at 260 and 280 nm in 0.1% TFA/H2O/acetonitrile, higher than that of EpiA [42]. This enzyme reaction was also detected using crude cell extracts of S. carnosus (pTepi14) and EpiA [40]. This clone has been used for heterologous expression of epidermin [3, 70]. With longer incubation times, two additional, less hydrophobic peptides with molecular masses of 5,524–5,528 Da (product 1; 102–105 Da less than EpiA) and 5,579–5,585 Da (product 2; 45–48 Da less than EpiA) were identified. Product 3 (oxidatively decarboxylated peptide) is unstable and is non-enzymatically converted to products 1 and 2. Product 2 has the same mass as product 3, but its absorbance at 260 and 280 nm is comparable to that of unmodified peptide EpiA. Based on its molecular mass, product 1 is either the peptide EpiA(M1-C51) (lacking the last cysteine residue; 103.2 Da less) or the peptide EpiA(M1-C51)-NH2 (104.1 Da less) [42]. All known lantibiotics are synthesized as pre-peptides with an N-terminal leader peptide. It has been proposed that all the processing signals are in the leader region of the pre-peptides [15]. Thus, the pre-peptides would be recognized as substrates by binding of the leader peptides to the enzymes involved in posttranslational modifications. To test this hypothesis, unmodified pro-epidermin (obtained by factor Xa cleavage of EpiA) was used as a substrate for the flavoprotein EpiD. Even with this substrate, the reaction occurs. Hence, a leader peptide is not required for substrate recognition by EpiD. The primary reaction product was analyzed by Edman degradation to detect unmodified amino acid residues. A pattern of pmol amounts of the PTH-amino acids Ile+1 to Tyr+20 similar to that of unmodified pro-epidermin was obtained, which indicates that amino acids 1 to 78

3.7 The flavoenzyme EpiD and formation of peptidyl-aminoenethiolates 20 are unmodified and that one of the last two cysteine residues (Cys+21, Cys+22) is modified [42]. Recently, a more detailed analysis of the reaction product has become possible using tandem mass spectrometry and NMR methods.

3.7.7 In vivo reaction of affinity-tag-labeled epidermin precursor peptide with the flavoenzyme EpiD The genes encoding the His-tag-labeled epidermin precursor peptide EpiA and the flavoenzyme EpiD or the mutant protein EpiD-G93D, which lacks the coenzyme, were co-expressed and the proteins were synthesized in vivo in E. coli. Only in the presence of EpiD was the precursor peptide converted to a reaction product with a decrease in mass of 44–46 Da. This result confirms the in vitro experiments carried out with purified EpiA and purified EpiD from S. epidermidis. In the presence of EpiD, the amount of purified (modified) peptide EpiA was several-fold higher than in the presence of EpiD-G93D, indicating that the stabilization of EpiA against proteolysis is due to an interaction with EpiD or to the presence of the C-terminal modification [39].

3.7.8 Determination of the substrate specificity of EpiD using mutant precursor peptides and chemically synthesized peptides The precursor peptide EpiAC52S, which is altered by gene mutation and contains a C-terminal serine, was used to test the production of enols by reaction with EpiD [31]. No reaction occurred, which indicates the necessity for a Cterminal cysteine residue. However, not all peptides with a C-terminal cysteine residue, e. g. the peptide EpiA M1-C51, were a substrate of EpiD. EpiAS49A was a substrate for oxidative decarboxylation, providing the first hint that EpiD has no absolute substrate specificity. The leader peptide of the precursor peptide EpiA has no significant influence on the reaction with EpiD. Synthetic peptides with increasingly larger deletions of the amino-terminus were used to determine the minimal size of the substrate. A weak reaction was even observed for the tetrapeptide SYCC. The heptapeptide SFNSYCC was used to study the substrate specificity of EpiD further (Table 3.3). The serine and cysteine residues of this heptapeptide form the Cterminal bicyclic structure of mature epidermin, and it could not be excluded that amino acid exchanges in this peptide have an influence on the reaction with EpiD [31]. Interestingly, the penultimate cysteine residue can be exchanged with at least a serine or threonine residue. Modification or exchange of the C-terminal cysteine residue results in loss of the reaction with EpiD. The peptides SFNSYCS, SFNSYCM, and SFNSYC were no substrates of EpiD, which confirms the results 79

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin Table 3.3: Determination of the substrate specificity of the flavoprotein EpiD [43]. Peptide

Reaction with EpiD

EpiA, EpiAR-1Q, K-EpiA EpiAS+19A EpiAC+22S EpiAdesC+22

+ + – –

proepidermin synthetic proepidermin AKTGSFNSYCC SFNSYCC FNSYCC NSYCC SYCC YCC

+ + + + + + + –(?)

AFNSYCC SANSYCC SFASYCC SFNAYCC SFNSACC SFNSYGC

+ + + + – –

SFNSYCS SFNSYC SFNSYCC-NH2 SFNSYCC(Et) SFNSYCHCy SFNSYCM

– – – – – –

SFNSYSC SFNSYTC

+ +

SFNSFCC SFNSWCC

+ +

SFNYYCC SYNSYCC SWNSYCC

+ + +

TLTSECIC

–

obtained with the mutant precursor peptides. The analogue with a C-terminal ethyl-thioether structure [SFNSYCC(Et)], the amide SFNSYCC-NH2, and the peptide SFNSYCHcy (C-terminal homocysteine residue) were not substrates of EpiD. Mersacidin is another lantibiotic containing a C-terminal -NH–CH=CH–Sgroup [61], probably formed by modification of a C-terminal cysteine residue. The C-terminal peptide TLTSECIC of the mersacidin precursor peptide is not a substrate of EpiD [43]. 80

3.7 The flavoenzyme EpiD and formation of peptidyl-aminoenethiolates

3.7.9 Tandem mass spectra of modified SFNSYTC and SFNSYSC The sequence and structure of peptides can be analyzed with tandem mass spectrometry (collision-induced dissociation). In collision-induced dissociation experiments, peptides are preferentially cleaved at the peptide bonds between NH and CO, resulting in amino-terminal Bn fragments and C-terminal Yn fragments [7, 8]. To verify that the C-terminal cysteine residue of the reaction products is modified, product ion scans of SFNSYTC and SFNSYSC and the corresponding reaction products were recorded. These peptides were used to exclude intramolecular disulfide bridge formation in the peptide SFNSYCC. The B1–B6 fragments of the peptide and its reaction product are identical, proving that the C-terminal amino acid residue is modified. SFNSYTC and SFNSYSC and their reaction products differ from each other by 14 mass units in their B6 fragments, showing the S/T exchange. The mass difference between the modified peptide and its B6 fragment is 75 mass units; the mass difference between the unmodified peptide and its B6 fragment is 121 mass units. It was, therefore, possible to identify the reaction products by neutral loss mass spectrometry [43].

3.7.10 The application of neutral loss mass spectrometry to determine the substrate specificity of EpiD Tandem mass spectrometry methods have already been used to determine the composition of synthetic multicomponent peptide mixtures. For example, O-tertbutylated by-products of peptide libraries were identified by neutral loss scans [49]. In the constant neutral loss scan, the first and second analyzer of the mass spectrometer are scanned together such that there is a constant mass difference between the ions transmitted by the two analyzers. Under these conditions, only ions that lose a neutral fragment with a mass corresponding to the chosen mass difference will be detected. Heptapeptide sublibraries with one variable amino acid residue at positions 1 to 7 of the peptide substrate S1FNSYCC7 were synthesized and incubated with EpiD. Peptides were identified by their masses using product ion scans and neutral loss scans, and by comparison of the mass spectra obtained after various incubation times. The heptapeptides with a single amino acid substitution at positions 1 to 4 are substrates of EpiD. Not all amino acid residue substitutions at positions 5 to 7 of the peptide led to active substrates; therefore, the last three amino acids determine the substrate specificity for EpiD. For the sublibraries SFNSX5CC and SFNSYX6C, the reaction products were identified by neutral loss mass spectrometry of the peptide mixtures. The tyrosine residue at position 5 can be replaced by the hydrophobic amino acid residues V, I/L, (M), F, and W; it is not possible to differentiate between Ile and Leu by ES-MS. The cysteine residue at position 6 can be replaced by A, S, V, (I/L), and T. Pep81

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin tides containing amino acid residues with acidic or basic side chains at position 5 or 6 are not substrates of EpiD. In position 7 of the peptide substrates, only cysteine is accepted [43].

3.7.11 Structure analysis of the reaction products by two-dimensional NMR methods Due to the complexity of the NMR spectra, the introduction of a 13C isotope in the C-terminal cysteine residues was necessary to obtain the interesting signals using heteronuclear correlation experiments. Hence, the peptide KKSFNSYTC was synthesized by solid-phase synthesis using cysteine labeled with 13C at the b-carbon atom. The two lysine residues at the N-terminus were introduced to increase the solubility of the substrate in water. Two crosspeaks occur in the HSQC spectrum of the educt KKSFNSYTC. The first signal is due to the 13C-labeled b-carbon, while the second is due to the methyl groups in Tris[tris(hydroxymethyl)aminomethane]. The NMR data, the UV-VIS spectra, the MS-MS experiments, the observed reaction product with Ellman’s reagent, and the mass difference between educt and product of 46 Da indicate that the product contains a C-terminal thioenol [30].

3.7.12 The pKa value of the enethiol side chain of the reaction products is lower than that of the thiol side chain of peptides The UV-VIS spectra of the reaction products of EpiD are pH-dependent, indicating that the enethiol side chain is converted to an enethiolate anion. The pKa value of the enethiol group was determined to be 6.0 and is substantially lower than the pKa value of the thiol side chain of cysteine residues. This increased acid strength of the enethiol side chain compared to that of the thiol group is attributed to the resonance stabilization of the negative charge of the anion [39].

3.7.13 Overview of the function of EpiD In conclusion, the formation of the lantibiotic epidermin from the precursor peptide EpiA includes the oxidative decarboxylation of the C-terminal cysteine residue of EpiA to a (Z)-enethiol catalyzed by the FMN-containing enzyme EpiD. This oxidative decarboxylation reaction was the first analyzed posttranslational 82

3.8 Incorporation of d-alanine into S. aureus teichoic acids confers resistance modification reaction involved in lantibiotic biosynthesis. Two reducing equivalents from the C-terminal cysteine residue of EpiA are removed, a double bond is formed, and the coenzyme FMN is reduced to FMNH2. The decarboxylation occurs spontaneously or is catalyzed by EpiD. The in vivo conversion of (His)6tag-labeled precursor peptide EpiA by EpiD was demonstrated. The (Z)-enethiol derivative is the intermediate in the formation of the C-terminal S-((Z)-2-aminovinyl)-D-cysteine residue of epidermin. The enethiol structure has been confirmed by UV-VIS spectroscopy, mass spectrometry, tandem mass spectrometry, two-dimensional NMR spectroscopy, and conversion of the enethiol to a mixed disulfide with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent). At physiological pH, the dominant form of the reaction product is the enethiolate anion (“peptidyl-aminoenethiolates”), and the pKa of the enethiol group is 6.0. EpiD has a low substrate specificity, and most of the peptides with the sequence (V/I/ L/(M)/F/Y/W)-(A/S/V/T/C/(I/L))-C at the carboxy terminus are substrates of EpiD, as elucidated by analysis of the reaction of EpiD with single peptides and peptide libraries. Amino acid residues of EpiD involved in FMN binding or substrate binding, or important for the catalytic action are currently identified by analyzing analogues generated by site-directed mutagenesis. In addition, the structure of EpiD has been determined by X-ray crystallography (Kupke et al. unpublished). The overall aim is the detailed molecular analysis of the flavoenzyme EpiD and related enzymes, and a more detailed investigation of the role of EpiD in epidermin biosynthesis. It is still not known how reduced EpiD is re-oxidized in vivo and whether EpiD forms a complex with the enzymes EpiB and EpiC. The chemistry of the peptidyl-aminoenethiolate reaction products has to be studied in order to develop these peptides as enzyme inhibitors.

3.8 Incorporation of d-alanine into S. aureus teichoic acids confers resistance to defensins, protegrins, and other antimicrobial peptides

Why some staphylococcal strains are more tolerant to gallidermin than other strains has always been of interest. In order to answer this question, Andreas Peschel [59] isolated S. aureus and S. xylosus transposon-insertion mutants that were hypersensitive to gallidermin, and found that genes hit by the transposon are involved in teichoic acid modification.

83

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin

3.8.1 Identification and sequence analysis of the S. aureus and S. xylosus dlt operon S. aureus Sa113 and the coagulase-negative S. xylosus C2 a have high innate tolerances towards several antimicrobial peptides. To investigate the resistance mechanism, S. xylosus C2 a was mutagenized with Tn917, and the resulting transposon-insertion mutants were analyzed for reduced growth on agar plates containing the antimicrobial peptide gallidermin. The nucleotide sequence upstream and downstream of the transposon from seven clones whose growth is specifically reduced in the presence of gallidermin was determined. In all seven mutants, the transposon had integrated into the same determinant of 4.6 kb, which encodes four open reading frames arranged in an operon-like structure, followed by a typical terminator (Fig. 3.10). The open reading frames showed sequence similarity to the Lactobacillus casei and B. subtilis dltABCD operons, which are responsible for esterification of teichoic acids with d-alanine [16, 55]. The growth rates in the absence of gallidermin and the microscopic appearance of the mutants were indistinguishable from those of the wild type strain.

Figure 3.10: cocci.

Genetic organization and proposed function of the dlt operon in staphylo-

3.8.2 Disruption of the S. aureus dlt operon and analysis of the d-alanine content in lipoteichoic acids (LTA) and wall teichoic acids (WTA) The dltA gene of S. aureus Sa113 was replaced by a spectinomycin resistance gene (spc) by homologous recombination, thereby producing the galliderminsensitive strain AG1. WTA and LTA of S. aureus and S. xylosus wild type strains and mutants were isolated, and the molar ratios of d-alanine to phosphorus were determined (Table 3.1). In the wild type strains, 75% (S. aureus) and 95% (S. xylosus) of the alditol phosphate residues in LTA are esterified with d-ala84

3.8 Incorporation of d-alanine into S. aureus teichoic acids confers resistance nine, while only 51% (S. aureus) and 15% (S. xylosus) are esterified in WTA. In the dlt mutants, no d-alanine is detected in LTA or WTA, indicating that the pathway for d-alanine incorporation is inactivated by the spectinomycin resistance gene and transposon insertions. When the mutant strains are complemented with plasmid pRBdlt1, which carries the dlt operon, normal or slightly increased amounts of d-alanine are found in LTA and WTA. Transformation of the wild type strains with pRBdlt1 results in an increase of d-alanine in LTA and WTA by 5–18% [59].

3.8.3 Sensitivity towards antimicrobial peptides The minimal inhibitory concentrations of gallidermin and of several other membrane-damaging antimicrobial peptides were determined for the S. aureus and S. xylosus wild type and mutant strains. The mutants are sensitive to a variety of antimicrobial peptides that bear a positive net charge [59]. The sensitivity of the S. aureus mutant to defensin from human neutrophils and to protegrins 3 and 5 from porcine leukocytes is at least 10- to 23-fold higher. Factors of 7–12 were determined with tachyplesin 1 and 3 from hemocytes of the horseshoe crab and to a variant of magainin II from the skin of the clawed frog. The tolerance towards the lanthionine-containing bacterial peptides gallidermin from S. gallinarum and nisin from L. lactis is 8–50-fold lower; very similar results were obtained with the S. xylosus strains. The increased sensitivity of dlt mutants seems to be restricted to cationic peptides since no considerable differences are observed in the inhibitory concentrations of the neutral peptide gramicidin D from Bacillus brevis. Furthermore, the mutants are not sensitive to cationic polylysine, indicating that cationic properties are not sufficient for activity of a peptide against staphylococci lacking d-alanine esters in their teichoic acids [59]. The results indicate that the general surface charge of staphylococci can be severely influenced by the degree of esterification with d-alanine. Loss of dalanine esterification is not deleterious to the bacterium, but has other pleiotropic effects. One such effect is that mutants become hypersensitive to a large variety of cationic peptides. Other effects are currently being studied. One can speculate whether the D-alanination of teichoic acids is a means whereby S. aureus overcomes the human defensin attack to which they are confronted during the first defense regimen.

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3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin

3.9 Conclusions

The collaborative research centre 323 allowed the lantibiotic research to boom, particularly in Tübingen. The fermentation group (H. Zähner and P. Fiedler), the organic chemistry group (G. Jung) and microbial genetics group (F. Götz) complemented each other ideally. The fermentation group optimized the fermentation process of the producing strains and provided sufficient amounts of the compounds to the chemists for structure determination and mass analysis, and the molecular biologists studied the biosynthetic genes and the functions of the corresponding enzymes together with the organic chemists. This concerted action was the secret of success. Quite a few of the Tübingen achievements can be regarded as milestones in lantibiotic research, such as: . Determination of the chemical structure of epidermin (Allgaier et al. 1986) . First finding of an antibiotic substance (later named gallidermin) in Staphylococcus gallinarum (F. Götz 1984) . First identification of the structural gene (epiA) encoding the lantibiotic precursor for epidermin; and the first proof that epidermin, and as we now know, all lantibiotics, are ribosomally synthesized (Schnell et al. 1989) . Determination of the NMR structure of gallidermin (Freund et al. 1991) . First cloning and nucleotide sequence of lantibiotic (epidermin) biosynthesis genes: epiB, C, D, P, and Q (Schnell et al. 1992, Augustin et al. 1992) . First characterization of a novel flavin-mononucleotide-dependent decarboxylase encoded by the epiD gene (Kupke et al. 1992) . Regulation of epidermin biosynthesis by the activator EpiQ (Peschel et al. 1993) . Isolation and characterization of genetically engineered gallidermin and epidermin analogues (Ottenwälder et al. 1995) . Further optimization of gallidermin production leading to a productivity of 300 mg/liter culture supernatant (Kempf et al. 1997) . Secretion mechanism of the lantibiotics epidermin and gallidermin (Peschel et al. 1997) . Unique producer self-protection against the lantibiotic epidermin by the ABC transporter EpiFEG (Peschel et al. 1996; Otto et al. 1998) . Nearly complete X-ray structure of the lantibiotic biosynthesis enzyme, the flavoprotein EpiD (Kupke et al., in progress) In Fig. 3.11 an overview of the biosynthetic pathway of epidermin (gallidermin) is presented. There are still many open questions that deserve attention. For example, the underlying enzyme mechanisms for the key reaction, the lanthionine formation, is still unsolved and it is not known which cofactors are involved. The environmental conditions under which the activator EpiQ is functional are unknown; there are no indications for a two-component system like that of nisin or subtilisin. Finally, the question remains why Gram-positive bac86

3.9 Conclusions

Figure 3.11: Pathway of epidermin biosynthesis.

teria produce such lantibiotics – what is their real function? It is unlikely that the antibiotic activity is the only function since it only becomes obvious when larger amounts of the lantibiotic are produced. Many isolates produce only minute amounts, which might, however, be sufficient for communication with other bacteria. Most of the lantibiotics interact with the cytoplasmic membrane; some, such as mersacidin and gallidermin, bind to the cell wall precursor lipid I and, as recently shown, interact with the teichoic acids to an extent that depends on the degree of negative charges. Perhaps they represent a measure for the fine tuning of cell wall structures. Since genes nearly identical to the epidermin biosynthesis genes are also present in S. aureus, one can ask whether they have an influence on infection and survival in the host. Where the lantibiotic biosynthesis genes originate is not known. The nucleotide sequences of most lantibiotic genes suggest that the genes are either plasmid encoded (e. g. epidermin) or part of a transposon (e. g. nisin). They might be relicts of bacteriophages that used lantibiotics for a better penetration of their genome into the target cells; in this respect, a pore-forming activity could be helpful. Whether any of the lantibiotics were once used as antibiotics or not, the study of these unique peptides sheds light on various areas of microbiology.

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3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin

References

1. Allgaier, H., Jung, G., Werner, R. G., Schneider, U., and Zähner, H. (1985) Elucidation of the structure of epidermin, a ribosomally synthesized, tetracyclic heterodetic polypeptide antibiotic. Angew. Chem. Int. Ed. Engl. 24, 1051–1053. 2. Allgaier, H., Jung, G., Werner, R. G., Schneider, U., and Zähner, H. (1986) Epidermin: sequencing of a heterodet tetracyclic 21-peptide amide antibiotic. Eur. J. Biochem. 160, 9–22. 3. Augustin, J. (1991) Dissertation. Eberhard-Karls-Universität Tübingen. 4. Augustin, J., Rosenstein, R.,Wieland, B., Schneider, U., Schnell, N., Engelke, G., Entian, K. D., and Götz, F. (1992) Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur. J. Biochem. 204, 1149–1154. 5. Benz, R., Jung, G., and Sahl, H.-G. (1991) Mechanism of channel formation by lantibiotics in black lipid membranes. In: Jung, G. and Sahl, H. G. (eds.), Nisin and novel lantibiotics. Leiden: Escom, pp. 359–372. 6. Berridge, N., Newton, N. J., and Abraham, E. P. (1952) Purification and nature of the antibiotic nisin. Biochem. J. 52, 529–535. 7. Biemann, H. P. and Erikson, R. L. (1990) Abnormal protein kinase C down regulation and reduced substrate levels in non-phorbol ester-responsive 3T3-TNR9 cells. Mol. Cell Biol. 10, 2122–2132. 8. Biemann, K. (1992) Mass spectrometry of peptides and proteins. Annu. Rev. Biochem. 61, 977–1010. 9. Bierbaum, G., Brötz, H., Koller, K. P., and Sahl, H. G. (1995) Cloning, sequencing and production of the lantibiotic mersacidin. FEMS Microbiol. Lett. 127, 121–126. 10. Bierbaum, G., Götz, F., Peschel, A., Kupke, T., van de Kamp, M., and Sahl, H.-G. (1996) The biosynthesis of the lantibiotics epidermin, gallidermin, pep5 and epilancin K7. Antonie Van Leeuwenhoek. 69, 119–127. 11. Bierbaum, G. and Sahl, H. G. (1987) Autolytic system of Staphylococcus simulans 22: influence of cationic peptides on activity of N-acetylmuramoyl-L-alanine amidase. J. Bacteriol. 169, 5452–5458. 12. Bolhuis, H., van Veen, H. W., Poolman, B., Driessen, A. J., and Konings, W. N. (1997) Mechanisms of multidrug transporters. FEMS Microbiol. Rev. 21, 55–84. 13. Brötz, H., Josten, M., Wiedemann, I., Schneider, U., Götz, F., Bierbaum, G., and Sahl, H. G. (1998) Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics. Mol. Microbiol. 30, 317–327. 14. Brückner, R. (1997) Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiol. Lett. 151, 1–8. 15. Buchman, G. W., Banerjee, S., and Hansen, J. N. (1988) Structure, expression and evolution of a gene encoding the precursor of nisin, a small protein antibiotic. J. Biol. Chem. 263, 16260–16266. 16. Debabov, D. V., Heaton, M. P., Zhang, Q., Stewart, K. D., Lambalot, R. H., and Neuhaus, F. C. (1996) The d-alanyl carrier protein in Lactobacillus casei: cloning, sequencing and expression of dltC. J. Bacteriol. 178, 2869–3876. 17. Devriese, L. A., Poutrel, B., Kilpper-Bälz, R., and Schleifer, K. H. (1983) Staphylococcus gallinarum and Staphylococcus caprea, two new species from animals. Int. J. Syst. Bact. 33, 480–486. 18. Freund, S., Jung, G., Gutbrod, O., Folkers, G., and Gibbons, W. A. (1991) The threedimensional solution structure of gallidermin determined by NMR-based molecular graphics. In: Jung, G. and Sahl, H.-G. (eds.), Nisin and novel lantibiotics. Leiden: Escom, pp. 91–102.

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References 19. Freund, S., Jung, G., Gutbrod, O., Folkers, G., Gibbons, W. A., Allgaier, H., and Werner, R. (1991) The solution structure of the lantibiotic gallidermin. Biopolymers 31, 803–811. 20. Geißler, S., Götz, F., and Kupke, T. (1996) Serine protease EpiP from Staphylococcus epidermidis catalyzes the processing of the epidermin precursor peptide. J. Bacteriol. 178, 284–288. 21. Gross, E., Kiltz, H., and Nebelin, E. (1973) Subtilin. VI. Die Struktur des Subtilins. Hoppe-Seyler’s Z. Physiol. Chem. 354, 810–812. 22. Gross, E. and Morell. J. L. (1971) The structure of nisin. J. Am. Chem. Soc. 93, 4634– 4635. 23. Hurst, A. (1966) Biosynthesis of the antibiotic nisin by Streptococcus lactis organisms. J. Gen. Microbiol. 44, 209–220. 24. Ingram, L. C. (1969) Synthesis of the antibiotic nisin: formation of lanthionine and bmethyl-lanthionine. Biochim. Biophys. Acta. 184, 216–219. 25. Jack, R., Benz, R., Tagg, J., and Sahl, H. G. (1994) The mode of action of SA-FF22, a lantibiotic isolated from Streptococcus pyogenes strain FF22. Eur. J. Biochem. 219, 699–705. 26. Jung, G. (1991) Lantibiotics – ribosomally synthesized biologically active polypeptides containing sulfide bridges and a,b-didehydroamino acids. Angew. Chem. Int. Ed. Engl. 30, 1051–1068. 27. Katz, E. and Demain, A. L. (1977) The peptide antibiotics of Bacillus: chemistry, biogenesis, and possible functions. Bacteriol. Rev. 41, 449–474. 28. Kellner, R., Jung, G., Hörner, T., Zähner, H., Schnell, N., Entian, K. D., and Götz, F. (1988) Gallidermin: a new lanthionine-containing polypeptide antibiotic. Eur. J. Biochem. 177, 53–59. 29. Kempf, M., Theobald, U., and Fiedler, H.-P. (1999) Economic improvement of the fermentative production of gallidermin by Staphylococcus gallinarum. Biotechnol. Lett. 21, 663–667. 30. Kempter, C., Kupke, T., Kaiser, D., Metzger, J. W., and Jung, G. (1996) Thioenols from peptidyl-cysteines: oxidative decarboxylation of a 13C labeled substrate. Angew. Chem. Int. Ed. Engl. 35, 2104–2107. 31. Kleerebezem, M., Quadri, L. E. N., Kuipers, O. P., and de Vos, W. M. (1997) Quorum sensing by peptide pheromones and two-component signal-transducing systems in Gram-positive bacteria. Mol. Microbiol. 24, 895–904. 32. Klein, C. and Entian, K. D. (1994) Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633. Appl. Environ. Microbiol. 60, 2793–2801. 33. Kleinkauf, H. and von Döhren, H. (1990) Nonribosomal biosynthesis of peptide antibiotics. Eur. J. Biochem. 192, 1–15. 34. Kordel, M. and Sahl, H. G. (1986) Susceptibility of bacterial, eukaryotic and artificial membranes to the disruptive action of the cationic peptide Pep5 and nisin. FEMS Microbiol. Lett. 34, 139–144. 35. Kordel, M., Schuller, F., and Sahl, H. G. (1989) Interaction of the pore forming-peptide antibiotics Pep 5, nisin and subtilin with non-energized liposomes. FEBS Lett. 244, 99–102. 36. Kuipers, O. P., Beerthuyzen, M. M., de Ruyter, P. G. G. A., Luesink, E. J., and de Vos, W. M. (1995) Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J. Biol. Chem. 270, 27299–27304. 37. Kuipers, O. P., Beerthuyzen, M. M., Siezen, R. J., and de Vos, W. M. (1993) Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis. Eur. J. Biochem. 216, 281–291. 38. Kuipers, O. P., Rollema, H. S., Yap, W. M., Boot, H. J., Siezen, R. J., and de Vos, W. M. (1992) Engineering dehydrated amino acid residues in the antimicrobial peptide nisin. J. Biol. Chem. 267, 24340–2436.

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3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin 39. Kupke, T. and Götz, F. (1997) The enethiolate anion reaction products of EpiD: pKa value of the enethiol side chain is lower than that of the thiol side chain of peptides. J. Biol. Chem. 272, 4759–4762. 40. Kupke, T. and Götz, F. (1996) Expression, purification, and characterization of EpiC, an enzyme involved in the biosynthesis of the lantibiotic epidermin, and sequence analysis of Staphylococcus epidermidis epiC mutants. J. Bacteriol. 178, 1335–1340. 41. Kupke, T. and Götz, F. (1996) Post-translational modifications of lantibiotics. Antonie van Leeuwenhoek. 69, 139–150. 42. Kupke, T., Kempter, C., Gnau, V., Jung, G., and Götz, F. (1994) Mass spectroscopic analysis of a novel enzymatic reaction. J. Biol. Chem. 269, 5653–5659. 43. Kupke, T., Kempter, C., Jung, G., and Götz, F. (1995) Oxidative decarboxylation of peptides catalyzed by flavoprotein EpiD: determination of substrate specificity using peptide libraries and neutral loss mass spectrometry. J. Biol. Chem. 270, 11282– 11289. 44. Kupke, T., Stevanovic, S., Ottenwälder, B., Metzger, J. W., Jung, G., and Götz, F. (1993) Purification and characterization of EpiA, the peptide substrate for posttranslational modifications involved in epidermin biosynthesis. FEMS Microbiol. Lett. 112, 43–48. 45. Kupke, T., Stevanovic, S., Sahl, H. G., and Götz, F. (1992) Purification and characterization of EpiD, a flavoprotein involved in the biosynthesis of the lantibiotic epidermin. J. Bacteriol. 174, 5354–5361. 46. Linnett, P. E. and Strominger, J. L. (1973) Additional antibiotic inhibitors of peptidoglycan synthesis. Antimicrob. Agents Chemother. 4, 231–236. 47. Liu, W. and Hansen, J. N. (1993) The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanism. Appl. Environ. Microbiol. 59, 648–651. 48. Liu, W. and Hansen, J. N. (1992) Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis. J. Biol. Chem. 267, 25078–25085. 49. Metzger, J. W., Kempter, C., Wiesmuller, K. H., and Jung, G. (1994) Electrospray mass spectrometry and tandem mass spectrometry of synthetic multicomponent peptide mixtures: determination of composition and purity. Anal. Biochem. 219, 261–277. 50. Molitor, E. and Sahl, H.-G. (1991) Applications of nisin: a literature survey. In: Jung, G. and Sahl, H.-G. (eds.), Nisin and novel lantibiotics. Leiden: Escom, pp. 434–439. 51. Moll, G. N., Konings, W. N., and Driessen, A. J. (1998) The lantibiotic nisin induces transmembrane movement of a fluorescent phospholipid. J. Bacteriol. 180, 6565–6570. 52. Moll, G. N., Roberts, G. C., Konings, W. N., and Driessen, A. J. (1996) Mechanism of lantibiotic-induced pore-formation. Antonie Van Leeuwenhoek. 69, 185–191. 53. Ottenwälder, B., Kupke, T., Brecht, S., Gnau, V., Metzger, J., Jung, G., and Götz, F. (1995) Isolation and characterization of genetically engineered gallidermin and epidermin analogs. Appl. Environ. Microbiol. 61, 3894–3903. 54. Otto, M., Peschel, A., and Götz, F. (1998) Producer self-protection against the lantibiotic epidermin by the ABC transporter EpiFEG of Staphylococcus epidermidis Tü3298. FEMS Microbiol. Lett. 166, 203–211. 55. Perego, M., Glaser, P., Minutello, A., Strauch, M. A., Leopold, K., and Fischer, W. (1995) Incorporation of d-alanine into lipoteichoic acid and wall teichoic acid in Bacillus subtilis. J. Biol. Chem. 270, 15598–15606. 56. Peschel, A., Augustin, J., Kupke, T., Stevanovic, S., and Götz, F. (1993) Regulation of epidermin biosynthetic genes by EpiQ. Mol. Microbiol. 9, 31–39. 57. Peschel, A. and Götz, F. (1996) Analysis of the Staphylococcus epidermidis genes epiF, E, and G involved in epidermin immunity. J. Bacteriol. 178, 531–536. 58. Peschel, A., Ottenwälder, B., and Götz, F. (1996) Inducible production and cellular location of the epidermin biosynthetic enzyme EpiB using an improved staphylococcal expression system. FEMS Microbiol. Lett. 137, 279–284.

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References 59. Peschel, A., Otto, M., Jack, R. W., Kalbacher, H., Jung, G., and Götz, F. (1999) Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J. Biol. Chem. 274, 8405–8410. 60. Peschel, A., Schnell, N., Hille, M., Entian, K.-D., and Götz, F. (1997) Secretion of the lantibiotics epidermin and gallidermin: sequence analysis of the genes gdmT and gdmH, their influence on epidermin production and their regulation by EpiQ. Mol. Gen. Genet. 254, 312–318. 61. Prasch, T., Naumann, T., Markert, R. L., Sattler, M., Schubert, W., Schaal, S., Bauch, M., Kogler, H., and Griesinger, C. (1997) Constitution and solution conformation of the antibiotic mersacidin determined by NMR and molecular dynamics. Eur. J. Biochem. 244, 501–512. 62. Rayman, M. K., Aris, B., and Hurst, A. (1981) Nisin: a possible alternative or adjunct to nitrite in the preservation of meats. Appl. Environ. Microbiol. 41, 375–380. 63. Reis, M., Eschbach-Bludau, M., Iglesias-Wind, M. I., Kupke, T., and Sahl, H. G. (1994) Producer immunity towards the lantibiotic Pep5: identification of the immunity gene pepI and localization and functional analysis of its gene product. Appl. Environ. Microbiol. 60, 2876–2883. 64. Reisinger, P., Seidel, H., Tschesche, H., and Hammes, W. P. (1980) The effect of nisin on murein synthesis. Arch. Microbiol. 127, 187–193. 65. Rogers, L. A. and Whittier, E. O. (1928) The inhibitory effect of Streptococcus lactis on Lactobacillus bulgarius. J. Bacteriol. 16, 321–325. 66. Ruhr, E. and Sahl, H. G. (1985) Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles. Antimicrob. Agents Chemother. 27, 841–845. 67. Sahl, H.-G., Jack, R. W., and Bierbaum, G. (1995) Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur. J. Biochem. 230, 827–853. 68. Sahl, H. G. (1991) Pore formation in bacterial membranes by cationic lantibiotics. In: Jung, G. and Sahl, H. G. (eds.), Nisin and novel lantibiotics. Leiden: Escom, pp. 347– 358. 69. Sahl, H. G. and Brandis, H. (1983) Efflux of low Mr substances from the cytoplasm of sensitive cells caused by the staphylococcin-like agent pep5. FEMS Microbiol. Lett. 16, 75–79. 70. Schnell, N., Engelke, G., Augustin, J., Rosenstein, R., Ungermann, V., Götz, F., and Entian, K. D. (1992) Analysis of genes involved in the biosynthesis of lantibiotic epidermin. Eur. J. Biochem. 204, 57–68. 71. Schnell, N., Entian, K. D., Götz, F., Horner, T., Kellner, R., and Jung, G. (1989) Structural gene isolation and prepeptide sequence of gallidermin, a new lanthionine containing antibiotic. FEMS Microbiol. Lett. 49, 263–267. 72. Schnell, N., Entian, K. D., Schneider, U., Götz, F., Zähner, H., Kellner, R., and Jung, G. (1988) Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature 333, 276–278. 73. Schüller, F., Benz, R., and Sahl, H. G. (1989) The peptide antibiotic subtilin acts by formation of voltage-dependent multi-state pores in bacterial and artificial membranes. Eur. J. Biochem. 182, 181–186. 74. Siegers, K. and Entian, K.-D. (1995) Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3. Appl. Environ. Microbiol. 61, 1082–1089. 75. Siezen, R. J., Kuipers, O. P., and de Vos, W. M. (1996) Comparison of lantibiotic gene clusters and encoded proteins. Antonie Van Leeuwenhoek. 69, 171–184. 76. Stock, J. B., Ninfa, A. J., and Stock, A. M. (1989) Protein phosphorylation and regulation of adaptive response in bacteria. Microbiol. Rev. 53, 450–490. 77. Vogel, H., Nilsson, L., Rigler, R., Meder, S., Boheim, G., Beck, W., Kurth, H. H., and Jung, G. (1993) Structural fluctuations between two conformational states of a trans-

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3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin membrane helical peptide are related to its channel-forming properties in planar lipid membranes. Eur. J. Biochem. 212, 305–313. 78. Weil, H. P., Beck-Sickinger, A. G., Metzger, J., Stevanovic, S., Jung, G., Josten, M., and Sahl, H. G. (1990) Biosynthesis of the lantibiotic Pep5: isolation and characterization of a prepeptide containing dehydroamino acids. Eur. J. Biochem. 194, 217–223.

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4 Fermentation of Lantibiotics Epidermin and Gallidermin Uwe Theobald*

4.1 Introduction

In recent years the research of the lantibiotics epidermin and gallidermin focused on two topics, mainly the more genetically based work on the biosynthesis of those antimicrobial peptides [1–3] and their production in bioreactors, respectively. Since antibiotics serve a vital role in modern medicine, fermentation technology represents a commercially significant part [4, 5]. Additionally, the need for an industrial production of new bioactive compounds become more and more important due to the increasing problem of multi-resistence in bacteria [6]. Both lantibiotics, gallidermin produced by Staphylococcus gallinarum Tü 3928 [7] and epidermin produced by Staphylococcus epidermidis Tü 3298 [8] exhibit strong activity against Gram-positive bacteria, particularly against propioni bacteria which are involved in the acne disease. Other favourable properties for a large-scale production are the comparable biological activity to renowned antibiotics in current clinical practice like erythromycin or fusidin because of the bactericidal impressiveness of both compounds against actively growing and non-dividing bacteria, the advantage for treatment of endocarditis, abscesses or skin infections. Finally, gallidermin or epidermin are eligible alternatives to vancomycin, so that these compounds gained pharmaceutical interest. The remarkable pharmacological properties led to extensive investigations during the last decade to optimise the production process of these drugs for industrial purpose [9–12]. However, each biotechnological production process is an unique operation. So, every step of this process needs optimisation for a promising conversion into an industrial standard. Major crucial points and optimisation steps known in general are the variation of media components or the design of special media compositions [13], the development of a suitable and reproducible process [14], the scale-up problems including oxygen transfer and mixing in the

* LSMW GmbH, Roßbachstraße 38, D-70499 Stuttgart

93 Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

4 Fermentation of Lantibiotics Epidermin and Gallidermin bioreactor [15], and the implementation of down stream processing steps to the process [16]. Hence, a complete summary of data concerning the fermentative production of gallidermin or epidermin has to reflect all optimisation steps of a fermentation – from strain cultivation to large scale production. Therefore, this chapter will focus on several different topics of an entire antibiotic production process that enabled efficient metabolite production.

4.2 Strains for gallidermin/epidermin production

The fermentative production of epidermin or gallidermin was possible with different microbial systems. Besides the mentioned wild type strains epidermin could be produced by a recombinant strain of Staphylococcus carnosus. In contrast to those wild type strains, Staphylococcus carnosus is a “food-grade” strain which is applied in starter cultures. Beyond that the strain Staphylococcus carnosus TM 300 was successfully used for heterologous expression of epidermin in a two-plasmid system. With the recombinant strain Staphylococcus carnosus TM 300 (pTepiMA+, pRBgepSI), containing the biosynthesis genes for epidermin production and immunity against its own antibiotic, nearly 70 % of the wild type production yield were observed. A problem of the two-plasmid system was the insufficient plasmid stability, so that most work on process development and optimisation was carried out with the wild type strains S. gallinarum Tü 3928 and S. epidermidis Tü 3298. According to the somewhat higher activity against living and non-dividing Gram-positive bacteria, this review will focus on the antibiotic gallidermin solely, although a lot of successful work was done for epidermin, too [17–21].

4.3 Disadvantages during gallidermin process development

Regarding the development of a gallidermin production process over a couple of years a few problems were generally noticed. First of all, a exceedingly strain instability concerning product excretion was observed, though biomass formation remained still stable. This fluctuating lantibiotic production yield was contradictory to a reproducible and standardised process. A second crucial point 94

4.4 Gallidermin – a lantibiotic and its way towards industrial production was the composition of the production medium. Several investigations dealt with the search for a defined and synthetic medium for staphylococci, but the biomass and product concentrations gained with the developed medium [22] could not compete with any of the tested complex media. Best production yield could be observed with a medium based upon meat extract. Meat extract was supposed to be the only suitable complex medium component that led to appropriate product yields [9] but is first of high costs for production or downstream processing and second carried a possible risk of prion infections such as bovine spongiform encephalopathy or Creutzfeld-Jakob-disease [23–25].

4.4 Gallidermin – a lantibiotic and its way towards industrial production

The basis for a microbial process is the stock culture for storage of the talented strain. This first step of the production could be identified as prime cause for the problem of the mentioned instability in product formation during fermentation [26]. A new mathematical parameter (hs-value) based upon the commonly analysed parameters biomass and product concentration was introduced to facilitate the optimisation of the media used for this cultivation of stock cultures on agar slants. The hs-value (high and stable product concentration) reduced the amount of data generated in optimisation experiments to one single value for each medium composition and allowed an assessment of any medium formulation with regard to reproducibility and product formation [26]. Figure 4.1 illustrates the time course of the product concentration over several passages found in the corresponding liquid cultures after 24 hours of incubation (A) and the corresponding hs-values (B) obtained exemplary from four different stock culture media (out of 53 different formulations tested) during several passages. Another indispensable optimisation step was the design of a new medium composition. An analysis of different complex compounds [13] suggested the possibility for a successful use of yeast extract (without risk of prion infection). In contrast to earlier results [9, 27] the investigations revealed that a special yeast extract could efficiently replace meat extract in the production medium of S. gallinarum Tü 3928. In addition to that the special yeast extract (Ohly KAT) applied in a three-fold lower concentration led to approximately 20 % higher product yields compared to the old medium composition [28]. Figure 4.2 shows a comparison of the gallidermin concentrations obtained with these media in batch fermentations. Since the Staphylococcus species are very halotolerant in general, the effect of salt (cation) is very important for growth and production [9–11, 27]. To optimise this parameter, different cations in various concentrations were 95

4 Fermentation of Lantibiotics Epidermin and Gallidermin

Figure 4.1: Gallidermin concentrations (A) and corresponding hs-values (B) obtained from a suitability test shown for four different stock culture media during several passages.

tested [12, 28–30]. Best results were gained with CaCl2 in very high concentrations of 30 to 40 g/l whereas the other cations like NaCl showed only slight influence. Figure 4.3 indicated, that there is an optimum in CaCl2 concentration whereas the NaCl concentration is only of slight influence upon product formation. However, Peschel and co-workers demonstrated that cationic teichonic acids at the staphylococcal cell surface are involved in gallidermin resistance [31]. This may be due to an ionic interaction between the teichonic acids and the gallidermin molecule that also has a positive net charge (3-fold). Although the mechanism of resistance is not yet clear in detail [1] it is possible that Ca2+ 96

4.4 Gallidermin – a lantibiotic and its way towards industrial production

Figure 4.2: Comparison of gallidermin formation by Staphylococcus gallinarum Tü 3928 grown in the previously used medium (open symbols) and the new developed production medium (full symbols). Both cultures were inoculated from the same agar plate.

Figure 4.3: Maximal gallidermin concentrations in the culture broth of Staphylococcus gallinarum Tü 3928 in dependency on different NaCl/CaCl2 proportions in the production medium.

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4 Fermentation of Lantibiotics Epidermin and Gallidermin probably may be involved in the immunity as well. Hence, the components of the new medium were roughly (qualitatively) adjusted. Final optimisation were carried out with a computer-aided procedure. Genetic algorithms are able to optimise simultaneously a multi-parameter system like a medium composition (quantitatively optimisation) [32]. Several fermentation modes (batch, fed-batch, continuous) were tested. Preliminary experiments in continuous cultivations resulted in lower gallidermin yields compared to those gained in batch or fed-batch cultures. Best results were determined in batch processes or fed-batch processes with pulses of several amino acids or maltose during the antibiotic production phase [33]. A comparison of different types of bioreactor (dialysis fermenter, airlift reactor, stirred tank and loop reactor) showed no significant differences in growth and production. The effect of dissolved oxygen tension seemed to be more critical [11] than the type of reactor. The dialysis reactor consisted of two chambers which are separated by a dialysis membrane with an exclusion limit of 10 000 Dalton [10]. This reactor has the advantage of on-line product separation during the production phase but has the crucial drawback that no scale-up procedure is possible. Hence, a transfer from laboratory scale to industrial scale seemed to be out of question at that time. Scale-up investigations were focused on the batch and fed-batch processes in stirred tank reactors, which enabled a reliable scale-up procedure from 0.2 litre to 200 litre scale [34]. Best results in large scale fermentations were achieved by addition of maltose during the late production phase as shown in Fig. 4.4.

Figure 4.4: Concentrations of gallidermin (full symbols) and cell dry weight (open symbols) observed during a scale-up from 20 to 200 litres bioreactor. The arrow marks the transition from 20 to 200 litres and the time when maltose is pulsed into the 200 litres reactor; the grey area shows the range of the maximal gallidermin concentration (including errors) expected in a 200 litres bioreactor without maltose addition.

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4.5 Conclusion The downstream processing (product recovery from the fermentation broth) was successfully optimised by product adsorption onto resin (Amberlite XAD-1180) [35]. An integration of the product separation step was performed by direct addition of resin into the bioreactor during fermentation or addition of an cross-flow filtration step between bioreactor and adsorber column. This resulted in a cell retention by the cross-flow module during the production phase. Product containing filtrate was harvested afterwards onto an adsorber column with XAD resin. So, the overall production time could be prolonged.

4.5 Conclusion Extensive investigations led to a new method for strain storage and cultivation, the design of a new production medium, process development and the ability for scale-up into technical scale. An integration of a filtration step during production permitted an on-line product harvesting. Finally, it should be emphasised that higher production yields were obtained (more than 300 mg/l) and costs for medium components were drastically reduced more than 15-fold from 72 Euro per gram gallidermin to approximately 4.6 Euro per gram. Figure 4.5 summarised the reduction of medium costs and the increase in gallidermin concentration during the three optimisation steps (replacement of ingredients, rough adjustment of salt concentrations and final optimisation using a computer program).

Figure 4.5: Evolution of medium costs (columns) and product yields during three optimisation steps (see text).

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4 Fermentation of Lantibiotics Epidermin and Gallidermin

References

1. Jack, R. W., Bierbaum, G., and Sahl, H.-G. (1998) Lantibiotics and related peptides. Springer-Verlag, Berlin. 2. Bierbaum, G., Götz, F., Peschel, A., Kupke, T., van de Kamp, M., and Sahl, H.-G. (1996) The biosynthesis of the lantibiotics epidermin, gallidermin, pep5 and epilancin K7. Antonie van Leeuwenhoek. 69, 119–127. 3. Kupke, T. and Götz, F. (1996) Post-translational modifications of lantibiotics. Antonie van Leeuwenhoek. 69, 139–150. 4. Strohl, W. R. (1997) Industrial antibiotics: Today and the future. In: Biotechnology of Antibiotics (ed. W. R. Strohl), Marcel Dekker, New York, pp 1–48. 5. Neijssel, O. M., Teixeira de Mattos, M. J., and Tempest, D. W. (1993) Overproduction of metabolites. In: Biotechnology (eds. H.-J. Rehm and G. Reed), Vol 1 Biological fundamentals (ed. H. Sahm), VCH, Weinheim, pp 163–188. 6. Dennensen, P. J. W., Bonten, M. J. M. and Weinstein, R. A. (1998) Multiresistant bacteria as a hospital epidemic problem. Annals of Medicine 30, 176–185. 7. Kellner, R., Jung, G., Hörner, T., Zähner, H., Schnell, N., Entian, K.-D., and Götz, F. (1988) Gallidermin: a new lanthionine-containing polypeptide antibiotic. Eur. J. Biochem. 177, 53–59. 8. Allgaier, H., Jung, G., Werner, R. G., Schneider, U., and Zähner, H. (1986) Epidermin: a sequencing of a heterodet tetracyclic 21-peptide amide antibiotic. Eur. J. Biochem. 160, 9–22. 9. Hörner, T., Ungermann, V., Zähner, H., Fiedler, H.-P., Utz, R., Kellner, R., and Jung, G. (1990) Comparative studies on the fermentative production of lantibiotics by staphylococci. Appl. Microb. Biotechnol. 32, 511–517. 10. Ungermann, V., Goeke, K., Fiedler, H.-P., and Zähner, H. (1991) Optimisation of fermentation and purification of gallidermin and epidermin. In: Nisin and novel lantibiotics (eds. G. Jung and H.-G. Sahl), Escom, Leiden, pp 410–421. 11. Kempf, M., Theobald, U., and Fiedler, H.-P. (1997) Influence of dissolved O2 on the fermentative production of gallidermin by Staphylococcus gallinarum. Biotech. Lett. 19, 1063–1065. 12. Breckel, A., Harder, M., Fiedler, H.-P., and Zähner, H. (1995) Production of gallidermin by Staphylococcus gallinarum Tü 3928. In: Biochemical Engineering 3 (ed. R. D. Schmid), Kurz & Co, Stuttgart, pp 62–66. 13. Greasham, R. L. (1993) Media for microbial fermentations. In: Biotechnology (eds. H.J. Rehm and G. Reed), Vol 3 Bioprocessing (ed. G. Stephanopoulos), VCH, Weinheim, pp 127–140. 14. Fiechter, A. (1986) Bioprocess development. In: Overproduction of microbial metabolism (eds. Z. Vanek and Z. Hostalek), Butterworths, Boston, pp 231–259. 15. Reuss, M. (1993) Oxygen transfer and mixing: scale-up implications. In: Biotechnology (eds. H.-J. Rehm and G. Reed), Vol 3 Bioprocessing (ed. G. Stephanopoulos), VCH, Weinheim, pp 185–217. 16. Spears, R. (1993) Overview of downstream processing. In: Biotechnology (eds. H.-J. Rehm and G. Reed), Vol 3 Bioprocessing (ed. G. Stephanopoulos), VCH, Weinheim, pp 39–55. 17. Jung, G., Allgaier, H., Kellner, R., Schneider, U., Hörner, T., Zähner, H., and Werner R. G. (1987) Isolation, purification and structure elucidation of epidermin, a ribosomally synthesized polypeptide antibiotic. In: Biochemical Engineering 1 (eds. H. Chmiel, W. P. Hammes and J. E. Bailey), Gustav Fischer, Stuttgart, pp 494–497. 18. Werner, R. G., Zähner, H., Jung, G., Allgaier, H., and Schneider, U. (1985) Antibio-

100

References

19. 20. 21.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34. 35.

tisches Polypeptid, Verfahren zu seiner Herstellung und seine Verwendung. European patent No. 0 181 578 B1. Werner, R. G., Zähner, H., Jung, G., Hörner, T., Kellner, R., and Fiedler, H.-P. (1989) Verfahren zur Gewinnung, Isolierung und Reinigung von Epidermin. European patent No. 0 350 810 B1. Allgaier, H., Hentschel, N., Walter, J. and Werner R. G. (1992) Isolation and purification of lantibiotics. European patent No. 0 508 371 A1. Ungermann, V., Hörner, T., Utz, R., Fiedler, H.-P., and Zähner, H. (1991) Comparative studies on the fermentation of lantibiotics, produced by Staphylococci. In: Biochemical Engineering 2 (eds. M. Reuss, H. Chmiel, E.-D. Gilles, and H.-J. Knackmuss), Gustav Fischer, Stuttgart, pp 301–305. Mollenkopf, F. (1998) Untersuchungen zur Epiderminbiosynthese bei Staphylococcus epidermidis Tü 3298 und zum Wachstum von Staphylokokken. Dissertation Universität Tübingen. Collee, J. G. and Bradley, R. (1997) BSE: A decade on – part 1. Lancet 349, 636–641. Collee, J. G. and Bradley, R. (1997) BSE: A decade on – part 2. Lancet 349, 715–721. Ironside, J. W. (1996) Creutzfeld-Jakob disease. Brain Path 6, 379–388. Theobald, U. and Kempf, M. (1998) A novel tool for medium optimisation and characterization in the early stages of a metabolite production process. Biotechnol. Techniques 12, 893–897. Jung, G., Kellner, R., Zähner, H., Götz, F., Hörner, T., Werner, R. G., and Allgaier, H. (1989) Antibiotic. European patent No. 0 342 486 B1. Kempf, M., Theobald, U., and Fiedler, H.-P. (1999) Economic improvement of the fermentative production of gallidermin by Staphylococcus gallinarum. Biotech. Lett. 21, 663–667. Ungermann, V. (1992) Untersuchungen zur Produktbildung und Scale-up der Produktion von Gallidermin einem Lanthioninhaltigen Peptidantibiotikum aus Staphylococcus gallinarum Tü 3928. Dissertation Universität Tübingen. Hörner, T. (1989) Fermentation und Isolierung Lanthioninhaltiger Polypeptidantibiotika aus Staphylococcen. Dissertation Universität Tübingen. Peschel, A., Otto, M., and Götz, F. (1998) Incorporation of D-alanine into staphylococcal teichonic acids confers resistance to antimicrobial peptides from bacteria, animals and humans. Lecture (No. KA021) held at the VAAM Jahrestagung, (22.–25. 3. 1998), Frankfurt. Holland, J.H. (1975) Adaption in natural and artificial systems. The University of Michigan Press Ann Arbor, Michigan. Kempf, M., Theobald, U., and Fiedler, H.-P. (1999) Correlation between the consumption of amino acids and the production of the antibiotic gallidermin by Staphylococcus gallinarum. Biotech. Lett. (accepted for publication). Kempf, M., Theobald, U., and Fiedler, H.-P. (2000) Production of the antibiotic gallidermin by Staphylococcus gallinarum – Development of a scale-up procedure. Biotech. Lett. (submitted for publication). Allgaier, H., Walter, J., Schlüter, M., and Werner, R. G. (1991) Strategy for purification of lantibiotics. In: Nisin and novel lantibiotics (eds. G. Jung and H.-G. Sahl), Escom, Leiden, pp 422–433.

101

5 Genetics of Nikkomycin Production in Streptomyces tendae Tü 901 Christiane Bormann*

5.1 Introduction: nikkomycins

Nikkomycins are peptidyl nucleoside antibiotics that are potent and specific inhibitors of chitin synthetases. They are structurally similar to UDP-N-acetylglucosamine, the natural substrate of these enzymes, and inhibition occurs in a competitive manner [1, 2]. Structures of nikkomycins are shown in Fig. 5.1. Nikkomycins X and Z, the main compounds produced by Streptomyces tendae Tü 901, exhibit high antifungal, insecticidal, and acaricidal activity [3]. Since their toxicity towards mammals and bees is very low or not detectable, and since nikkomycins are easily degraded in nature, they are potentially useful in agriculture or as therapeutic antifungal agents in humans. Nikkomycin Z shows significant activity towards the highly chitinous, pathogenic, dimorphic fungi Coccidioides immitis and Blastomyces dermatitidis [4]. Polyoxins, antibiotics related to nikkomycins, are commercially produced by Streptomyces cacaoi and applied as agricultural fungicides in Japan (for a review, see [5]). Nikkomycins X and Z are composed of the unusual amino acid hydroxypyridylhomothreonine (HPHT; nikkomycin D) and a peptidically linked nucleoside moiety. The nucleoside moiety comprises an aminohexuronic acid N-glycosidically linked to 4-formyl-4-imidazolin-2-one, forming nikkomycin Cx , or to uracil, forming nikkomycin Cz. Minor components of the culture filtrate of S. tendae Tü 901 are nikkomycins I and J, which are structures analogous to nikkomycins X and Z that contain glutamic acid peptidically bound to the 6'-carboxyl group of the aminohexuronic acid. Nikkomycins Cx , Cz, and D are also detected in culture filtrates and arise by hydrolytic cleavage of nikkomycins X and Z. These three compounds neither act as chitin synthetase inhibitors nor display biological activity. Nikkomycins Kx, Kz, Ox, and Oz (Fig. 5.1), which will be dis-

* Mikrobiologie/Biotechnologie, Universität Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen

102 Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

5.1 Introduction: nikkomycins

R3 C O R2 NH CH O R 1

OH OH A

O

CHO

HN

R1

O

HN

N

N

O

OH

R2

HO

OH

NH2

CH CH CH C N

C

B

CH3

H

O

NH2

CH CH2 CH C N

O

D

OH

R2

HO

NH2

CH CH2 CH C N

O

nikkomycin

R1

R2

R3

X

A

A

OH

Z

B

A

OH

I

A

A

Glu

J

B

A

Glu

Cx Cz

A B

B B

OH OH

Kx

A

C

OH

Kz

B

C

OH

Ox

A

D

OH

Oz

B

D

OH

D

HO

OH

NH2

CH CH CH C OH N

CH3

O

Figure 5.1: Structures of nikkomycins.

103

5 Genetics of Nikkomycin Production in Streptomyces tendae Tü 901 cussed in a later section, are synthesized by mutants derived from S. tendae Tü 901/8 c by UV treatment and chemical mutagenesis [6]. Biosynthesis of nikkomycins can be divided into two parts: the nucleoside and peptidyl moieties are synthesized in separate pathways and then are linked by peptide bonds [6]. Figure 5.2 summarizes the steps of the nikkomycin biosynthetic pathway based on isotopic labeling and chemical characterization of pathway intermediates and shunt products. It also includes data on the biosynthesis of the polyoxin nucleoside, which contains an aminohexuronic acid moiety identical to that of nikkomycin nucleosides. Isono and coworkers [7] have shown that the polyoxin nucleoside arises from uridine and carbon-3 of phosphoenolpyruvate, and they proposed the reaction mechanism as a condensation of uridine and phosphoenolpyruvate to octofuranuloseuronic acid as the intermediate, followed by oxidative elimination of carbon-7' and carbon-8' and introduction of an amino group on carbon-5'. Isolation of octosyl acids, shunt metabolites derived from the postulated intermediate, supports this hypothesis. Analogues of octosyl acids, nikkomycins Sx and Sz, have been isolated from nikkomycin-producing S. tendae, and therefore the same biosynthetic pathway has been suggested for the nikkomycin nucleosides nikkomycins Cx and Cz [8]. Histidine is the precursor of the imidazolone base [9]; l-lysine is incorporated via picolinic acid into the pyridyl moiety and the attached carbon atom of nikkomycin D [10, 11]. Pyridylhomothreonine (PHT, nikkomycin E) and 4-pyridyl-2-oxo4-hydroxy-isovalerate (POHIV), which have been isolated from S. tendae culture filtrate [12, 13], are potential biosynthetic precursors of HPHT.

5.2 Isolation of nikkomycin biosynthetic genes

A number of approaches have been successfully used to isolate genes of antibiotic biosynthetic pathways from several Streptomyces strains. The strategy of cloning antibiotic-resistance genes, which are usually clustered with antibiotic biosynthetic genes, is not applicable to nikkomycin genes because the producing strain lacks the nikkomycin target site. Complementation of S. tendae mutants blocked in nikkomycin synthesis has led to the isolation of a 9.4-kb fragment that complements a non-producing mutant to nikkomycin Cx, Cz, and Kx synthesis [14]. However, structural genes of the nikkomycin pathway have not been identified. Therefore, the nikkomycin genes were cloned by identifying gene products involved in nikkomycin synthesis, microsequencing the N-termini to design oligonucleotide probes, and cloning the corresponding genes using these probes [15]. Nikkomycin biosynthetic enzymes were identified in gene expression studies using two-dimensional gel electrophoresis to separate cellular proteins. Initially, gene expression was analyzed in S. tendae wild type and mutant strains 104

Figure 5.2: Nikkomycin biosynthetic pathway based on the incorporation of labeled precursors and chemical characterization of pathway intermediates and shunt products; included are data on the biosynthesis of the polyoxin nucleoside (for references, see text). POHIV, 4-pyridyl-2-oxo-4-hydroxyisovalerate; PHT, pyridylhomothreonine.

105

5 Genetics of Nikkomycin Production in Streptomyces tendae Tü 901

Figure 5.3: Two dimensional gels of silver-stained cell extracts prepared from wild type Tü 901/8 c (A) and nikkomycin non-producing mutant Tü 901/NP13 (B). Mycelia were harvested in the stationary phase after 27.5 h of incubation. Proteins P1–P10 observed in the Tü 901/8c extract (A) and their corresponding positions in the Tü 901/NP13 extract (B) are indicated by arrows. These proteins were identified on the basis of three experiments performed with each producing and non-producing S. tendae strain. Positions of protein molecular mass standards are shown on the left. The acidic end of the gel is on the left, and the basic on the right.

blocked in nikkomycin biosynthesis [6]. Mutants which were able to synthesize nikkomycins but which differed from the wild type in the spectrum of nikkomycin structures formed had a protein pattern identical to that of the wild type. In contrast, protein profiles of mutants which did not synthesize any known nikkomycin structure differed from the wild type pattern as well as from each other. Selection of protein spots that were present in the producing strains and absent in each non-producing mutant led to the identification of ten gene products (P1–P10) (Fig. 5.3). As nikkomycin biosynthesis is regulated by the growth phase, the kinetics of the appearance of proteins P1–P10 was investigated during growth of S. tendae Tü 901/8 c in production medium (Fig. 5.4). Growth of S. tendae is characterized by a biphasic growth curve with two phases of rapid growth separated by a 1.5 to 2 h period in which growth slows down. Nikkomycin production begins in the transition to the stationary phase, after approximately 27 h after inoculation. Proteins P1–P6 and P10 were detected in extracts of mycelia harvested in the second exponential growth phase after 22.5 h of inoculation, and the amount of all of proteins P1–P10 increased to maximum levels at the early stationary phase and remained constant throughout the stationary phase. Synthesis of proteins P1–P10 preceded nikkomycin production, as would be expected for nikkomycin biosynthetic enzymes. N-terminal amino acid sequences were obtained for six of the ten identified proteins. Oligonucleotide probes designed from the N-terminal sequences of proteins P4, P5, and P8 gave positive signals of similar intensities with more 106

5.2 Isolation of nikkomycin biosynthetic genes

Figure 5.4: Time course of nikkomycin production (A) and gene expression (B) in S. tendae Tü 901/8 c cultivated in production medium. Nikkomycin Z plus X (g) in the culture filtrate compared to growth as determined by dry weight (&). Arrows indicate time points at which mycelia were harvested to prepare protein extracts for 2-D gel electrophoresis. (B) Enlarged sections of silver-stained 2-D gels of extracts of S. tendae Tü 901/8c harvested in the exponential phase (17.5 h), the late exponential phase (22.5 h), and the stationary phase (27.5 h). The position of proteins P1, P2, P3, P4, P7, P8, and P9 are marked by arrows.

than ten bands of digested genomic DNA and therefore were unsuitable for isolating the corresponding genes. A mixture of oligonucleotide probes designed from proteins P1 and P2, which have identical N-terminal sequences except that protein P1 has three additional amino acid residues at its N-terminus, and probes designed from protein P6 led to the isolation of genomic DNA fragments containing the binding sites for the probes used. The isolated fragments, an 8-kb BamHI fragment and a 6.5-kb PvuII fragment, appeared to overlap by 1.5-kb, and the former contained the binding sites for both oligonucleotide probes used. DNA sequence analysis revealed that the 8-kb BamHI fragment contains two open reading frames (ORFs) whose deduced N-terminal amino acid sequences are identical to that of proteins P1/P2 and that of protein P6. As gene-disruption mutants in the ORF encoding proteins P1/P2 (designated nikJ) and encoding protein P6 (designated nikI) failed to synthesize nikkomycins, it was evident that nikkomycin biosynthesis genes had been isolated. 107

5 Genetics of Nikkomycin Production in Streptomyces tendae Tü 901

5.3 Isolation of the nikkomycin gene cluster and expression in Streptomyces lividans

In bacteria, antibiotic biosynthetic genes are usually clustered. In order to isolate the entire nikkomycin gene cluster, a genomic library of S. tendae Tü 901/8 c was constructed in the Escherichia coli/Streptomyces shuttle cosmid pKC505 [16] and screened with the 8-kb BamHI fragment containing the nikI and nikJ genes. Hybridizing cosmids containing inserts of approximately 30 kb were mapped with restriction enzymes. Cosmids p24/32 and p9/43 carried the recognition sites for oligonucleotide probes designed from the N-terminal amino acid sequences of proteins P1/P2, P4, P5, P6, and P8, and of proteins P1/P2, P5, P6, and P8, respectively. S. lividans TK23, which does not produce nikkomycins, was used as a host to express the cloned nikkomycin biosynthetic genes. The nik cluster was not isolated on a single plasmid, as shown by the lack of synthesis of nikkomycins I, J, X, and Z by S. lividans TK23 transformants containing one of the recombinant plasmids. The nik cluster was cloned on two plasmids, p24/32 and p9/43, which carry inserts of about 31 and 27 kb, respectively, 15 kb of which overlap (Fig. 5.5). To facilitate selection for both plasmids, the apramycin resistance gene of p9/43 was removed by restriction with XhoI, which did not cut within the insert, and replaced with the aphII gene from Tn5. One of the apramycin- and neomycin-resistant S. lividans TK23 transformants synthesized relatively large amounts of nikkomycins I, J, X, and Z, approximately 50 % of that synthesized by S. tendae Tü 901/8 c. This transformant contained a large (>100-kb) plasmid that had formed by recombination between homologous regions of the two plasmids, either the vector pKC505 regions or the homologous regions of the inserts [17]. 27 kb

p9/43 31 kb

p24/32

8 kb

probe

B

S

S B

S

BS

B

S

BB

B

B BS

S

B

S

genome VM5 VM8

VM4

orfR

nikV U T

S R Q P2

P1

A B C

VM6 VM1/2

D

E

F G

I

J

K

LM N

O

Figure 5.5: Organization of the nik gene cluster and restriction map of the cloned chromosomal region containing the nik cluster. The structural genes (nik) and the regulatory gene (orfR) of the nik cluster are indicated by arrows. Wavy arrows indicate transcripts. The boxes below the genome map indicate the recognition sites for oligonucleotide probes VM1/2, VM4, VM5, VM6, and VM8, designed from proteins P1/2, P4, P5, P6, and P8. The hybridization probe used to screen the S. tendae gene library and the inserts of cosmid p24/32 and p9/43 are also indicated. B, BamHI; S, SacI.

108

5.4 Organization of the nikkomycin gene cluster

5.4 Organization of the nikkomycin gene cluster

The nucleotide sequence of an approximately 36-kb genomic region containing the nik cluster has been determined, and a series of 23 ORFs comprising a region of 29 kb identified. The translational start points of the ORFs have been tentatively located using the following criteria: (a) the G+C content in the third position of codons, (b) the location of a potential ribosome binding site at a suitable distance from the putative translation start [18], and (c) the observed similarities of the deduced amino acid sequence with proteins in databases. Each of the deduced proteins of the nikJ, nikS, nikA, nikI, and nikC genes had an N-terminal amino acid sequence identical to that determined for proteins P1/P2, P4, P5, P6, and P8, respectively. The most relevant features of the nik cluster and the encoded proteins deduced from the nucleotide sequence are summarized in Table 5.1, and the organization of the nik cluster is shown in Fig. 5.5. The ORFs of the nik cluster are arranged in three sets of adjacent genes with an intergenic spacing of 95 % of cells were still swimming backward 2 min after the increase of K+. This dramatic effect required a 15 min preincubation with okadaic acid and, as in metazoans, higher concentrations of okadaic acid compared to those needed to inhibit protein phosphatase type 1 in 308

18.6 Downstream of second messengers

Figure 18.4: Okadaic acid, a specific protein phosphatase type 1 inhibitor, impairs Ca2+channel closure in Paramecium in vivo. Cells were equilibrated in buffer containing 1 mM KCl, 50 µM CaCl2, 10 mM MOPS, pH 7.2. Okadaic acid (final concentration 34 µM in 1% DMSO) was added 15 min prior to stimulation. Swimming behavior was monitored microscopically. Cells were depolarized by addition of KCl (20 mM). Okadaic acid sustained the Ca2+-influx as measured by the prolonged backward swimming response. Addition of 75 µM EGTA abruptly ended the avoiding reaction due to chelation of external calcium (from [14]).

vitro. This may be due to higher local phosphatase concentrations in vivo compared to those present in in vitro assays, as well as to slow and insufficient diffusion of okadaic acid across the thickly protein-coated surface of Paramecium. The effect of okadaic acid most likely was directed at the voltage-operated Ca2+channel because chelation of external Ca2+ by addition of EGTA 2 min after K+depolarization in the presence of okadaic acid caused an immediate termination of backward swimming and resumption of forward movement, i. e. okadaic acid treatment enhanced the influx of Ca2+ and did not affect the motile apparatus of the cilia itself [14]. The effects of okadaic acid in Paramecium are compatible with a model for regulation of the voltage-operated Ca2+-channel in the ciliary membrane, which accounts for all the behavioral, electrophysiological and biochemical observations. With the membrane potential at rest the Ca2+-channel is in a closed state, which is susceptible to activation by depolarization. Depolarization triggers a conformational change of the channel converting it to an open state within 1–2 ms, which leads to a depolarizing Ca2+-influx and drives the reversal of the ciliary beat, hence backward swimming, by an okadaic acid-insensitive mechanism. We propose that the Ca2+-channel is phosphorylated in the closed groundstate, which is inaccessible to dephosphorylation. By contrast, the open state of the channel is dephosphorylated rapidly converting it to an inactivated, dephosphorylated state, which does not permit further Ca2+-influx. This 309

18 Second Messenger Systems in Paramecium reaction sequence is responsible for termination of the backward swimming response after about 10 s in the absence of okadaic acid. In the presence of okadaic acid protein phosphatase 1 is inhibited, dephosphorylation attenuated and a Ca2+-inward current is sustained, which is sufficient to support ciliary reversal unless Ca2+-influx is prevented by chelation of extracellular Ca2+. The reconversion of the inactivated dephosphorylated state to the closed phosphorylated state, which resensitizes the channel to depolarizing stimuli, is a slow process requiring 5–10 min at ambient temperature. These data, though encouraging, also demonstrate that characterization of a molecular target prior to in vivo screening can be a most useful way to define in vivo reaction cascades and support the tendency to approach drug development by reverse pharmacology. The extent of overall conservation among protein phosphatase 2 C enzymes from different phyla, for which sequence data are available, is limited. The considerable sequence disparities may indicate a functional diversity in different phyla. In Paramecium, the protein phosphatase 2C is membrane-associated, a considerable fraction of the enzyme is localized to the cilia, the highly specialized motile organelle of the ciliate [58]. Microsequencing of six tryptic peptides of the purified enzyme revealed a relationship to other PP2C isoforms. The Paramecium PP2C gene was obtained using degenerate oligonucleotide primers. The gene coded for a 33 kDa-protein with 300 amino acids, i. e. it is one of the smallest PP2C isoforms [58]. A C-terminal truncation by about 80 amino acids is responsible for the small size of the Paramecium PP2C compared to isoforms from other organisms. We defined three core regions of high conservation to be present in all PP2C enzymes. These account for about 25% of the protozoan primary sequence. After mutation of nine ciliate Q codons (TAA) to CAA the Paramecium gene was expressed as an active protein in E. coli. The catalytic core region was defined by N- and C-terminal deletions to represent 284 amino acids, i. e. the ciliate PP2C mainly comprises the catalytic core [59]. The data support the notion that the three-dimensional structure of the Paramecium PP2C is identical to that determined with the recombinant human PP2C despite an overall meager amino acid conservation (31%), which is more or less restricted to the three conserved regions mentioned above. In the X-ray structure an unordered loop region is in front of the b9-strand [60]. We introduced a factor Xa protease cutting site (IEGRA) in this domain to define the catalytically active center by generation of proteolytically truncated versions of the PP2C. Surprisingly, this construct, in which the sequence QLII (212–215) was changed to IEGR, thus generating in conjunction with Ala216 a factor Xa cutting site, was inactive. Individual amino acid exchanges at each of the changed positions showed that exclusively the I214G conversion was responsible for the loss of function (Table 18.1). In an I214A variant 62% of wild type activity was retained, and an I214L product was as active as the wild type enzyme. Strikingly, the adjacent isoleucine 215 was not crucial for enzymatic activity. Neither an I215R nor an I215G conversion abolished phosphatase activity. Similar mutation experiments were then carried out with the bovine type 2Ca protein phosphatase, which contains a hydrophobic valine at an equivalent position. A V215G conversion also inactivated the mammalian enzyme (Table 18.1). In this respect our 310

18.7 In vivo screening of bacterial secondary metabolites Table 18.1: A single amino acid replacement (I214G) in a region without defined secondary structure [60] abolishes enzymatic activity of Paramecium protein phosphatase 2C. A corresponding replacement in the bovine type 2Ca isoform (V215G) also leads to an inactive enzyme (adapted from [59]). Enzyme variant

Specific activity (mU/mg)

PtPP2C wild type PtPP2C L213E PtPP2C I214G PtPP2C I214A PtPP2C I214L PtPP2C I215R

15.7 12.3 0 9.7 19.2 11.2

bov. PP2Ca wild type bov. PP2Ca V215G

2.5 0

data clearly go beyond the picture, which has emerged from the crystal structure of PP2C. We have no clue why a mutation that replaces a hydrophobic amino acid at position 214 (Paramecium PP2C numbering) with glycine results in inactivation. The loss of a hydrophobic side chain may alter the stability or a directed movability of this part of the protein, which has no defined tertiary structure in the crystal, such that the active site centers cannot sufficiently stabilize an enzyme/substrate transition state. In contrast to PP1/PP2A, there are currently no specific inhibitors available for protein phosphatase 2C. So far, we have tested a number of synthetic and biological compounds, yet could not unequivocally identify a specific inhibitor of PP2C, which would aid to pinpoint a specific physiological function for this protein phosphatase like okadaic acid did for PP1. We also examined the subcellular localization of PP2C [59]. The major fraction is cytosolic, partly it is associated with the macronucleus, and a minor part is structurally bound to the cilia where it was associated with microtubulus and dynein, i. e. it was clearly targeted to the ciliary motor, implicating a motor component as one of the PP2C substrates. It seems reasonable to consider that the cytoplasmic and nuclear localization of PP2C may be related to processes involving cellular cargo transport [59].

18.7 In vivo screening of bacterial secondary metabolites

We tested more than 100 bacterial secondary metabolites for their toxicity and registered qualitatively and quantitatively by digitized motion analysis changes in the swimming behavior of Paramecium. In part, the assays were marred by 311

18 Second Messenger Systems in Paramecium the poor solubility of the compounds and the need to use organic solvents, mainly dimethylsulfoxide, for bath application. Although we have identified several compounds, e. g. depsichlorin, Tü 3580 and Tü 3586, to distinctly affect evoked swimming patterns of the ciliate, clear-cut actions were not obtained, which would have permitted to deduce a mechanism of action on a single component of the second messenger signal transduction pathway.

Acknowledgments

Supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. Sequence data for P. falciparum chromosome 13 was obtained from The Sanger Centre website at http://www.sanger.ac.uk/Projects/ P_falciparum/. Sequencing of P. falciparum chromosome 13 was accomplished as part of the Malaria Genome Project with support by The Wellcome Trust.

References

1. Kung, C., Chang, S.-Y., Satow, Y., van Houten, J., and Hansma, H. (1975) The genetic dissection of behavior in Paramecium. Science 188, 898–904. 2. Hinrichsen, R. D. and Schultz, J. E. (1988) Paramecium: A model system for the study of excitable cells. Trends in Neurosciences 11, 27–32. 3. Machemer, H. (1989) Cellular behavior modulated by ions: electrophysiological implications. J. Protozool. 36, 463–487. 4. Machemer, H. and de Peyer, J. (1977) Swimming sensory cells: electrical membrane parameters, receptor properties and motor control of ciliated protozoa. Verh. Dtsch. Zool. Ges. 1977, 86–110. 5. Thiele, J., Honer-Schmid, O., Wahl, J., Kleefeld, G., and Schultz, J. E. (1980) A new method for axenic mass cultivation of Paramecium tetraurelia. J. Protozool. 27, 118– 121. 6. Schönefeld, U., Alfermann, F. W., and Schultz, J. E (1986) Economic mass cultivation of Paramecium tetraurelia on a 200 liter scale. J. Protozool. 33, 222–225. 7. Machemer-Röhnisch, S. and Machemer, H. (1989) A Ca paradox: electric and behavioral responses of Paramecium following changes in external ion concentration. Eur. J. Protist. 25, 45–59. 8. Schultz, J. E., Boheim, G., Gierlich, D., Hanke, W., von Hirschhausen, R., Kleefeld, G., Klumpp, S., Otto, M. K., and Schönefeld, U. (1984) Cyclic nucleotides and calcium in Paramecium: a neurobiological model organism. Hormones and Cell Regulation 8, 99–112.

312

References 9. Schultz, J. E., Grünemund, R., von Hirschhausen, R., and Schönefeld, U. (1984) Ionic regulation of cyclic AMP levels in Paramecium tetraurelia “in vivo”. FEBS Lett. 167, 113–116. 10. Schultz J. E., Pohl, T., and Klumpp, S. (1986) Voltage-gated calcium entry into Paramecium coupled to intraciliary cyclic GMP formation. Nature 322, 271–273. 11. Schultz, J. E., Klumpp, S., Benz, R., Schürhof-Goeters, W., and Schmid, A. (1992) Regulation of adenylyl cyclase from Paramecium by an intrinsic potassium conductance. Science 255, 600–603. 12. Schultz, J. E. and Schade, U. (1989) Ca2+-channel activation and inactivation in Paramecium biochemically measured by cyclic GMP production. J. Membr. Biol. 109, 259–267. 13. Klumpp, S., Jung, G., and Schultz, J. E. (1984) Activation of the calcium/calmodulindependent guanylate cyclase from Paramecium by polypeptide antibiotics and melittin. Biochim. Biophys. Acta 800, 145–151. 14. Klumpp, S., Cohen, P., and Schultz, J. E. (1990) Okadaic acid, an inhibitor of protein phosphatase 1 in Paramecium causes sustained Ca2+-dependent backward swimming in response to depolarizing stimuli. EMBO J. 9, 685–690. 15. Hoff, H., Drautz, H., Fiedler, H.-P., Zähner, H., Schultz, J. E., Keller-Schierlein, W., Philipps, S., Ritzau, M., and Zeeck, A. (1992) Metabolic products of microorganisms. 261. Obscurolides, a novel class of phosphodiesterase inhibitors from Streptomyces. I. Production, isolation, structural elucidation and biological activity of obscurolides A1 to A4. J. Antibiotics 45, 1096–1107. 16. Schulz, S., Yuen, P. S., and Garbers, D. L. (1991) The expanding guanylyl cyclases. Trends Pharmacol. Sci. 12, 116–120. 17. Sunahara, K. S., Dessauer, C. W., and Gilman, A. G. (1996) Complexity and diversity of mammalian adenylyl cyclases. Annu. Rev. Pharmacol. Toxicol. 36, 461–480. 18. Kung, C. and Saimi, Y. (1982) The physiological basis of taxes in Paramecium. Annu. Rev. Physiol. 44, 519–544. 19. Oertel, D., Schein, S. J., and Kung, C. (1977)Separation of membrane currents using a Paramecium mutant. Nature 268, 120–124. 20. Saimi, Y. and Kung, C. (1987) Behavioral genetics of Paramecium. Annu. Rev. Genet. 21, 47–65. 21. Hinrichsen, R. D. and Saimi, Y. (1984) A mutation that alters properties of the Ca2+channel in Paramecium tetraurelia. J. Physiol. Lond. 271, 397–410. 22. Hinrichsen, R. D., Saimi, Y., and Kung, C. (1984) Mutants with altered Ca2+-channel properties in Paramecium tetraurelia: isolation, characterization and genetic analysis. Genetics 108, 545–558. 23. Klumpp, S. and Schultz, J. E. (1982) Characterization of a Ca2+-dependent guanylate cyclase in the excitable ciliary membrane from Paramecium. Eur. J. Biochem. 124, 317–324. 24. Klumpp, S., Kleefeld, G., and Schultz, J. E. (1983) Calcium/Calmodulin regulated guanylate cyclase of the excitable ciliary membrane from Paramecium: Dissociation of calmodulin by La3+: Calmodulin specificity and properties of the reconstituted guanylate cyclase. J. Biol. Chem. 258, 12455–12459. 25. Schultz, J. E. and Schönborn, C. (1994) Cyclic AMP formation in Tetrahymena controlled by a K+-conductance. FEBS Lett. 356, 322–326. 26. Richard, E. A., Hinrichsen, R. D., and Kung, C. (1985) A single gene mutation that affects a potassium conductance and resting membrane potential in Paramecium. J. Neurogenet. 2, 239–252. 27. Krupinski, J., Coussen, F., Bakalyar, H., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R., and Gilman, A. G. (1989) Adenylyl cyclase amino acid sequence: Possible channel- or transporter-like structure. Science 244, 1558–1564. 28. Barzu, O. and Danchin, A. (1994) Adenylyl cyclases: A heterogenous class of ATP-utilizing enzymes. Progr. Nucl. Acid Res. and Mol. Biol. 49, 241–283.

313

18 Second Messenger Systems in Paramecium 29. Brownlie, R. M., Coote, J. G., Parton, R., Schultz, J. E., Rogel, A., and Hanski, E. (1988) Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis. Microb. Pathol. 4, 335–344. 30. Rogel, A., Schultz, J. E., Brownlie, R. M., Coote, J. G., Parton, R., and Hanski, E. (1989) Bordetella pertussis adenylate cyclase: Purification and characterization of the toxic form of the enzyme. EMBO J. 8, 2755–2760. 31. Tang, W.-J. and Hurley, J. H. (1998) Catalytic mechanism and regulation of mammalian adenylyl cyclases. Mol. Pharmacol. 54, 231–240. 32. Buck, J., Sinclair, M. L., Schapal, L., Cann, M. J., and Levin, L. R. (1999) Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc. Natl. Acad. Sci. USA 96, 79–84. 33. Hinrichsen, R. D., Fraga, D., and Russell, C. (1995) The regulation of calcium in Paramecium. Adv. Second Messenger Phosphoprotein Res. 30, 311–338. 34. Zhang, G., Liu, Y., Ruoho, A. E., and Hurley, J. H. (1997) Structure of the adenylyl cyclase catalytic core. Nature 386, 247–254. 35. Tesmer, J. J. G, Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Crystal structure of the catalytic domains of adenylyl cyclase in a complex with GsaGTPgS. Science 278, 1907–1916. 36. Tucker, C. L., Hurley, J. H., Miller, T. R., and Hurley, J. B. (1998) Two amino acid substitutions convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase. Proc. Natl. Acad. Sci. USA 95, 5993–5997. 37. Sunahara, R. K., Beuve, A., Tesmer, J. J. G., Sprang, S. R., Garbers, D. L., and Gilman, A. G. (1998) Exchange of substrate and inhibitor specificities between adenylyl and guanylyl cyclases. J. Biol. Chem. 273, 16332–16338. 38. Stokes, D. L., Taylor, W. R., and Green, N. M. (1994) Structure, transmembrane topology and helix packing of P-type ion pumps. FEBS Lett. 346, 32–38. 39. Zhang, P. J., Toyoshima, C., Yonekura, K., Green, N. M., and Stokes, D. L. (1998) Structure of the calcium pump from sarcoplasmic reticulum at 8-Å resolution. Nature 392, 835–839. 40. Henikoff, S. and Henikoff, J. G. (1992) Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 89, 10915–10919. 41. Fagan, M. J. and Saier, M. J. Jr (1994) P-type ATPases of eukaryotes and bacteria: sequence analyses and construction of phylogenetic trees. J. Mol. Evol. 38, 57–99. 42. Allen, G. and Green, N. M. (1976) A 31-residue peptide from the active site of the [Ca2+]-transporting adenosine triphosphatase of rabbit sarcoplasmic reticulum. FEBS Lett. 63, 188–191. 43. Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Ca2+-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 265, 14088–14092. 44. Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) Functional consequences of alterations to amino acids located in the nucleotide binding domain of the Ca2+-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 265, 22223–22227. 45. Elwess, N. L. and van Houten, J. L. (1997) Cloning and molecular analysis of the plasma membrane Ca2+-ATPase gene in Paramecium tetraurelia. J. Eukaryot. Microbiol. 44, 250–257. 46. Hauser, K., Pavlovic, N., Kissmehl, R., and Plattner, H. (1998) Molecular characterization of a sarco(endo)plasmic reticulum Ca2+-ATPase gene from Paramecium tetraurelia and localization of its gene product to sub-plasmalemmal calcium stores. Biochem. J. 334, 31–38. 47. Preer, J. R. Jr., Preer, L. B., Rudman, B. M., and Barnett, A. J. (1991) Deviation from the universal code shown by the gene for surface protein 51A in Paramecium. Nature 314, 188–190. 48. Linder, J. U., Engel, P., Reimer, A., Krüger, T., Plattner, H., Schultz, A., and Schultz, J.

314

References

49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

E. (1999) Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium. EMBO J. 18, 4222–4232. Coudart-Cavalli, M. P., Sismeiro, O., and Danchin, A. (1997) Bifunctional structure of two adenylyl cyclases from the myxobacterium Stigmatella aurantiaca. Biochimie 79, 757–767. Schultz, J. E., Uhl, D., and Klumpp, S. (1987) Characterization of an ionically regulated adenylate cyclase from the excitable ciliary membrane of Paramecium. Biochem. J. 246, 187–192. Schultz, J. E. and Jantzen, H. M. (1980) Cyclic nucleotide-dependent protein kinases from cilia of Paramecium tetraurelia: Partial purification and characterization. FEBS Lett. 116, 75–78. Eistetter, H., Seckler, B., Bryniok, D., and Schultz, J. E. (1983) An electrophoretic analysis of phosphorylation of endogenous proteins of cilia from Paramecium tetraurelia. Eur. J. Cell Biol. 31, 220–226. Hochstrasser, M. and Nelson, D. L. (1989) Cyclic AMP-dependent protein kinase in Paramecium tetraurelia. J. Biol. Chem. 264, 14510–14518. Bonini, N. M., Evans, T. C., Miglietta, L. A. P., and Nelson, D. L. (1991) The regulation of ciliary motility in Paramecium by Ca2+ and cyclic nucleotides. Adv. Second Messenger and Phosphoprotein Res. 23, 227–272. Son, M., Gundersen, R. E., and Nelson, D. L. (1993) A second member of the novel Ca2+-dependent protein kinase family from Paramecium tetraurelia. J. Biol. Chem. 268, 5940–5948. Bialojan, C. and Takai, A. (1988) Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem. J. 256, 283–290. Friderich, G., Klumpp, S., Russell, C. B., Hinrichsen, R. D., Kellner, R., and Schultz, J. E. (1992) Purification, characterization and structure of protein phosphatase 1 from the cilia of Paramecium tetraurelia. Eur. J. Biochem. 209, 43–49. Klumpp, S., Hanke, C., Donella-Deana, A., Beyer, A., Kellner, R., and Schultz, J. E. (1994) A membrane-bound protein phosphatase type 2C from Paramecium: Purification, characterization and cloning. J. Biol. Chem. 269, 32774–32780. Grothe, K., Hanke, C., Momayezi, M., Kissmehl, R., Plattner, H., and Schultz, J. E. (1998) Functional characterization and localization of protein phosphatase 2C from Paramecium. J. Biol. Chem. 273, 19167–19172. Das, A. K., Helps, N. R., Cohen, P. T. W., and Bartford, D. (1996) Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 angstrom resolution. EMBO J. 15, 6798–6809.

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Chemical Synthesis and Structure Elucidation

Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites Roderich D. Süßmuth, Jörg Metzger, and Günther Jung*

19.1 Introduction

The introduction of the collaborative research centre 323 opened a fruitful and interesting cooperation between biologists and chemists, wherefrom both sides have greatly benefited. This successful cooperation profited from the expertise of the co-working chemists in the fields of biochemical analytics and chemical synthesis. From the chemist’s view three achievements extraordinarily contributed to the work of the collaborative research centre 323. The first was the introduction of automated parallel peptide synthesis. The development of robotic systems made the synthesis of hundreds of different peptides within several days feasible and enormously accelerated the production of peptide libraries and peptidomimetics. The second important innovation was the electrospray mass spectrometer (ESI-MS) installed in 1989, and the successive development and performance of so called hyphenated analytical techniques, such as HPLC-ESI-MS. The introduction of the electrospray mass spectrometer at that time completely revolutionized the possibilities of analyzing biological samples and extracts. Finally, the installation of the 600 MHz NMR spectrometer enabled the determination of peptide and small protein 3D-structures. However, the impetus by these developments was given not only to the groups directly cooperating within the collaborative research centre 323, but also to new cooperations in related fields e. g. immunology (collaborative research centre 510) and finally the introduction of combinatorial chemistry in Tübingen (BMBF project 03 D 0037). The resonance towards the introduction of these new techniques reached far beyond the cooperating research groups of

* Institut für Organische Chemie, Universität Tübingen, Auf der Morgenstelle 18, D-72076 Tübingen.

319 Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites microbiology in and outside of Tübingen. Due to the early introduction of these new methods in Tübingen, some key reviews [1–4], scientific contributions and books were published, which had impact on the research of related fields. The following gives an overview only of the most significant experimental scientific contributions within the collaborative research centre 323.

19.2 Development of methods

19.2.1 Peptide synthesis Shortly after the invention of the solid phase synthesis by Nobel laureate R. B. Merrifield in 1963, the Institute of Organic Chemistry introduced this new technique at the University of Tübingen (G. Jung, E. Bayer in 1965). Automated peptide synthesizers were introduced in 1989. As one of the first academic groups in Europe G. Jung’s group used these synthesizers for parallel synthesis of hundreds of peptides in mg-quantities. As a consequence of this development and after the introduction of the library concept, partly or fully randomized peptide libraries were synthesized. From the rapidly established key technologies in Tübingen, cooperating research groups in biochemistry, immunology, and sensorics gained considerable profit from protein and substrate mapping experiments, the finding of sequence motifs of MHC ligands and T cell epitopes, and chemosensor developments. Peptides or peptide libraries synthesized in parallel were extensively used in the collaborative research centre 323, e. g. with the groups of V. Braun [5] and F. Götz [6], but also in cooperation with the immunology group of H.-G. Rammensee and many external research groups within Europe. A further important branch was the expertise on the synthesis of unusual peptides, e. g. alamethicin in 1984 and lipopeptides vaccines in 1986. This continuous knowledge was decisive for the success of several projects, e. g. synthesis of heterocyclic backbone modifying amino acids as a part of the microcin B17 structure [7] and the total synthesis of microcin B17 in 1996 [8].

320

19.2 Development of methods

19.2.2 Characterization of peptide libraries with electrospray mass spectrometry The collaborative research centre 323 obtained the first ESI-MS instrument in Europe. This opened new ways for the mass spectrometric characterization of natural products, especially of peptides (Fig. 19.1) and proteins, but also of oligonucleotides, oligosaccharides, and polymers. In the projects of J. Metzger (project C3) and G. Jung (project C2) several hyphenated techniques, e. g. HPLC-MS (Fig. 19.2) and autosampler-ESI-MS were established. The first mass spectrometric investigation of randomized peptide libraries has been performed [9] and further extensive investigations of these libraries confirmed ESI-MS as a method of choice for synthesis control of mixtures containing few to several thousands of components [10]. Moreover, the first LC-ESI-MS characterization of a 9-mer peptide library was performed, revealing side reactions, incomplete side chain deprotection, etc. resulting from peptide synthesis and cleavage from the resin [10, 11]. It was shown that mass spectrometric tandem experiments such as daughter ion scan, parent ion scan and neutral loss scan are suitable methods for the detection of undesired side reactions and byproducts. In further studies mass spectra of the synthetic peptide libraries were compared with theoretically expected

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Figure 19.1: Identification of tert-butylated peptides with electrospray-tandem mass spectrometry in the octapeptide library SNYTFX1X2X3(X1 = T,I,E,S)(X2 = N,K,Q)(X3 = L,M,I,V). Top: Q1-spectrum of the mixture displaying the [M+H]+-signals of the theoretically expected 48 peptides and the tert-butylated byproducts. Bottom: Selective detection of the tert-butylated byproducts using neutral loss scan (loss of isobutene –56).

321

Rel. intensity [%]

Rel. intensity [%]

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites

100 75 50 25 0 100 75 50 25 0 0.0

total ion chromatogram m/z 800-1200

16.1 16.4

ion chromatogram m/z 1041

5.0

15.2 14.5 10.0

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Figure 19.2: Total ion chromatogram of an HPLC-MS run containing a 48 peptide mixture. The peptides were separated on a nucleosil C-18-column (2 6 100 mm, 5 µm; linear gradient 5–20 % B in 40 min; A 0,1% TFA aq., B 0.1% TFA in acetonitrile). Top: The total ion chromatogram displays the elution of 48 peptides in the mass range of m/z 800–1200. Bottom: The ion chromatogram of m/z 1041 displays the elution of eight peptides with identical nominal mass (isobaric peptides).

mass distributions, using computer-assisted programs, which have been developed especially for this purpose [12]. With the help of these programs, which implement a basis set of proteinogenic amino acids, protecting groups, as well as user-defined amino acid residues, the purity of a peptide library can be qualitatively estimated. These fundamental investigations on peptide library analysis formed the basis for subsequent contributions of other groups, e. g. on combinatorially synthesized small molecule libraries reported by Rebek et al. [13–15].

19.2.3 Automated sequential Edman degradation combined with ESI-MS detection The major focus of the group of G. Jung and his coworkers has been the structure determination of complex peptides. One of the most advanced instrumental sequencing methods is Edman degradation, which is still of essential importance in determining the primary structure of lantibiotics. However, post-translational modifications, e. g. N-terminal blocking and intra-chain bridging via lanthionines as they occur in lantibiotics made the application of standard protocols impossible. In the case of lanthionines, e. g. nisin, bearing a,b-didehydroamino acids sequence abortion occurs due to desamination, thus resisting routine Edman degradation. As a consequence important information about sequences could not be obtained. A second problem arose for lanthionine amino acids, because for these amino acids no PTH-derivative could be detected. To circumvent 322

19.2 Development of methods both problems, a chemical derivatization procedure prior to Edman sequencing was developed [16], which enabled the complete determination of the amino acid sequences of the lantibiotics gallidermin and Pep5. However, analysis of other peptides with unusual amino acids with standard Edman degradation faced problems since the obtained information was not sufficient for compound characterization because no PTH-standards are available for modified amino acids, and their Edman degradation products are often overseen or misinterpreted. The routine identification by HPLC retention times is only standardized for the 19 essential amino acids. As a consequence, the sequencer-ESI-MS-coupling was developed for more sophisticated sequence analysis [17] (Fig. 19.3). The continuous effluent of the HPLC of the Edman se-

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Figure 19.3: Sequencer-ESI-MS coupling. UV-absorbance chromatogram and total ion chromatogram (TIC) of an optimized gradient of PTH-standard amino acids (despite Cys).

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Figure 19.4: Sequencer-ESI-MS coupling: UV chromatogram of an Edman degradation cycle of 3-chloro-b-hydroxy-tyrosine (Cht) and corresponding mass spectra of identified PTH-derivatives.

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19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites

19.3 Structure elucidation quencer was directly introduced into the ESI source. The on-line coupling guarantees a complete overview over all products after the degradation cycle. As an additional and most important information, the total ion chromatogram (TIC) contains the molecular mass data of the PTH-amino acids. With this method, peptides bearing unusual amino acids, e. g. 4-hydroxyphenylglycine (Hpg), 3,5dihydroxy-phenylalanine, ornithine, lanthionine, [2,3]-didehydro-asparagine, etc. have been analyzed. This method turned out to be practicable for a great variety of linear and cyclic peptides, e. g. lantibiotics or even microheterogenous peptides. Furthermore, because of the extremely low amounts of sample required this method is suitable for the analysis of natural metabolic peptides of microbial and mammalian origin. The sequencing method based on mass spectrometric detection was of importance in the structure elucidation of CDA, a calcium dependent antibiotic [18], confirming its primary structure, which was not clearly solved from the sequence information obtained by two-dimensional HMBC- and NOESY-NMR spectra. Moreover, this method is generally applied for the structure elucidation of new peptides bearing post-translational modifications. The application of the sequencer-ESI-MS coupling sometimes leads to surprising results which are, however, clearly explicable through the mass spectrometric data. For example, Fig. 19.4 displays the degradation cycle of 3-chloro-bhydroxy-tyrosine (Cht), an amino acid which was found during the structure elucidation of intermediates of the balhimycin biosynthesis [19]. Almost no PTH-derivative of 3-chloro-b-hydroxy-tyrosine was detected. Instead, as a major product PTH-glycine and minor amounts of the 2,3-didehydro-3-chloro-tyrosine PTH-derivative were unequivocally identified. We could explain these results by assuming a retro-aldol reaction under basic conditions (formation of the PTC-peptide) and a dehydration under acid conditions (formation of the ATZ-amino acid and conversion to the PTH-amino acid) during Edman degradation (Fig. 19.5).

19.3 Structure elucidation

Over the past years several dozen structures of a variety of different metabolites from microbial sources have been determined. They comprise molecules of a wide range of compound classes, e. g. antibiotics, siderophores, and signaling molecules. They were either isolated in the course of a targeted screening by the cooperating groups, or represented biosynthetic intermediates produced by mutants genetically generated with the methods of molecular biology. As tools for structure elucidation a variety of analytical methods have been used: electrospray mass spectrometry (ESI-MS) and respective hyphenation techniques, 2-dimensional NMR spectroscopy, GC- and LC-chromatograpy, chiral GC-MS, amino acid analysis, Edman degradation and CD spectroscopy. 325

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites

Figure 19.5: Retro-aldol and dehydration reaction for the Edman degradation of 3-chlorob-hydroxy-tyrosine.

19.3.1 Structure elucidation of metabolites of microbial origin The following chapter comprises a selection of some of the structures belonging to different classes of metabolites. Compounds presented had no greater impact on the main research projects which mainly concerned siderophores and lantibiotics. However, they represent interesting structures and some of these compounds developed into research fields of other groups. The following peptidic compounds have been characterized (Fig. 19.6): the rhizocticins, small phosphono-oligopeptides [20], the fengycins lipopeptide antibiotics [21] and the chlorotetains, which all were isolated from Bacillus subtilis strains [22], the lipoglycopeptidic herbicolins from Erwinia herbicola [23], echinoserin from Streptomyces tendae [24], and aborycin, a tricyclic 21-peptide anti326

19.3 Structure elucidation

Figure 19.6:

Peptidic metabolites isolated from different microorganisms.

biotic from Streptomyces griseoflavus [25]. For the latter antibiotic the complete solution 3D-structure was also determined by NMR spectroscopy. In addition, the structures of non-peptidic compounds (Fig. 19.7), e. g. of polyol macrolide antibiotic kanchanamycin from Streptomyces olivaceus [26], the antifungal lactam antibiotic maltophilin produced by Stenotrophomonas maltophilia [27], spirofungin from Streptomyces violaceusniger [28], and boophiline from Boophilus microplus [29] were determined. The early structure determinations were done with the help of FAB-MS, and 1H- and 13C-NMR spectroscopy. However, with the availability of ESI-MS and straightforward 2-dimensional NMR-techniques, the structure elucidation was facilitated and speeded up significantly.

327

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites

Figure 19.7:

328

Non-peptidic metabolites isolated from different microorganisms.

19.3 Structure elucidation 19.3.1.1 Siderophores Rhizoferrin from Rhizopus microsporus [30], ferrirhodin from Botrytis cinerea [31], yersiniabactin from Yersinia enterocolitica [32], and staphyloferrin A from Staphylococcus hyicus [33] were isolated and their structure determined. Staphyloferrin A consists of two citric acid moieties and D-ornithin (Fig. 19.8). From dicitrylputrescin contained in rhizoferrin, a number of analogs has been obtained by directed fermentation [34]. The chirality of these analogs and their iron complexation properties have been characterized spectroscopically. The complexation modes of citryl-containing carboxylate siderophores for a number of different transition metal ions have been studied with rhizoferrin as a model compound [35]. From Ralstonia pickettii DSM 6297, the enantiomer of fungal rhizoferrin has been isolated [36, 37]. This opened up a new field of research on the chirality in citric acid-containing carboxylate siderophores and comparative studies on the biosynthetic pathways in fungal and bacterial siderophore producers. New developments in the structure elucidation, synthesis, and function of peptide siderophores have been summarized in a recent review by H. Drechsel and G. Jung [38].

Figure 19.8:

Isolated and characterized siderophores.

329

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites 19.3.1.2 Lantibiotics The lantibiotics represent a class of antimicrobials extensively studied at the University of Tübingen through the cooperation of the groups of Prof. Jung, Prof. Entian, Prof. Götz, and Prof. Zähner. They are polypeptide antibiotics, produced by Gram-positive bacteria which contain intra-chain thioether bridges formed by lanthionine or methyllanthionine. These covalent thioether links impose conformational constraints and stability against protease degradation. Within the collaborative research centre 323 the lantibiotics Pep5 [39], epidermin [40], actagardine [41], gallidermin [42], and SA-FF22 [43], were elucidated as shown in Fig. 19.9.

S

S

Ala Ser Gly Trp Val Ala Abu Leu Abu Leu Glu Ala Gly Abu Val Ile Ala Ala Ala S

S

Actagardin

S

O

S

S

Ile Ala Ala Lys Phe Ile Ala Abu Pro Gly Ala Ala Lys Dhb Gly Ala Phe Asn Ala Tyr Ala

H N S

Epidermin S

S

S

Ile Ala Ala Lys Phe Leu Ala Abu Pro Gly Ala Ala Lys Dhb Gly Ala Phe Asn Ala Tyr Ala

H N S

Gallidermin S

O

S

Ala Gly Pro Ala Ile Arg Ala Ala Val Lys Gln Ala Gln Lys Dhb Leu Lys Ala Dhb Arg Leu Phe Abu Val Ala Ala Lys Gly Lys Asn Lys Ala Lys O S

Pep5 Figure 19.9: Sequences and thioether bridging patterns of lantibiotics elucidated by chemical and enzymatic degradation, mass spectrometry, and 2-dimensional NMR spectroscopy.

The structures of epidermin and Pep5 had to be determined by enzymatic degradation and derivatization to smaller, sequencable subfragments. With 2-dimensional NMR-experiments, using a 600 MHz NMR-spectrometer, in addition to ESI-MS and sequencer-MS, 3-dimensional solution structures of duramycin B, C [44], actagardin [45], and gallidermin [46] were determined by distance geometry and restrained-molecular-mechanics calculations (Fig. 19.10). 330

19.3 Structure elucidation

Figure 19.10:

Solution structures of the lantibiotics gallidermin and actagardine.

Knowledge of the lantibiotics structures prompted microbiologists to investigate the biosynthesis of the lantibiotics which is described in Chapter 3.

19.3.1.3 Microcin B17 The structure determination of the 43-peptide antibiotic microcin B17 (Fig. 19.11) from E. coli revealed the structure of the first known gyrase inhibitor (topoisomerase II inhibitor) of peptidic nature. The structure elucidation turned out to be extremely sophisticated because of eight backbone modifications and a nonaglycine sequence. The post-translational backbone modifications [47, 48] of bicyclic oxazole/thiazole-rings protected the major part of peptide from Edman degradation. Fully 13C,15N-labeled microcin B17 had to be prepared for crucial heteronuclear 2D-NMR experiments on the backbone of the heterocyclic polypeptide. In subsequent work, synthesis of the novel oxazole and thiazole amino acids had to be achieved [7], which ended up in the total synthesis of microcin B17 in 1996 [8]. The total synthesis of microcin B17 was performed on solid support using fragment condensations in order to form the nonaglycine sequence, which was not accessible by stepwise successive standard solution phase chemistry. The purity of the crude 43-peptide was about 50 %. Chemical data and antibiotic activity were identical with those of natural microcin B17 [8]. In addition to the natural gyrase inhibitor some analogs have been synthesized for structure-activity studies. The structure of microcin B17 formed the basis to postulate a biosynthesis mechanism for the post-translational modification leading to the oxazole/thiazole rings [47, 48]. The investigation of the microcin biosynthesis is still pursued [49–51].

N

O VGIGGGGGGGGG

S

GGQGG N

N

S

G N

S

N

S

O

GGNG N

SN N

O

G N

O

Figure 19.11: Structure of microcin B17 [48].

331

GSHI

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites 19.3.1.4 CDA (Calcium Dependent Antibiotic) Further interesting secondary metabolites are the peptides of the CDA (Calcium Dependent Antibiotic) group, isolated from Streptomyces coelicolor. These cyclic peptides inhibit the growth of Gram-positive bacteria in the presence of Ca2+-ions. The cyclic 11-mer peptide antibiotic (Fig. 19.12) consists of seven proteinogenic and four non-proteinogenic amino acids: D-tryptophan, 4-D-hydroxyphenylglycine, phospho-asparagine and D-methylglutamic acid [18]. Initial attempts to perform Edman degradation failed due to N-terminal blocking with an epoxyhexanoyl group which was identified in a later period of structure elucidation by NMR. Therefore, the peptide was treated with BNPS-skatol, which cleaves the peptide at the N-terminal bond of tryptophan residues. The peptide fragments obtained were further investigated by Edman degradation using the above mentioned sequencer-ESI-MS coupling. The structure elucidation of CDA required the combination of a sophisticated set of analytical methods (HPLC-MS, MS/MS, 2D-NMR) and chemical modification reactions. At present the biosynthesis of CDA is investigated by the groups of Hopwood and Marahiel [52].

Figure 19.12:

Structure of CDA [18].

19.3.1.5 Peptide pheromones of Staphylococcus epidermidis The agr quorum-sensing system (accessory gene regulator) is responsible for the regulation of several virulence factors in staphylococci. Novick et al. [53, 54] characterized the agr-system of S. aureus strains and in accordance with the genomic sequence data, a released peptide factor was presumed to activate the agr-system. Indeed, small amounts of peptides have been isolated from S. aureus strains to perform Edman degradation and mass spectrometry. A conserved cysteine was presumed to form a thiollactone with the carboxy terminus of the peptide. However, insufficient amounts were obtained for a full structure and function analysis. Since we and F. Götz’s group were also unable to isolate sufficient amounts from S. epidermidis strains, we decided to synthesize the proposed molecule. We showed that the cyclic thiollactone DSVc[CASYF] (Fig. 19.13) indeed activated the agr-system in nanomolar concentrations [55]. Non-cyclic peptides were completely inactive, and shortened or elongated cyclic thiollactones 332

19.3 Structure elucidation

Figure 19.13:

Structure of the quorum sensing signal peptide DSVc[CASYF] [55].

(GDSVc[CASYF] and SVc[CASYF]) showed a far lower activity as DSVc[CASYF]. Furthermore, we showed that the S. epidermidis thiollactone peptide DSVc [CASYF] inhibited the activation of the agr-system of S. aureus strains [55]. The replacement of the bridging cysteine, forming the thiollactone bond, by serine (DSVc[SASYF]) or 1,3-diamino propionic acid (DSVc[DprASYF]) had only little influenced the inhibition or activation of the agr-system. Administration of such thiollactone peptides against S. aureus infected mice reduced the infection [56]. It remains to be shown whether such quorumsensing blockers are attractive lead structures for the development of antibiotic drugs aimed at treating staphylococcal infections.

19.3.2 Investigation of biosyntheses 19.3.2.1 Nikkomycin biosynthesis Nikkomycin biosynthesis has been of major interest, because nikkomycins act as competitive inhibitors of chitin synthetases from fungi and insects. Nikkomycins might be attractive drugs and highly diverse lead structures for the agrochemical and pharmaceutical industries. Earlier works on structure elucidation and synthesis of nikkomycine derivatives have been published by W. A. König and H. Hagenmaier [57, 58] in cooperation with H. Zähner’s group. More recently, in cooperation with C. Bormann, biosynthesis of nikkomycins, produced by Streptomyces tendae Tü 901 was investigated by characterization of novel biosynthetic intermediates formed by mutants inactivated in specific nikkomycin-biosynthesis genes. Additionally, mutasynthetically generated compounds from genetically engineered mutants have been characterized. Within this cooperation the function of the nikF nikkomycin biosynthesis gene encoding a P450 monooxygenase was elucidated [59]. Nikkomycins Lx 333

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites

Figure 19.14:

Structure of isolated nikkomycins Lx [59] and Bx [60].

and Lz produced by NikF inactivated mutants are analogous structures to nikkomycins X and Z formed by the parent strain, but lack the hydroxy group at the pyridyl residue (Fig. 19.14). The structure of the biosynthetic intermediate formed by a nikO-inactivated mutant was determined as ribofuranosyl-4-formyl4-imidazolone, which represents a novel nucleoside. This finding indicated that the nikO encoded putative enolpyruvyl transferase catalyzes the initial step in the biosynthesis of the nucleoside moieties of nikkomycin. Structure elucidation of two biosynthetic intermediates isolated from the culture filtrate of mutants blocked in the biosynthesis of the nucleoside moieties is in progress. The structure of a novel intermediate produced by a nikK-mutant that is unable to introduce the amino group to the nucleoside moiety was determined as 4-formyl-4imidazolin-2-one. This is the base which is incorporated to yield nikkomycins containing this base. Feeding benzoic acid to a mutant deficient in the nikC gene which encodes lysine-2-aminotransferase catalyzing the initial step in the biosynthesis of the peptidyl moiety led to production of nikkomycin Bx and Bz [60]. These nikkomycins, which are the most potent compounds among known nikkomycins revealed good activity against Candida albicans.

19.3.2.2 Epidermin biosynthesis The characterization and structure elucidation of lantibiotics had an impact on the microbiologists within and outside the collaborative research centre 323. The gene cluster for the biosynthesis of epidermin, epiA-D, epiP, and epiQ was sequenced. The prepeptide EpiA, consisting of 52 amino acids, is ribosomally 334

19.3 Structure elucidation synthesized and transformed into the biologically active 22mer peptide by several post-translational modifications, including oxidative decarboxylation, dehydration, formation of thioether bridges, and cleavage of the leader peptide. In cooperation with T. Kupke and F. Götz the enzyme EpiD has been isolated and characterized. With EpiA substrates it has been shown that EpiD catalyzes the oxidative decarboxylation (–H2, –CO2) of C-terminal cysteine residues to form S-[(Z)-2-aminovinyl]-D-cysteine [61]. This hitherto unknown enzymatic oxidative decarboxylation reaction of EpiA substrates with EpiD has been detected by electrospray mass spectrometry. Subsequently, the substrate specificity of this reaction has been determined with precursor peptides of mutants and chemically synthesized 7mer peptides and peptide libraries [62]. The substrate specificity of EpiD was clearly shown by neutral loss scan experiments using randomized peptide libraries. This was the first application of neutral loss scan to identify products of an enzymatic reaction. Moreover, kinetic studies of the conversion reaction have been performed with electrospray mass spectrometry. The enzymatic reaction of EpiD with 13C-labeled peptide substrate has been followed by HSQC-NMR spectroscopy [63] (Fig. 19.15).

O H H C N C COOH 13

H C H O H

SH

H

C N C 13

HS

C H

675 min 375 min 195 min 75 min 0 min

δ / ppm

120

100

80

60

40

20

Figure 19.15: Reaction of the 13C-labeled substrate KKSFNSYTC with the enzyme EpiD in projections along the F1 (13C) axis of the HSQC spectra recorded during the course of the reaction. The product signal at d(13C) = 120.5 ppm increases in intensity with time [63].

335

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites 19.3.2.3 Biosynthesis of glycopeptides: balhimycin Paying attention to the dramatically increasing number of antibiotic resistant microorganisms, vancomycin and the related family of glycopeptides constitute antibiotics of the last resort, especially in the case of infections caused by methicillin-resistant staphylococci. The cyclic glycopeptides are also of interest as model receptors [64] for the investigation of ligand-receptor interactions, and they are widely used as chiral selectors in chromatography [65]. Considering the stereochemistry of the glycopeptides, the total synthesis of these molecules is one of the most challenging enterprises for synthetically working chemists [66, 67]. The target molecule of the group of W. Wohlleben and S. Pelzer is balhimycin, a vancomycin-type glycopeptide antibiotic, produced by Amycolatopsis mediterranei, from which the biosynthesis gene cluster was sequenced [68]. After identification of the biosynthesis gene cluster, in 1998 the first gene-dis-

Figure 19.16: Structures of the linear biosynthesis intermediates of balhimycin, a vancomycin type tricyclic glyco-peptide antibiotic [19].

336

19.4 Summary ruption mutants were cloned in the oxygenase genes oxyA and oxyB and in the glycosyltransferase gene bgtfB. In contrast to the glycosyltransferase mutant, the oxygenase mutants showed no antibiotic activity. The culture filtrates from both types of mutants were investigated, and according to the characteristic isotopic pattern of the two-fold chlorination of the detected compounds they were assigned to the expected biosynthesis intermediates. The compounds were isolated and investigated with ESI-MS, Edman degradation, amino acid analysis, and 2-dimensional NMR experiments [19]. The compounds produced by the oxygenase mutants revealed the first known linear peptide intermediates of glycopeptides (Fig. 19.16). They give a first insight into the biosynthesis pathway of the glycopeptide antibiotics [19, 68] (Fig. 19.17). The compounds produced by the bgtfB-mutant showed a complete lack of glycosylation. In-frame mutations of each single gene, of the oxygenases OxyA/B/C and the glycosyltransferases BgtfA/B/C are currently generated. The culture filtrates from the mutant strains are investigated with HPLC-ESI-MS for the presence of the biosynthesis intermediates, and the structures of these metabolites are determined after purification. The goal of the ongoing cooperation is to inactivate the halogenase gene bhaA to unravel the so far unknown function of a haloperoxidase and to demonstrate its substrate specificities.

19.4 Summary

During the past decade of the collaborative research centre 323, efficient analytical and synthetic methods were developed and applied, e. g. ESI-MS and coupling techniques, multiple peptide synthesis, 2-dimensional NMR spectroscopy, and molecular modeling. With these new methods it was possible to perform a high level of biological and biochemical research. Frequently, cooperation with biologically working groups showed that an expertise in both analytical chemistry and synthesis was extremely valuable or even decisive for the success of a project. Either the synthetic and analytical work supported the value and meaning of biological results or paved the way for the biological research.This fruitful constellation, the close cooperation between microbiology and chemistry resulted in numerous journal publications. With the installation of the Fourier-transform-ion-cyclotron resonance mass spectrometer in 1998 it has been once more possible to establish a modern analytical technique at the Institute of Organic Chemistry [69]. The installation of these key technologies is extremely important to keep pace with the rapidly growing requirements of biologists for powerful analytical methods.

337

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites

O HO

polyketidsynthase “?” aminotransferase “?”

NH2

Cl

Cl

OH

HO

HO

OH HO

OH

OH O

HO

3,5-dihydroxyphenylglycine

O H N

N H

O H N

N H

O

O

NH2

N H O

H2N O

SP-969

OH

peptidesynthetases “?“ Cl

Cl

OH

HO

HO

OH O

OH O

H N

HO

O H N

N H

N H

O

HO

O H N

O

O

H2N O

OH OH

oxygenases oxyA/B/C

NH2

N H

SP-1134

-6H R1 = -H / -CH3 R2 = -H / -OH

Cl

OH O

O R2

HO Cl O

O H N

O N H

O

HN HO2C

R1

O H N

N H

NH

N H O

H2N O

glycosyltransferases bgtfA/B/C

HD-1112/1126 HD-1128/1142

OH HO

OH

HO HO

HO

O

O

CH2OH Cl

O

H2N

O

O H3C CH 3

O

O

OH Cl O

O H N

O N H HN HO2C

O

O H N

N H

N H

CH3 NH

O

H2N O

OH HO

OH

Balhimycin

Figure 19.17: Proposed biosynthesis pathway of glycopeptides with the example of balhimycin.

338

References

References

1. Jung, G. (1991) Lantibiotics, ribosomally synthesized biologically active polypeptides containing sulfide bridges and a,b-didehydroamino acids. Angew. Chem. 103, 1067– 1084; Angew. Chem. Int. Ed. Engl. 30, 1051–1068. 2. Jung, G. and Sahl, H.-G. (1991) (eds.) Nisin und novel lantibiotics, Escom, Leiden. 3. Jung, G. and Beck-Sickinger, A. G. (1992) Multiple peptide synthesis methods and their applications. Angew. Chem. 104, 375–391; Angew. Chem. Int. Ed. Engl. 31, 367– 383. 4. Früchtel, J. and Jung, G. (1996) Organic chemistry on solid supports. Angew. Chem. 108, 19–46; Angew. Chem. Int. Ed. Engl. 35, 17–42. 5. Killmann, H., Videnov, G., Jung, G., Schwarz, H., and Braun, V. (1995) Identification of receptor binding sites by competitive peptide mapping: phages T1, T5, and 0/80 and colicin M bind to the gating loop of FhuA. J. Bacteriol. 177, 694–698. 6. Peschel, A., Otto, M., Jack, R. W., Kalbacher, H., Jung, G., and Götz, F. (1999) Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J. Biol. Chem. 274, 8405–8410. 7. Videnov, G., Kaiser, D., Kempter, C., and Jung, G. (1996) Synthesis of naturally occurring, conformationally restricted oxazole and thiazole containing di- and tripeptide mimetics. Angew. Chem. 108, 1604–1607; Angew. Chem. Int. Ed. Engl. 35, 1503– 1506. 8. Videnov, G., Kaiser, D., Kempter, C., Brooks, M., and Jung, G. (1996) Synthesis of the DNA gyrase inhibitor Microcin B17, a 43-peptide antibiotic with eight heteroaromatic rings in the backbone. Angew. Chem. 108, 1607–1609; Angew. Chem. Int. Ed. Engl. 35, 1506–1508. 9. Stevanovic, S., Wiesmüller, K.-H., Metzger, J., Beck-Sickinger, A. G., and Jung, G. (1993) Natural and synthetic peptide pools: characterization by sequencing and electrospray mass spectrometry. Bioorg. Med. Chem. Lett. 3, 431–436. 10. Metzger, J. W., Wiesmüller, K.-H., Gnau, V., Brünjes, J., and Jung, G. (1993) Ionspraymass spectrometry and high performance liquid chromatography-mass spectrometry of synthetic peptide libraries. Angew. Chem. 105, 901–903; Angew. Chem. Int. Ed. Engl. 32, 894–896. 11. Metzger, J. W., Kempter, C., Wiesmüller, K.-H., and Jung, G. (1994) Electrospray mass spectrometry and tandem mass spectrometry of multi-component peptide mixtures: determination of composition and purity. Anal. Biochem. 219, 261–277. 12. Kienle, S., Wiesmüller, K.-H., Brünjes, J., Metzger, J. W., and Jung, G. (1997) MSPep: A computer program for the interpretation of mass spectra of peptide libraries. Fresenius J. Anal. Chem. 359, 10–14. 13. Carell, T., Wintner, E. A., Sutherland, A. J., Rebek Jr., J., Dunayevskiy, Y. M., and Vouros, P. (1995) New promise in combinatorial chemistry: synthesis, characterization, and screening of small-molecule libraries in solution. Chem. Biol. 2, 171–183. 14. Dunayevskiy, Y., Vouros, P., Carell, T., Wintner, E. A., and Rebek Jr., J. (1995) Characterization of the complexity of small-molecule libraries of electrospray ionization mass spectrometry. Anal. Chem. 67, 2906–2915. 15. Dunayevskiy,Y. M.,Vouros, P., Wintner, E. A., Shipps, G. W., Carell, T., and Rebek Jr., J., (1996) Application of capillary electrophoresis-electrospray ionization mass spectometry in the determination of molecular diversity. Proc. Natl. Acad. Sci. USA 93, 6152–6157. 16. Meyer, H. E., Heber, M., Eisermann, B., Korte, H., Metzger, J. W., and Jung, G. (1994) Sequence analysis of lantibiotics: chemical derivatization procedures allow a fast access to complete Edman degradation. Anal. Biochem. 223, 185–190.

339

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites 17. Walk, T. B., Süßmuth, R., Kempter, C., Gnau, V., Jack, R. W., and Jung, G. (1999) Identification of unusual amino acids in peptides using automated sequential Edman degradation by electrospray-ionization mass spectrometry. Biopolymers 49, 329–340. 18. Kempter, C., Kaiser, D., Haag, S., Nicholson, G., Gnau, V., Walk, T., Gierling, K.-H., Decker, H., Zähner, H., Jung, G., and Metzger, J. W. (1997) CDA: Calcium-dependent peptide antibiotics from Streptomyces coelicolor A3(2), containing unusual residues. Angew. Chem. 109, 510–513; Angew. Chem. Int. Ed. Engl. 36, 498–501. 19. Süßmuth, R., Pelzer, S., Nicholson, G., Walk, T., Wohlleben, W., and Jung, G. (1999) New advances in the biosynthesis of glycopeptide antibiotics of the vancomycin type from Amycolatopsis mediterranei. Angew. Chem. 111, 2096–2099; Angew. Chem. Int. Ed. Engl. 38, 1976–1979. 20. Rapp, C., Jung, G., Kugler, M., and Loeffler, W. (1988), Rhizocticins – new phosphono-oligopeptides with antifungal activity. Liebigs Ann. Chem., 655–661. 21. Vanittanakom, N., Loeffler, W., Koch, U., and Jung, G. (1986), Fengycin – a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29–3. J. Antibiot. 39, 888–901. 22. Rapp, C., Jung, G., Katzer, W., and Loeffler, W. (1988) Chlorotetain from Bacillus subtilis, a antifungal dipeptide with an unusual chlorinated amino acid, Angew. Chem. 100, 1801–1802; Angew. Chem. Int. Ed. Engl. 27, 1733–1734. 23. Aydin, M., Lucht, N., König, W. A., Lupp, R., Jung, G., and Winkelmann, G. (1985) Structure elucidation of the peptide antibiotics herbicolin A and B. Liebigs Ann. Chem., 2285–2300. 24. Blum, S., Fiedler, H.–P., Groth, I., Kempter, C., Stephan, H., Nicholson, G., Metzger, J. W., and Jung, G. (1995) Biosynthetic capacities of Actinomycetes. Echinoserin, a new member of the quinoxaline group, produced by Streptomyces tendae. J. Antibiot. 48, 619–625. 25. Potterat, O., Stephan, H., Metzger, J. W., Gnau, V., Zähner, H., and Jung, G. (1994), Aborycin – a tricyclic 21-peptide antibiotic isolated from Streptomyces griseoflaveus. Liebigs Ann. Chem., 741–743. 26. Stephan, H., Kempter, C., Metzger, J. W., Jung, G., Potterat, O., Pfefferle, C., and Fiedler, H.-P. (1996) Kanchanamycins, new polyol macrolide antibiotics produced by Streptomyces olivaceus Tü 4018, II. Structure elucidation. J. Antibiotics 49, 765–769. 27. Jakobi, M., Winkelmann, G., Kaiser, D., Kempter, C., Jung, G., Berg, G., and Bahl, H. (1996) Maltophilin: A new antifungal compound produced by Stenotrophomonas maltophilia R3089. J. Antibiotics 49, 1101–1104. 28. Höltzel, A., Kempter, C., Metzger, J. W., Jung, G., Groth, I., Fritz, T., and Fiedler, H.P. (1998) Spirofungin, a new antigungal antibiotic from Streptomyces violaceusniger Tü 4113. J. Antibiotics 51, 699–707. 29. Potterat, O., Hostettmann, K., Höltzel, A., Jung, G., Dichl, P.-A., and Petrini, O. (1997) Boophiline, an antimicrobial sterol amide from the cattle tick Boophilus microplus. Helv. Chim. Acta 80, 2066–2072. 30. Drechsel, H., Metzger, J., Freund, S., Jung, G., Boelaert, J. R., and Winkelmann, G. (1991). Rhizoferrin – a novel siderophore from the fungus Rhizopus microsporus var. rhizopodiformis. BioMetals 4, 238–243. 31. Konetschny-Rapp, S., Jung, G., Huschka, H.-G., and Winkelmann, G. (1988) Isolation and identification of the principal siderophore of the plant pathogenic fungus Botrytis cinerea. BioMetals 1, 90–98. 32. Drechsel, H., Stephan, H., Lotz, R., Haag, H., Zähner, H., Handtke, K., and Jung, G. (1995). Structure elucidation of yersiniabactin, a siderophore from highly virulent yersinia strains. Liebigs Ann. Chem., 1727–1733. 33. Konetschny-Rapp, S., Jung, G., Meiwes, J., and Zähner, H. (1990) Staphyloferrin A: A structurally new siderophore from Staphylococci. Eur. J. Biochem. 191, 65–74. 34. Tschierske, M., Drechsel, H., Jung, G., and Zähner, H. (1996) Production of rhizofer-

340

References

35.

36. 37.

38. 39. 40. 41. 42. 43.

44. 45. 46. 47. 48. 49. 50. 51. 52.

rin and new analogues obtained by directed fermentation. Appl. Microbiol. Biotechnol. 45, 664–670. Carrano, C. J., Drechsel, H., Kaiser, D., Jung, G., Matzanke, B., Winkelmann, G., Rochelle, N., and Albrecht-Gary, A. M. (1996) Coordination chemistry of the carboxylate type siderophore rhizoferrin: The iron(III) complex and its metal analogs. Inorg. Chem. 35, 6429–6436. Drechsel, H., Jung, G., and Winkelmann, G. (1992) Stereochemical characterization of rhizoferrin and identification of its dehydration products. BioMetals 5, 141–148. Münzinger, M., Taraz, K., Budzikiewicz, H., Drechsel, H., Heymann, P., Winkelmann, G., and Meyer, J.-M. (1999) S,S-Rhizoferrin (enantio-rhizoferrin) – a siderophore of Ralstonia (Pseudomonas) pickettii DSM 6297 – the optical antipode of R,R-rhizoferrin isolated from fungi. BioMetals 12, 189–193. Drechsel, H. and Jung, G. (1998) Peptide siderophores. J. Peptide Science 4, 147–181. R. Kellner, G. Jung, M. Josten, C. Kaletta, K.-D. Entian, and H.-G. Sahl (1989) Pep5: Structure elucidation of a large lantibiotic. Angew. Chem. 101, 618–621; Angew. Chem. Int. Ed. Engl. 28, 616–619. Allgaier, H., Jung, G., Werner, R. G., Schneider, U., and Zähner, H. (1985), Structure elucidation of epidermin, a ribosomally synthetized, heterodet tetracyclic polypeptide antibiotic. Angew. Chem. 97, 1052–1054; Angew. Chem. Int. Ed. Engl. 24, 1051–1053. Zimmermann, N., Metzger, J. W., and Jung, G. (1995), The tetracyclic lantibiotic actagardine, 1H-NMR and 13C-NMR assignments and revised primary structure. Eur. J. Biochem. 228, 786–797. Kellner, R., Jung, G., Hörner, T., Zähner, H., Schnell, N., Entian, K.-D., and Götz, F. (1988) Gallidermin, a new lanthionine-containing polypeptide antibiotic. Eur. J. Biochem. 177, 53–59. Jack, R. W., Carne, A., Metzger, J., Stevanovic, S., Sahl, H.-G., Jung, G., and Tagg, J. (1994), Elucidation of the structure of SA-FF22, a lanthionine-containing antibacterial peptide produced by Streptococcus pyogenes strain FF22. Eur. J. Biochem. 220, 455– 462. Zimmermann, N., Freund, S., Fredenhagen, A., and Jung, G. (1993) Solution structures of the lantibiotics duramycin B and C. Eur. J. Biochem. 216, 419–428. Zimmermann, N. and Jung, G. (1997) The three-dimensional solution structure of the lantibiotic murein-biosynthesis inhibitor actagardine determined by NMR. Eur. J. Biochem. 246, 809–819. Freund, S., Jung, G., Gutbrod, O., Folkers, G., Gibbons, W. A., Allgaier, H., and Werner, R. (1991) The solution structure of the lantibiotic gallidermin. Biopolymers 31, 803–811. Bayer, A., Freund, S., Nicholson, G., and Jung, G. (1993) Posttranslational backbone modification in the ribosomal biosynthesis of the glycine-rich antibiotic microcin B17. Angew. Chem. 105, 1410–1413; Angew. Chem. Int. Ed. Engl. 32, 1336–1339. Bayer, A., Freund, S., and Jung, G. (1995) Post-translational heterocyclic backbone modifications in the 43-peptide antibiotic microcin B17, structure elucidation and NMR study of a 13C, 15N-labelled gyrase inhibitor. Eur. J. Biochem. 234, 414–426. Genilloud, O., Moreno, F., and Kolter, R. (1989) DNA sequence, products, and transcriptional pattern of the genes involved in production of the DNA replication inhibitor microcin B17. J. Bacteriol. 171, 1126–1135. Li, Y.-M., Milne, J. C., Madison, L. L., Kolter, R., and Walsh, C. T. (1996) From peptide precursors to oxazole-thiazole containing peptide antibiotics. Science 274, 1188–1193. Roy, R. S., Allen, O., and Walsh, C. T. (1999) Expressed protein ligation to probe regiospecifity of heterocyclization in the peptide antibiotic microcin B17. Chem. Biol. 6, 789–799. Chong, P. P., Podmore, S. M., Kieser, H. M., Redenbach, M., Turgay, K., Marahiel, M., Hopwood, D. A., and Smith, C. P. (1998) CDA: Physical identification of a chromoso-

341

19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites

53. 54. 55. 56. 57. 58. 59. 60. 61.

62. 63. 64. 65. 66. 67. 68.

69.

mal locus encoding biosynthetic genes for the lipopeptide calcium-dependent antibiotic (CDA) of Streptomyces coelicolor A3(2). Microbiology 144, 193–199. Ji, G., Beavis, R., and Novick, R. P. (1995) Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc. Natl. Acad. Sci. 92, 12055–12059. Ji, G., Beavis, R., and Novick, R. P. (1997) Bacterial interference caused by autoinducing peptide variants. Science 276, 2027–2030. Otto, M., Süßmuth, R., Jung, G., and Götz, F. (1998) Structure of the pheromone peptide of the Staphylococcus epidermidis agr system. FEBS Letters 424, 89–94. Mayville, P., Ji, G., Beavis, R., Yang, H., Goger, M., Novick, R. P., and Muir, T. W. (1999) Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence. Proc. Natl. Acad. Sci. 96, 1218–1223. Hagenmaier, H., Keckeisen, A., Zähner, H., and König, W. A. (1979) Structure elucidation of the nucleoside antibiotic nikkomycin X, Liebigs Ann. Chem. 1494–1502. Zimmermann, G., Hass, W., Faasch, H., Schmalle, H., and König, W. A. (1985) Synthesis of pure stereoisomers of the N-terminal amino acid of Nikkomycin B. Liebigs Ann. Chem. 2165–2177. Bormann, C., Lauer, B., Kalmanczhelyi, A., Süssmuth, R., and Jung, G. (1999) Novel nikkomycins Lx and Lz produced by genetically engineered Streptomyces tendae Tu901. J. Antibiotics 52, 582–585. Bormann, C., Kalmanczhelyi, A., Süssmuth, R., and Jung, G. (1999) Production of nikkomycins Bx and Bz by mutasynthesis with genetically engineered Streptomyces tendae Tu901. J. Antibiotics 52, 102–108. Kupke, T., Kempter, C., Gnau, V., Jung, G., and Götz, F. (1994) Mass spectrometric analysis of a novel enzymatic reaction: Oxidative decarboxylation of the lantibiotic precursor peptide EpiA catalyzed by the flavoprotein EpiD. J. Biol. Chem. 269, 5653– 5659. Kupke, T., Kempter, C., Jung, G., and Götz, F. (1995) Oxidative decarboxylation of peptides catalyzed by flavoprotein Epi D: Determination of substrate specificity using peptide libraries and neutral loss mass spectrometry. J. Biol. Chem. 270, 11282–11289. Kempter, C., Kupke, T., Kaiser, D., Metzger, J. W., and Jung, G. (1996) Thioenols from peptidyl-cysteines: Oxidative decarboxylation of a 13C-labeled substrate. Angew. Chem. 108, 2235–2238; Angew. Chem. Int. Ed. Engl. 35, 2104–2107. Chu, Y.-H., Dunajevskiy, Y.M., Kirby, D. P., Vouros, P., and Karger, B. L. (1996) Affinity capillary electrophoresis-mass spectrometry for screening combinatorial libraries. J. Am. Chem. Soc. 118, 7827–7834. Armstrong, D. W., Rundlett, K. L., and Chen, J. R. (1994) Evaluation of the macrocyclic antibiotic vancomycin as a chiral selector for capillary electrophoresis. Chirality 6, 496–509. Evans, D. A., Wood, M. R., Trotter, B. W., Richardson, T. I., Barrow, J. C., and Katz, J. L. (1998) Total syntheses of vancomycin and eremomycin aglycons. Angew. Chem. Int. Ed. Engl. 37, 2700–2704. Nicolaou, K. C., Boddy, C. N. C., Bräse, S., and Winssinger, N. (1999) Chemistry, biology, and medicine of the glycopeptide antibiotics. Angew. Chem. Int. Ed. Engl. 38, 2097–2152. Pelzer, S., Süßmuth, R., Heckmann, D., Recktenwald, J., Huber, P., Jung, G., and Wohlleben, W. (1999) Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908. Antimicrob. Agents Chemother. 43, 1565–1573. Walk, T. B., Trautwein, A. W., Richter, H., and Jung, G. (1999), ESI Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS): A rapid high-resolution analytical method for combinatorial compound libraries. Angew. Chem. 111, 1877– 1880. Angew. Chem. Int. Ed. Engl. 38, 1763–1765.

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Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

20 Documentation of the Collaborative Research Centre 323

20.1 List of institutes involved

Lehrstuhl für Mikrobiologie/Biotechnologie, Universität Tübingen Lehrstuhl für Mikrobiologie/Membranphysiologie, Universität Tübingen Lehrstuhl für Mikrobielle Genetik, Universität Tübingen Lehrstuhl für Physiologische Chemie/Biochemie, Universität Tübingen Lehrstuhl für Pharmazeutische Chemie, Universität Tübingen Lehrstuhl für Pharmazeutische Biologie, Universität Tübingen Medizinisch-Naturwissenschaftliches Forschungszentrum, Universität Tübingen Max-Planck-Institut für Entwicklungsbiologie, Abt. Biochemie, Tübingen Max Planck-Institut für Biologie, Abt. Infektionsbiologie, Tübingen Institut für Organische Chemie, Universität Tübingen Lehrstuhl für Hydrochemie und Hydrobiologie, Universität Stuttgart Robert Koch-Institut des Bundesgesundheitsamtes, Wernigerode

20.2 List of supported project areas

Project area A TP A1 TP A2

Mikrobieller Sekundärstoffwechsel Prof. Ing. Hans Zähner, Mikrobiologie/Biotechnologie Fermentation, Aufarbeitung und Analytik niedermolekularer Metabolite Fermentationstechnik, Naturstoffisolierung, Naturstoffanalytik

1986–1996 1986–1988 1989–1990 345

Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright # 2001 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30615-3

20 Documentation of the Collaborative Research Centre 323 Grundlagen der Produktionsoptimierung und Analytik mikrobieller Sekundärmetabolite 1991–1993 Fermentation und Produktionsoptimierung mikrobieller Naturstoffe (Mikrobiologie/Biotechnologie) 1994–1999 Prof. Dr. Hans-Peter Fiedler, Mikrobiologie/Biotechnologie TP A3

Biotechnologische Produkte von Peptiden in Hefe PD Dr. Karl-Dieter Entian, Biochemie

TP A4

Genetik und Biochemie der Nikkomycin-Biosynthese in Streptomyces tendae Genetische und biochemische Charakterisierung eines antifungisch wirksamen Proteins aus Streptomyces tendae (Mikrobielle Genetik) Biosynthese des Antibiotikums Nikkomycin und extrazelluläre Proteine bei Streptomyceten Dr. Christiane Bormann, Mikrobiologie/Biotechnologie

TP A5

Hopanoid-Cyclasen Synthese und Funktion von Hopanoiden Prof. Dr. Karl Poralla, Mikrobiologie/Biotechnologie

TP A10 Molekulargenetische und biochemische Analyse der Sekundärmetabolit-Biosynthese in Aktinomyceten Prof. Dr. Wolfgang Wohlleben, Mikrobiologie/ Biotechnologie TP A11 Molekularbiologische Untersuchungen zur Biosynthese des Cytostatikums Landomycin A, gebildet von Streptomyces cyanogenus (DSM 5087) – Untersuchungen zum Aufbau der Zuckerseitenkette – Untersuchungen zur Biosynthese der Desoxyzucker L-Rhodinose und D-Olivose Dr. Andreas Bechthold, Pharmazeutische Biologie TP A12 Kontrolle der autolytischen Enzyme in Escherichia coli Prof. Dr. Joachim-Volker Höltje, MPI für Entwicklungsbiologie, Abt. Biochemie

1986–1988

1991–1993

1997–1999 1991–1996 1997–1999

1997–1999

1997–1999 1997–1999

Project area B TP B1

346

Metabolische Wechselwirkungen zwischen Pround Eukaryonten 1986–1996 Eisen als Umweltsignal Prof. Dr. Volkmar Braun und PD Dr. Klaus Hantke, Mikrobiologie/Membranphysiologie Regulierte Transport- und Signaltransfer-Kanäle in der äußeren Membran Gram-negativer Bakterien 1997–1999 Prof. Dr. Volkmar Braun, Mikrobiologie/Membranphysiologie

20.2 List of supported project areas TP B2

TP B3

TP B4

TP B5

TP B6

Regulation der interzellulären Kommunikation in Zellen des Nervensystems durch eu- und prokaryontische Wirkstoffe Auffindung, Isolierung und Charakterisierung von second messenger-Systeme regulierenden bakteriellen Wirkstoffen Prof. Dr. Bernd Hamprecht, Biochemie Molekulare Mechanismen der Aktivierung von Makrophagen zur Phagosytose und Bakterizidie durch mikrobielle Oberflächenkomponenten und synthetische Analoga Prof. Dr. Wolfgang Bessler, Mikrobiologie/ Membranphysiologie Chemie der Reizverarbeitung in Paramecium Prof. Dr. Joachim Schultz, Pharmazie PD Dr. Susanne Klumpp, Pharmazie Funktion von second messengern in Paramecium Prof. Dr. Joachim Schultz, Pharmazeutische Chemie Molekularbiologische Charakterisierung und Regulation von Exoproteinen und Exopeptiden bei Stapylokokken Genetische und biochemische Charakterisierung von Exopeptiden und Staphylokokken Biosynthese und Regulation von Exoproteinen, Exopeptiden und Membranpigmenten bei Staphylokokken Genetische und biochemische Charakterisierung von Exopeptiden/Exoproteinen und Carotinoiden bei Staphylokokken Prof. Dr. Friedrich Götz, Mikrobielle Genetik Eisentransport bei Gram-positiven und Gramnegativen Bakterien Prof. Dr. Klaus Hantke, Mikrobiologie/ Membranphysiologie

TP B7

Proteinphosphatasen in Paramecium PD Dr. Susanne Klumpp, Pharmazeutische Chemie

TP B8

Analyse der IgA Protease b-Domäne, ein Vehikel für den Proteinexport durch die äußere Membran von Gram-negativen Bakterien Mechanismus und Bedeutung der natürlichen Transformationskompetenz bei Neisserien PD Dr. Thomas Meyer, MPI Biologie

1986–1990

1991–1991

1986–1988

1986–1990

1997–1999

1988–1990 1991–1993

1994–1996

1997–1999

1991–1999

1991–1995

1991–1993 1994–1996

347

20 Documentation of the Collaborative Research Centre 323 TP B9

TP B10

Intrazelluläre Freisetzung und Transport von A-Proteinen pathogener Neisserien in eukaryontischen Zellen PD Dr. Johannes Pohlner, MPI Biologie Genetische und biochemische Charakterisierung des Cytotoxins von Helicobacter pylori Dr. Rainer Haas, MPI Biologie

1991–1993

1994–1996

Project area C – Chemical structure elucidation TP C2

Chemie von Antibiotika und Immunmodulatoren Chemie der Mikroorganismen Peptid- und Proteinchemie der Mikroorganismen Prof. Dr. Günther Jung, Organische Chemie

1986–1990 1991–1996 1997–1999

TP C3

Naturstoffaufklärung Analytik und Strukturaufklärung von Naturstoffen PD Dr. Jörg Metzger, Organische Chemie, später Hydrochemie/Hydrobiologie

1991–1993 1994–1999

TP YE1 Dr. Reissbrodt, RKI Wernigerode

1992–1993

TP YW1 Prof. Braun, Tübingen, Mikrobiologie

1992–1993

20.3 Promotion of members of the collaborative research centre

The following project leaders left for other positions outside the collaborative research centre: Annette Beck-Sickinger, C4, University of Basel. Wolfgang Bessler, C3, Immunology, University of Freiburg. Karl-Dieter Entian, C3, Microbiology, University of Frankfurt. Rainer Haas, C3, Medical Microbiology, University of Munich. Knut Heller, C2, Microbiology, University of Konstanz. Susanne Klumpp, C3, Pharmaceutical Chemistry, University of Marburg. Jörg Metzger, C4, University of Stuttgart. Thomas F. Meyer, C4, Infection Biology, MPI Infection Biology, Berlin. Johannes Pohlner, Evotec BioSystems GmbH, Hamburg. Oliver Potterat, C2, University of Lausanne.

348

20.5 Alphabetical list of members and participants

20.4 Recruitment of new project leaders

The project leaders who left were replaced by the following new project leaders: Andreas Bechthold, Pharmaceutical Biology, University of Tübingen. Christiane Bormann, Microbial Genetics/Microbiology-Biotechnology, University of Tübingen. Klaus Hantke, Microbiology-Membrane Physiology, University of Tübingen. Joachim-Volker Höltje, Biochemistry, MPI of Developmental Biology, Tübingen. Karl Poralla, Microbiology-Biotechnology, University of Tübingen. Wolfgang Wohlleben, Microbiology-Biotechnology, University of Tübingen.

20.5 Alphabetical list of members and participants

Name

Prename

Allgaier*, Hermann Alvarado*, Maria Andres*, Nikolaus Angerer*, Annemarie Anton*, Helga Auge, Ulrike Augustin*, Johannes Bauch, Angela Baumgartner*, Angelika Barten*, Roland Bayer*, Anja Bayer, Ernst Bechthold, Andreas Beck-Sickinger, Annette Beitz*, Eric Bessler, Wolfgang Beyer*, Angelika Bielecki, Jarek Blanck*,Wolfgang Blind*, Brigitte Bös*, Christoph Bormann, Christiane** Bormann, Christiane Braun*, Dieter Braun,Volkmar Breisch*, Monika

Acad. degree

Institute

Project area

Participation

Dipl. Chem. Dipl. Biol. Dipl. Biol. Dipl. Biol. Apothekerin Dipl. Biol. Dipl. Biol. Dipl. Biol. Apothekerin Dipl. Biol. Dipl. Biol. Dr. rer. nat. PD Dr. rer. nat. Dipl. Chem. Apotheker Prof. Dr. Apothekerin Dr. rer. nat. Dipl. Biol. Dipl. Ökotroph. Dipl. Chem. Dr. rer. nat. Dr. rer. nat. Dipl. Biol. Prof. Dr. Dipl. Biol.

Organ. Chemie Biologie Biologie Biologie Pharmazie Biologie Biologie MPI, Infektions. Biologie Biologie Organ. Chemie Phys. Chem. Pharmazeut. Bio. Organ. Chemie Pharmazie Biologie Pharmazie Biologie Biologie Phys. Chem. Biologie Biologie Biologie Biologie Biologie Phys. Chem.

C2 A1 A1 B1 B4 B5 B5 B9 B4 B8 C2 B2 A11 C2 B4 B3 B4 A1/GW A1 B2 B1 A1 A4 A1 B1 B2

86–90 86–89 88–89 88–91 91–95 99 88–90 93 86– 94–96 88–94 86–87 97–99 88 94–95 86–88 89 86 86–87 87–91 96–97 86–90 91–99 90–93 86–99 86

349

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Name

Prename

Acad. degree

Institute

Project area

Participation

Britten* Uwe Brooks*, Mark Brückner, Reinhold Bruntner*, Christina Brutsche*, Sandra Burckhard*, Renate Cebulla*, Ingeborg Chehadeh*, Heidi Choi*, Ok-Byung Cullmann*, Hans-Jürgen Decker*, Heinrich Demleitner*, Gaby Deres*, Karl Domann*, Silvie Drautz, Hannelore Drechsel*, Hartmut Dringen*, Ralf Dürr, Hansjörg Eick-Helmrich*, Katrin Engel*, Peter Entian**, Karl-Dieter Enz*, Sabine Fauth*, Ursula Faust*, Bettina Feil*, Corinna Fels*, Johannes Flechsler*, Insa Fleckenstein*, Burkhard Fiedler**, Hans-Peter Fiedler,* Waltraud Fischer, Eckhard Franz, Brigitte Freund*, Stefan Freund*, Wolf-Dietrich Friedl, Anette Freund, Stefan Freund*, Wolf-Dieter Fridrich*, Gerald Frosch, Ingrid Früchtel*, Jörg Fussenegger*, Martin Gaisser*, Sabine Gierlich, Doris Götz, Friedrich Groeger*,Wolfram Groß, Matthias Grothe*, Kirsten Gombert*, Frank Groß*, Patricia Günther*, Karola Guo*,Yinglan

Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dr. rer. nat. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dr. rer. nat. Dipl. Chem. Dipl. Biochem. Dipl. Chem. Dipl. Biol. Dipl. Biol. Dr. rer. nat. Dr. rer. nat. Dr. rer. nat. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Chem. Dipl. Biochem. Prof. Dr. Dipl. Biol. Dr. rer. nat. Dipl. Chem. Dipl. Chem. Dipl. Biochem. Dipl. Biochem. Dipl. Chem. Dipl. Biochem. Apotheker Dr. rer. nat. Dipl. Chem Dipl. Biol. Dipl. Biol. Apothekerin Prof. Dr. Dipl. Biol. Dipl. Biol. Apothekerin Dipl. Biochem. Dipl. Biol. Dr. rer. nat. Dipl. Chem.

Biologie Organ. Chemie Biologie Biologie Biologie Biologie Biologie Biologie Biologie Biologie Biologie Biologie Organ. Chemie Biologie Biologie Organ. Chemie Phys. Chemie Organ. Chemie Biologie Pharmazie Phys. Chem. Biologie Biologie Pharmazie Biologie Biologie Organ. Chemie Organ. Chemie Biologie Biologie Biologie Organ. Chemie Organ. Chemie Pharmazie Phys. Chem. Organ. Chemie Pharmazie Pharmazie Biologie Organ. Chemie Biologie Biologie Pharmazie Biologie Biologie Biologie Pharmazie Organ. Chemie Biologie Biologie Pharmazie

A1 C2 B5 A4 B1 B1 A1 B1 A5 A1 A1 B5 C2 A11 A1 C2 B2 C2 B1 B4 A3 B1 A1 A11 A5 A1 C2 C2 A2 B1 B1 C2 C2 B4 B2 C2 B4 B4 A1 C2 B9 B6 B4 B5 B1 B5 B4 C2 B6 B1 B4

89–91 94–99 89–99 95–97 97–97 86 92–95 86 92–95 92–94 86–95 88–92 88–92 97–98 86–92 89–99 88–90 Stip. 86–89 95 86–88 95–99 86–90 97–98 93–96 91–93 93–98 93–99 86–99 86–87 86 88 88–93 87–91 86–87 88 86–91 89 86–87 94–97 93–95 91–93 86 86–99 98–98 98–99 95–98 90–91 93–95 87–89 86–99

350

20.5 Alphabetical list of members and participants

Name

Prename

Acad. degree

Institute

Project area

Participation

Haag*, Hubert Haag*, Sabine Habeck*, Martina Häsler*, Peter Hambach, Kristina Hamprecht, Bernd Handschuh, Dieter Hansen*, Martin Hantke**, Klaus Hantke, Klaus Harder, Michael, Harkness, Robin Hartjen*, Uwe Hatzelmann, Armin Heckmann*, Dorothee Heidel, Martina Hertle, Ralf Heuermann*, Dorothee Hilger*, Martina Hille*, Matthias Hobbie*, Silke Höltje, Joachim-Volker, Höltzel*, Alexandra Hörner*, Thomas Hörr*, Ingmar Hoff*, Hubert Hofmann,* Hans-Joachim Hoffmann*, Helmut Hoffmann*, Thomas Holz, Bärbel Huhn*, Wolfgang Hummel*, Rolf-Peter Hwang-Kim*, In-Sook Ihlenfeldt*, Hans-Georg Isselhorst-Scharr*, Caroline Jack, Ralph-Wilson Jung, Günther Jung*, Oliver Kabatek* Ursula Kaiser*, Dietmar Kammler*, Meike Kannenberg, Elmar Kapitza, Susanne Katzer*,Werner Kellner*, Roland Kempf, Markus Kempter*, Christoph Kern, Armin Kies, Stefanie Killmann*, Helmut

Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Apothekerin Prof. Dr. Dr. rer. nat. Apotheker Prof. Dr. Prof. Dr. Dr. rer. nat. Dr. rer. nat. Dipl. Biol. Dipl. Biochem. Dipl. Biol. Dipl. Biol. Dr. rer. nat. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Prof. Dr. Dipl. Chem. Dr. rer nat. Dipl. Biol. Dipl. Biol. Apotheker Dipl. Biol. Dipl.Chem. Dipl. Biol. Dipl. Biol. Dipl. Biochem. Dipl. Biol. Dipl. Chem.

Biologie Biologie Biologie Biologie Pharmazie Biochemie Biochemie Pharmazie Biologie Biologie Biologie Biologie Biologie Pharmazie Biologie Pharmazie Biologie Biologie Biologie Biologie Biologie Biochemie Organ. Chemie Biologie Organ. Chemie Biologie Pharmazie Biologie Pharmazie Biologie Biologie Organ. Chemie Biologie Organ. Chemie

A1 A1 B1 A1 B4 B2 B2 B4 B1 B6 A2 B1 A2 B4 A10 B4 B1 B10 B1 B5 B1 A12 C2 A2 C2 A1 B4 B1 B4 A1 A1 C2 B1 C2

89–92 92–95 98–98 86–88 98–98 86–91 86–91 98–99 86–90 91–99 94–95 86–88 89–90 86 95–98 86 96–99 95–96 94–95 96–99 90–93 97–99 95–99 86–90 95–99 88–90 86–89 86 95–97 90–94 86–87 87 91–93 90–95

Dipl. Biol. Dr. rer. nat. Prof. Dr. Dipl. Biol Dipl. Biol. Dipl. Chem. Dipl. Biol. Dr. rer. nat. Dipl. Biol. Dipl. Biol. Dipl. Chem. Dipl. Biol. Dipl. Chem. Dr. rer. nat. Dipl. Biol. Dr. rer. nat.

Biologie Organ. Chemie Biochemiker Biologie Organ. Chemie Organ. Chemie Biologie Biologie Organ. Chemie Biologie Organ. Chemie Biologie Organ. Chemie Organ. Chemie Biologie Biologie

A1 C2/GW C2 A1 C2 C2 B6 A5 C2 A1 C2 A2 C2 C2 B5 B1

88–90 91–99 86–99 91–94 86–87 95–98 92–94 98–99 98–99 87–91 86–89 97 93–96 86 98–99 90–99

351

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Name

Prename

Kim*, In-Sook Klauser, Thomas Kleemann*, Gisela Klumpp**, Susanne Knigge, Michael Koebnik*, Ralf Könninger*, Ulrich Köster*,Wolfgang Krämer, Joachim Kremer*, Stephan Krismer*, Bernhard Koch*, Ulrike Köster**,Wolfgang Konetschny-Rapp*, Silvia Koss, Dieter Krüger*, Thomas Kugler*, Martin Kühn*, Sabine Kupke*, Thomas Langenberg, Uwe Langer*, Monika Lauer*, Bettina Lechner*, Max Leipert, Dietmar Linder*, Jürgen Lipps*, Hans-Peter Lohmann, Susanne Maerker, Christian Mahnke*, Marion Marquardt*, Udo Mayer*, Irene Merkofer, Thorsten Mehrkühler, Christian Meiwes*, Johannes Mende*, Jasmin Metzler*, Monika Metzger**, Jörg Metzger, Jörg Meyer, Thomas Möhrle*,Volker Möller, Andreas Müller*, Kerstin Müller, Judith Mutard, Denise Muth, Günther Neubauer, Heike Noda, Shigeru Ochs*, Dietmar Ochs*, Martina Odenbreit, Stefan Ölschläger*, Thobias

352

Acad. degree

Institute

Dipl. Biol. Biologie Dr. rer. nat. MPI-Biologie Dipl. Biol. Biologie Dr. rer. nat. Pharmazie Dipl. Biol. Biologie Dipl. Biol. Biologie Dipl. Biol Biologie Dipl. Biol. Biologie Dipl. Biol. MPI Infekt. Biol. Dipl. Biol. Biologie Dipl. Biol. Biologie Dipl. Chem. Organ. Chemie Dr. rer. nat. Biologie Dr. rer. nat. Organ Chemie Dipl. Biol. Biologie Dipl. Chem. Pharmazie Dipl. Biol. Biologie Dipl. Biol. Biologie Dipl. Biochem. Biologie Dipl. Biochem. MPI Infekt. Biol. Dipl. Biol. Biologie Dipl. Biol. Biologie Dipl. Biol Biologie Dipl. Chem. Organ. Chemie Dipl. Chem. Pharmazie Apotheker Pharmazie Dr. rer. nat. Phys. Chem. Dipl. Biol. MPI Infekt. Biol. Dipl. Biol. Biologie Dipl. Chem. Organ. Chem. Dipl. Biol. Biologie Dipl. Biol. Biologie Dipl. Biol. Phys. Chemie Dipl. Biol. Biologie Dipl. Biol. Biologie Dipl. Biol. Biologie Dr. rer. nat Organ. Chemie Dr. rer. nat. Organ. Chemie Dr. rer. nat. MPI Infekt. Biol. Dipl. Biol. Biologie Dipl. Chem. Phys. Chem. Dipl. Biol. Biologie Dipl.Lebensm.Ing. MPI Biochem. Apothekerin Pharmazie Dr. rer. nat. Biologie Dipl. Biol. Biologie Dr. med. vet. Pharmazie Dipl. Biol. Biologie Dipl. Biol. Biologie Dipl. Biol. MPI, Biochem. Dipl. Biol. Biologie

Project area

Participation

B1 B8 A5 B4 A5 B1 B1 B1 B8 A1 B5 C2 B1 C2 B6 B4 A1 B1 B5 B9 A1 A4 B5 C2 B4 B4 B2 B9 A1 C2 B1 A5 B2 A1 B1 A1 C2 C3 B8 A4 B2 B6 A12 B4 A10 A4 B4 A5 B1 B10 B1

93 91–93 89–92 91–95 99 90–91 94–98 86–87 91–93 87–90 95–99 86–88 86–90 86–90 94–97 93–97 86–86 91–95 89–95 91 92–96 95–99 89 94–98 98–99 89 86 91 86–87 98–99 89–90 96–98 89–91 87–90 86–87 86–89 86–90 91–99 91 91–95 86–90 94–97 99 91 95–99 96 86 90 91–95 94–95 86–90

20.5 Alphabetical list of members and participants

Name

Prename

Ötzelberger, Karin Ondrazcek, Roland Ottenwälder, Birgit Otto*, Michael Otto*, Susanne Pantel, Iris Patzer*, Silke Peschel, Andreas Perzl*, Michael Petersen*, Frank Pfefferle*, Uwe Pfeiffer, Brigitte Pilsl*, Holger Pfeifer,Volker Plaga*, Armin Plantoer*, Stefan Pohlner**, Johannes Potterat, Oliver Poralla, Karl Preßler*, Uwe Pultar*, Thomas Probst, Katrin Rabenhorst*, Jürgen Rak, Gabriele Rauch*, Beatrix Rapp* Claudius Rechenberg*, Moritz von Reeger, Eva Reissbrodt, Rolf Reuschenbach,*, Peter Reuschenbach*, Petra Reutter*, Felix Rexer, Hans-Ulrich Richter*, Monika Röhl*, Franz Rösch, Hartmut Rohling*, Anette Roos*, Margareta Roos*, Ulrich Rose, Andreas Rosenstein, Ralph Ruan,Yuan Russwurm*, Roland Sauer*, Martin Sauter, Simon Schade, Uwe Schäffer*, Sven Scharr*, Jürgen Schaude*, Renate Schiller*, Max Schiffer*, Guido

Acad. degree

Institute

Project area

Participation

Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Chem. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dr. rer. nat. Dr. rer. nat. Dipl. Biochem. Dr. rer. nat. Dipl. Biol. Dr. rer. nat. Dr. rer. nat. Prof. Dr. Dipl. Biol. Dipl. Biol. Dipl. Chem. Dipl. Biol. Dr. rer. nat. Dipl. Biol. Dipl. Chem. Dipl. Biochem. Dipl. Biol. Dr. rer. nat. Dipl. Biol. Dipl. Biol. Dipl. Chem. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biochem. Dr. rer. nat. Dipl. Biol Dipl. Biol. Dipl. Biol. Dipl. Biol. Apotheker Dipl. Biol. Dipl. Biol. Dipl. Biochem. Dipl. Biol. Dr. rer. nat.

MPI Biochem. Biologie Biologie Biologie Pharmazie Biologie Biologie Biologie Biologie Biologie Biologie Phys. Chemie Biologie Biologie Biologie Biologie MPI Biochem. Biologie Biologie Biologie Biologie Organ. Chemie Biologie Biologie Biologie Organ. Chemie MPI Biochem. Biologie R. Koch Inst. Biologie Biologie Organ. Chemie Biologie Biologie Biologie Pharmazie Biologie Biologie Biologie Phys. Chem. Biologie Biologie Biologie Biologie Biologie Pharmazie Biologie Biologie Organ. Chemie Phys. Chemie Biochemie

B9 B1 B5 B5 B7 A4 B6 B5 A5 A1 A2 B2 B1 A10 A2 B1 B9 A1 A5 B1 A1 C2 A1 A1 A1 C2 A12 B6 YE1 A2 A1 C3 A10 A2 A1 B4 A10 A1 B5 A3 B5 B1 A4 B1 B1 B4 B1 A1 C2 B2 A12

91–93 91 95–96 93–97 90–93 95 94–99 94–95 93–99 88–91 89–90 86–90 94–98 99 86–90 98 91–93 91–94 91–99 86 86–88 99 86–87 87–90 89–91 86–88 97–99 97 91–93 86–88 86 96–99 97–99 95 86–87 90– 97 89–92 91–93 86 86–99 92 97–99 86–87 99 86–90 86–87 87–91 87–89 86–90 97–98

353

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Name

Prename

Schmidt, Günther Schmitz, Susanne Schneider*, Ursula Schneider, Richard Schnell, Norbert Schöffler*, Harald Schön*, Claudia Schönborn, Christoph Schönefeld*, Ulrich Schönherr*, Roland Schuerhoff-Göters*, Wilhelm Schüz*, Traugott Schüz*, Traugott Schultz*, Gabriele Schultz, Joachim Schwarz*,Wolfgang Schwartz, Dirk Seiffert*, Andreas Selke*, Dagmar Sommer*, Patricia Sorg, Gerhard Sprengler, Siegried Stahl, Bernd Stanger*, Andrea Staudenmaier*, Horst Steinlen*, Siegfried Stephan*, Holger Stefanovic*, Stefan Stegmann*, Evi Stiefel*, Alfred Stümpfel*, Joachim Süßmuth*, Roderich Surovoy, Andrey Tappe*, Cord-Henning Tejmar-Kolar, Liana Templin, Markus Teufel*, Pia Theobald, Uwe Thumm*, Günter Tippelt*, Anette Traub*, Irene Trefzer, Axel Troeger*,Wilfried Tschen, Shu Yuan Tschierske*, Martin Ungermann*,Volker Völkel*; Helge Voges, Brigitte Voges, Klaus-Peter Vollmer*,Waldemar

354

Acad. degree

Institute

Project area

Participation

Dipl. Biochem. Dipl. Biochem. Apothekerin Dipl. Biol. Dipl. Biochem. Dipl. Biol. Dipl. Biol. Apotheker Dipl. Biochem. Dipl. Biol.

Phys. Chemie Biologie Biologie Biologie Phys. Chemie Biologie Biologie Pharmazie Pharmazie Biologie

A3 A5 A1 B1 A3 B1 B1+B6 B4 B4 B1

89 97–99 86 94–95 89 86–89 87–90 90–91 86–88 92–93

Apotheker Dipl. Biol. Dr. rer. nat. Dipl. Biol. Prof. Dr. Dipl. Biol. Dr. rer. nat. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Chem. Dipl. Chem. Dipl. Biochem. Dipl. Biol. Dipl. Biol. Dipl. Biochem. Dipl. Chem. Dipl. Biochem. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Chem. Dr. rer. nat. Dipl. Biochem. Dr. rer. nat. Dr. rer. nat. Dipl. Biol. Dr. Ing. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Chem. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biochem. Dr. rer. nat. Dr. rer. nat Dr. rer. nat.

Pharmazie Biologie Biologie Biologie Pharmazie Biologie Biologie Biologie Pharmazie Biologie Organ. Chemie Phys. Chemie Phys. Chemie Biologie Biologie Pharmazie Organ. Chemie Organ. Chemie Biologie Biologie Biologie Organ. Chemie Organ. Chemie Botan. Instit. Biologie Biochemie Biologie Biologie Biologie Biologie Biologie Pharmazie Organ. Chemie Biologie Biologie Biologie Pharmazie Organ. Chemie Organ. Chemie Biochemie

B4 A1 A2 B1 B4 A4 A10 A1 B4 A4 C3 B2 B2 A1 B1 B4 C2 C2 A10 B1 A1 C2 C2/GW A5 A1 A12 B5 A2 B5 A5 B1 A11 C2 A1 A1 A1 B4 C2 C2 A12

86–90 86 87–94 88–92 86–99 97–99 95–99 90–92 91–95 94–95 98–99 86–88 86–87 86 86 89–92 91–95 89–92 98–99 97–99 86–88 95–99 90–97 91–93 86 97–99 89–92 95–99 88–93 96–97 90–92 98–99 89–91 91 92–94 88–90 92–96 86–88 86–88 97–99

20.6 Support of young scientists

Name

Prename

Van hove*, Brundhild von der Mülbe, Florian Veitinger*, Sabine Vierling*, Silke Videnov, Georgi Walker, Georg Walz*, Franz Wang-Tschen*, Shu-Yuan Wasiliu*, Michal Weber, Tillmann Weitnauer, Gabriele Welz*, Dietrich Wieland*, Bernd Wieland*, Karsten-Peter Wiesinger, Heiner Wiesmüller*, Karl-Heinz Witke, Claudia Woelk*, Uwe Wohlleben,Wolfgang Zähner, Hans Ziegelmaier-Kemmling,* Dagmar Zimmermann, Luitgard Zimmermann, Norbert

Acad. degree

Institute

Project area

Participation

Dipl. Biol. Dipl. Biochem. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dr. rer. nat. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dipl. Biol. Dr. rer. nat. Dipl. Chemiker Dipl. Biol. Dipl. Biol. Prof. rer. nat. Prof. Dr. Ing.

Biologie Organ. Chemie Biologie Biologie Organ. Chemie Biologie Biologie Biologie Biologie Biologie Biologie Biologie Biologie Biologie Phys. Chem. Organ. Chemie Biologie MPI Infektions. Biologie Biologie

B1 C2 B1 A10 C2/GW B1 A2 B1 A1 A10 A11 B1 B5 B5 B2 C2 B5 B8 A10 A1

89 97–99 89–92 97–99 91–95 97–99 86–88 91–92 86–86 99 98–99 92–95 89–93 96–99 86–91 86–92 91–94 95–96 95–99 86–96

Dipl. Biol. Dr. rer. nat. Dipl. Chem.

Biologie Biologie Organ. Chemie

A1 B1 C2

86–90 86 91

* Graduation with PhD during the collaborative research centre 323. ** Habilitation during the collaborative research centre 323.

20.6 Support of young scientists

List of promotions resulting from the collaborative research centre TP A1 Alvarado, Maria (1990): Monomeres und dimeres Cinnachinon aus Streptomyces griseoflavus (Tü 2482). Andres, Nikolaus (1989): Hormaomycin, ein Peptidlacton mit morphogener Wirkung auf Streptomyceten. Blank, Wolfgang (1987): Untersuchungen zur biologischen Modifikation der Bafilomycine. Braun, Dieter (1993): Enzymatische Halogenierung von mikrobiellen Metaboliten und Etablierung eines Photokonduktivitätsscreening. 355

20 Documentation of the Collaborative Research Centre 323 Cebulla, Ingeborg (1995): Gewinnung komplexbildender Substanzen mittels Amycolatopsis orientalis. Cullmann, Hans Jürgen (1994): Depsichlorine und andere Metabolite aus Streptomyces antibioticus Tü 1661. Decker, Heinrich (1989): Untersuchung zur Struktur-Wirkungsbeziehung der Nikkomycine und Isolierung neuer Nikkomycine. Fauth, Ursula (1987): Galbonolid A und B, neue antifungische Makrolid-Antibiotika. Fels, Johannes (1994): Suche nach Chitinase-Inhibitoren und Charakterisierung der Hemmstoffe aus Streptomyces tendae Tü 2774 und Streptomyces sp. Tü 3566. Haag, Hubert (1992): Neue Eisenkomplexbildner aus Staphylokokken und Yersinia enterocolitica. Screening, Fermentation, Isolierung und Charakterisierung. Haag, Sabine (1996): Gentransfer zur Produktion neuer Naturstoffe am Beispiel der Tetracenomycine und Urdamycine und CDA – ein Calcium-abhängiges Antibiotikum aus Streptomyces coelicolor A3(2). Häsler, Peter (1988): Streptonolide. Sekundärmetabolite aus Streptomyces olivaceus Tü 2108. Hoff, Hubert (1990): Obskurolide: neue Butyrolactone aus Streptomyceten. Huhn, Wolfgang (1986): Maduraferrin, ein neues Siderophor aus Actinomadura madurae. Screening, Isolierung und Fermentation. Isselhorst-Scharr, Caroline (1995): Tü 3010/1. Ein neues Thiolactonantibiotikum aus Streptomyces olivaceus ssp. gelaticus Tü 3010. Jung, Oliver (1993): Screening nach neuen Siderophoren und die Charakterisierung eines hochaffinen Eisenaufnahmesystems bei Staphylococcus aureus. Katzer, Werner (1991): Bromo- und Chlorotetain aus Bacillus amyloliquefaciens. Kremer, Stefan (1990): Untersuchungen zur Biosynthese von ungewöhnlichen Macrolidantibiotika am Beispiel der Bafilomycine. Kugler, Martin (1986): Rhizocticin. Ein neues Antibiotikum aus Bacillus subtilis ATCC 6633. Langer, Monika (1996): Biotinantagonisten und Siderophore aus Streptomyces griseoflavus ssp. griseoflavus. Mahnke, Marion (1990): Inhibitoren der Lysin-N6-Hydroxylase aus Escherichia coli MM 128. Screening, System und Charakterisierung. Meiwes, Johannes (1989): Neue Siderophore von Staphylococcen und Streptomyceten. Metzler, Monika (1989): Untersuchungen an sekundären Metaboliten verschiedener Streptomyceten. Petersen, Frank (1991): Germicidin B – ein autoregulatorischer Keimungshemmstoff aus Streptomyces viridochromogenes. Plaga, Armin (1990): Studien zur mikrobiologischen Produktion von Phosphinothricin aus Streptomyces viridochromogenes Tü 494. Pultar, Thomas (1988): Lysolipin X und I. Fermentation, Isolierung, biologische Wirkung und Interaktion mit Mg2+. Rabenhorst, Jürgen (1986): Valclavam – ein antifungisches b-Lactam. 356

20.6 Support of young scientists Rauch, Beatrix (1992): Hormaomycin und andere differenzierungsaktive Substanzen aus Streptomyceten. Reuschenbach, Peter (1986): Studien zur Fermentation der phosphorsäuretriesterhaltigen Lactone aus Tü 1718. Röhl, Franz (1986): Untersuchungen zur Wirkungsweise von Clavam Antibiotica. Roos, Margareta (1994): Untersuchungen zur Differenzierung bei Streptomyces griseus und Streptomyces antibioticus. Scharr, Jürgen (1993): Hohe Zelldichten bei Bacillus thuringiensis var. israelensis. Schüz, Traugott (1990): Pelletbildung bei Streptomyces tendae Tü 901/S2566 und verfahrenstechnische Optimierung der Nikkomycin-Fermentation. Seiffert, Andreas (1992): Untersuchungen zum Eisentransport bei verschiedenen Bakterien. Stümpfel, Joachim (1988): Pyrrolam. Ein gamma-Lactam aus Streptomyces olivaceus Tü 3082. Fermentation, Isolierung und biologische Wirkung. Tschierske, Martin (1994): Studien zur Produktion von Rhizoferrin und analogen Verbindungen mit Cunninghamella elegans. Ziegelmaier-Kemmling, Dagmar (1990): Vergleichende Untersuchungen zur Wirkungsweise von Tetramsäure-Verbindungen. TP A2 Incorporated in TP A1 TP A4 Bruntner, Christina (1997): Molekularbiologische Untersuchungen zur Biosynthese von Nikkomycin D in Streptomyces tendae TÜ 901. Lauer, Bettina (1997): Charakterisierung von Nikkomycin-Biosynthesegenen und genetische Manipulation des Biosyntheseweges in Streptomyces tendae TÜ 901. Möhrle, Volker (1995): Klonierung und Charakterisierung von Nikkomycin-Biosynthesegenen aus Streptomyces tendae TÜ 901. Roos, Ulrich (1993): Histidin-Aminotransferase-Aktivität in Streptomyces tendae: Korrelation zur Nikkomycin-Produktion, Reinigung und Charakterisierung. Schwarz, Wolfgang (2000): Untersuchungen zur transkriptionellen Regulation der Nikkomycin Synthese in Streptomyces tendae Tü 901. Sommer, Patricia (1995): Klonierung, Sequenzierung und Charakterisierung eines Lipasegens aus Streptomyces cinnamomeus TÜ 89. Russwurm, Roland (2000): Genetische Untersuchungen zur Nikkomycin-Biosynthese in Streptomyces tendae TÜ 901. Tesch, Cornelia (1995): Klonierung, Sequenzierung und Charakterisierung eines Esterasegens aus Streptomyces diastatochromogenes TÜ 20. TP A5 Choi, Ok-Byung (1995): Experimente zur Klonierung, Sequenzierung und Expression der Squalen-Hopen-Cyclase aus Rhodopseudomonas palustris und Alicyclobacillus acidoterrestris. 357

20 Documentation of the Collaborative Research Centre 323 Feil, Corinna (1996): Ortsspezifische Mutagenese zur Identifizierung katalytisch aktiver Aminosäuren der Squalen-Hopen-Cyclase von Alicyclobacillus acidocaldarius. Kleemann, Gisela (1992): Hopanoidgehalt und Fettsäuremuster zweier Rhodopseudomonas-Arten und Reinigung der Squalen-Hopen-Cyclase aus Rhodopseudomonas palustris. Perzl, Michael (1996): Biochemische und molekularbiologische Untersuchungen zur Hopanoid-Biosynthese in Bradyrhizobium und zur Tetrahymanol-Biosynthese in dem Ciliaten Tetrahymena. Schmitz, Susanne (2000): Charakterisierung des Hopanoidbiosynthese-Operons aus Bradyrhizobium japonicum und der Squalen-Hopen-Cyclase aus Alicyclobacillus acidocaldarius. Tappe, Cord Henning (1993): Squalen-Hopen-Cyclasen: Reinigung, Charakterisierung und Inhibitor-Experimente. TP A10 Burger, Annette (1997): Isolierung und Charakterisierung einer Genregion mit Zellteilungs- und Differenzierungsgenen aus Streptomyces coelicolor A3(2). Maas, Ruth Maria (1997): Molekulargenetische Analyse des Plasmids pSG5 aus Streptomyces ghanaensis DSM 2932. Schwartz, Dirk (1997): Molekulargenetische Analyse der Phosphinothricin-Tripeptid-Biosynthese in Streptomyces viridochromogenes Tü 494. Stegmann, Efthimia (1999): Molekulargenetische und biochemische Untersuchungen des EDDS-Produzenten Amycolatopsis japonicum MG417-CF17. Vierling, Silke (2000): Molekulargenetische Analyse des recA/recX-Operons in Streptomyces lividans TK 64. TP A11 Gaisser, Sibylle (1998): Molekularbiologische und biochemische Untersuchungen zur Avilamycin-Biosynthese und Resistenz in Streptomyces viridochromogenes Tü57. Doman, Silvie (1998): Untersuchungen zum Wirkmechanismus der Landomycine und zur Biosynthese der Desoxyzucker D-Olivose und L-Rhodinose in den Angucyclin-Antibiotika Landomycin A und Urdamycin A. Faust, Bettina (1998): Untersuchungen zur Biosynthese von Urdamycin A und Herstellung neuer Naturstoffe mittels molekularbiologischer Methoden. TP A12 Schiffer, Guido (1998): Identifizierung und funktionale Charakterisierung des Penicillin-Bindeproteins 1C als Mureinpolymerase in Escherichia coli. Rechenberg, Moritz von (1998): Untersuchungen der Protein-Protein Wechselwirkungen zwischen Murein Hydrolasen und Penicillin-bindenden Proteinen in Escherichi coli. Vollmer, Waldemar (1998): Identifizierung und Charakterisierung eines Strukturproteins in Multienzymkomplexen aus Mureinsynthasen und Mureinhydrolasen in Escherichia coli. 358

20.6 Support of young scientists TP B1 Hoffmann, Helmut (1986): Proform und reifes FhuA Rezeptorprotein von E. coli K-12: Reindarstellung und biologische Eigenschaften. Köster ,Wolfgang (1986): Eisenhydroxamattransport von E. coli: Nukleotidsequenz des fhuB Gens. Identifizierung und Lokalisierung des FhuB Proteins. Ölschläger, Tobias (1986): Genetische Analyse des colM Lokus und Nukleotidsequenz des Colicin M Immunitätsproteins. Burkhardt, Renate (1987): Molekulare Charakterisierung der Gene fhuA, fhuC und fhuD des Ferri-Hydroxamat-Transportsystems bei E. coli. Fiedler, Waltraud (1987): Physiologische Bedeutung periplasmatischer Oligosaccharide (membrane derived oligosaccharides, MDO) bei Escherichia coli K-12: Untersuchungen an Mutanten der MDO Biosynthese. Preßler, Uwe (1987): Genetische Charakterisierung des Bindeprotein-abhängigen Eisen-Dicitrattransports. Eick-Helmerich, Katrin (1989): Untersuchungen zur Struktur und Funktion der Gene exbB und exbD bei Escherichia coli K-12. Sauer, Martin (1989): Eisen(III)-Aufnahme von Escherichia coli K12: Nukleotidsequenz des fhuE Gens und Untersuchungen zur Funktion konservierter Bereiche bei TonB-abhängigen Rezeptoren. Schäffer, Sven (1989): Untersuchung des Fur-Eisenrepressors von Escherichia coli K12. Schöffler, Harald (1989): Transport durch die äußere Membran von Escherichia coli. Staudenmaier, Horst (1989): Exogene Induktion des Eisendicitrat-Transportsystems von Escherichia coli. Günter, Karola (1990): Zur Intermembran-Kopplungsfunktion des TonB Proteins von Escherichia coli. Mende, Jasmin (1990): Colicin B: Untersuchungen zum Exportverhalten Colicin-produzierender Zellen und zur TonB-abhängigen Aufnahme durch die äußere Membran. Schön, Claudia (1990): Untersuchung des Aufnahmesystems für zweiwertiges Eisen bei Escherichia coli. Van hove, Brunhilde (1990): Bindeprotein-abhängiger Transport und Transmembranregulation des Eisen(III)Dicitrat-Transportsystems. Chehade, Heidi (1990): Untersuchungen zur Wechselwirkung von Escherichia coli Wildstämmen und Mutanten in umweltregulierten Genen bei bakteriziden Komponenten von Humanserum. Angerer, Annemarie (1991): Ein neues Eisentransportsystem in Serratia marcescens. Gaisser, Sabine (1992): Das TonB Gen aus Serratia marcescens. Koebnik, Ralf (1992): Ferrichrom-Aufnahme in Bakterien: Membrantopologie des Ferrichromrezeptors von Escherichia coli und Struktur des Ferrichromrezeptors und des TonB Proteins von Yersinia enterocolitica. Schultz-Hauser, Gabriele (1992): FhuC, die konservierte Komponente des Eisen(III) Hydroxamat-Transports. Traub, Irene (1992): Untersuchung funktioneller Domänen des TonB-Proteins von Escherichia coli. 359

20 Documentation of the Collaborative Research Centre 323 Veitinger, Sabine (1992): Das Eisen(III)-Dicitrat-Transportssystem: Kartierung auf dem Chromosom von Escherichia coli K-12 und Untersuchungen zur Transmembran-Regulation. Hobbie, Silke (1993): Untersuchungen zur Struktur und Funktion des ShlB Proteins, der transportierenden und aktivierenden Komponente des Hämolysins von Serratia marcescens. Killmann, Helmut (1993): Eisen(III)Hydroxamat-Transport in Escherichia coli. Funktionsdomänen des Rezeptor-Proteins FhuA und Untersuchungen zur dreidimensionalen Grundstruktur des Proteins in der äußeren Membran. Schönherr, Roland (1993): Aktivierung und Sekretion des Hämolysins von Serratia marcescens. Ochs, Martina (1994): Untersuchungen zur Regulation des Eisen-Dicitrat-Transportsystems von Escherichia coli K-12. Kim, In-Sook (1995): Exogene Signaltransduktion des Eisen(III)-Dicitrat-Transportsystems von Escherichia coli K-12. Kühn, Sabine (1995): Rhizoferrin-Aufnahme in Morganella morganii. Groß, Patricia (1996): Untersuchungen zur Struktur und Funktion des Colicin M-Immunitätsproteins (Cmi). Pilsl, Holger (1996): Domänenstruktur, Evolution und Immunität der Colicindeterminanten 5, 10 und K von Escherichia coli und der Pesticindeterminanten von Yersinia pestis. Bös, Christof (1997): In vivo Charakterisierung des Ferrichrom-Rezeptor-Proteins FhuA aus Escherichia coli mittels thiolspezifischer Reagenzien. Brutsche, Sandra (1997): SigX – ein neuer Sigmafaktor der ECF-s70 Subfamilie aus Bacillus subtilis. Enz, Sabine (1997): Transkriptionsregulation des Eisen(III) Dicitrat-Transportsystems von Escherichia coli K-12. Welz, Dieter (1997): Funktion und Lokalisierung der Induktionsproteine FecI und FecR von Escherichia coli K-12. Groeger, Wolfram (1998): Eisen(III)-Hydroxamat-Transport in Escherichia coli: Topologie des integralen Transportproteins FhuB. Habeck, Martina (1998): Energiekopplung durch TonB im Eisen(III)DicitratTransportsystem von Escherichia coli K-12.

TP B4 Hirschhausen, Heinrich Reginhard von (1986): Die Phosphodiesterasen in Paramecium. Schade, Uwe (1988): Zur physiologischen Rolle von cyclischem 3',5'-Guanosinmono-phosphat in Paramecium tetraurelia. Hofmann, Hans-Joachim (1990): Anreicherung und Charakterisierung einer membranständigen, ciliären Guanylatcyclase aus Paramecium tetraurelia. Schürhoff-Goeters, Wilhelm (1990): Zur Entstehung cyclischen AMPs infolge hyperpolarisierender Pufferveränderungen bei Paramecium tetraurelia. Freund, Wolf-Dietrich (1991): Inositol-Phospholipide und Inositol-Phosphate in Paramecium tetraurelia. 360

20.6 Support of young scientists Steinlen, Siegfried (1992): Untersuchungen zur Regulation der partikulären Guanylatcyclasen der Ciliaten Paramecium und Tetrahymena durch Calmodulin. Friderich, Gerald (1992): Reinigung und Charakterisierung der Proteinphosphatase Typ 1 aus den Cilien von Paramecium tetraurelia. Völkel, Helge (1992): Adenylatcyclasen aus ciliären Geweben und dem retinalen Pigmentepithel. Beyer, Angelika (1993): Proteinphosphatase Typ 2C aus Paramecium tetraurelia: Lokalisation, Isolierung, Teilsequenzierung und Charakterisierung. Schönborn, Christoph (1993): Untersuchungen zur Regulation von cyclischem Adenosin 3',5'-monophosphat bei Paramecium tetraurelia und Ionenkanalmutanten. Otto, Susanne (1994): Reinigung und Charakterisierung einer Adenylatcyclase der Retina. Völkel, Helge (1995): Adenylatcyclasen aus ciliären Geweben und dem retinalen Pigmentepithel. Selke, Dagmar (1995): Proteinphosphatase 2C aus der Rinderretina – Aufreinigung, Charakterisierung, Klonierung und Expression. Hanke, Cordula (1996): Proteinphosphatase Typ 2C aus Paramecium tetraurelia. Guo, Yinglan (1996): Zur Ca2+-abhängigen Regulation der Bildung von cGMP, des Schwimmverhaltens und der Lokalisation von Ca-Kanälen in Paramecium tetraurelia. Anton, Helga (1996): Proteinphosphatase Typ1 aus der Rinderretina – Reinigung, Charakterisierung und Expression. Linder, Jürgen (1997): Klonierung einer Adenylatcyclase aus Paramecium. Beitz, Eric (1997): Zur cAMP-Signaltransduktion des retinalen Pigmentepithels des Rindes und des Innenohrs der Ratte. Krüger, Thomas (1997): Klonierung von Adenylatcyclasen aus dem Ciliaten Tetrahymena pyriformis. Grothe, Kirsten (1998): Mutationsanalyse und Lokalisation der Proteinphosphatase Typ 2C aus Paramecium tetraurelia. Hoffmann, Thomas (1999): Membranständige Guanylatcyclasen aus Paramecium und Tetrahymena: Klonierung und bakterielle Expression der katalytischen Bereiche. Engel, Peter (1999): Klonierung und Expression einer Guanylatcyclase aus Paramecium tetraurelia. TP B5 Demleitner, Gabi (1992): Die Exolipase von Staphylococcus hyicus: Identifizierung der katalytisch aktiven Aminosäuren und Untersuchungen zur Funktion des Propeptids. Krismer, Bernhard (1999): Studium der Funktion der sekretierten Proteine SceA und SceB, Analyse des Galaktoseoperons galRKET und Kontruktion von Sekretions- und Expressionsvektoren in Staphylococcus carnosus. Kupke, Thomas (1992): Posttranslationelle Modifikation bakterieller Peptide – proteinchemische Untersuchungen zur Epiderminbiosynthese. Lechner, Max (1989): Klonierung und Charakterisierung des Gens für die Phosphatidylinositol-spezifische Phospolipase C von Bacillus thuringiensis. 361

20 Documentation of the Collaborative Research Centre 323 Otto, Michael (1997): Epidermin: Biochemische Untersuchungen zur Biosynthese, Regulation und Immunität. Teufel, Pia (1993): Isolierung; Sequenzierung und Charakterisierung einer Metalloprotease aus Staphylococcus epidermidis und Sekretions- und Regulationsstudien bei Staphylococcus carnosus. Thumm, Günther (1996): Molekularbiologische Charakterisierung von Lysostaphin und Lysostaphin-Immunitäts-Faktor (Lif). Wieland, Bernd (1993): Der xylA Promotor aus Staphylococcus xylosus als Grundlage der transkriptionellen Regulation von Genen in Staphylococcus carnosus. Wieland, Karsten-Peter (1999): Organisation und Genexpression der Carotinoid-Biosynthesegene aus Staphylococcus aureus Newman und Untersuchungen zur Funktion von Staphyloxanthin. TP B6 Kammler, Meike (1994): Sequenzierung und Charakterisierung des Aufnahmesystems für zweiwertiges Eisen von Escherichia coli. Müller, Kerstin (1997): FhuF, ein neuartiges, eisenreguliertes Eisen-SchwefelProtein von Escherichia coli. Patzer, Silke (1999): Regulation durch Metallionen in Escherichia coli, insbesondere Identifizierung und Charakterisierung des hochaffinen Zink-Aufnahmesystems ZnuABC und des zinkabhängigen Regulators Zur. TP C2 Bayer, Anja (1993): Produktion, Isolierung und Strukturaufklärung des glycinreichen Polypeptidantibiotikums Microcin B17. Brooks, Marc (1999): Neue DNA-Gyrase und Humane Type II DNA-Topisomerase-Inhibitoren. Deres, Karl (1992): MHC-Klasse-1-restringierte Peptide. Drechsel, Hartmut (1993): Strukturaufklärung neuer Siderophore vom Carboxylat-Typ und Synthese von Siderophor-Antibiotika-Konjugaten. Flechsler, Insa (1998): Transfektion von Nukleinsäuren mit neuen kationischen Lipiden und Vergleich verschiedener Antisense-Strategien. Fleckenstein, Burkhard (1997): Kombinatorisch aufgebaute Peptidkollektionen zum Studium der HLA-Klasse II-Peptid-Interaktion und der Antigenerkennung autoreaktiver, humaner T-Zellen. Höltzel, Alexandra (1999): Structure Elucidation of Secondary Metabolites and of the Loop Sequence 316–333 of the ThuA Receptor. Hörr, Ingmar (1999): RNA-Vakzine zur Induktion von spezifischen cytotoxischen T-Lymphozyten und Antikörpern. Ihlenfeldt, Hans-Georg (1995): Die B-und T-Zell-Epitope des Hepatitis C-Virus. Kabatek, Ursula (1987): Eine neuartige Teichonsäure aus Streptomyces venezuelae. Kaiser, Dietmar (1998): Strukturaufklärung und Konformationsanalyse biologisch aktiver Sekundärmetabolite. Kellner, Roland (1989): Lantibiotika – ribosomal synthetisierte Polypeptidantibiotika mit Sulfidbrücken und Dehydroaminosäuren. 362

20.6 Support of young scientists Kempter, Christoph (1996): Strukturaufklärung mikrobieller Sekundärmetabolite durch Elektrospray-Massenspektrometrie und mehrdimensionale Kernresonanzspektroskopie. Koch, Ulricke (1988): Fengycin, Strukturaufklärung eines mikroheterogenen Lipopeptolidantibiotikums. Konetschny-Rapp, Sylvia (1990): Neue mikrobielle Eisenkomplexbildner, Screening, Isolierung, Strukturaufklärung und komplexchemische Untersuchungen. Metzger, Jörg (1988): Immunstimulierende Lipopeptide als Membrananker für Haptene und biologisch aktive Wirkstoffe. Rapp, Claudius (1988): Neue antifungische Phosphono- und Chloro-Oligopeptide aus Bacillus subtilis und ein neues Thiolactonantibiotika aus Streptomyces olivaceus – Isolierung und Strukturaufklärung. Stephan, Holger (1995): Strukturaufklärung mikrobieller Sekundärstoffwechselprodukte durch mehrdimensionale Kernresonanzspektroskopie. Stevanovic, Stefan (1992): Multiple Sequenzanalyse – Ein neuer Ansatz in der Peptidsequenzierung: Isolierung, Synthese und Strukturaufklärung von mikrobiellen Metaboliten aus Amycolatopsis mediterranei, Staphylococcus epidermidis und Streptomyces lividans. List of habilitations resulting from the collaborative research centre Bormann, Christiane (1997): Biosynthese des Antibiotikums Nikkomycin. Brückner, Reinhold (1997): Katobolitrepression in Staphylokokken. Bechthold, Andreas (1998): Streptomycetengenetik, Grundlage für die Herstellung neuer Antibiotika: Klonierung und Charakterisierung der Biosynthesegencluster von Avilamycin, Landomycin, Urdamycin und Granaticin. Fiedler, Hans-Peter (1988): Isolierung und Analytik niedermolekularer mikrobieller Sekundärmetabolite. Klumpp, Susanne (1989): Charakterisierung von Phosphatasen und Entdeckung eines Inhibitor-Proteins. Kupke, Thomas (1998): Mikrobielle Enzyme – neuartige Reaktionen und Katalysemechanismen. Metzger, Jörg (1994): Naturstoffanalytik und Strukturaufklärung mit modernen Methoden der Massenspektrometrie. Graduiertenkollegs – „Mikrobiologie“ – „Analytische Chemie“ – „Zellbiologie in der Medizin“

363

20 Documentation of the Collaborative Research Centre 323

20.7 Alphabetical list of guests

Barnet, James Prof. Bielecki, Jarek Dr., Univ. Warsaw Focareta, Antonio Dr.,University of Adelaide Howard, Stephen Peter, University of Regina Kálmanánczhelyi, Attila, Bukarest Kim, In-Sook Dr. rer. nat Jack, Ralph-Wilson Dr. rer. nat., Univ. of Otago Milla, Paola, Univ. Turin Noda, Shigeru Dr. rer. nat., Univ. Gießen Potterat, Olivier, Dr. rer. nat. Reinhard, Peter Dr. rer.nat., Univ. Canberra Sarem, Aslani Dr. rer. nat., Univ. Würzburg Shi, Liangru Dr. Smaijs, David Dr., Masaryk University Stojiljkovic, Igor Dr. med., Univ. Zagreb Surovoy, Andrey Dr. med., Shemaykin-Inst. Moscow Tschen, Shu-Yuan Dr. rer. nat., Chengdu Videnov, Georgi Dr. rer. nat., Molecular Biology, Sofia

A1-Zähner A1-Zähner B1-Braun B1-Braun A4-Bormann B1-Braun C2-Jung A5-Poralla B4-Schultz A1-Zähner B2-Hamprecht B2-Hamprecht A1-Zähner B1-Braun B1-Braun C2-Jung A1-Zähner

USA Poland Australia Canada Bulgaria South-Korea N. Zealand Italy (China) Switzerland Australia Egypt P. R. China Czech Rep. Croatia Russia P. R. China

87–88 86 89–90 99 91 91 90–2000 98 86 91–92 86 88 90 97 92–93 90–97 91

C2-Jung

Bulgaria

91–95

20.8 International cooperation

(A5) Prof. M. Rohmer, University of Strasbourg, France Prof. L. Cattel, Institute of Applied Pharmacie, University of Turin, Italy Prof. G. D. Prestwich, Dep. Chemistry, University at Stony Brook, New York (A11) Prof. Dr. J. A. Salas, Oviedo, Spain Prof. Dr. P. Leadlay, Cambridge, UK Prof. Dr. K. Ichinose, Tokyo, Japan Prof. Dr. H. G. Floss, Seattle, USA Eli-Lilly and Company Limited, Hampshire, UK Combinature-Biopharm AG, Berlin, Germany Glaxo Wellcome, Stevenage, UK (A12) Prof. Dr. Miguel A. de Pedro, Laboratory for Cell Envelopes, Centro de Biologia Molecular „Severo Ochoa“, Facultad de Ciencias UAM, Campus de Cantoblanco, 28049 Madrid, Spain 364

20.10 Funding (C2) Nijmegen SON Research Center for Molecular Structure, Design and Synthesis University of Nijmegen, The Netherlands Shemyakin Institute of Bioorganic Chemistry, University of Moscow, Russia Laboratoire Nationale de Santé, Luxembourg Laboratory of Microbial Technology, Agricultural University of Norway, As, Norway Ecole Polytechnique Federale de Lausanne, Department de Chimie, EPFLEcublens, Lausanne, Switzerland Groupe RdMN, Institut Pasteur de Lille, Lille, France German-Israel Foundation for Scientific Research & Development, Weizmann Institute Rehovot, Israel

20.9 International conferences

Sekundärmetabolite aus Mikroorganismen. Tübingen 1989. Microbial Secondary Metabolism. Interlaken 1994. „Vectorial Transport Across Bacterial Membranes“. Tübingen 1995. 1. Import, Export and Sorting 2. Protein and Peptide Channels Biologie der Actinomyceten. Tübingen 1996. Plasmide und Genregulation. Blaubeuren 1997. Tübinger/Göttinger Gespräche zur Chemie von Mikroorganismen. Blaubeuren, each year.

20.10 Funding

The collaborative research centre 323 has been supported by grants of the Deutsche Forschungsgemeinschaft totalling DM 35 678 000 in the period 1986– 1999.

365