Zeitschrift für Allgemeine Mikrobiologie: Band 22, Heft 5 1982 [Reprint 2021 ed.] 9783112538869, 9783112538852

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ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS HEFT 5 • 1982 • BAND 22

W EVP 20,— M

AKADEMIE-VERLAG • BERLIN ISSN 0044-2208

34112

CONTENTS

OF N U M B E R 5

Enzyme induction in Streptomyces hydrogenans. Comparison of the effects of different steroids to increase the activity of 3a, 20(3-hydroxysteroid dehydrogenase and of 3(3, 17(J-hydroxysteroid dehydrogenase

BRIGITTE BAUER a n d L . TRÄGER

287

P-1.3-1.4-Glucanasein spore-forming microorganisms. VI. Genetic instability of (3-glucanase production in a high-producer strain of Bacillus amyloliquefaciens grown in a chemostat

R . BORRISS, D . NOACK a n d R . GEUTHER

293

Binding of a bacteriophage to wall-membrane adhesions in Proteus mirabilis

S . A . COLE a n d D . G. SMITH

299

Microbial transformation of bile acids. A unified scheme for bile acid degradation, and hydroxylation of bile acids

S . HAYAKAWA

309

Factors regulating the steroid 11-hydroxylation by non-germinating spores of Cunninghamella ele-

A . JAWORSKI, L . SEDLACZEK, D . WILMAÄSKA, A . SASIAK AND A . STRYCHARSKA

327

S . KRETSCHMER

335

ments (LENDNER)

Dependence of the mycelial growth pattern on the individually regulated cell cycle in Streptomyces granaticolor Short N o t e New anthracycline antibiotics produced by interspecific recombinants of streptomycetes. IV. Antimicrobial activity of iremycin

W . F . FLECK, AND W . IHN

BRIGITTE

SCHLEGEL 349

Book Reviews

355

INHALTSVERZEICHNIS HEFT 5

Enzyminduktion bei Streptomyces hydrogenans. Wirkung von verschiedenen Steroiden auf die Aktivitätssteigerung der 3a, 20ß-Hydroxysteroid-Dehydrogenase und der 3ß, 17ß-Hydroxysteroid-Dehydrogenase

BRIGITTE BAUER u n d L . TRÄGER

ß-l,3-l,4-Glucanase in sporenbildenden Mikroorganismen. VI. Genetische Instabilität der ß-Glucanasebildung in Chemostatenkulturen eines Hochleistungsstammes von Bacillus amyloliquefaciens

R . BORRISS, D . NOACK UND R . GEUTHER 293

Bindung eines Bakteriophagen an Wand-MembranAdhäsionen bei Proteus mirabilis

S . A . COLE u n d D . G . SMITH

299

Mikrobielle Transformation von Gallensäuren. Abbau und Hydroxylierung von Gallensäuren

S . HAYAKAWA

309

Regulation der 11-Hydroxylierung von Steroiden durch nichtkeimende Sporen von Cunninghamella elegans (LENDNER)

A . JAWORSKI, L . SEDLACZEK, D . WILMAI&SKA, A . SASIAK UND A . STRYCHARSKA

327

Abhängigkeit des mycelialen Wachstumsmusters von Streptomyces granaticolor vom Zellzyklusgeschehen

S . KRETSCHMER

335

Kurze Originalmitteilung Neue Anthracyclin-Antibiotica aus interspezifischen Streptomyceten-Rekombinanten. IV. Antimikrobielle Aktivität von Iremycin Buchbesprechungen

W . F . FLECK, UND W . IHN

BRIGITTE

287

SCHLEGEL 349

355

ZEITSCHRIFT FÜR ALLGEMEINE MIKRO- BIOLOGIE A N INTERNATIONAL JOURNAL ON

H E R A U S G E G E B E N VON

F. Egami, Tokio G. F. Gause, Moskau 0 . Hoffmann-Ostenhof, Wien A. A. Imseneckii, Moskau R. W. Kaplan, Frankfurt/M. F. Mach, Greifswald 1. Malek, Prag C. Weibull, Lund

unter der Chefredaktion von W. Schwartz, Braunschweig

MORPHOLOGY, PHYSIOLOGY, GENETICS,

und

AND ECOLOGY OF MICROORGANISMS

U. Taubeneck, Jena U N T E R MITARBEIT VON

J . H. Becking, Wageningen H. Böhme, Gatersleben M. Girbardt, Jena S. I. Kusnecov, Moskau 0 . Necas, Brno C. H. Oppenheimer, Port Aransat N. Pfennig, Göttingen I. L. Rabotnova, Moskau A. Schwartz, Wolfenbüttel REDAKTION

HEFT 5 • 1982

BAND 22

AKADEMIE-VERLAG BERLIN

U. May, Jena

Die Zeitschrift f ü r Allgemeine Mikrobiologie soll dazu beitragen, Forschung u n d internationale Zusammenarbeit auf dem Gebiet der Mikrobiologie zu fördern. E s werden Manuskripte aus allen Gebieten der allgemeinen Mikrobiologie veröffentlicht. Arbeiten über Themen aus der medizinischen, landwirtschaftlichen, technischen Mikrobiologie u n d aus der Taxonomie der Mikroorganismen werden ebenfalls aufgenommen, wenn sie Fragen von allgemeinem Interesse behandeln. Zur Veröffentlichung werden angenommen: Originalmanuskripte, die in anderen Zeitschriften noch nicht veröffentlicht worden sind und in gleicher F o r m auch nicht in anderen Zeitschriften erscheinen werden. Der Umfang soll höchstens 1% Druckbogen (24 Druckseiten) betragen. Bei umfangreicheren Manuskripten müssen besondere Vereinbarungen mit der Schriftleitung u n d dem Verlag getroffen werden. Kurze Originalmitteilungen über wesentliche, neue Forschungsergebnisse. U m f a n g im allgemeinen höchstens 3 Druckseiten. Kurze Originalmitteilungen werden beschleunigt veröffentlicht. Kritische Sammelberichte u n d Buchbesprechungen nach Vereinbarung mit der Schriftleitung. Bezugsmöglichkeiten der Zeitschrift f ü r Allgemeine Mikrobiologie: Bestellungen sind zu richten — in der DDK an den Postzeitungsvertrieb, an eine Buchhandlung oder an den AkademieVerlag, DDR-1086 Berlin, Leipziger Str. 3—4 — im sozialistischen Ausland an eine Buchhandlung f ü r fremdsprachige Literatur oder an den zuständigen Postzeitungsvertrieb — in der BRD und Berlin (West) an eine Buchhandlung oder an die Auslieferungsstelle K U N S T U N D W I S S E N , Erich Bieber OHG, D-7000 S t u t t g a r t 1, Wilhelmstraße 4 - 6 — in den übrigen westeuropäischen Ländern an eine Buchhandlung oder an die Auslieferungsstelle K U N S T U N D W I S S E N , Erich Bieber GmbH, CH-S008 Zürich, Dufourstiaße 51 — im übrigen Ausland an den Internationalen Buch- und Zeitschriftenhandel; den Buchexport, Volkseigener Außenhandelsbetrieb der Deutschen Demokratischen Republik, DDR-7010 Leipzig, Postfach 160, oder an den Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3—4. Zeitschrift f ü r Allgemeine Mikrobiologie Herausgeber: I m Auftrag des Verlages von einem internationalen Wissenschaftlerkollektiv herausgegeben. Verlag: Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3 - 4 ; Fernruf 2 2 3 6 2 2 9 oder 2236221• Telex-Nr.' 114420; B a n k : Staatsbank der D D R , Berlin, Kto.-Nr.: 6836-26-20712. Chefredaktion: Prof. Dr. U D O T A T J B E N E C K , Prof. Dr. W I L H E L M S C H W A R T Z . Anschrift der R e d a k t i o n : Zentralinstitut f ü r Mikrobiologie und experimentelle Therapie der Akademie der Wissenschaften, DDR-6900 J e n a , Beutenbergstr. 11; F e r n r u f : J e n a 885614; TelexNr. 058621. Veröffentlicht unter der Lizenznummer 1306 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: V E B Druckerei „Thomas Müntzer", DDR-5820 Bad Langensalza. Erscheinungsweise: Die Zeitschrift f ü r Allgemeine Mikrobiologie erscheint jährlich in einem B a n d mit 10 Heften. Bezugspreis je Band 250, — M zuzüglich Versandspesen (Preis f ü r die D D R 200, —M) Preis je H e f t 2 5 , - M (Preis f ü r die D D R 2 0 , - M). Urheberrecht: Alle Rechte vorbehalten, insbesondere die der Ubersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translations into foreign languages). No p a r t of this issue may be reproduced in a n y form, b y photoprint, microfilms or any other means, without written permission from the publishers. Erscheinungstermin: J u n i 1982 Bestellnummer dieses Heftes 1070/22/5 g 1982 b y Akademie-Verlag Berlin. P r i n t e d in t h e German Democratic Republic. A N (EDV) 75218

]

Zeitschrift für Allgemeine Mikrobiologie

22

5

1982

287—292

(Gustav-Embden-Zentrum der Biologischen Chemie, Arbeitsgruppe Biochemie der Hormone Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, geschäftsführender Direktor: Prof. Dr. L . TRÄGES)

E n z y m e induction in Streptomyces

hydrogenans

Comparison, of the effects of different steroids to increase the activity of 3a,20ß-hydroxysteroid dehydrogenase and of 3ß,17ß-hydroxysteroid dehy drogena se1)2) BRIGITTE BAUER a n d L . TRÄGER

(Eingegangen

am 1.

10.1981)

After cultivation of Streptomyces hydrogenans in the presence of different steroids the activity of both 3a,20/j-hydroxy steroid dehydrogenase and 3/S,17/3-hydroxy steroid dehydrogenase was determined in the cell homogenate of the microorganism. B y comparing the efficacy of the steroids to increase enzyme activities, steroids could be divided into 3 groups: a) steroids which stimulated preferentiallly the activity of 3«,20/S-hydroxysteroid dehydrogenase (e. g., corticosterone), b) steroids which stimulated preferentially 3/?,17/9-hydroxy steroid dehydrogenase (estradiol-17/S), and c) those behaving intermediately (e. g., progesterone, 5 Zl4-3-oxo bile acids -»• -*- C16 or C18 perhydroindane derivative (at least in two ways) -> (4e)-4-methyl-5oxo-octanedioic acid (at least in three ways) -*• -> C02 and H 2 0. A microbial hydroxylation method for the preparation of bile acid samples was investigated which could be used as reference standards in the analysis of bile acids in biological materials and also as materials for studying the function of bile acids. The particular fungi, Curcularia lunata

N R R L - 2 3 8 0 , Helicostylum

piriforme

ATTC-8992 a n d Pestalotia

foedans

ATCC-11817 effec-

ted the 1/3-, 11/3-, 12/?-, 15a- or 15/3-hydroxylation of certain bile acids and gave the following products: 1/3,3a-, 3a, 12/?- and 3a,15/3-dihydroxy-5/3-cholan-24-oic acids, 3a,12/3,15a- and 3a,12/3, 15/3-trihydroxy-5/J-cholan-24-oic acids and 12/3,15/3-dihydroxy-3-oxo-5/3-cholan-24-oic acid fromlithocholic acid; 1/8,3a, 12a- and 3a,12a,15/3-trihydroxy-5/3-cholan-24-oic acids and 3a,ll/3-dihydroxy-12-oxo-5/3-cholan-24-oic acid from deoxycholic acid; 3a,7a,12/3-trihydroxy-5/3-cholan-24oic acid and 3a,7a,12/3,15a-tetrahydroxy-5/3-cholan-24-oic acid from chenodeoxycholic acid; 3a6a,12/3- and 3a,6a,15/?-trihydroxy-5/J-cholan-24-oic acids from hyodeoxycholic acid; 3a,7/3,12/3 trihydroxy-5j3-cholan-24-oic acid from ursodeoxycholic acid; 3a,12/3-dihydroxy-7-oxo-5/3-cholan24-oic acid from 3a-hydroxy-7-oxo-5/3-cholan-24-oic acid. Some of these products were new compounds and their structures were determined. As a practical working model for the prediction of possible intermediates in the bile acid metabolism in vivo I started this research under the direction of the late Prof. T A Y E I SHIMIZU at Okayama Medical School about 30 years ago. From 1920 until 1923 and again from 1929 until 1931 SHIMIZU was a pupil of the late Prof. HEINRICH W I E L A N D who was an outstanding German organic chemist and a winner of the Nobel prize for chemistry in 1927. SHIMIZU is well known as the founder of the chief Japanese school of studies on bile acids. In the course of our early studies we proposed that the formation of a Zl 4 -3-one structure in the bile acid molecule is important for the microbial degradation of bile acids including a side chain shortening ( H A Y A K A W A et al. 1957a) and that at least two degradative pathways involve in the microbial degradation of cholic acid: (1) cholic acid —• —• C24 bile acid derivatives having a Zl 4 -3-one structure —» a C22 dinor bile acid derivative having a Zl 4 -3-one structure —» further degradation and (2) cholic acid —• —» C24 bile acid derivatives having a J 4 -3-one structure —» C24 bile acid derivatives having a Zl 4 ' 6 -3-one structure —• further degradation (HAYAKAWA et al. 1958). Then we imagined that such transformations might occur in our bodies. During the course of these studies, much of the work on bile acid metabolism in vivo by the BERGSTROM'S group using 14 C-labelled bile 1 ) Presented at the 2nd Symposium on Biochemical Aspects of Steroid Research, September 1 4 - 1 9 , 1981, Weimar/GDR

310

S . HAYAKAWA

acids have been reported. According to their papers reported until near 1960, it seemed unlikely that the degradation of cholic acid in vivo to a C22 dinor bile acid derivative and its further degradation products could occur in our bodies ( B E R G S T R O M et al. 1960). However, we continued our studies on the complete degradation of bile acids to carbon dioxide and water, since we believed that even though certain reactions found in the degradation of bile acids in vitro could not occur in our bodies, defining the intermediates and reaction sequence involved in the degradation of bile acids in Nature is an interesting object of steroid biochemistry. Recently, the microbial transformation of bile acids in vitro has been extensively investigated by many investigators in connexion with the original hypothesis of H I L L et al. (Aries et al. 1969, H I L L et al. 1971) that intestinal bacteria produce colon carcinogens from bile acids. An outline of these studies has been described and discussed in my recent review ( H A Y A K A W A 1980). In the present paper a unified scheme for the microbial degradation of bile acids is proposed through our own studies reported so far and the recent work of other investigators. This report also summarizes our recent study which deals with the microbial hydroxylation of bile acids. Side chain shortening and slight modification

of ring

Among the structure of many products formed from cholic acid (1) by Arthrobacter simplex, Corynebacterium equi, Streptomyces gelaticus and Streptomyces rubescens, most of those having the steroid nucleus are shown in Scheme 1 ( H A Y A K A W A 1973). A.-simplex produced 7(%,12a-dihydroxy-3-oxo-5/?-cholan-24-oic acid (.3), 7a, 12adihydroxy-3-oxo-4-cholen-24-oic acid (5), 12a-hydroxy-3-oxo-4,6-choladien-24-oic acid (7), 12a-hydroxy-3-oxo-4-cholen-24-oic acid (11) and 12a-hydroxy-3-oxo-l,4choladien-24- oic acid (12); C. equi 3, 5, 7a-hydroxy-3,12-dioxo-4-cholen-24-oic

rubescens

Microbial transformation of bile acids

311

acid (6), (20tS')-7a,12a-dihydroxy-3-oxo-4-pregnene-20-carboxylic acid (9) and (20$)7a-hydroxy-3,12-dioxo-4-pregnene-20-carboxylic acid (10); S. gelacticus 3, 7«-hydroxy-3,12-dioxo-5/?-cholan-24-oic acid (4), 6 and 10; S. rubescens 5, 6, 7 and 3,12-dioxo4,6-choladien-24-oic acid (8) ( H A Y A K A W A 1973). Deoxycholic acid (2) was not utilized by these organisms as their carbon sources but this acid was able to be utilized by A. simplex in the presence of cholic acid, probably due to the enzymes induced by cholic acid ( H A Y A K A W A 1973). Intermediates involved in the degradation of deoxycholic acid with this organism are not isolated yet but at least three of them are probably 11, 12 and 12a-hydroxy-3-oxo-5/3-cholan-24-oic acid (13) as discussed later. This possibility is supported by the work of L E P P I K (1981a, 1981b) and BILTON etal. (1981) who isolated and identified 11, 12, 13 and many other products formed from 2 by Pseudomonas spp. Taxonomical differences in species between both the Pseudomonas spp. strains used by these investigators are unknown. The further degradation of the dinor acid (9) is not studied yet by the author's group but T E N N E S O N et al. (1979b) have recently isolated and identified four 3-oxo1,4-androstadiene derivatives which were formed from 1 by Pseudomonas sp. and correspond to the further degradation products of 9. This compound (9) itself is also obtained in their studies. I t is therefore conceivable t h a t C. equi and S. gelaticus can degrade 1 to androstane derivatives through a path, l-^3-+5-+-^9—> further degradation. The occurrence of such type of a side chain shortening in the degradation of 2 has been recently reported by B A R N E S et al. (1976), L E P P I K (1981a, 1981b) and B I L T O N et al. (1981) who isolated and identified many of C19, C22 and C24 compounds having a A4- or Zl1'4-3-one structure as the degradation products formed from 2 by Pseudomonas spp. Thus the results of these investigators support our early proposal t h a t in the microbial degradation of cholic acid, a Zl4-3-one structure is a t first introduced into the molecule before the side chain shortening ( H A Y A K A W A et al. 1957a). Also it seems likely t h a t since in the degradation of bile acids with these Pseudomonas spp. a side chain shortening precedes a ring cleavage, the biodegradability of these strains for bile acids is similar to t h a t of G. equi and S. gelaticus. Although it is not conclusive, the side chain shortening of bile acids probably occurs in a manner analogous to the conventional f a t t y acid ^-oxidation mechanism. This possibility is supported by the recent work of L E P P I K (1981b) who isolated and identified (222?)-12a-hydroxy-3-oxo-l,4,22-cholatrien-24-oic acid and (17 J B)-12a-hydroxy3-oxo-l,4,17(20)-pregnatriene-20-carboxylic acid as the degradation products formed from 2 with Pseudomonas sp. That is, these ^ - u n s a t u r a t e d carboxylic acids correspond to certain metabolic intermediates involved in the /3-oxidation of 12 and its dinor derivative formed from 2. L E P P I K (1981b) has also isolated and identified dinor deoxycholic acid and its 3-dehydro derivative as the metabolites formed from 2 by Pseudomonas sp. These products are probably not the respective /9-oxidation products of 2 and its 3-dehydro derivative but the reduction products of once-formed C22 Zl4-3-oxo acids, since the possible occurrence of such reduction in the degradation of 1 by S. gelaticus has been reported by HAYAKAWA et al. (1957b). In connexion with the ^-oxidation of bile acids, the MASON'S group ( B A R N E S et al. 1976, T E N N E S O N et al. 1979b, B I L T O N et al. 1981) and L E P P I K (1981a, 1981b) have postulated t h a t the ^-oxidation of Zl1'4-3-oxo bile acids and Zl1'4-3-oxo dinor bile acids can occur in their organisms. Perhaps such reactions also participate in the microbial degradation of bile acids. There is no doubt t h a t the degradation of 11 formed from 1 and 2 follows a path, 11 —• 12 -> 9ac-hydroxylated 12(15) —* further degradation including the formation of perhydroindane derivatives or 11 -* 9a-hydroxylated 11(14) —• 15 -* further degradation, since it is well known t h a t many other steroids having a Zl4-3-one structure can undergo such reactions by microorganisms (SIH et al. 1968). A mechanism for the

312

S . HAYAKAWA

degradation of 15 will be described later. It is therefore conceivable that A. simplex and S. rubescens are able to degrade 1 through a pathway, l-+3-»5—>7-+ll—• 12{or 14) —* 15 —» further degradation. It is also assumed that the degradation of 2 by these organisms follows a path, 2 —• 13 —• 11 —• 12(or 14) —• 15 —• further degradation, although further study must be made as to whether the latter organism, which could not utilize 2 as its carbon source, can degrade 2 in the presence of 1 as well the former. Regarding the location of 4, 6, 8 and 10 in a pathway for the degradation of 1, we have thought that in certain organisms such as S. gelaticus and S. rubescens, the C22 and C24 degradative intermediates having the intact steroid nucleus and a 12«hydroxy group are probably in equilibrium with the corresponding 12-oxo derivatives. This is shown as 3 4, 5 6, 7 8, 9 10 in Scheme 1. This may be true for three sets of the products reported by B I L T O N et al. (1981), namely 11, 12 and 13 and their corresponding 12-oxo derivatives. This means that in the degradation of 1, a 7«hydroxy group in 1 seems to be removed easily as water through a path, 5 -* 7 11, before a ring fission but a 12a-hydroxy group in 1 and 2 is never removed in their degradative pathways unless the steroid nucleus is intact. A proposal for the facility of removal of a 7a-hydroxy group is supported by the recent work of T E N N E S O N et al. (1979a, 1979b) who obtained 12/?-hydroxy-l,4,6-androstatriene-3,17-dione, which corresponds to 7 in structures, as one of the cholic acid metabolites. Since they obtained some 7a-hydfoxy-l,4-androstradine derivatives in addition to this 1,4,6triene, however, the question has been raised as to whether a 7«-hydroxy group in 1 is necessarily removed before a ring fission. The answer to this question should wait for further study on this problem. A proposal for the more difficulty of removal of a 12«-hydroxy group compared to a 7a-hydroxy group is also supported by evidence that there are several compounds having a 12«-, 12/?- or 12-oxo group in all steroidnucleus-containing products isolated as the degradative intermediates of 1 and 2 by the MASON'S group ( B A R N E S et al, 1976, T E N N E S O N et al. 1979b, B I L T O N et al. 1981) and L E P P I K (1981a, 1981b). This is further supported by the fact that a 12/J-hydroxy group is still present in a 9,10-seco-androstane derivative formed form 2 by Pseudomonas sp. ( L E P P I K 1981a). This significance of a 12/3-hydroxy group in their products will be discussed later. Although the representation of Scheme 1 proposed for the microbial degradation of 1 and 2 is somewhat hypothetical, the following points should be noted. First, the first step of degradation of 1 and 2 by the microorganisms including the Pseudomonas spp. strains of other investigators is the formation of 3-oxo bile acids such as 3 and 13 and the subsequent one is that of J 4 -3-oxo bile acids such as 5 and 11. Secondly, the further degradation of the Ai-3-oxo bile acids thus formed proceeds through at least two pathways: in one of them a side chain shortening precedes a ring fission as in a path, 5 —• —• 9 —• —• 7a-hydroxy-3-oxo-4-cholen-24-oic acid (29) —• 3-oxo-4,6choladien-24-oix acid (24) —• 25 — 21 —> 22 —• 23, is quite similar to that postulated for the degradation of cholic acid (1) with this organism, namely this pathway consists of the following steps: (1) oxidation of a 3• a postulated intermediate (33) —• 34 or 30 —> 32 —» 33 -» 34. The last step yielding the seco-phenol (34) is believed to occur by a spontaneous, probably nonenzymatic, reverse aldol reaction. Compound 34 is then transformed into a perhydroindane derivative, 37, and a C6

Microbial transformation of bile acids

315

unit, 38, via 35 and 36. Compound 38 thus formed is further degraded to pyruvic acid (40) and propionaldehyde (41) via the /?-hydroxycarboxylic acid (39) or it is aminated to the amino acid (42). In the degradation of bile acids, however, 1-dehydrogenation such as a path, 11 -» 12 in Scheme 1 or 25 —• 27 in Scheme 2, perhaps precedes 9a-hydroxylation such as a path, 11 —>• 14 in Scheme 1, since there is no compound having a 9a-hydroxyZl4-3-one structure in all of the microbial degradation products isolated so far by the author's group and other investigators. A. simplex converted cholic acid (1) into (4J?)4-[4a-(2-carboxyethyl)-7a-methyl--5-oxo-3a«,7a/3-perhydromdan-l/J-yl]valericacid(46') and (4ii)-4-methyl-5-oxo-octanedioic acid (47) in Scheme 4 in addition to 3, 5, 7, 11 and 12 in Scheme 1. Under certain conditions 46 was obtained in about 4 0 % yield. On the basis of the mechanism illustrated in Scheme 3, there is no doubt that the degradation of 1 to 46 by this organism follows a path, 1 —>• —» 15 —• 43 —• —• 46. I t should be noticed, however, that in this degradation a 12a-hydroxy group in 15 must be removed before yielding 46. A clue to this problem was obtained by confirming that the chemically synthesized 7«-hydroxy derivative of 46, namely 44 was metabolized to 46 by this organism via Ae-46, namely 45 (HAYAKAWA 1973). However, there is a possibility that these dehydration and hydrogenation reactions begin with 43 and subsequently, the resulting 12-deoxy-4• 57 — 65 — malate- ( • ) and citrate-fed ( ¡ \ ) spores of C. elegans in the course of cortexolone transformation

The sum alanine + glutamic acid remains constant throughout the starving period. In citrate-fed spores the overall pool is diminished after 24 h, this being caused mainly by the decrease of alanine + glutamic acid content. Glutamic acid can be degraded either by a specific dehydrogenase using N A D P H as cofactor (PATEMAN and KINGHOEN 1 9 7 6 ) , or via the pathway described recently by SCHMIT and BRODY (1975) in N. crassa where, besides NADPH, NADH is formed.

Data obtained in the present paper may indicate that the dehydrogenation of glutamate and some tricarboxylic acid cycle intermediates provides the required reduced coenzyme for steroid hydroxylations, although the suggested NADPH-generation reactions have to be confirmed in C, elegans spores.

332

A . JAWOKSKI, L . SEDLACZEK, D . WILMANSKA, A . SASIAK a n d A . STRYCHARSKA

Fig.2. Photomicrographs of C. elegans sporangiospores k e p t for 24 h in water (a)or 1 % sodium citrate (b). Magnification 1600 x Table 3 : Changes of the free amino acid pools of starved and citrate-fed spores of C. elegans Time of maintenance of spores in water

Amino acid 0

2

in 1% citrate (h) 12

24

0

2

12

24

Concentration of amino acid (¡¿moles/g dry weight) Lysine Histidine Arginine Aspartate Threonine \ Serine / Glutamate Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine -Phenylalanine Total

9.35 2.62 14.74 16.42

9.60 2.37 15.28 5.01

9.20 2.68 14.16 10.41

9.95 2.97 14.78 17.30

9.35 2.62 14.74 16.42

8.64 2.12 12.48 5.84

8.57 2.21 14.03 11.87

8.62 1.83 8.77 17.41

28.50 31.98 tr 1.10 49.56 tr 10.51 0.82 1.41 3.52 1.86 0.00

26.90 38.73 tr 3.38 37.57 tr 9:30 1.10 1.68 2.94 1.34 1.23

32.39 47.81 tr 2.78 26.92 tr 8.93 0.89 1.33 1.67 0.93 0.73

38.32 54.14 tr 4.37 23.41 tr 9.38 1.56 1.44 1.61 1.38 0.93

28.50 31.98 tr 1.10 49.56 tr 10.51 0.82 1.41 3.52 1.86 0,00

18.27 43.84 tr 2.42 37.73 tr 7.90 1.05 1.18 1.99 0.95 0.00

25.23 53.53 tr 2.58 28.53 tr 7.88 1.11 1.36 1.19 0.88 0:37

25.56 37.55 tr 2.31 17.76 tr 7.41 1.21 1.18 2.11 1.36 0.73

172.37

156.45

160.8

181.5

144.4"

159.35

172.4

| 133.8

Transformation of steroids b y C. elegans spores

333

E x p e r i m e n t s w i t h f a c t o r s a c t i n g o n t h e G. elegans s p o r e e n v e l o p e s ( H e l i x pomatia d i g e s t i v e j u i c e , K O H , E D T A ) i n d i c a t e t h a t i r r e s p e c t i v e of it c h e m i c a l n a t u r e e a c h c o m p o u n d c a u s i n g s w e l l i n g of s p o r e s , a n d a d i s t i n c t d e c r e a s e i n a l a n i n e + g l u t a m i c a c i d c o n c e n t r a t i o n , s t i m u l a t e s t h e s t e r o i d h y d r o x y l a t i o n a c t i v i t y of C. elegans s p o r e s . R e s u l t s of t h e s e e x p e r i m e n t s are b e i n g n o w p r e p a r e d f o r p u b l i c a t i o n .

A

cknowledgements

These investigations were supported b y t h e Grant M R — I I —17. References BARNES, L . D . , MCGUIBE, J . J . a n d ATKINSON, E . E . , 1972. Y e a s t d i p h o s p h o p y r i d i n e n u c l e o t i d e

specific isocitrate dehydrogenase. Regulation of a c t i v i t y a n d unidirectional catalysis. Biochemistry, 11, 4322. CASSELTON, P . J . , 1976. Anaplerotic p a t h w a y s . I n : The Filamentous Fungi, Vol. I I . , p . 125, (ed. b y J . E . S M I T H a n d D . R . B E B B Y ) . E . Arnold L t d . London. DLTTGONSKI, J . a n d S E D L A C Z E K , L . , 1 9 8 1 . Regulation of steroid 16A-hydroxylation in Streptomyces olivoviridis. Z. Allg. Mikrobiol., 21, 499 — 506. DUNN-COLEMAN, N. S. a n d PATEMAN, J . A., 1977. In vivo a n d in vitro studies of n i t r a t e reductase regulation in Aspergillus nidulans. Mol. Gen. Genet., 152, 285. H A W K E B , L . E . , THOMAS, B . a n d B E C K E T T , A., 1 9 7 0 . An electron microscope s t u d y of s t r u c t u r e a n d germination of conidia of Cunninghamella elegans (LENDNER). J . gen. Microbiol., 60, 181 to 189. HÖBHOLD, C., HÜLLER, E . u n d ROSE, G., 1979. S t e r o i d u m w a n d e l n d e E n z y m e a u s M i k r o o r g a n i s -

men. X I I . Merkmale der I n d u k t i o n der 4-En-3-oxosteroid: (Akzeptor)-1-en-oxidoreduktase in Nocardia opaca. Z. Allg. Mikrobiol., 19, 731 — 739.

HÖRHOLD, C., HÜLLEB, E . u n d ROSE, G . , 1980. S t e r o i d u m w a n d e l n d e E n z y m e a u s M i k r o o r g a n i s -

men. X I I I . Beziehung zwischen Steroidstruktur u n d I n d u k t i o n der 4-En-3-oxo-steroid: (Akzeptor) -1-en-oxidoreduktase in Nocardia opaca. Z. Allg. Mikrobiol., 20, 23 — 32.

JAWORSKI, A . , DLUGONSKI, J . , WILMANSKA, D . a n d SEDLACZEK, L . , 1 9 7 6 . C h a n g e s i n t h e

cellu-

lar content of t h e pool constituents of Monosporium olivaceum — a steroid hydroxylating mould. Acta microbiol. polon., 25, 329 — 337. LEHNINGEB, A. L., 1979. Biochemistry, p. 332. Polish edition, P W R L , Warszawa. PATEMAN, J . A. a n d KINGHORN, J . R., 1976. Nitrogen metabolism. I n : The Filamentous Fungi, Vol. I I . , p. 188 (Edited b y J . E . SMITH a n d D. R . BEBBY). E . Arnold L t d . London. S C H M I T , J . C . a n d B B O D Y , S . , 1 9 7 5 . Neurospora crassa conidial germination: role of endogenous amino acid pools. J . Bacteriol., 124, 232 — 242. SCRUTTON, M .C., 1971. Assay of enzymes of C0 2 metabolism. I n : Methods in Microbiology, Vol. 6A, p. 479 — 541 (ed. b y J . R . N O R R I S a n d D. W . R I B B O N S ) . Academic Press London. S E D L A C Z E K , L . , J A W O R S K I , A . , S A S I A K , A . a n d ZAJACZKOWSKA, E . , 1 9 7 9 . T h e

oxidation-reduction

state of t h e nicotinamide nucleotides a n d t h e steroid l l a - h y d r o x y l a s e a c t i v i t y in rium olivaceum ATCC 36300. Acta microbiol. polon., 28, 111 — 121.

Monospo-

SEDLACZEK, L . , JAWORSKI, A . a n d WILMANSKA, D . , 1981. T r a n s f o r m a t i o n of s t e r o i d s b y f u n g a l

spores. I. Chemical changes of Cunninghamella elegans spores a n d mycelium during cortexolone hydroxylation. E u r o p . J . Appl. Microbiol. Biotech., 13, 155 — 160. VEZINA, C. a n d SINGH, K . , 1975. Transformation of organic compounds b y f u n g a l spores. I n : The Filamentous F u n g i , Vol. I . , p . 1 5 8 (edited b y J . E . S M I T H a n d D . R . B E B B Y ) . E . Arnold L t d . London. Mailing address: D r . A. J A W O B S K I I n s t i t u t e of Microbiology, University of Lodz B a n a c h a 12/16 90-237 Lodz, Poland

23

Z. Allg. Mikrobiol.. Ud. 22, H. 5

Zeitschrift für Allgemeine Mikrobiologie

22

1982

335-347

(Akademie der Wissenschaften der DDR, Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und experimentelle Therapie, Jena, Direktor: Prof. Dr. U. TAUBENECK)

Dependence of the mycelial growth pattern on the individually regulated cell cycle in Streptomyces granaticolor S . KRETSCHMER

(Eingegangen am 21. 9.1981)

The growth behaviour of Streptomyces granaticolor ETH 7437 was studied by the microculture technique. The kinetics of growth and branching were recorded and, since elongation was found to be restricted to apical elongation sites (e-sites), the rate of elongation per site (a) was determined as well. The mycelia grew exponentially. Initially the growth was dependent on a of the germ tube, but after the start of branching, growth paralleled the exponential increase of the number of branches while a attained a constant average value. Further, for liquid grown mycelia showing about the same growth kinetics the cellular structure was determined after cell wall staining. Three types of cells could be distinguished: apical branchless cells (20%), non-apical branchless cells (20%) and non-apical cells with one branch each (60%). Since both the apical and the branched cells possessed an e-site, 80% of the cells must have been growing at the time of sampling. Combining detailed data obtained from both the alive and the stained mycelia a model was elaborated, which may reflect the events taking place on the cellular level during mycelial growth. The model is based on the assumption that each cell behaves as an independent unity with respect to its cell cycle. But, in contrast to the behaviour of single cell bacteria, in mycelia the two daughter cells formed upon division are neither equivalent nor uniform. Here, the sister cells differ in length, shape and posssession of an e-site. Only one of the daughter cells receives the e-site of the mother cell, while the other starts its own cell cycle by generating a new e-site at the cylindrical part of its envelope. Regarding the length of sister cells the degree of heterogeneity increases with the age of the corresponding region of the mycelium, and eventually some cells lose the ability to generate an e-site, i. e. to grow. With this model the kinetic and structural peculiarities of the mycelial growth of Streptomyces granaticolor can be explained. Studying the growth behaviour of some unrelated actinomycetes strains (SCHUHand BERGTER 1 9 7 6 , KRETSCHMER 1 9 7 8 , 1 9 8 1 ) it has become evident that mycelial growth underlies common rules. Unlimited mycelial growth is characterized by t w o features: exponential increase of the total length parallel to the number of branches, and elongation being restricted to elongation sites (e-sites) ( B E G G and D O N A C H I E 1977) situated at each hyphal tip. The exponential increase of mycelial length (SCHUHMANN and BERGTER 1 9 7 6 , KRETSCHMER 1 9 7 8 , 1 9 8 1 ) resp. of the biomass (HILLIGER and R I E S E N B E R G 1 9 7 6 ) indicates that the cytoplasm is exponentially synthesized equally throughout the whole mycelium. Indeed, with Streptomyces hygroscopicus growing in a chemostat it was demonstrated autoradiographically that protein synthesis is not restricted to the hyphal tips, and that the portion of mycelial regions with high synthetic activity rises with increasing growth rate ( R I E S E N B E R G and BERGTER 1 9 7 9 ) . Also, pulse labelling the D N A of S. hygroscopicus, S. granaticolor and Thermoactinomyces vulgaris by 3 H-thymidine, STROHBACH and K U M M E R (pers. communication) found autoradio-

MANN

23*

336

S. KRETSCHMEE

graphically that DNA-replication extends over all parts of the mycelia. But according to the mode of hyphal elongation (BROWN and CLARK 1966, SCHUHMAIW and BERGTEK 1976, KRETSCHMER 1978, 1981) it must be concluded that growth of the envelope occurs only at distinct, apically situated sites and there generally in a non-exponential manner. Until now, there is no idea how to explain the balanced cooperation of the syntheses of the cytoplasm and the envelope in mycelia, since each have their own peculiarities concerning site and kinetics. Seeking for a possible mechanism responsible for the balanced cooperation, it seemed useful to apply the rules of cell cycle to mycelia. Thus, the aim of our study was to elucidate the cellular structure of mycelia and to analyse the behaviour of their individual cells. S. granaticolor ETH 7437 was found to be a suitable organism to study the mycelial growth behaviour on the cellular level. Materials

and methods

The strain used throughout was Streptomyces granaticolor E T H 7347, described by filfiicovi. and HEHACEK (1968) and kindly supplied by K . MIKTTLIK (Prague). The medium consisted of K H 2 P 0 4 0.13%, N a 2 H P 0 4 • 2 H 2 0 0.28%, NaCl 0.1%, MgS0 4 • 7 H 2 0 0.05%, CaC0 3 0.0005%, F e S 0 4 • 7 H 2 0 0.001%, glucose 0.5% (glu), proteose peptone No. 3 (DIFCO) (pep) 0.6% and aqua dest. (pH 7.0). Only in some experiments the peptone was replaced by (NH 4 ) 2 S0 4 . For the microcultures the medium was solidified by adding 1.5% agar. Growth occurred a t 30 °C, liquid cultures were shaken in a water bath. Methods used for microcultivation on agar and phase contrast observation were described by KBETSCHMEB (1978). I n liquid cultures growth was controlled b y measuring the optical density (ELPHO, V E B CAEL ZEISS Jena). Mycelia grown exponentially for at least 6 doublings in liquid medium were prepared for wall staining. Immediately after sampling, growth was stopped by addition of 0.8% formaldehyde. Smears on slides were placed in 5 % tannic acid for 10 min at 50 °C. After washing, a 0.5% aqueous solution of crystal violet was added and allowed to remain 4 min on the smears. The slides were washed again and flooded with 0.5% aqueous solution of congo red for 1 min, then washed and blotted dry. Our staining regimen followed a method d e s c r i b e d b y WEBB (1954).

From the mycelia the following parameters were determined: a) from series of photographs taken during mycelial growth on agar (microcultures): d^: doubling time of mycelial length (L), (min) djy: doubling time of the number of elongation sites (hyphal tips ) (N), (min) L/N: mean hyphal length (jim) a : elongation rate per elongation site (¡xmh -1 ) b) from liquid-grown, stained mycelia: 5. Lc: length of cells (length enclosed by a continuous envelope), (¡im) 6. Ls: interseptum length (distance between 2 neighbouring septa, resp. a tip and the nearest septum), (|J.m) 7. length of branches (in non-apical cells the part of a cell formed by its elongation site), (¡xm) 1. 2. 3. 4.

8. Ln: distance between neighbouring branches a t a superordinate hypha, ((¿m). I n unbranched cells L c equals L s .

Results The growth behaviour of S. granaticolor was studied with respect to the kinetics as well as to the structure, especially the cellular structure. The kinetics of growth can be determined only with living materials, while the elucidation of the cellular structure was possible only with stained, i. e. dead material. Furthermore, for observation of living mycelia, microcolonies growing on agar are most suitable, but for staining liquid grown material. Thus, for gaining a complete picture of the mycelial growth pattern at first the behaviour under both growing conditions was compared.

Cell cycle in Streptomyces

337

Fig. 1. Part of a series of photographs taken of S. granaticolor growing on glu-pep-agar. The interval is 30 min, the growth from the 1.5th to the 5th hour is presented. (Phase contrast)

The development of a mycelium on agar can be followed on Fig. 1. The kinetics of growth on glu-pep-agar is presented in Fig. 2. The mycelial grew exponentially. Initially the growh rate was equal to the elongation rate of the germ tube, but once branching had started growth followed the exponential increase of the number of e-sites (hyphal tips) (Fig. 2). Concerning the morphological changes during growth on agar the formation of branches is most interesting (Fig. 1). At distinct areas within older parts of the mycelium new e-sites arose, starting elongation in that way, that theyself became placed at the tip of the nascent hypha (branch). The rate of elongation of each new-born e-site increased until a maximum value, which then was maintained by the apparently potentially immortal e-site (Fig. 2). The structural behaviour of the growing mycelia revealed that the envelope is synthesized by the e-sites, and no doubt only there. With liquid-grown mycelia the kinetics of growth — expressed by increase of the optical density — could be determined only in the glu-pep-medium, since in the glu(NH 4 ) 2 S0 4 -medium mycelia aggregated and formed pellets. In the former medium the OD increased' exponentially. Morphologically the liquid-grown mycelia resembled the agar-grown ones. For comparison, some growth parameters of the differently grown mycelia were listed in Table 1. It is obvious that concerning their structure and growth kinetics liquid and agar-grown mycelia do not differ. S e a r c h f o r s t r u c t u r a l r e g u l a r i t i e s in g r o w i n g m y c e l i a Starting from our finding that mycelial growth is well controlled we looked for structural regularities responsible for establishing and maintaining the regular growth. First, the pattern of branching was determined by correlating the frequency of bran-

rsoo

Hours after

germination

Fig. 2. Growth parameters of a mycelium growing on glu-pep-agar (microculture). L : length of the mycelium (o), N : number of e-sites (•),(*: elongation rate of the first formed e-site. (germ tube) ( x ) , o f the e-site of the 2nd branch (A), and average value of all e-sites ( + )

Cell cycle in Streptomyces

339

Table 1 Parameters of S. granaticolor mycelia growing exponentially under different conditions Growth condit ions Source of C

medium

N

agar agar liquid

glu (NH 4 ) 2 S0 4 glu pep glu pep

LIN (average)

di min

¡¿m

75 50 60

measured (im

16.8 18.2 19.1

x (average) calculated (after BERGTER 1978)

h-1

u.m h - 1

9.7 16.0

9.3 15.2 13.2

ches to the age of the hyphal part from where they emerged. A broad variation of the distance between neighbouring branches was observed, but generally in older hyphal parts the branches were more frequent (Fig. 3A). A similar but more tight correlation was found regarding the frequency of septa (Fig. 3B).

80

•

60

•:

r'

;

• • •

» •\ :«.• •

1

:• •

40 *

•

»•«

20

OL L. 15

Ln (jjuTi)

20 0

10 4 (Jim)

15

Fig. 3. Frequency of branches (A) resp. of septae (B) in dependence on the age of hyphal parts. The age is expressed by the distanoe to the hyphal tip, the frequency of branches by the distance to the neighbouring distal branch, and the frequency of septation by the distance to the neighbouring distal septum

Then, regarding the structure of individual cells three types could be distinguished (Fig. 4): apical branchless cells (20%), non-apical branched cells (60%) and non-apical branchless cells (20%). From the observation of growing mycelia we learned that each hyphal tip harbours a growing e-site. Thus, the two cell types mentioned first must have been growing at the time of sampling, while the non-apical branchless were not. The non-growing cells were scattered between the 8 0 % of the growing ones.

340

S . KRETSCHMER

Fig. 4. Mycelia grown in liquid glu-pep-medium. Cell wall staining, magnif. 1200:1. At the left younger at the right older mycelial parts

Concerning the length of individual cells a broad variation between 2 and about 30 pirn was observed. Looking for a correlation between length and shape of the mycelial cells, we found the apical cells to be the longest and the non-apical branchless cells to be the shortest (Fig. 5). But the heterogeneity in size of each cell type was too large to find any regulatory mechanism directly determining the cell size. In contrast to Lc the interseptum length (Ls) seemed to be a more valuable parameter. L s showed a correlation to the distance to the hyphal tip (Fig. 3B) as well as to the length of its branches (Lb) (Fig. 6). According to these figures the non-apical cells could be classified into at least two groups. In the vicinity of the hyphal tip cells had long .¿¡.-values (about 9 — 15 [Am), but short branches (up to 7 ¡j.m) (Fig. 6A), whereas in older hyphal regions cells had short (3—9[i.m) Ls~, but in average long

341

Cell cycle in Streptomyces

F i g . 5. Distribution of cell length of liquid grown mycelia. A l l 300 cells: only the branchless non-apical cells: A , branched non apical cells: o . The apical cells are represented b y the difference of • — ( o + A ) and are found also at F i g . 7. Values are given as moving average

L(,-values (up to 20 ¡i.m) (Fig. 6B). The apical cells which do not appear within Fig. 3B and Fig. 6 then would represent a third class of cells, the youngest, having the largest Ls-values and no branch at all. According to this characterization the frequency of each cell class was determined (Fig. 7). Furthermore, these three cell classes were described quantitatively (Table 2). The most important finding was that the ¿„-values of each subsequent class were about half the size of the former (Table. 2). This fact indicates that during ageing septation proceeded by placing new cross walls approximately intermediate between the preexisting ones. This means that our classification reflects the sequence of cell divisions, and thus we replaced the term "class" by the term "generation", being aware that, in mycelia, it can only be used with restrictions. The question, if there were also "generation" 4-cells, could not be answered, since the old parts of the mycelia lysed, and its proposed L ¿.-value of 3 ¡i.m was also met within the "generation" 3-cells.

M o d e l of m y c e l i a l g r o w t h b a s e d on c e l l u l a r

behaviour

The combination of the various data enabled us to construct a model reflecting the events taking place during growth of S. granaticolor (Fig. 8). Starting with an ascent apical cell the repeated sequence of elongation and division is as follows: 1. A young apical cell is about 16 [Am long. A t the one cellular pole an e-site is situated which continuously synthesizes envelope, the rate being in balance to the doubling of the cytoplasm. 2. When this apical cell attains a length of about 28 ¡j.m it divides by septation. The apical daughter cell — which always retains the e-site — is in average 16 |j,m long, and the subapical, e-site-less daughter cell in average 11.8 ¡j.m. Assuming that the signal for septation is given at the moment where the mother cell is twice as long as the subapical daughter cell, the time needed from initiation to completion of the septum can be calculated. The difference of length of both daugther cells at the time of completion of septation indicates that this process would take 19 min. T;he apical daughter cell continuously elongates as described above, then divides again and so on. 3. The subapical daughter cell initially is handicapped since it has not got an e-site necessary for elongation. But after a lag period it creates a new e-site of its own 24 Z. Allg. Mikrobiol., Bd. 23 , H. 5

S. Kretschmer

342

within the cylindrical part of its envelope. Since 2 5 % of the non-apical cells lack a branch, i. e. an e-site, we m a y conclude that the lag f o r the formation of an e-site takes 2 5 % of the doubling time of the mycelium, i. e. about 12 min. T h e place where the e-site arises is not f i x e d (Fig. 3A, 4). T h e branch produced b y the new

B 20

-

\

I 15

\

10 •

•

•

\

X 2

_L

_L 4

6

8

10 Interseptum

12 length

_L n

(jim)

Fig. 6. Morphology of the branched cells of liquid grown mycelia, expressed by the correlation between interseptum and branch length. According to the branch length cells fall into 2 groups: A (branches much shorter than a/doubling time = 11 ¡¿m) and B (branches in average longer). The dashed lines mark the obvious maximum cell length of both groups (Lb + Ls = 18.5 [j.m (A), resp. 22 ¡xm (B))

Cell cycle in Streptomyces

343

Interseptum length (jim) Fig. 7. Distribution of Ls of mycelial cells ordered according to their suggested membership to different "generations". "Generation" 1 (apical cells): x , "generation" 2: "generation" 3: O. Values are given as moving average 1

3E

Fig. 8. Proposed pattern of cellular elongation and cell division during mycelial growth of S. granaticolor. Numbers indicate the membership to the corresponding "generation". # marks cells, which are no longer able to elongate. Hatched cells temporarily lack elongation.

344

S . KRETSCHMER

m 1>