Zeitschrift für Allgemeine Mikrobiologie: Band 23, Heft 10 1983 [Reprint 2021 ed.] 9783112582145, 9783112582138

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ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS BAND 23 • 1983 • HEFT 10

AKADEMIE-VERLAG • BERLIN ISSN 0044-2208

Z. allg. Mikrobiol., Berlin 88 (1983) 10, 605 - 668

EVP 2 0 , - M

Instructions

to

Authors

1. The journal publishes original papers, short communications, and review articles. Submission of a paper implies that it has not been published or has not been submitted for publication elsewhere. Manuscripts should be sent in duplicate with one set of the original illustrations to the Editor-in-Chief: Prof. Dr. U. Taubeneck, DDR-6900 Jena, Beutenbergstr. 11. 2. Manuscripts should preferably been written in English but may also be submitted in German. They should be typed double-spaced and be accompanied by a title page comprising: name and address(es) of the institution(s) where the work was done, title of the paper, and the complete name(s) of the author(s). Each paper must begin with a brief summary in English. Original papers should be divided into sections headed: Introduction, Materials and Methods, Results, Discussion, Acknowledgements, and References. A short title for use as running head should be provided. The exact mailing address of the author to whom correspondence, reprint requests etc. are to be addressed must be given at the end of the paper. 3. Tables, illustrations, and descriptive legends of the illustrations must be submitted on separate sheets. Each table should have a heading. The size of illustrations should not exceed the maximum printing area of 12 X 19 cm or 4.7 X 7.5 inches, respectively. 4. Literature citations in the text should be by author and year of publication. If there are more than two authors, only the first should be named, followed by „et. al". References should include only publications cited in the text. They should be given in alphabetical order: a) Books: Family name(s) and initials of authors(s), year of publication. Title of the book. Volume and Edition, Publisher and place of publication, e.g.: BERTHELIN, J., BELGY, G. and MAGNE, R., 1977. Some aspects of the mechanism of solubilization and insolubilization of uranium from granites by heterotrophic microorganisms. I n : Bacterial Leaching Conference (W. SCHWARTZ, Editor), pp. 261 —270. Verlag Chemie Weinheim. b) Journals: Family (name(s) and initials of authors(s), year of publication. Title of the paper. Abbreviated name of the journal, volume (underlined), number of the first and last page, e.g.: KÄPPELI, O., MÜLLER, M. a n d FIECHTER, A., 1978. Chemical a n d s t r u c t u r a l alte-

rations at the cell surface of Candida tropicalis, substrate. J . Bacteriol., 138, 952—958.

induced by hydrocarbon

5. Galley proofs will be sent to the author in duplicate. One of them should be corrected and returned to the Editor-in-Chief or to Redaktion der Zeitschrift für Allgemeine Mikrobiologie, DDR-6900 Jena, Beutenbergstr. 11, as soon as possible. 6. 100 reprints of each paper are provided free of charge. Additional reprints may be purchased at cost price.

ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN

INTERNATIONAL

JOURNAL

ON

M O R P H O L O G Y , PHYSIOLOGY, G E N E T I C S , AND ECOLOGY OF MICROORGANISMS

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

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 und 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 Aransas N. Pfennig, Göttingen I. L. Rabotnova, Moskau A. Schwartz, Wolfenbüttel

REDAKTION

HEFT 10

1983

BAND 23

AKADEMIE-VERLAG • BERLIN

U. M a y , J e n a

Die Zeitschrift f ü r Allgemeine Mikrobiologie soll dazu beitragen, Forschung und internationale Zusammenarbeit auf dem Gebiet der Mikrobiologie zu fördern. Es werden Manuskripte aus allen Gebieten der allgemeinen Mikrobiologie veröffentlicht. Arbeiten über Themen aus der medizinischen, landwirtschaftlichen, technischen Mikrobiologie und aus der Taxonomie der Mikroorganismen werden ebenfalls aufgenommen, wenn sie Fragen von allgemeinem Interesse behandeln.

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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 ; F e r n r u f : 2236229 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 U 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 Redaktion: 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 852200; Telex-Nr.: 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 Band mit 10 Heften. Bezugspreise je Band 250,— M zuzüglich Versandspesen (Preis f ü r die D D R 2 0 0 , - 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 Übersetzung. 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 any form, by photoprint, microfilms or any other means, without written permission from t h e publishers.

Erscheinungstermin: Dezember 1983

Bestellnummer dieses H e f t e s : 1070/23/10 © 1983 by Akademie-Verlag Berlin. Printed in t h e German Democratic Republic. AN (EDV) 75218

Zeitschrift f ü r Allgemeine Mikrobiologie

1983

23

10

607-619

( I n s t i t u t e of Biological Physics of t h e U S S R A c a d e m y of Sciences, Pushchino, Moscow Region, 142292, U S S R )

Formation of additional contacts of chromosome with membrane in the process of D N A repair synthesis in bacterial cells V . G . BEZLEPKIN, Y U . Y U . MALINOVSKY a n d A . I . GAZIEV

(Eingegangen

am 1. 2. 1983)

A n increase in t h e a m o u n t of m e m b r a n e - b o u n d D N A was f o u n d in B. subtilis cells with UVinduced D N A repair synthesis as compared t o u n t r e a t e d cells. I t was shown t h a t D N A repair synthesis occurred in D N A m e m b r a n e complexes (DMC) f o r m e d during UV-irradiation. UV-induced f o r m a t i o n of DMC was observed in cells of wild t y p e strains which were capable of repairing d a m a ged D N A b u t n o t in a m u t a n t defective in DNA-polymerase I . I t was d e m o n s t r a t e d t h a t D N A polymerase I is located on t h e m e m b r a n e of B. subtilis cells. This suggested a participation of D N A polymerase I in binding of t h e chromosome t o t h e m e m b r a n e in UV-irradiated cells. UV-induced DMC did n o t dissociate w h e n t h e cells were t r e a t e d with inhibitors of DNA-gyrase. I t , therefore, was qualitatively different f r o m t h e DMC f o u n d during replication. T h e m e c h a n i s m s of binding of t h e d a m a g e d D N A t o t h e m e m b r a n e in UV-irradiated cells of B. subtilis are discussed. C o m p a c t n e s s a n d a h i g h d e g r e e of o r d e r of t h e b a c t e r i a l c h r o m o s o m e a r e t h e r e s u l t of t h e c o n f o r m a t i o n a l p r o p e r t i e s of D N A a s w e l l a s i t s i n t e r a c t i o n w i t h p r o t e i n s , R N A , a n d m e m b r a n e s t r u c t u r e s . T h i s s t r u c t u r a l o r g a n i z a t i o n is of g r e a t i m p o r t a n c e i n reg u l a t i o n of g e n e t i c p r o c e s s e s ( L E I B O W I T H a n d S C H A E C H T E R 1 9 7 5 , L Y D E R S E N a n d PETTIJOHN 1 9 7 7 , SUGINO a n d BOTT

1980).

I t is b e l i e v e d t h a t a s s o c i a t i o n of t h e D N A w i t h t h e m e m b r a n e is n e c e s s a r y f o r n o r m a l D N A r e p l i c a t i o n a n d s e g r e g a t i o n of c h r o m o s o m e s ( L E I B O W I T H a n d S C H A E C H T E R 1 9 7 5 , YAMAGUCHI and YOSHIKAWA 1 9 7 7 ) . R e c e n t l y we have proposed that the D N A repair t a k e s place a t t h e m e m b r a n e in bacterial cells (CHEFRANOVA a n d GAZIEV 1 9 7 9 , G A Z I E V et al. 1 9 8 0 ) . W e d e r i v e d t h i s c o n c l u s i o n f r o m t h e i n c r e a s e i n t h e a m o u n t o f m e m b r a n e - b o u n d D N A w h i c h w a s o b s e r v e d in cells treated w i t h N - m e t h y l - N - n i t r o s o u r e a o n l y i n s t r a i n s of B. subtilis c a p a b l e of r e p a i r i n g D N A d a m a g e b u t n o t in s t r a i n s d e f e c t i v e i n g e n e t i c c o n t r o l of v a r i o u s s t e p s of D N A r e p a i r ( C H E F R A N O V A a n d G A Z I E V 1979).

I n t h i s c o m m u n i c a t i o n w e r e p o r t o n t h e U V - i n d u c e d f o r m a t i o n of a s p e c i f i c D M C a n d i t s r o l e i n t h e D N A r e p a i r s y n t h e s i s i n d u c e d b y U V - i r r a d i a t i o n i n B. subtilis.

Materials

and

methods

Bacterial s t r a i n s : Bacillus subtilis 168 (leu" m e t " ) ; GSY 228 (trpC, metB 4 ); GSY 1026 (trpC 2 m e t B ? ) ; GSY 1025 (metB 4 t r p C , r e c A ~ ) ; GSY 1027 (metB 4 trpC 2 uvr_ 1 ); 1306 (leu" m e t " polA~) were supplied b y I . I . SAMOILENKO f r o m t h e Gamalei I n s t i t u t e of Epidemiology a n d Microbiology. Cultivation of strains a n d radioactive labelling of proteins, D N A , a n d lipids h a v e been described previously (GAZIEV et al. 1980). D N A repair synthesis in bacterial cells was induced b y UV-irradiation of a cell suspension (5 • 107 — 10 8 cells/ml) in 0.1 M p o t a s s i u m - p h o s p h a t e b u f f e r ( p H 7.3) a t 2 — 4 °C in open PETRI dishes (15 cm in diameter) b y using a s t a n d a r d U V - l a m p (254 n m , 0.6 J / m 2 • sec). To inhibit t h e replicative D N A synthesis, t h e cells were t r e a t e d for 25 min a t 37 °C with nalidixic acid (NA) or novobiocin (NB) in 0.1 M potassium-phosphate b u f f e r ( p H 7.3) a t concentrations of 200 ¡i.g/ml or 40 (¿g/ml, respectively (COZZARELLI 1977, STAUDENBAUER 1978). T h e r a p i d l y s e d i m e n t i n g D N A w a s i s o l a t e d b y t h e m e t h o d o f SUEOKA a n d HAMMERS ( 1 9 7 4 ) . A s 41*

Zeitschrift f ü r Allgemeine Mikrobiologie

1983

23

10

607-619

( I n s t i t u t e of Biological Physics of t h e U S S R A c a d e m y of Sciences, Pushchino, Moscow Region, 142292, U S S R )

Formation of additional contacts of chromosome with membrane in the process of D N A repair synthesis in bacterial cells V . G . BEZLEPKIN, Y U . Y U . MALINOVSKY a n d A . I . GAZIEV

(Eingegangen

am 1. 2. 1983)

A n increase in t h e a m o u n t of m e m b r a n e - b o u n d D N A was f o u n d in B. subtilis cells with UVinduced D N A repair synthesis as compared t o u n t r e a t e d cells. I t was shown t h a t D N A repair synthesis occurred in D N A m e m b r a n e complexes (DMC) f o r m e d during UV-irradiation. UV-induced f o r m a t i o n of DMC was observed in cells of wild t y p e strains which were capable of repairing d a m a ged D N A b u t n o t in a m u t a n t defective in DNA-polymerase I . I t was d e m o n s t r a t e d t h a t D N A polymerase I is located on t h e m e m b r a n e of B. subtilis cells. This suggested a participation of D N A polymerase I in binding of t h e chromosome t o t h e m e m b r a n e in UV-irradiated cells. UV-induced DMC did n o t dissociate w h e n t h e cells were t r e a t e d with inhibitors of DNA-gyrase. I t , therefore, was qualitatively different f r o m t h e DMC f o u n d during replication. T h e m e c h a n i s m s of binding of t h e d a m a g e d D N A t o t h e m e m b r a n e in UV-irradiated cells of B. subtilis are discussed. C o m p a c t n e s s a n d a h i g h d e g r e e of o r d e r of t h e b a c t e r i a l c h r o m o s o m e a r e t h e r e s u l t of t h e c o n f o r m a t i o n a l p r o p e r t i e s of D N A a s w e l l a s i t s i n t e r a c t i o n w i t h p r o t e i n s , R N A , a n d m e m b r a n e s t r u c t u r e s . T h i s s t r u c t u r a l o r g a n i z a t i o n is of g r e a t i m p o r t a n c e i n reg u l a t i o n of g e n e t i c p r o c e s s e s ( L E I B O W I T H a n d S C H A E C H T E R 1 9 7 5 , L Y D E R S E N a n d PETTIJOHN 1 9 7 7 , SUGINO a n d BOTT

1980).

I t is b e l i e v e d t h a t a s s o c i a t i o n of t h e D N A w i t h t h e m e m b r a n e is n e c e s s a r y f o r n o r m a l D N A r e p l i c a t i o n a n d s e g r e g a t i o n of c h r o m o s o m e s ( L E I B O W I T H a n d S C H A E C H T E R 1 9 7 5 , YAMAGUCHI and YOSHIKAWA 1 9 7 7 ) . R e c e n t l y we have proposed that the D N A repair t a k e s place a t t h e m e m b r a n e in bacterial cells (CHEFRANOVA a n d GAZIEV 1 9 7 9 , G A Z I E V et al. 1 9 8 0 ) . W e d e r i v e d t h i s c o n c l u s i o n f r o m t h e i n c r e a s e i n t h e a m o u n t o f m e m b r a n e - b o u n d D N A w h i c h w a s o b s e r v e d in cells treated w i t h N - m e t h y l - N - n i t r o s o u r e a o n l y i n s t r a i n s of B. subtilis c a p a b l e of r e p a i r i n g D N A d a m a g e b u t n o t in s t r a i n s d e f e c t i v e i n g e n e t i c c o n t r o l of v a r i o u s s t e p s of D N A r e p a i r ( C H E F R A N O V A a n d G A Z I E V 1979).

I n t h i s c o m m u n i c a t i o n w e r e p o r t o n t h e U V - i n d u c e d f o r m a t i o n of a s p e c i f i c D M C a n d i t s r o l e i n t h e D N A r e p a i r s y n t h e s i s i n d u c e d b y U V - i r r a d i a t i o n i n B. subtilis.

Materials

and

methods

Bacterial s t r a i n s : Bacillus subtilis 168 (leu" m e t " ) ; GSY 228 (trpC, metB 4 ); GSY 1026 (trpC 2 m e t B ? ) ; GSY 1025 (metB 4 t r p C , r e c A ~ ) ; GSY 1027 (metB 4 trpC 2 uvr_ 1 ); 1306 (leu" m e t " polA~) were supplied b y I . I . SAMOILENKO f r o m t h e Gamalei I n s t i t u t e of Epidemiology a n d Microbiology. Cultivation of strains a n d radioactive labelling of proteins, D N A , a n d lipids h a v e been described previously (GAZIEV et al. 1980). D N A repair synthesis in bacterial cells was induced b y UV-irradiation of a cell suspension (5 • 107 — 10 8 cells/ml) in 0.1 M p o t a s s i u m - p h o s p h a t e b u f f e r ( p H 7.3) a t 2 — 4 °C in open PETRI dishes (15 cm in diameter) b y using a s t a n d a r d U V - l a m p (254 n m , 0.6 J / m 2 • sec). To inhibit t h e replicative D N A synthesis, t h e cells were t r e a t e d for 25 min a t 37 °C with nalidixic acid (NA) or novobiocin (NB) in 0.1 M potassium-phosphate b u f f e r ( p H 7.3) a t concentrations of 200 ¡i.g/ml or 40 (¿g/ml, respectively (COZZARELLI 1977, STAUDENBAUER 1978). T h e r a p i d l y s e d i m e n t i n g D N A w a s i s o l a t e d b y t h e m e t h o d o f SUEOKA a n d HAMMERS ( 1 9 7 4 ) . A s 41*

608

V . G . B E Z L E P K I N , Y U . Y U . MALINOVSKY a n d A . I . GAZIEV

has been shown previously (GAZIEV et al. 1980), UV-irradiation of cells led t o changes in t h e a m o u n t of D N A in DMCs while t h e a m o u n t of m e m b r a n e lipids was not significantly altered. Therefore, t h e results of t h e comparative studies were presented as 3 H-DNA/ 1 4 C-lipid or 3 H-DNA/ 3 6 S-protein ratios, where n u m e r a t o r a n d denominator represented t h e radioactivity of nucleic acid a n d membrane material sedimenting t h r o u g h n e u t r a l sucrose density gradient (NSDG) onto t h e cushion of dense CsCl-sucrose mixture. The a m o u n t of this rapidly sedimenting material was expressed as percent of t o t a l radioactivity of [ 3 H] thymidine-labelled D N A a n d [ 14 C]glycerol- (or [ 3 6 S]methionine)-labelled m e m b r a n e lipids (or proteins) distributed over t h e gradient. Membrane vesicles containing t h e " a n c h o r " D N A (DNA f r a g m e n t s b o u n d to t h e m e m b r a n e and, therefore, inaccessible t o D N A a s e I ) were obtained according to K O N I N G S et al. ( 1 9 7 3 ) . D N A a n d H-labelled D N A were isolated f r o m B. subtilis 2 2 8 by t h e m e t h o d of M A B M U R ( 1 9 6 1 ) . The purified preparations which showed a ratio of D 2 m I D . m = 1.85 h a d a specific radioactivity of 1.1 — 1.5 x 1 0 4 cpm per [xg of D N A . " A c t i v a t e d " D N A was obtained b y DNAase I (SIGMA Chemical Co.) t r e a t m e n t of t h e D N A isolated f r o m B. subtilis G S Y 2 2 8 as described b y A P O S H I A N a n d K O R N B E R G ( 1 9 6 2 ) . D e n a t u r a t i o n was carried out b y heating t h e D N A solution two times for 1 0 min in boiling w a t e r b a t h a n d t h e subsequent cooling on ice. 3

The a m o u n t of single- a n d double-stranded f r a g m e n t s in t h e preparations of " a n c h o r " D N A was determined b y c h r o m a t o g r a p h y on a 13.5 cm colums packed with h y d r o x y l a p a t i t e Bio-Gel H T P (Bio-Rad) a t 60 °C according t o T I B B E T T S et al. (1973). Sedimentation analysis of " a n c h o r " D N A was performed on linear alkaline sucrose density gradients (ASDG, 3 — 15%, 4.8 ml) according t o M C G R A T H a n d W I L L I A M S (1966). Gradients were centrifuged in a swinging bucket-rotor No. 40 of VAC-601 ultracentrifuge (JANETZKI GDR) a t 40.000 r p m for 10 h. After centrifugation gradients were f r a c t i o n a t e d a n d samples were collected o n t o filter paper discs (2.5 cm in diameter, FN-16, FILTRAR). The filters were t h e n dried a n d washed in 5 % trichloroacetic acid, ethanol a n d ether, a n d eventually counted in 5 ml of a toluene-based scintillation cocktail. In vitro binding of D N A t o t h e m e m b r a n e vesicles was determined b y t h e m e t h o d of J O E N J I E et al. (1974). The 36 S-labelled m e m b r a n e f r a g m e n t s were i n c u b a t e d with 3 H - D N A in 0.1 M potassiu m - p h o s p h a t e buffer ( p H 7.0) for 25 min a t 30 °C. T h e n an aliquote of t h e incubated sample (120 ¡xl) was layered o n t o p of a discontinuous neutral sucrose gradient (1.8 ml of 5 % per 3 ml of 15% sucrose) a n d centrifuged for 25 min a t 20° C in a B E C K M A N SW 50.1 rotor a t 15,000 r p m . To e s t i m a t e the a m o u n t of D N A b o u n d t o t h e m e m b r a n e vesicles t h e ratio of 3 H - D N A to 3 5 S-protein was determined in t h e complex sedimenting t o t h e b o t t o m of t h e centrifuge tube. To estimate t h e D N A - d e g r a d a t i n g (nuclease) activity of m e m b r a n e vesicles, we measured t h e acid-insoluble radioactivity retained in t h e samples a f t e r incubation with t h e p r e p a r a t i o n s of 3 H - D N A f r o m B. subtilis 2 2 8 ( J O E N J I E et al. 1 9 7 4 ) . The DNA-polymerase activity of m e m b r a n e vesicles (in vitro) was determined b y measuring t h e a m o u n t of t h e acid-insoluble radioactivity formed during incubation of an aliquote of t h e vesicle suspension (150 ¡xg of protein) in a reaction m i x t u r e (the f i n a l volume 130 ¡xl) containing 70 mM HEPES ( p H 7.4), 5 MM Tris-HCl ( p H 7.4), 35 MM NaCl, 0.2 MM E D T A , 80 MM KC1, 5 MM MgCl.,, 5 MM A T P , 5 MM 2-mercaptoethanol, 6 % glycerol, 3.5 MM d A T P , 3.5 MM dCTP, 3.5 MM d G T P , 0.005 mM [ 3 H ] T T P . I n c u b a t i o n was carried o u t for 40 min a t 37 °C in centrifuge tubes. Termination was accomplished successively adding 0.5 ml of cold 1 N HC10 4 , containing 0.05 M N a 4 P 2 0 7 , 0.2 ml of a solution of B. subtilis D N A (0.5 mg/ml), a n d 1.5 ml of cold water. After 60 min of incub a t i o n a t 2—4 °C a pellet was formed b y centrifugation. This pellet was resolved in 0.2 ml of 0.2 N N a O H a n d subsequently formed again b y adding 0.5 ml of H C 1 0 4 - N a 4 P 2 0 7 m i x t u r e a n d centrifugation. Finally, t h e pellet was redissolved in 0.2 ml of 0.2 N N a O H , a n d 0.5 ml of 10% trichloroacetic acid was added. The precipitate formed was q u a n t i t a t i v e l y transferred o n t o W H A T M A N GF/C glass-fiber filters. T h e filters were washed several times with 5 % trichloroacetic acid containing Na 4 P„0 7 , t h e n dried a n d eventually t h e radioactivity was counted. I n some experiments Nethylmaleimide (N-EMI), ^-chloromercuribenzoate (pCMB) or KC1 were added t o t h e DNA-polymerase reaction mixture. In vitro D N A synthesis a t t h e m e m b r a n e of intact a n d UV-irradiated cells was measured b y t h e incorporation of [ 3 H ] t h y m i d i n e into acid-insoluble material in t h e m e m b r a n e fraction. For this purpose the NA-treated cells suspension (5 • 107 cells/ml in 0.1 M potassium-phosphate buffer, p H 7.3) was irradiated b y UV-light a t a dose of 40 J / m 2 a n d subsequently i n c u b a t e d for 30 min a t 37 °C in t h e presence of 370 K B q / m l of [ 3 H]thymidine. I n pulse labelling experiments [ 3 H]thymidine was a d d e d t o t h e N A - t r e a t e d cell suspension immediately after UV-irradiation a n d t h e samples were incubated a t 37 °C for 2 min. P a r t of the suspension was used t o obtain t h e m e m b r a n e vesicles a n d t h e rest was used t o obtain DMCs by sedimentation t h r o u g h N S D G ( S U E O K A a n d H A M M E R S 1974). The radioactivity of t h e samples on filters was counted in a toluene-based scintillation cocktail. A M U L T I - M A T liquid scintillation system with computer (Intertechnique) was used.

609

DNA repair synthesis in bacterial cells

Results A n a l y s i s of c h r o m o s o m e b i n d i n g to t h e m e m b r a n e during U V - i n d u c e d DNA r e p a i r s y n t h e s i s The ability of different B. subtilis strains to form UV-induced DMCs was compared by using the sedimentation method (SUEOKA and HAMMERS 1 9 7 4 ) . The data obtained indicated that UV-induced binding took place only in the strains capable of repairing DNA damage, i.e., in wild type and recA~ strains (Fig. 1). In the recA~ strain binding was measured in a narrower dose-range of UV-radiation. In the uvr_1 mutant which carried a defect in the incision stage of repair essentially no changes were found. However, the mutant defective in DNA-polymerase I showed a detachment of the chromosome from the membrane as UV-doses increased.

10

hO

60 UV-dose (3-m'2)

80

100

Fig. 1. The amount of DNA in fast sediinenting DMCs of UV-irradiated B. subtilis

strains.

• GSY 228 w. t., o 168 w.t., V GSY 1025 recA~, A GSY 1027 uvr_v • 1306 polA'

These results allowed us to propose the existence of a functional relationship between the processes of DNA-membrane interaction and DNA-repair synthesis. To further confirm this conclusion we carried out experiments in the course of which the level of the DNA binding to the membrane and the intensity of the UV-induced DNA synthesis (in the presence of the DNA-replication inhibitor — NA) were measured simultaneously in UV-irradiated cells. The data presented in Fig. 2 show that NA practically completely inhibited the DNA synthesis in the untreated cells, while, in the UV-irradiated cells the DNA synthesis remains at the original level. I t is noteworthy that the amount of DNA associated with the membrane remained high in the UV-irradiated cells even at NA concentrations up to 200 ji.g/ml. In contrast, in intact cells the amount of DNA bound to the membrane decreased as the NA concentration in the incubation medium was increased. Thus, a correlation was found between DNA binding to the membrane and the level of UV-induced DNA repair synthesis in B. subtilis cells. It was, therefore, assumed that the DNA repair synthesis occurred in the DMC of bacterial cells. Short-term incubation of UV-irradiated B. subtilis cells in a medium containing high activity of [ 3 H]thymidine (pulse labelling) demonstrated that DNA repair synthesis really occurred in the DMC (Fig. 3). In intact cells, i.e., cells

610

V. G. Bezlepkin-, Yu. Yu. Malxnovsky and A. I. Gaziev

10

-o 50

100>

150 200 Nalidixic acid (jig¡m!)

Fig. 2. The effect of NA on the DNA synthesis and the amount of DNA in the fast sedimenting DMC of UV-irradiated B. subtilis 168. V and o : DNA synthesis and 3H-DNA/14C-lipid ratio in the non irradiated cells; • and • : DNA synthesis and 3H-DNA/14C-lipid ratio in UV-irradiated cells

not treated with NA a substantial part of the newly synthesized DNA as well as a great part of the totally labelled DNA sedimented together with the membrane fraction. NA-treatment of intact cells caused DMC dissociation and inhibited incorporation of pulse label. Additional binding of totally labelled DNA to the membrane and incorporation of pulse label into the membrane-bound DNA was observed in contrast to intact cells (Fig. 3). Both processes were resistant to NA-treatment in the UV-irradiated cells, i.e., the DMC formed in such cells proved to be resistant to the inhibitor of the replicative DNA synthesis and DNA-gyrase (Cozzarelli 1977). A s s o c i a t i o n of " a n c h o r " D N A w i t h t h e m e m b r a n e The increase in the amount of membrane-bound DNA which we have observed may be explained by an increase in the quantity of DNA sites interacting with the membrane. In this case one should expect an increase in the amount of the "anchor" DNA in membrane vesicles. Indeed, UV-irradiation of cells of the wild type strains increased the amount of "anchor" DNA in membrane vesicles with the maximum being at 40 J/m a . However, in membrane vescicles of UV-irradiated cells defective in DNA-polymerase -I no increase of the "anchor" DNA amount was found (Fig. 4 A). These results were in a good agreement with those obtained by the sedimentation method. Treatment of the cells with inhibitors of DNA-gyrase (NA and NB) caused a decrease of the amount of "anchor" DNA bound to the membrane. However, in the UV-irradiated cells this decrease occurred to a lesser extend (Fig. 4B). This indicated that the DMC induced by UV-light was resistant to NA and NB. As it was shown above (Fig. 3) the UV-induced DNA repair synthesis was associated with the rapidly sedimenting DMC. These results were also confirmed by the isolation of membrane vesicles (Fig. 4C), which showed that a short-time incubation of cells with [ 3 H]thymidine resulted in the incorporation of radioactive label into acid-in-

DNA repair synthesis in bacterial cells

10 Fraction

20 number

from

611

SO bottom

Fig. 3. Sedimentation of B. subtilis 168 DNA in neutral sucrose gradient. The cells were grown in the presence of [ 14 C]thymidine and treated with NA. After UV-irradiation (40 J/m 2 ) they were incubated for 2 min in the presence of NA and [ 3 H]thymidine; then protoplasts were obtained and lysed. Finally, sedimentation analysis of the cell lysates was carried out. A — without UV-irradiation and NA-treatment; B — treatment with NA; C — UV-irradiation + treatment with NA; 1, 3, and 5 - the label of [ 14 C]thymidine; 2, 4, and 6 — the label of [ 3 H]thymidine. The arrow indicates the border of CsCl-sucrose dense shelf

soluble material bound to the membrane, i.e., the synthesis occurred at the membrane. It is noteworthy that DNA synthesis at the membrane of unirradiated cells was completely inhibited by NA, while the UV-induced DNA synthesis at the membrane was not sensitive to the action of this inhibitor. It should be pointed out that the increase in the amount of DNA bound to the membrane in the UV-irradiated cells was accompanied by an increase in the sedimentation rate of "anchor" DNA fragments in ASDG (Fig. 5). The increase in the size of "anchor"

612

V. G. BEZLEPKIN, YTJ. YU. MALINOVSKY and A. I. GAZIEV

300

100

40

60 SO UV-dose fJ-m'2 )

60 90 120 30 Incubation time (seconds) Fig. 4. The amount of "anchor" D N A in the membranes (A, B) and D N A synthesis on the membrane (C) of B. subtilis irradiated by UV-light and treated with DNA-gyrase inhibitors. A — the change of 3 H-DNA/ 1 4 C-lipid ratio (% of control) depending on the dose of UV-light (J/m 2 ): 1 —228 w. t.; 2 — 1306 polA'. B — the change of 3 H-DNA/ 3 6 S-protein ratio (% of control) depending on the conditions of cell treatment (228, w. t. strain); 3 — control; 4 — UV-irradiation; 5 — NA-treatment; 6 — UV-irradiation + NA-treatment; 7 — NB-treatment; 8 — UV-irradiation + NB-treatment. C — the amount of newly synthesized D N A ( 3 H-DNA/ 3 5 S-protein ratio is normalized to the value at 0 s incubation) depending on the incubation time (228, w. t. strain): 1 — control; 2 — UV-irradiation; 3 — NA-treatment; 4 — UV-irradiation + NA-treatment

DNA fragments in newly formed complexes as well as the resistance of "repair" DMC to NA and NB indicated a qualitative difference between the DMC of UV-irradiated and intact cells. It should also be mentioned that the ratio of single-stranded to double-stranded "anchor" DNA fragments in membrane vesicles of UV-irradiated (40 J/m a ) and intact cells was approximately 1:6 and 1:11, respectively (data not shown). An increased level of the UV-induced DNA binding to the membrane of wild type cells as compared to the polA~ mutant may indicate a specificity of DNA binding to the membrane which was probably due to peculiarities of the membrane structure of these cells. To check this, we performed in vitro experiments with membrane vesicles isolated from B. subtilis wild type and polA~ strains in order to their ability to bind homologous DNA (Fig. 6). In preliminary experiments we determined the dependence of DNA binding on the pH of the incubation mixture and on the concentration of EDTA and free Mg ++ ions in the medium by using the method of J O E N J I E et al. ( 1 9 7 4 ) . The results presented in Fig. 6 show that at optimal concentrations of EDTA and MgS0 4 (20 MM and 15 MM, respectively) membrane vesicles derived from cells of a wild type strain bound much more DNA (native, activated or denaturated) than membrane vesicles obtained from a strain defective in DNA-polymerase I.

5

10

15

20

Fraction number from

25 bottom

Fig. 5. Sedimentation of " a n c h o r " DNA derived from the membrane vesciles of UV-irradiated (40 J/m 2 ) B. subtilis 228 w. t . strain (A) and 1306 pol A- strain (B). 1 and 3 — control; 2 and 4 — UV-irradiation

5

•3? I

1

Fig. 6. In vitro DNA binding to the membrane vesicles ( 3 H-DNA/ 3 5 S protein ratio) of B. subtilis 228 w. t . (A) and 1306 polA~ (B). 1 - native D N A ; 2 - " a c t i v a t e d " D N A ; 3 - denaturated D N A

614

V . G . B E Z L E P K I N , Y U . Y U . MALINOVSKY, a n d A . I . GAZIEV

Thus, the results obtained suggested an involvement of DNA-polymerase I in the formation of additional DNA complexes with the membrane and allowed us to propose that in B. subtilis this enzyme is associated with the cytoplasmic membrane. D N A - p o l y m e r a s e a c t i v i t y of m e m b r a n e v e s i c l e s The finding that membrane vesciles from a wild type strain showed a higher in vitro activity of DNA synthesis than membrane vesciles from a polA~ mutant was an indirect indication for an association of DNA-polymerase I with the membrane in B. subtilis (Fig. 7 A). The results obtained also demonstrated that there was no difference in the DNA-degrading activity of membrane vesicles from these bacteria (Fig. 7B). The higher level of the DNA-polymerase activity in wild type cells coincided with the increased ability to bind homologous DNA in vitro (Fig. 6). Moreover, our experiments showed resistance of the DNA synthesis observed on the isolated cell membranes of the wild type strain to pCMB and N-EMI (Table 1). Conversely, the polymerase activity of membranes from the cells of a polA" strain was essentially inhibited by these substances. The resistance to thiolic substances is one of the features to discri-

Fig. 7. DNA-polymerase (A) and DNA-degrading (B) activity of membrane vesicles of B. subtilis 228 w. t. and 1306 polA' A — DNA synthesis (cpm • 10"3/1 mg of protein) without addition of DNA (1) and with addition of native (2) or "activated" DNA (3) of B. subtilis 228 w. t.; left — 228 w. t. strain, right — 1306 polA~ strain. B — dependence of degradation of 3 H-DNA from B. subtilis 228 on the incubation time (acid insoluble 3 H-radioactivity, % of initial): 4, 6 —228 w. t.; 5, 7 — 1306 polA~.

DNA repair synthesis in bacterial cells

615

minate between bacterial DNA-polymerase I and polymerases II and III (KORNBERG and KORNBERG 1 9 7 4 ) . Partial inhibition of the polymerase activity at a high K C 1 concentration in the reaction mixture (Table 1) was probably caused by DMC dissociation which was shown to occur in solutions of a high ionic strength (HEIDRICH and OLSEN 1975). Thus, these data suggested that DNA-polymerase I participating in the DNA repair synthesis (KORNBERG and KORNBERG 1 9 7 4 ) was really located in the DNA-containing membrane vesciles and was likelay to be a DNA-binding protein during the UV-induced formation of DMC of the "repair" type. Supposed the DNA synthesis occurred at the sites of DNA binding to the membrane then the absence of a noticeable stimulation of DNA synthesis in vitro by the addition of exogenous DNAtemplate could be explained by a relatively small amount of free sites available for DNA binding in the isolated membrane vesicles (Table 2). A stimulation of the DNA synthesis after the addition of exogenous DNA would, therefore, be expected if the endogenous DNA had been released from the binding sites before the addition of exogenous DNA. To check this, we used the DNA-gyrase inhibitors NA and NB to dissociate DMCs by incubation of intact cells with these agents (Fig. 2, 4). When we used membrane vesciles obtained from the cells treated in this way for initiating the DNApolymerase reaction, we really found a substantial increase in the DNA synthesis of samples with the added exogenous DNA as compared to preparations incubated without DNA addition (Table 2). When the membranes were isolated from the cells irradiated by UV-light after treatment with the antibiotics practically no* stimulation Table 1 The in vitro effect of KC1, N - E M I and y C M B on the DNA-synthesizing activity of the membrane vesicles from B. subtilis. The amount of [ 3 H]thymidine incorporation (cpm/mg protein) over a 4 0 min incubation period was expressed as per cent of control level in the samples with the addition of thiolic poisons and KC1. The average d a t a of 5 experiments are presented. D N A synthesis, % of control

Experimental conditions

228 (w.t.)

Control KC1, 350 m M KC1, 350 mM + N - E M I , 5 mM KC1, 350 mM + í>CMB, 1 mM N - E M I , 5 mM

1306

100 48.5 25.7 20.9 78.3 92.8

PCMB, 1 mM

(polk~) 100 18.7 10.4 6.8 14.3 11.4

Table 2 In vitro DNA synthesis by the membrane vesicles from B. subtilis cells treated with nalidixic acid or novobiocin and irradiated with UV-light. The table presents the average data of 6 independent measurements of DNA synthesis in the presence ( + DNA) and in the absence (— DNA) of activated B. subtilis 228 DNA in the reaction mixture. H-incorporation for 4 0 min incubation, cpm/mg protein

3

Experimental conditions

+DNA

-DNA Control UV-irradiation Nalidixic acid NA + U V Novobiocin N B + UV

24406 16493 13243 10869 14578 11495

± ± ± ± ± ±

1220 792 1073 826 1385 793

26573 16727 22076 11391 24873 12095

± ± ± ± ± ±

1599 820 1634 954 1915 1330

616

V . G. BEZLEPKIN, YIT. Y U . MALINOVSKY a n d A . I . GAZIEV

of the DNA synthesis following the addition of exogenous DNA was observed. This was probably due to a saturation of DNA-membrane association sites as a consequence of binding of UV-damaged DNA to the membrane during the repair process.

Discussion The increase in the amount of DNA associated with the membrane in UV-irradiated B. subtilis cells as observed in our experiments can be explained either by the formation of DNA-protein cross-links or by additional chromosome binding to the membrane. The first explanation seems less reasonable. Within the dose interval of UV-irradiation used (from 0 to 80 J/m a ) for the induction of DNA repair synthesis the formation of DNA-protein cross-links is characterized by a linear dependence on the UV-dose ( S I G A E V A et al. 1 9 8 1 ) . The kinetics of DNA binding to the membrane observed in our experiments (Fig. 1, 4) was different and showed a distinct maximum. Moreover, an equal amount of the DNA-protein cross-links was formed in UV-irradiated cells of polA+ and polA~ strains. Induction of DNA repair synthesis in B. subtilis by N-methyl-N-nitrosourea has been demonstrated previously ( C H E F R A N O V A and G A Z I E V 1 9 7 9 ) to result in an increase in the amount of DNA bound to the membrane. Under these conditions no additional DNA binding to the membrane was observed in the cells of polA~ mutant. It should be noted that there was no increase of DNA in DMC after UV-irradiation of the uvr_1 strain (Fig. 1). Thus, from our point of view, the second explanation is more preferable. The results obtained allowed us to propose the existence of a functional relationship between the DNA repair svnthesis in the UV-irradiated cells of B. subtilis and the increase in the amount of DNA associated with the membrane. The correlation between the increase of DNA binding to the membrane and the intensity of DNA repair synthesis (Fig. 2) as well as the localization of the newly synthesized DNA in DMCs in the cells with inhibited DNA replicative synthesis (Fig. 3) confirmed that the DNA repair synthesis occurred in DMC. It is interesting that inhibition of replicative DNA synthesis by NA and NB treatment is accompanied by the DMC dissociation in intact cells (Figs. 2—4). Since NA and NB have been shown to be inhibitors of DNA-gyrase in bacterial cells (CozZ A R E L L I 1 9 7 7 , S T A U D E N B A U E R 1 9 7 8 ) one may conclude that the latter phenomenon was caused by irreversible conformational changes of DNA-membrane binding sites and a disturbance of chromosome packing. It is believed that binding of the DNA to the membrane results in a topological fixation of the DNA. This is necessary for enzymes to function among them DNA-gyrase or other topoisomerases ( G E L L E R T et al. 1 9 7 6 ) forming a superhelical DNA structure. I t is believed that such a superhelical structure is necessary for both the initiation of DNA synthesis and the elongation of polynucleotide chains during the replicative DNA synthesis ( S T A U D E N B A U E R 1 9 7 6 ) . The UV-induced contacts of DNA with the membrane were not destroyed by NA or NB treatment. This was to be expected when newly formed DMCs were the sites of repair synthesis. It should be pointed out that their formation and functioning did not depend on the activity of DNA-gyrase. This was in agreement with data from other authors who showed that the inhibitors of replicative DNA synthesis and DNA-gyrase such as NA, NB as well as oxolinic acid and coumermycin did not inhibit the DNA repair synthesis ( C O Z Z A R E L L I 1 9 7 7 , S T A U D E N B A U E R 1 9 7 6 , 1 9 7 8 , R Y A N 1 9 7 6 ) , although a recovery of high molecular weight DNA was significantly delayed in the presence of these substances ( E B E R L E and M A S K E R 1 9 7 1 , R Y A N 1 9 7 6 ) . An increase in the amount and size of "anchor" DNA fragments in membrane vesicles obtained from the UV-irradiated cells of wild type strains can be explained by the

DNA repair synthesis in bacterial cells

617

changes in the steric accessibility of these DNA fragments to DNAase I during the preparation of vesicles. The mechanism of such phenomenon may be as follows. The new contacts of DNA with the membrane were formed at membrane sites situated near the DMCs which preexisted in intact cells. Therefore, both the DNA fragments directly bound to the membrane proteins and the DNA regions between two neighbouring binding sites were protected from nuclease degradation. However, it is as well possible that the sites of additional binding were clearly distinct from the DMCs which preexisting in intact cells. In this case, there would be not mutual overlapping of zones for a protection of the bound DNA fragments by the membrane structures. The increase in the size of "anchor" DNA fragments observed in this case would be caused solely by the conformational peculiarities of new DMCs within which a considerably larger part of the DNA fragments is blocked by the membrane structures than with the complexes of intact cells. I t , therefore, is reasonable to assume that the DNA binding to the membrane in the complexes existing in intact cells and in DMCs of the "repair" type is provided by different proteins. Thus, in UV-irradiated B. subtilis cells DMCs which are qualitatively different from the complexes in intact cells were formed. They are believed to be essential in the repair synthesis of UV-induced DNA damages. Direct evidence for the repair synthesis occurring at the membrane was obtained from pulse incorporation of [ 3 H]thymidine into DNA which was associated with the membrane and inaccessible for DNAse I action. During these pulse labelling experiments the replicative DNA synthesis was inhibited (Fig. 4C). A comparison of in vitro DNA-polymerase activity of B. subtilis membrane vesicles of polA~ and wild type strains showed that the vesicles of wild type strain were characterized by a higher level of activity (Fig. 7 A). The difference in the activity could not be attributed to various levels of nuclease activity in the isolated membranes since they were practically equal in comparable control strains (Fig. 7 B ) . The data of these experiments indicated that DNA-polymerase I was located within the membrane vesicles to which the DNA was bound. This was also confirmed by the resistance to PCMB and N-EMI of in vitro DNA synthesis at isolated membranes from cells of a wild type strain. In contrast, DNA-polymerase activity at the membranes from the cells of polA~ strain was strongly inhibited by these agents (Table 1). Our data agreed with the results of ZERIAL et al. (1978) and MANOIL et al. (1977). Thus, it can be proposed that in B. subtilis cells DNA-polymerase I is the membrane-associated protein which specifically binds the chromosome to the membrane during the UV-induced DNA repair synthesis. The results obtained allowed us to propose the following model of UV-induced DMC formation in B. subtilis. The DNA damages induced by UV-light in a cell are recognized by a UV-specific endonuclease that makes an incision break near the damage. Such breaks result in a relaxation of superhelical chromosomal DNA and in a 'release' of DNA loops. According to HECHT (1976) these loops released from superhelical chromosomal DNA may have contour lenght'of up to 30 ixrri. This allows an interaction of the relaxed chromosome with the membrane and eventually leads to the formation of the additional DMC (as compared to the intact cell) by participation of single-stranded DNA fragments at. the sites of the incision break and polymerase I localized at the membrane. The formation of UV-induced DMCs of the "repair type" and initiation of repair synthesis evidently did not depend on DNA-gyrase activity as they were resistant to the inhibitors of this enzyme. I t is reasonable to expect that at the end of repair synthesis the remaining breaks are ligated by DNA-ligase. The presence of DNA-ligase at the membrane of bacterial cells has been shown by GREEN and FIRSHEIN (1976) and

618

V . G . B E Z L E P K I N , Y U . Y U . MALINOVSKY a n d A . I . G A Z I E V

MANOIL et al. ( 1 9 7 7 ) . A f t e r l i g a t i o n t h e D N A is r e l e a s e d f r o m t h e c o m p l e x a n d t h e c h r o m o s o m e is c o n v e r t e d t o a c o m p a c t f o r m b y t h e a c t i o n of D N A - g y r a s e . I n h i b i t i o n of D N A - g y r a s e w i t h N A r e s u l t s i n t h e r e l a x a t i o n of t h e f o l d e d c h r o m o s o m e a s i t w a s s h o w n b y DRLICA a n d SNYDER ( 1 9 7 8 ) . Thus, the results obtained allowed us t o state t h a t D N A - m e m b r a n e interactions in U V - i r r a d i a t e d B. subtilis cells were a n e c e s s a r y s t e p in D N A repair synthesis. D M C s of t h e " r e p a i r " t y p e q u a l i t a t i v e l y d i f f e r e d f r o m D M C s of i n t a c t cells. T h e s i n g l e - s t r a n d ed D N A fragments and the membrane-associated D N A polymerase I appeared to be i n v o l v e d in mediating contact b e t w e e n the c h r o m o s o m e d a m a g e d sites a n d the multienzyme complex located at the membrane.

References APOSHIAN, H . V . and KORNBERG, Chem., 287, 5 1 9 - 5 2 5 .

A., 1 9 6 2 . E n z y m a t i c synthesis of deoxyribonucleic acid. J . biol.

CHEFRANOVA, O. A. a n d GAZIEV, A. I., 1979. T h e s t u d y of D N A - m e m b r a n e interaction u p o n induction of repair synthesis b y m e t h y l nitrosourea. Proc. Acad. Sci. U S S R , 1244, 755—758. COZZARELLI, N. R . , 1977. T h e mechanism of action of inhibitors of D N A synthesis. A n n . R e v . Biochem., 46, 6 4 1 - 6 6 8 .

M. a n d A V E R B E C K , D . , 1 9 8 0 . D N A - m e m b r a n e complex restoration in Micrococcus radiodurans a f t e r X-irradiation : relation t o repair D N A synthesis a n d D N A degradation. I n t e r . J . R a d i a t . Biol., 3 8 , 3 1 — 5 2 , 1 9 8 0 . Some factors influencing D N A - m e m b r a n e complex restoration in Micrococcus radiodurans a f t e r X-irradiation. I n t . J . R a d i a t . Biol., 38, 109. D R L I C A , K . a n d S N Y D E R , M . , 1 9 7 8 . Superhelical Escherichia coli D N A : relaxation b y coumermycin. J . molecular Biol., 120, 145 — 154. E B E R L E , H . a n d M A S K E R , W . , 1 9 7 1 . E f f e c t of nalidixic acid on semiconservative replication a n d repair synthesis a f t e r ultraviolet irradiation in Escherichia coli. J . Bacteriol., 105, 9 0 8 — 9 1 2 . E L K I N D , M . M . a n d C H A N G - L I U , C . - M . , 1972. R e p a i r of D N A complex f r o m X - i r r a d i a t e d Chinese h a m s t e r cells. I n t . J . R a d i a t . Birl. 22. 75 — 90. Actinomycin D inhibition of repair of a D N A complex f r o m Chinese h a m s t e r cells. I n t . J . R a d i a t . Biol 22, 313—324. DARDALHOJJ-SAMSOKOFF,

G A Z I E V , A . I . , CHEFRANOVA, O . A . , B E Z L E P K I N , V . G . a n d Z A K R Z H E V S K A Y A , D . T . , 1 9 8 0 . D N A - m e m -

b r a n e interactions in t h e repair of D N A in Bacillus subtilis. I n : D N A - R e c o m b i n a t i o n , I n t e r actions a n d R e p a i r ( E d i t o r s : S . ZADRAZIL a n d J . S P O N A R ) , pp. 519—529. P e r g a m o n Press Oxford a n d New Y o r k . G E L L E R T , M . , M I Z U U C H I , K . , O ' D E A , M . H . a n d N A S H , H . A., 1 9 7 6 . D N A gyrase: A n enzyme t h a t introduces superhelical t u r n s into D N A . Proc. n a t . Acad. Sci. USA, 73, 3 8 7 2 — 3 8 7 6 . G R E E N , M . a n d F I R S H E I N , W . , 1 9 7 6 . Role of deoxyribonucleic acid ligase in a deoxyribonucleic acid m e m b r a n e fraction e x t r a c t e d f r o m pneumococci. J . Bacteriol., 126, 777 — 784. H E C H T , R . M., 1 9 7 6 . Autoradiographic visualization a n d sedimentation properties of unfolded bacterial nucleoid D N A . I n : Molecular Mechanisms in t h e Control.of Gene Expression ( E d i t o r s : D. P . N I E R L I C H , V . J . B U T T E R a n d C. F . Fox), pp. 4 5 — 5 0 . New York. H E I D R I C H , H.-G. a n d O L S E N , W . L., 1975. Deoxyribonucleic acid envelope complexes f r o m Escherichia coli. A complex-specific protein a n d its possible f u n c t i o n for t h e stability of t h e complex. J . Cell. Biol., 67, 4 4 4 - 4 6 0 . J O E N J I E , H . , K O N I N G S , W . N. a n d V E N E M A , G., 1 9 7 4 . I n t e r a c t i o n between exogenous deoxyribonucleic acid m e m b r a n e vesicles isolated f r o m Bacillus subtilis 1 6 8 . J . Bacteriol, 119, 7 8 4 — 7 9 4 . KONINGS, W . N . , BISSCHOP, A . , V E E N H U I S , M . a n d VERMEULEN, C. A . , 1 9 7 3 . N e w p r o c e d u r e f o r t h e

isolation of m e m b r a n e vesicles of Bacillus subtilis a n d a n electron microscopy s t u d y of their u l t r a s t r u c t u r e . J . Bacteriol., 116, 1456 — 1465. KORNBERG, T . a n d KORNBERG, A., 1974. Bacterial DNA-polymerases. I n : T h e E n z y m e s (3rd Edition), Vol. 10 ( E d i t o r : P . D . BOYER), p p . 119 — 144. Academic Press New York a n d London. LYDERSEN, B. K . a n d PETTIJOHN, E . D., 1977. I n t e r a c t i o n s stabilizing D N A t e r t i a r y s t r u c t u r e in t h e Escherichia coli chromosome investigated with ionizing radiation. Chromosoma, 62,199—215. M A N O I L , C., S I N H A , N . a n d A L B E R T S , B . , 1 9 7 7 . Intracellular DNA-protein complexes f r o m bacteriophage T 4 infected cells isolated b y a r a p i d t w o step procedure. Characterization a n d identification of t h e protein components. J . biol. Chem., 2 5 2 , 2 7 3 4 — 2 7 4 1 . MARMUR, J . , 1961. A procedure for t h e isolation of deoxyribonucleic acid f r o m microorganisms. J . molecular Biol., 3, 2 0 8 - 2 1 8 . MCGRATH, R . A. a n d WILLIAMS, R . W., 1966. Reconstruction in vivo of irradiated Escherichia coli deoxyribonucleic acid: t h e rejoining of broken pieces. N a t u r e , 212, 534—535.

DNA repair synthesis in bacterial cells OGASAWARA, N . , SEIKI, M . a n d YOSHIKAWA, H . , 1 9 8 1 . I n i t i a t i o n of D N A r e p l i c a t i o n i n

619 Bacillus

subtilis. V. Role of DNA gyrase and superhelical structure in initiation. Mol. Gen. Genet., 181, 332-337.

RYAN, M. J., 1976. Coumermycin A T : a preferential inhibitor of replicative DNA synthesis in Escherichia coli. I. In vivo characterization. Biochemistry, 15, 3769 — 3782. SIGAEVA, V. A., MALININA, E. A. and GAZIEV, A. I., 1981. UV-induced DNA-protein cross-links and possibility of elimination thereof. Radiobiol. (USSR), 21, 568—571. STAUDENBAUER, W. L., 1976. Replication of Escherichia coli DNA in vitro. Inhibition by oxolinic acid. Europ. J . Biochem., 62, 4 9 1 - 4 9 7 . STAUDENBAUER, W. L., 1978. Novobiocin — a specific inhibitor of semiconservative DNA replication in permeabilized Escherichia coli cells. J . molecular Biol., 96, 201 — 205. STTEOKA, N. and HAMMERS, J . M., 1974. Isolation of DNA-membrane complexes in Bacillus subtilis. Proc. nat. Acad. Sci. USA, 71, 4787—4791. SUGIITO, A. and BOTT, K. P., 1980. Bacillus subtilis deoxyribonucleic acid gyrase. J . Bacteriol., 141, 1 3 3 1 - 1 3 3 9 . TIBBETTS, C., JOHANSSON, K . a n d PHILIPSON, L . , 1 9 7 3 . H y d r o x y a p a t i t e c h r o m a t o g r a p h y a n d f o r -

mamide denaturation of adenovirus DNA. J . Virol., 12, 218—225. YAMAGUSHI, K. and YOSHIKAWA, H., 1977. Chromosome-membrane association in Bacillus subtilis. I I I . Isolation and characterization of a DNA-protein complex carrying replication origin markers. J . molecular Biol., 110, 219—235. ZERIAL, A., GELMAN, J . and FIRSHEIN, W., 1978. Glycolipids stimulate DNA-polymerase activity in DNA-membrane fraction and in partly purified polymerase system extracted from pneumococci. J . Bacteriol., 135, 7 8 - 8 9 . Mailing address : Dr. V. G. BEZLEPKIN Department of Radiobiology Institute of Biological Physics The USSR Academy of Sciences Pushchino, Moscow Region, 142292, USSR

Zeitschrift f ü r Allgemeine Mikrobiologie

23

1983

10

621-624

(Departments of Chemistry and Agricultural Biology, University of Ibadan, Ibadan, Nigeria).

Production of oxalic acid by some fungi infected tubers 0 . FABOYA, T . IKOTUN a n d 0 . S. FATOKI

(Eingegangen

am 8. 4. 1983)

Oxalic acid (as oxalate) was detected in four tubers commonly used for food in Nigeria — Dioscorea rotundata (White yam), Solatium, tuberosum (Irish potato), Ipomoea batatas (Sweet potato), and Manihot esculenta (cassava). Whereas healthy I. batata had the highest oxalic acid content, healthy M. esculenta contained the lowest. When all tubers were artificially inoculated with four fungi — Penicillium oxalicum CURIE and THOM, Aspergillus niger VAN TIEOH, A. flavus and A. tamarii KITA, there was an increase in oxalate content/g of tuber tissue. The greatest amount of oxalate was produced by P. oxalicum in D. rotundata tuber. Consistently higher amounts of oxalate were produced by the four fungi in infected sweet potato tuber t h a n in any other tuber and consistently lower amounts of oxalate were produced by the four fungi in Irish potato tuber. Differences in the carbohydrate type present in the tubers and in the biosynthesis pathway are thought to be responsible for variation in the production of oxalate in the different tubers by the four fungi used. Oxalic a c i d has b e e n s h o w n t o b e p r o d u c e d b y f u n g i in vitro ( M A X W E L L a n d B A T E MAN 1 9 6 8 , M A X W E L L a n d L U M S D E N 1 9 7 0 , M A X W E L L 1 9 7 3 ) a n d in vivo in i n f e c t e d p l a n t s (BATEMAN a n d B E E R 1 9 6 5 , IKOTUN unpublished, MAXWELL and LUMSDEN 1 9 7 0 ) . I t h a s a l s o b e e n d e t e c t e d in s o m e c o m m o n g r e e n l e a f y v e g e t a b l e s c o n s u m e d in N i g e r i a (FABOYA, u n p u b l i s h e d ) . T h e p r e s e n c e of o x a l a t e s in o t h e r p l a n t p a r t s u s e d for f o o d in N i g e r i a has, h o w e v e r , n o t b e e n d o c u m e n t e d . A p a r t f r o m its i n f l u e n c e o n p l a n t t i s s u e d e g r a d a t i o n a n d in p a t h o g e n e s i s b y i n v a d i n g microorganisms (BATEMAN and B E E R 1 9 6 5 , LUMSDEN 1 9 6 9 , MAXWELL a n d LUMSDEN 1970) its e f f e c t o n t u b e r s a n d f o o d s t u f f d e r i v e d f r o m t h e m has n o t b e e n c o n s i d e r e d , a t l e a s t in Nigeria. T h i s is a report of t h e s t u d y carried o u t o n t h e p r o d u c t i o n of o x a l i c a c i d in s o m e t u bers c o m m o n l y u s e d for f o o d a n d in t h e p r e p a r a t i o n of f e e d s t u f f for a n i m a l c o n s u m p t i o n in N i g e r i a . T h e b i o s y n t h e s i s of o x a l a t e a n d t h e e f f e c t s of o x a l a t e o n t i s s u e degrad a t i o n , o n f o o d a n d e v e n t u a l l y o n a n i m a l s are discussed.

Materials

and

methods

Four fungi used in this study namely Penicillium oxalicum, Aspergillus niger, A. flavus, and A. tamarii were isolated from naturally infected white yam (Dioscorea rotundata) on plain agar and t h e n subcultured on potato dextrose agar. Tubers of white yam (D. rotundata), Irish potato (Solanurn tuberosum), sweet potato (Ipomoea batatas), and cassava (Manihot esculenta) were bought in an open market, washed clean in running t a p water and rinsed twice in distilled water. All tubers were surface-sterilized by swabbing with cotton wool soaked in 70% alcohol. Tubers weighing 100 —150 g were selected, then 8 mm-diameter and 20 mm-deep holes were bored into each using a sterile size 4 corkborer. Fungal cultures (96 hr-old) on potato dextrose agar medium were cut into 6 mm-diameter discs using sterile size 3 corkborers and were used to inoculate the holes bored into the various tubers. The hole was closed with a plug of tuber tissue scooped out with the corkborer. Tubers so inoculated were then incubated in separate polythene bags for seven days to maintain a high relative humidity at room temperature (27 °C). After seven days, infected tubers were removed from the polythene bags, grated to tiny pellets, ovendried at 60 °C and then ground into fine powder. Extraction of oxalic acid and subsequent 42

Z. Allg. Mikrobiol., Bd. 23, H. 10

622

0 . FABOYA, T . IKOTUN a n d O. S. FATOKI

estimations in infected and healthy (control) tubers were carried out by following an improved method of A N D R E W S and V I S E R ( 1 9 5 1 ) , as follows: Two grams of ground samples from each of the infected tubers were transferred into a 25 ml glass-stoppered graduated test tube, 20 ml of 30% HC1 were added and mixed by gently inverting the tube at regular intervals for 5 min. Then about 4 g ammonium sulphate was added until the solution was half saturated. The tube were set aside for 30 min. The supernantant (acid extract) was filtered into a 25 ml standard flask and was made up to 25 ml mark with 30% HC1. 10 ml-aliquotes of this extract were then pipetted into the inner tube of a modified C L A U S E N extractor. The acid solution was then extracted with ether for 5 hrs. The ether extract was transferred quantitatively into a 250 ml Erlenmeyer flask and the outer extractor tube was rinsed with distilled water. Ether in the extract was evaporated off and the remaining aqueous solution was filtered into a 50 ml centrifuge tube. The pH of the solution was adjusted to 7.0 with 0.75 M NH,OH and acetic acid, then saturated calcium chloride solution was added in an amount that was double the equivalent expected oxalate content of the samples. The solution was maintained at 60 °C, overnight, in an oven. After centrifuging at 8,000 X g for 30 min, the supernatant was discarded and the precipitate was dissolved in 2 N sulphuric acid. The solution was transferred into a 250 ml Erlenmeyer flask and titrated hot with standard (0.008 M) potassium permanganate (KMn0 4 ). Amount of oxalate present was calculated as % oxalic acid (PIERCE and HAENISCH 1954).

Results and

discussion

Results given in Fig. 1 show that the highest amount of oxalic acid was produced in D. rotundata (white yam) by P. oxalicum, followed by A. niger, A. flavus, and A. tamarii in descending order. Results also show that D. rotundata tubers naturally contained about 0.09% oxalic acid/g dry wt of tuber. Statistically, there was no significant difference (at 5% level) between the different amounts of oxalic acid produced in D. rotundata tuber by the various fungi but there was a significant difference between the amount produced by P. oxalicum and the amount occurring naturally in healthy D. rotundata (control). Fig. 2 shows that the amount of oxalic acid produced/g dry wt of infected tubers of S. tuberosum was higher in P. oxalicum-iniected tubers than in the tubers infected by other fungi, although they were not statistically different. A. tamarii did not seem to produce oxalic acid in Irish potato tubers. Irish potato appears to be unsuitable for colonization by these rot-causing organisms, hence the low oxalic acid content of the infected tubers. In infected Ipomoea batatas tubers, A. flavus produced the highest amount of oxalic acid followed by A. tamarii, then A. niger. P. oxalicum produced the least. Healthy I. batatas (control) appears to contain the highest amount of oxalic acid of all the tubers (Fig. 3). Fig. 4 shows that in infected M. esculenta tubers, A. tamarii produced the highest amount of oxalic acid whereas A. niger produced the least. The amounts of oxalic acid produced by A. flavus and P. oxalicum were not significantly different from the amount produced by A. tamarii, but were significantly different from that produced by A. niger and in the control tuber. The highest amount of oxalic acid/g dry wt of tuber was produced in D. rotundata by P. oxalicum. All microorganisms produced the least amount of oxalic acid in Irish potato. Healthy sweet potato tubers contained the highest amount of oxalic acid whereas healthy cassava tubers contained the least. I t seems that plants whose glucose catabolism employs the tricarboxylic acid (TCA) cycle are capable of producing oxalic acid as a by-product of glyoxylate produced from isocitrate. This scheme has been proposed for Sclerotium rolfsii ( M A X W E L L and B A T E MAN 1968). However, the splitting of oxaloacetate to oxalate and acetate was proposed for A. niger ( H A Y A I S H I et al. 1956). The mechanism of oxalate biosynthesis is not the aim of this study. This study has shown that tubers of plants from different bo-

Production of oxalic acid

623

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te 0MI 0.02£ 0.00

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A.N

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Fig. 1. Percentage of oxalic acid/g infected yam tuber (A), Irish potato tuber (B), sweet potato tuber (C), and cassava tuber (D) produced by the various fungi. P. 0 . — Pénicillium oxalicum, A. N. — Aspergillus niger, A. F . — A.flavus, A. T. — A. tamarii, C — control

tanical families (Dioscoreaceae, Solanaceae, Convolvulaceae, and Euphorbiaceae) naturally contained (though low) some oxalic acid which increased as soon as they were invaded by rot-causing microorganisms. FABOYA (unpublished) has shown that many green leafy vegetables commonly consumed in Nigeria also contained some oxalic acid. Thus oxalic acid production is not restricted only to pathogenic fungi. Since the carbon source may influence how much oxalic acid can be produced in vitro (MAXWELL and BATEMAN 1 9 6 8 ) and probably in vivo the differences in the amount of oxalic acid produced in the tubers used for this study may have arisen from the fact that the different carbohydrates (carbon source) in the tubers may influence how much oxalic acid may be produced by healthy and by diseased tubers. Another cause of differences in oxalic acid production in the different tubers by the fungi used may be due to the differences in the oxalic acid biosynthesis pathway. Oxalic acid produced by pathogens such as Sclerotium rolfsii, Sclerotinia sclerotiorum has been shown to play an essential role in their pathogenic capabilities and for S. rolfsii, oxalic acid has been shown to contribute synergistically to the destruction of bean tissue (BATEMAN and B E E R 1 9 6 5 , MAXWELL and LUMSDEN 1 9 6 8 ) . Oxalic acid is known to sequester calcium and magnesium in the middle lamella of plant cell walls rendering 42»

624

0 . FABOYA, T . IKOTUN a n d 0 . S . FATOKI

t h e p e c t a t e l i a b l e t o h y d r o l y s i s b y cell w a l l - d e g r a d i n g e n z y m e s (BATEMAN a n d BEER 1 9 6 5 ) . A l s o o x a l i c a c i d l o w e r s t h e p H of t h e t i s s u e t o a l e v e l s u i t a b l e f o r t h e a c t i v i t y of the macerating e n z y m e also produced b y the pathogen. O x a l i c a c i d is a l s o i m p o r t a n t in f o o d s a n d f e e d s t u f f s f e d t o a n i m a l s . W h e r e t h e o x a l i c a c i d c o n t e n t of f o o d s is h i g h , t h e o x a l a t e c a n c o m b i n e w i t h c a l c i u m a n d m a g n e s i u m to form calcium a n d magnesium oxalate. These c o m p o u n d s so formed cannot be u t i l i z e d h e n c e t h e y a r e n o t a b s o r b e d i n t h e i n t e s t i n e (ADOLF a n d LIANG 1 9 4 2 ) . S u c h a n i mals as well as m a n m a y develop w e a k bones a n d teeth. Continuous feeding on such f o o d s m a y r e s u l t in o x a l u r i a (FINCKE a n d S H E R M A N 1 9 3 5 ) .

References ADOLF, W . H . a n d LIANG, C., 1942. F a t e of oxalic acid administered to r a t . J . biol. Chem., 146, 497-502.

ANDREWS, J . C. a n d VISER, E . T., 1951. T h e oxalic acid c o n t e n t of some common foods. Food Res., 16, 3 0 6 - 3 1 2 . BATEMAN, D. F. a n d BEER, S. V., 1965. Simultaneous production a n d synergistic action of oxalic acid a n d polygalacturonase during pathogenesis b y Sclerotium rolfsii. P h y t o p a t h o l o g y , 55, 204 - 2 1 1 .

FINCKE, M. L. a n d SHERMAN, H . C., 1935. T h e availability of calcium f r o m some tropical foods. J. biol. Chem., 110, 4 2 1 - 4 2 8 . H A Y A I S H I , O . , SHIMAZONO, H . , KITAGIRI, M . a n d SAITO, Y . , 1 9 5 6 . E n z y m a t i c f o r m a t i o n of o x a l a t e

a n d a c e t a t e f r o m oxaloacetate. J . Amer. Chem. Soc., 78, 5126—5127. LTJMSDEN, R . D., 1969. Sclerotinia sclerotiorum infection of bean a n d t h e production of cellulase. P h y t o p a t h o l o g y , 59, 6 3 5 - 6 5 7 .

MAXWELL, D. P . , 1973. Oxalate f o r m a t i o n in Whetzelinia sclerotiorum b y oxaloacetate acetylhydrolase. Physiol. P l a n t . P a t h . , 3, 2 7 9 - 2 8 8 . MAXWELL, D. P . a n d BATEMAN, D. F., 1968. Oxalic acid biosynthesis b y Sclerotium rolfsii. P h y topathology, 58, 1 6 3 5 - 1 6 4 2 . MAXWELL, D. P . a n d LUMSDEN, R . D., 1970. Oxalic acid production b y Sclerotinia sclerotiorum in infected bean a n d in culture. P h y t o p a t h o l o g y , 60, 1395 — 1398. PIERCE, W . C. a n d HAENISCH, E . L., 1954. Q u a n t i t a t i v e Analysis. J o h n Wiley New York, p. 520. M a i l i n g a d d r e s s : D r . TUNDE IKOTUN

D e p a r t m e n t of Agricultural Biology, University of I b a d a n I b a d a n , Nigeria

23

Zeitschrift f ü r Allgemeine Mikrobiologie

10

1983

625-633

(Akademie der Wissenschaften d e r D D R , Forschungszentrum fiir Molekularbiologie u n d Medizin, Zentralinstitut f u r Mikrobiologie u n d experimentelle Therapie, J e n a , D i r e k t o r : Prof. Dr. U. TAUBEKECK)

Ultrastructural characterization of core structures and paracrystalline inclusion bodies in L-form cells of streptomycetes J . GUMPERT

(Eingegangen

am 8. 3. 1983)

P r o t o p l a s t t y p e L-form cells of Streptomyces hygroscopicus a n d 8. griseus contain different t y p e s of inclusion bodies. Cytoplasmic cores a n d paracrystalline structures are peculiar inclusions which could not be observed in normal p a r e n t bacteria. T h e cytoplasmic cores are 1 —4 pim long a n d 0.05—0.25 iim b r o a d straight a n d stiff n o n - t u b u l a r structures consisting of homogeneous moderate electron opaque material. Paracrystalline inclusions have side-lengths between 0.2 a n d 0.5 (im and show a characteristic p a t t e r n of 15—20 nm thick straight d a r k lines and electron lucent intervening spaces of 20—30 n m . Both cytoplasmic cores a n d paracrystalline inclusions are a p p a r e n t l y proteins. Their occurrence in L-form cells indicates an altered synthesis of one or several proteins in these cell types. U n d e r a p p r o p r i a t e c o n d i t i o n s l y s o z y m e p r o t o p l a s t s f r o m Streptomyces strains are a b l e t o p r o p a g a t e in t h e p r o t o p l a s t s t a t e a s L - f o r m s . L - f o r m cells g r o w o n s o l i d a g a r m e d i a f o r m i n g characteristic colonies a n d in liquid m e d i u m as single spherical cells or a g g r e g a t e s (BAUDLER a n d GUMPERT 1979, GUMPERT 1 9 8 2 ) . E l e c t r o n m i c r o s c o p i c i n v e s t i g a t i o n s of L - f o r m c e l l s r e v e a l e d a g r o s s f i n e s t r u c t u r e s i m i l a r t o p r o t o p l a s t s of s t r e p t o m y c e t e s a n d L - f o r m s of o t h e r g r a m p o s i t i v e b a c t e r i a (GUMPERT 1 9 8 2 , GILPIN et al. 1973, COLE 1 9 6 8 ) c h a r a c t e r i z e d b y s p h e r i c a l or p l e o m o r p h i c s h a p e , r i b o s o m e - r i c h c y t o p l a s m , m o r e or l e s s d i s p e r s e d n u c l e o i d a r e a s , a n d b y a b s e n c e of cell w a l l s a n d mesosomes. Additionally, ultrastructural peculiarities occur which we could not obs e r v e in t h e p a r e n t b a c t e r i a or i n L - f o r m s of o t h e r b a c t e r i a . T h e s e p e c u l i a r s t r u c t u r e s c o n c e r n i n c l u s i o n b o d i e s a n d f o r m a t i o n of i n t r a c e l l u l a r a n d e x t r a c e l l u l a r m e m b r a n e s . I n t h i s p a p e r o c c u r r e n c e a n d p r o p e r t i e s of c o r e s a n d p a r a c r y s t a l l i n e i n c l u s i o n b o d i e s will b e d e s c r i b e d a n d d i s u c s s e d .

Materials

and

methods

The isolation procedure for L-forms f r o m lysozyme protoplasts on a n complex L-form induction m e d i u m (LIM), t h e f u r t h e r cultivation and selection of stable L-forms on a n L-form cultivation m e d i u m (LCM), a n d t h e a d a p t a t i o n t o grow in liquid media are described in detail in a previous p a p e r (GUMPERT 1982).

The p a r e n t bacteria of t h e L-forms were Streptomyces hygroscopicus NG-33-354 ( I M E T J e n a 40668) a n d Streptomyces griseus ( I M E T J A 5142) from t h e collection of t h e Zentralinstitut f ü r Mikrobiologie u n d experimentelle Therapie J e n a . Colonies of t h e stable S. hygroscopicus L-form and of an unstable S. griseus L-form grown for 6 — 12 days on LCM agar a n d L-form cells grown for 6 — 10 days in liquid LCM were investigated b y using u l t r a t h i n sections. Cells f r o m liquid cultures were e m b e d d e d in 1 % LCM agar before f i x a t i o n . Agar blocks containing L-form cells or colonies were f i x e d in glutaraldehyde (5% in solution B, 16 h, 4 °C), washed in solution B, f i x e d in 0 s 0 4 (1% in solution B, 4.5 h, room temperature), washed again in solution B, d e h y d r a t e d in a s c e n d a n t concentrations of acetone, a n d finally

23

Zeitschrift f ü r Allgemeine Mikrobiologie

10

1983

625-633

(Akademie der Wissenschaften d e r D D R , Forschungszentrum fiir Molekularbiologie u n d Medizin, Zentralinstitut f u r Mikrobiologie u n d experimentelle Therapie, J e n a , D i r e k t o r : Prof. Dr. U. TAUBEKECK)

Ultrastructural characterization of core structures and paracrystalline inclusion bodies in L-form cells of streptomycetes J . GUMPERT

(Eingegangen

am 8. 3. 1983)

P r o t o p l a s t t y p e L-form cells of Streptomyces hygroscopicus a n d 8. griseus contain different t y p e s of inclusion bodies. Cytoplasmic cores a n d paracrystalline structures are peculiar inclusions which could not be observed in normal p a r e n t bacteria. T h e cytoplasmic cores are 1 —4 pim long a n d 0.05—0.25 iim b r o a d straight a n d stiff n o n - t u b u l a r structures consisting of homogeneous moderate electron opaque material. Paracrystalline inclusions have side-lengths between 0.2 a n d 0.5 (im and show a characteristic p a t t e r n of 15—20 nm thick straight d a r k lines and electron lucent intervening spaces of 20—30 n m . Both cytoplasmic cores a n d paracrystalline inclusions are a p p a r e n t l y proteins. Their occurrence in L-form cells indicates an altered synthesis of one or several proteins in these cell types. U n d e r a p p r o p r i a t e c o n d i t i o n s l y s o z y m e p r o t o p l a s t s f r o m Streptomyces strains are a b l e t o p r o p a g a t e in t h e p r o t o p l a s t s t a t e a s L - f o r m s . L - f o r m cells g r o w o n s o l i d a g a r m e d i a f o r m i n g characteristic colonies a n d in liquid m e d i u m as single spherical cells or a g g r e g a t e s (BAUDLER a n d GUMPERT 1979, GUMPERT 1 9 8 2 ) . E l e c t r o n m i c r o s c o p i c i n v e s t i g a t i o n s of L - f o r m c e l l s r e v e a l e d a g r o s s f i n e s t r u c t u r e s i m i l a r t o p r o t o p l a s t s of s t r e p t o m y c e t e s a n d L - f o r m s of o t h e r g r a m p o s i t i v e b a c t e r i a (GUMPERT 1 9 8 2 , GILPIN et al. 1973, COLE 1 9 6 8 ) c h a r a c t e r i z e d b y s p h e r i c a l or p l e o m o r p h i c s h a p e , r i b o s o m e - r i c h c y t o p l a s m , m o r e or l e s s d i s p e r s e d n u c l e o i d a r e a s , a n d b y a b s e n c e of cell w a l l s a n d mesosomes. Additionally, ultrastructural peculiarities occur which we could not obs e r v e in t h e p a r e n t b a c t e r i a or i n L - f o r m s of o t h e r b a c t e r i a . T h e s e p e c u l i a r s t r u c t u r e s c o n c e r n i n c l u s i o n b o d i e s a n d f o r m a t i o n of i n t r a c e l l u l a r a n d e x t r a c e l l u l a r m e m b r a n e s . I n t h i s p a p e r o c c u r r e n c e a n d p r o p e r t i e s of c o r e s a n d p a r a c r y s t a l l i n e i n c l u s i o n b o d i e s will b e d e s c r i b e d a n d d i s u c s s e d .

Materials

and

methods

The isolation procedure for L-forms f r o m lysozyme protoplasts on a n complex L-form induction m e d i u m (LIM), t h e f u r t h e r cultivation and selection of stable L-forms on a n L-form cultivation m e d i u m (LCM), a n d t h e a d a p t a t i o n t o grow in liquid media are described in detail in a previous p a p e r (GUMPERT 1982).

The p a r e n t bacteria of t h e L-forms were Streptomyces hygroscopicus NG-33-354 ( I M E T J e n a 40668) a n d Streptomyces griseus ( I M E T J A 5142) from t h e collection of t h e Zentralinstitut f ü r Mikrobiologie u n d experimentelle Therapie J e n a . Colonies of t h e stable S. hygroscopicus L-form and of an unstable S. griseus L-form grown for 6 — 12 days on LCM agar a n d L-form cells grown for 6 — 10 days in liquid LCM were investigated b y using u l t r a t h i n sections. Cells f r o m liquid cultures were e m b e d d e d in 1 % LCM agar before f i x a t i o n . Agar blocks containing L-form cells or colonies were f i x e d in glutaraldehyde (5% in solution B, 16 h, 4 °C), washed in solution B, f i x e d in 0 s 0 4 (1% in solution B, 4.5 h, room temperature), washed again in solution B, d e h y d r a t e d in a s c e n d a n t concentrations of acetone, a n d finally

626

J . GTJMPERT

embedded in Mikropal (FERRAK). Solution B is a Na-acetate veronal buffer supplemented with HC1 and CaCl2 according to R Y T E R and KELLENBERGER (1958). An Ultrotome I I I (LKB) was used for preparing ultrathin sections. They were poststained with lead citrate according to R E Y N O L D S ( 1 9 6 3 ) and investigated in a SIEMENS Elmiskop I operating at 80 kV.

Results C y t o p l a s m i c core s t r u c t u r e s In peripheric and central regions of L-form colonies from 8. hygroscopicus and S. griseus as well as in liquid cultures cells can be observed containing characteristic core structures (Fig, 1, 2, 3). The frequency of such core-containing cell sections can reach up to 2% in corresponding colony regions. Normally, cores occur in spherical cells of 1—4(xm in diameter showing an intact ultrastructure. Cytoplasmic core structures are characterized by their shape, fine structure, and position within the cell. The shape is like a straight and stiff stick, 0.05—0.25 ¡¿m broad and 1—4 ¡.im in length. Thicker and irregular forms are exceptions. In ultrathin sections cores consist of a homogeneous moderate electron-opaque material (Fig. 4a and b). Discernible subunits were not observed. In some cases broader cores seem to be composed of smaller parts of 0.05 to 0.075 fxm in diameter but of the same orientation in length (Fig. 4b). In most cases cytoplasmic cores are situated in the central part of the cells, extending from one cell side to the other. Often they are larger than the cell diameter leading to local lengthening and deformations of the cell surface (Fig. 2). Independent of their position within the cell cytoplasmic cores are always surrounded by cytoplasm without direct contacts to nucleoid areas. As shown in Figs .2, 4, and 6 the ends of the cores are mostly in close contact with the cytoplasmic membrane. Some core structures showed a close connection with lineary arranged paracrystalline structures (Fig. 3).

Fig. 1. L-form cells of Streptomyces hygroscopicus in the periphery of a surface colony containing core structures. Phase contrast micrograph, 629:1

Nucleoid cores In many L-form cells core-like structures can be seen within the nucleoid areas in longitudinal and cross sections (Fig. 5 and 6). In comparison with the cytoplasmic cores nucleoid cores are thinner, more irregular and less homogeneous. In cross sections

Inclusions in Streptomyc.es

L-forms

627

Fig. 3. Cytoplasmic core (CC) connected w i t h a paracrystalline inclusion b o d y ( P J B ) in an L - f o r m cell of Streptomyces hyyroscopicus

628

J . GUMPERT

Fig. 4. Cytoplasmic cores showing the homogeneous moderate electron opaque fine structure and the attachment to cytoplasmic membrane. Longitudinal subunits are visible in b

Fig. 5. Longitudinal section of a nucleoid core in a cell of Streptomyces griseus grown in agar medium

Inclusions in Streptomyces

L-forms

629

they look circular and can be followed in up to 10 serial sections. Filamentous threads proceed from these structures. Larger cells with dispersed nucleoid areas can contain several nucleoid cores. Paracrystalline inclusion

bodies

Many L-form cells contain inclusion bodies characterized by a t y p i c a l periodic linear structure (Fig. 7). T h e y appear as rectangular areas with side-lengths between 0.2 to 0.5 [xm, and they resemble paracrystals (DEMAKTIKI et al. 1976, POPE et al. 19(58).

Fig. 6. Large L-form cell of Streptomyces hygroscopicus grown in liquid LCM medium. CM ¡cytoplasmic membrane; NUC: nucleoid core; CC: cytoplasmic core; V : vacuole; I B : inclusion body

630

J . GUMPERT

Fig. 7. Paracrystalline inclusion bodies in L-form cells of Streptomyces hygrocospicus. b) is an enlarged part from F i g . 3, a) shows a close a t t a c h m e n t of paracrystalline inclusion body with the cytoplasmic membrane (CM)

T h e electron dense straight lines are 15 — 20 nm thick and 200—300 nm long. They are separated by electron lucent intervening spaces of 20—30 nm. Similar to the cytoplasmic cores these structures occur in intact L-form cells, and they are usually surrounded by cytoplasmic material. As shown in Fig. 3 paracrystalline inclusion bodies are sometimes closely connected with cytoplasmic cores, or they occur side by side within one cell.

Discussion L-form cells of streptomycetes contain different types of inclusion bodies. Typical moderately contrasted ribosome-free areas, normally closely connected with the cytoplasmic membrane, and spherical, electron lucent bodies without membraneous borderings are most frequent. They can be observed also in protoplasts and vegetative hyphal cells of the corresponding strains ( G U M P E R T 1 9 8 2 ) . The cytoplasmic cores and paracrystalline structures, however, are ultrastructural peculiarities of the Streptomyces L-forms. No similar structures could be observed in very extended electron

Inclusions in Streptomyces L-forms

631

microscopic investigations of vegetative hyphal cells and spores of S. hygroscopicus and other actinomycetes. Using similar methods of cultivation and the same light and electron microscopic techniques we never found such cores and paracrystalline inclusion bodies in L-form cells of Proteus mirabilis, Escherichia coli, and Bacillus subtilis. However, core structures have been found in different L-forms by other authors. Three types can be differentiated: non-tubular cores, tubular membraneous cores, and tubular non-membraneous cores. The cytoplasmic cores in streptomycetes are of the non-tubular type. Their characteristic properties are: — — — — —

a stiff, straight shape with diameters between 0.05—0.25 ^m, they occur in intact viable cells, mostly one core per cell, they are surrounded by cytoplasm, they are attached to the cytoplasmic membrane at one or both ends, their length can project beyond the cell body in one or both directions without piercing the cytoplasmic membrane, — they are homogeneously contrasted without recognizable subunits.

Cores showing a similar characteristic were found in bacterial and L-form cells of group D streptococci (MCCANDLES et al. 1 9 6 8 , COHEN et al. 1 9 6 8 , COLEMAN and B L E I W E I S 1 9 7 7 ) and in L-form cells of Pseudomonas aeruginosa ( H U B E R T et al. 1 9 7 1 ) . Tubular membraneous cores were observed in L-forms of streptococci (COBFIELD and S M I T H 1968, COHEN et al. 1968, E D A et al. 1979a), Staphylococcus ( E D A et al. 1977) and E. coli (EDA et al. 1979 b). They represent a different type being smaller in their diameter, surrounded by a trilamellar membrane, and containing cytoplasmic material with ribosomes. The core structures have been interpreted as artifacts of fixation, as special mesosomal structures, as products of degeneration processes, as disregulated cell wall deposits, or as storage bodies. Non-tubular cytoplasmic cores in streptomycetes and in streptococci were found in cells fixed by different methods including glutaraldehyde, osmium tetroxide, and freeze-etching as well as in living cells by phase contrast microscopy. This shows clearly that they are no artifacts. The well-known fact that mesosomes are special formations of the periplasmic space which disappears during protoplast formation and that mesosomes are absent in protoplasts and protoplast type L-forms, exclude the assumption that cores are products of mesosomal material ( C O R F I E L D and S M I T H 1 9 6 8 ) . Furthermore, no clear correlation between mesosomes and cores could be found in streptococcal cells (COLEMAN a n d B L E I W E I S

1977).

The facts that cores occur in intact L-form cells only, that they never could be observed in degenerating normal hyphal cells, and that similar cores can be present in up to 9 0 % of StrejJtococcus cells from which they disappear during 5 min after transfer into fresh medium (COHEN et al. 1968) show that they are not products of degenerative processes. Because the fine structure of the non-membraneous cores is very similar to those of gram positive cell walls some authors suggest that they may be deposits of cell wall material containing the group D antigene formed as a consequence of a disturbed wall synthesis (COHEN el al. 1 9 6 8 , MCCANDLESS et al. 1 9 6 8 ) . I t was shown by COLEMAN and B L E I W E I S ( 1 9 7 1 , 1 9 7 7 ) that the typical cores in D streptococci are proteins containing no cell wall components. They are stable at pH 6; and their formation can be inhibited by chloramphenicol. Obviously also in Streptomyces L-form cells they are labile repositories for cellular proteins.

632

J.

GUMPERT

The fine structure of the periodic non membraneous lamellated inclusion bodies and their sometimes close connection with cytoplasmic cores allows the supposition that they consist of proteins, too. Similar periodic structures are known as paracrystals formed under special conditions for instances by lipoproteins (DEMARTINI et al. 1976) and other proteineous material. Comparable paracrystal structures were found also in unstable L-forms of Proteus mirabilis (KANDA et al. 1977), Streptococcus (COEFIELD a n d SMITH 1 9 7 0 ) a n d s p e r m i n e - t r e a t e d E.

coli

cells (CHO a n d D O Y 1 9 7 2 ) .

Obviously neither cytoplasmic cores nor paracrystals are incomplete defective phage structures. The nucleoid cores differ from the cytoplasmic cores by their smaller and irregular shape and their position within the cell. They are present in about 5 0 % of all cell sections mostly cross sectioned. I t is not yet clear whether these nucleoid cores are artificial aggregates of chromosomal proteins and nucleic acids due to glutaraldehyde fixation or wheter they represent those protein cores within the chromosomes responsible for the integrity and folding of the chromosomal DNA (WORCEL and BURGI 1972).

In the folded chromosome isolated from vegetative cells of S. hygroscopicus a high content (30%) af a characteristic histone-like protein could be found (SARFERT et al. 1982).

The formation of cytoplasmic cores and paracrystals must be considered as a result of alteration in the cellular protein synthesis. I t needs further investigations to clarify whether they are products of a new proteins (s) or whether they are formed by overproduction of usual proteins. A

cknowedgement

Many thanks to Mrs. MONIKA VOLKEL for excellent technical assistance.

References BAUDLER, E . and GUMPERT, J . , 1979. Isolation of a protoplast type L-form from Streptomyces hygroscopicus. Z. allg. Microbiol., lit, 363—365. CHO, K . Y . and DOY, C. H., 1972. Ultrastructure of spermine-treated Escherichia coli, including a polar organelle concerned with envelope synthesis. Aust. J . biol. Sci., 25, 543—551. COHEN, M . , M C C A N D L E S S , R . G . , KALMANSON, G . M . a n d G U Z E , L . B . ,

1968. Core-like

structures

in transitional and protoplast forms of Streptococcus faecalis. I n : GUZE, Microbial Protoplasts, Spheroplasts and L-Forms. William Wilkins Comp. Baltimore, p. 94-109. COLE, R . M., 1968. The structure of the group A streptococcal cell and its L-form. In: CARAVANO, Current Research on Group A Streptococcus. Medica Found Amsterdam — New York, p. 5 — 4 2 . COLEMAN, S. E . and BLEIWEIS, A. S., 1971. Cytochemistry of core-like structures in group D Streptococcae. Bacteriol Proc., 1971, 48. COLEMAN, S. E . a n d BLEIWEIS, A. S., 1 9 7 7 . U l t r a s t r u c t u r a l , physiological, a n d c y t o c h e m i c a l c h a -

racterization of cores in group D streptococci. J . Bacteriol., 12'), 445—456. CORFIELD, P. S. and SMITH, D. G., 1968. Microtubular structures in group D streptococcal L-forms. Arch. Mikrobiol., (53, 356 — 361. CORFIELD, P . S. and SMITH, D. G., 1970. Ultrastructural changes during propagation of a group D streptococcal L-form. Arch. Mikrobiol., 75, 1—9. DEMARTINI, M., INOUYE, S. a n d INOUYE, M., 1 9 7 6 . U l t r a s t r u c t u r e o f p a r a c r y s t a l s o f a lipoprotein

from the outer membrane of Escherichia

coli. J . Bacteriol., 127, 564—571.

EDA, T . , KANDA, Y . , MORI, CH. a n d KIMURA, S., 1 9 7 7 . M i c r o t u b u l a r s t r u c t u r e s in a stable sta-

phylococcal L-form. J . Bacteriol., 132, 1024—1026. EDA, T., KANDA, Y . , MORI, CH. and KIMURA, S., 1979a. Core-like structures in a stable L-form of Streptococcus pyogenes. Current Microbiol., 2, 211—214. EDA, T., KANDA, Y . , MORI CH. and KIMURA, S., 1979b. Core-like and microtubular structures in a stable L-form of Escherichia coli. Microbiol. Immunol., 23, 915—920.

Inclusions in Streptomyces

L-forms

G I L P I N , R . W . , Y O U N G , F . E . and C H A T T E R J E E , A . N . , 1 9 7 3 . of Bacillus subtilis 1 6 8 . J . Bacteriol., 1 1 3 , 4 8 6 — 4 9 9 .

633

Characterization of a stable L-form

GUMPERT, J . , 1982. Growth characteristics and ultrastructure of protoplast type L-forms f r o m streptomycetes. Z. allg. Mikrobiol., 22, 617—627. H U B E R T , E . G . , POTTER, C. S . , H E N S L E Y , T . J . , COHEN, M . , KALMANSON, G . M . a n d GUZE, L .

B.,

1971. L-forms of Pseudomonas

aeruginosa. Infection a n d I m m u n i t y , 4, 60—72. K A N D A , Y . , E D A , T . , M O R I , C H . a n d K I M U R A , S . , 1 9 7 7 . Phage-like particles in an unstable L-form of Proteus mirabilis. J . Electron. Microsc., 2 6 , 2 1 5 — 2 1 7 . MCCANDLESS, R . , COHEN, M . , KALMANSON, G . M . a n d GUZE, L . B . , 1968. C o r e s , m i c r o b i a l o r g a n e l -

les possibly specific to group D streptococci. J . Bacteriol., 96, 1400—1412. and R O D E , L . J . , 1 9 6 8 . Crystalline inclusions of Clostridium cochlaerium. J . Bacteriol., 96, 1859 — 1862. REYNOLDS, E . S., 1963. The use of lead citrate a t high p H as an electron opaque stain in electron microscopy. J . Cell Biol., 17, 208—212. R Y T E R , A . et K E L L E N B E R G E R , E., 1 9 5 8 . E t u d e au microscope électronique de plasmas contenant de l'acide deoxyribonucleique. Z . Naturforsch., 1 3 b , 5 9 7 — 6 0 5 . S A R P E R T , E., Z I M M E R , C H . , G U M P E R T , J . a n d L A N G , H., 1983. Folded chromosome structure a n d DNA-binding protein of Streptomyces hygroscopicus. Biochim. biophysica Acta, 740,118 —124. W O R C E L , A . a n d B U R G I , E . , 1972. On t h e structure of t h e folded chromosome of Escherichia coli. J . Mol. Biol., 71, 1 2 7 - 1 4 7 . P O P E , L . , YOLTON, D . P .

Mailing address : Dr. J . GUMPERT Zentralinstitut f ü r Mikrobiologie u n d experimentelle Therapie der A d W DDR-6900 J e n a , Beutenbergstr. 11

Zeitschrift für Allgemeine Mikrobiologie

23

1983

10

635—644

(Akademie der Wissenschaften der D D R , Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Ernährung, Potsdam-Rehbrücke, Direktor: Prof. Dr. H. SCHMANDKE)

Wirkung von Ölen und Fettsäuren auf Wachstum und Enzymbildung bei Thermoactinomyces vulgaris III. Einfluß von Kulturgefäßen, Stammaterial und Medienzusammensetzung A . LEUCHTENBERGER u n d H . R U T T L O F F

(Eingegangen

am 8.

4.1983)

The influence of rape-oil as antifoam agent on growth and protease synthesis of T. vulgaris was studied. The turbulence and the aeration conditions caused by the culture vessel have a fundamental importance for the so-called oil effect. A higher aeration rate induces an enhanced lysis of the mycelium and consequently a reduced enzyme synthesis. The tentency to lysis of the mycelium at different selectants is variable. The addition of oil to the culture medium reduces this tendency and promotes the protease production. The oil effect on the enzyme synthesis is higher in deficient medium than in full one.

Analysiert m a n die in der L i t e r a t u r angegebenen Befunde verschiedener Autoren über die Wirkung von Ölen oder aber von F e t t s ä u r e n auf den mikrobiellen Stoffwechsel ( L E U C H T E N B E R G E R U. R U T T L O F F 1 9 7 9 ) k o m m t man zu dem Ergebnis, daß Ausmaß und Art ihrer Wirksamkeit sowohl von den bestehenden Kulturbedingungen als auch von Qualität und Q u a n t i t ä t des eingesetzten Lipids abhängig sind. U m den aktivitätsfördernden Einfluß von Entschäumerölen beobachten und diesen f ü r die technische Herstellung von E n z y m e n nutzen zu können, ist eine Untersuchung verschiedener F a k t o r e n notwendig. Von besonderer Bedeutung sind die Belüftungsverhältnisse. Über die Wirkung von Ölzusätzen auf W a c h s t u m und Produktsynthese in Abhängigkeit von der Belüftungsintensität berichten z. B. ARAVINA et al. ( 1 9 7 6 ) , G A L Y N K I N et al. ( 1 9 7 8 ) sowie P A C A et al. ( 1 9 7 8 ) . D a ß Öle wie auch F e t t s ä u r e n bei Fermentationsansätzen unter Einsatz von Mikroorganismen nahe verwandter, ja sogar gleicher Arten, unterschiedliche Effekte zeigen können, wird aus der Gegenüberstellung von Ergebnissen einiger Autoren (LEUCHTENBERGER U. R U T T L O F F 1 9 7 9 ) sowie aus Vergleichsansätzen mit S t ä m m e n gleicher Art ( YAKOVLEVA et al. 1 9 8 2 ) ersichtlich. Auch über enge Beziehungen zwischen dem Nährstoffgehalt eines K u l t u r m e d i u m s und der Ölwirkung wird berichtet (YAMAMOTO et al.

1 9 6 4 , K O B A Y A S H I U. S U Z U K I 1 9 7 2 , G A L Y N K I N et al.

1 9 7 8 , F R A N Z K E et

al.

1979).

E s ist Anliegen dieser Arbeit, den Öleinfluß auf W a c h s t u m und Proteasesynthese von Thermoactinomyces vulgaris (T. vulgaris) in Abhängigkeit von der Art der K u l t u r gefäße, vom Stammaterial u n d von der Medienzusammensetzung zu untersuchen. Material

und

Methoden

Stammhaltung und -Konservierung: Verschiedene Selektanten von T. vulgaris werden in Form von Schrägagarkulturen auf Maisquellwasser (MQW) I-Agar bis zu 4 Wochen als Sporenkonserven mit Luvos Heilerde über lange Zeit gelagert (KLINGENBERG et al. 1979, LEUCHTENBERGER et al. 1979).

Zeitschrift für Allgemeine Mikrobiologie

23

1983

10

635—644

(Akademie der Wissenschaften der D D R , Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Ernährung, Potsdam-Rehbrücke, Direktor: Prof. Dr. H. SCHMANDKE)

Wirkung von Ölen und Fettsäuren auf Wachstum und Enzymbildung bei Thermoactinomyces vulgaris III. Einfluß von Kulturgefäßen, Stammaterial und Medienzusammensetzung A . LEUCHTENBERGER u n d H . R U T T L O F F

(Eingegangen

am 8.

4.1983)

The influence of rape-oil as antifoam agent on growth and protease synthesis of T. vulgaris was studied. The turbulence and the aeration conditions caused by the culture vessel have a fundamental importance for the so-called oil effect. A higher aeration rate induces an enhanced lysis of the mycelium and consequently a reduced enzyme synthesis. The tentency to lysis of the mycelium at different selectants is variable. The addition of oil to the culture medium reduces this tendency and promotes the protease production. The oil effect on the enzyme synthesis is higher in deficient medium than in full one.

Analysiert m a n die in der L i t e r a t u r angegebenen Befunde verschiedener Autoren über die Wirkung von Ölen oder aber von F e t t s ä u r e n auf den mikrobiellen Stoffwechsel ( L E U C H T E N B E R G E R U. R U T T L O F F 1 9 7 9 ) k o m m t man zu dem Ergebnis, daß Ausmaß und Art ihrer Wirksamkeit sowohl von den bestehenden Kulturbedingungen als auch von Qualität und Q u a n t i t ä t des eingesetzten Lipids abhängig sind. U m den aktivitätsfördernden Einfluß von Entschäumerölen beobachten und diesen f ü r die technische Herstellung von E n z y m e n nutzen zu können, ist eine Untersuchung verschiedener F a k t o r e n notwendig. Von besonderer Bedeutung sind die Belüftungsverhältnisse. Über die Wirkung von Ölzusätzen auf W a c h s t u m und Produktsynthese in Abhängigkeit von der Belüftungsintensität berichten z. B. ARAVINA et al. ( 1 9 7 6 ) , G A L Y N K I N et al. ( 1 9 7 8 ) sowie P A C A et al. ( 1 9 7 8 ) . D a ß Öle wie auch F e t t s ä u r e n bei Fermentationsansätzen unter Einsatz von Mikroorganismen nahe verwandter, ja sogar gleicher Arten, unterschiedliche Effekte zeigen können, wird aus der Gegenüberstellung von Ergebnissen einiger Autoren (LEUCHTENBERGER U. R U T T L O F F 1 9 7 9 ) sowie aus Vergleichsansätzen mit S t ä m m e n gleicher Art ( YAKOVLEVA et al. 1 9 8 2 ) ersichtlich. Auch über enge Beziehungen zwischen dem Nährstoffgehalt eines K u l t u r m e d i u m s und der Ölwirkung wird berichtet (YAMAMOTO et al.

1 9 6 4 , K O B A Y A S H I U. S U Z U K I 1 9 7 2 , G A L Y N K I N et al.

1 9 7 8 , F R A N Z K E et

al.

1979).

E s ist Anliegen dieser Arbeit, den Öleinfluß auf W a c h s t u m und Proteasesynthese von Thermoactinomyces vulgaris (T. vulgaris) in Abhängigkeit von der Art der K u l t u r gefäße, vom Stammaterial u n d von der Medienzusammensetzung zu untersuchen. Material

und

Methoden

Stammhaltung und -Konservierung: Verschiedene Selektanten von T. vulgaris werden in Form von Schrägagarkulturen auf Maisquellwasser (MQW) I-Agar bis zu 4 Wochen als Sporenkonserven mit Luvos Heilerde über lange Zeit gelagert (KLINGENBERG et al. 1979, LEUCHTENBERGER et al. 1979).

636

A . L E U C H T E N BERGER u n d H . R U T T L O F F

N ä h r m e d i e n : M Q W I - A g a r - M e d i u m : 1 0 g Maisstärke, 1 0 g Maisquellwasser ( 5 0 % i. T . ) , 5 g NaCl, 0 , 5 g CaCl 2 , 2 0 g A g a r - A g a r ; m i t L e i t u n g s w a s s e r a d 1 0 0 0 m l ; p H 7 , 0 . M Y P - M e d i u m : 3 g Trockenhefe, 5 g P e p t o n , 5 g M a g e r m i l c h p u l v e r ; m i t L e i t u n g s w a s s e r a d 1000 ml; p H 7,0. M Q W I - F l ü s s i g m e d i u m : W i e M Q W I - A g a r - M e d i u m , j e d o c h ohne Z u s a t z v o n A g a r - A g a r . MQW II-Flüssigmedium: Komponenten von MQW I-Medium, ergänzt mit 0,3 g Trockenhefe, 0 , 5 g Magermilchpulver, 0 , 5 g P e p t o n u n d 3 g 1 N E s s i g s ä u r e ; m i t L e i t u n g s w a s s e r a d 1 0 0 0 m l ; p H 7,0. MQW V-Flüssigmedium: 10 g Stärkehydrolyseprodukt ( S H P mit a-Amylase hydrolysierte K a r t o f f e l s t ä r k e , SCHIERBAUM et al. 1 9 7 7 ) , 5 0 g M Q W - H y d r o l y s a t (MQW 1 S t d . bei 5 0 °C m i t einem P r o t e a s e - A m y l a s e - G e m i s c h g e s c h ü t t e l t , a u f p H 7 , 0 eingestellt, filtriert), 5 g NaCl, 0 , 5 g C a C l 2 ; mit Leitungswasser ad 1000 m l ; p H 7,0. Kulturgefäße: 500-ml-Einstichkolben (E-Kolben): In 500-ml-Stehrundkolben mit langem H a l s werden 4 S c h i k a n e n ( D u r c h m e s s e r 3 c m , Tiefe 0 , 5 cm, ökantig) a n g e b r a c h t . Die K u l t i v a t i o n erfolgt a u f einem R u n d s c h w i n g t i s c h v o m T y p M K 5 6 / 1 5 des V E B FAKAL/Bad F r a n k e n h a u s e n . 2-1F e r m e n t o r : K o n s t r u k t i o n u n d B a u der A n l a g e d u r c h V E B A r z n e i m i t t e l w e r k Dresden sowie F a . W o L F / R i e s a u n t e r V e r w e n d u n g v o n 3-1-Glasgefäßen des V E B J e n a e r G l a s w e r k e / J e n a . 3 0 - 1 - F e r m e n t o r : K o n s t r u k t i o n u n d B a u der A n l a g e d u r c h den V E B E x c e l s i o r w e r k e H e i d e n a u u n t e r E i n s a t z v o n 30-1-Glasgefäßen des V E B J e n a e r G l a s w e r k e / J e n a . V o r k u l t u r u n d A r b e i t s k u l t u r : I n A b h ä n g i g k e i t v o m v e r w e n d e t e n K u l t u r g e f ä ß werden diese n a c h dem in Abbildung 1 dargestellten A b l a u f a n g e s e t z t . A n a l y t i k : W a c h s t u m s m e s s u n g : Z e n t r i f u g a t i o n v o n 3 ml K u l t u r m e d i e n u n d P r o t e i n b e s t i m m u n g im S e d i m e n t (LEUCHTENBERGER et al. 1 9 8 1 ) . B e s t i m m u n g der P r o t e a s e a k t i v i t ä t : Sie erfolgt n a c h einer modifizierten A N S O N - M e t h o d e . U n t e r V e r w e n d u n g einer l % i g e n Caseinlösung wird 2 0 m i n bei 5 5 °C inkubiert (KLINGENBERG et al. 1 9 7 9 ) . Die nachfolgenden E r g e b n i s s e sind D u r c h s c h n i t t s w e r t e v o n mindestens 3 V e r s u c h s a n s ä t z e n .

Ergebnisse Vergleich verschiedener Kulturgefäße Nach Ermittlung des positiven Einflusses von Rapsöl auf die Proteasebildung im 30-1-Fermentor ( L E U C H T E N B E R G E R et al. 1979) werden nunmehr Versuche zu dieser Problematik im Schüttelkolben durchgeführt. Hierbei zeigt sich, daß die aktivitätsfördernde Wirkung von Öl sehr durch die F o r m der verwendeten Kulturgefäße bestimmt wird. Vergleichende Fermentationsansätze in verschiedenen Kulturkolben (Abb. 2) sowie in Fermentoren führen zu Ergebnissen gemäß Abbildung 3. Danach liefern 100-ml-Kulturkolben mit steiler, glatter Gefäßwand absolut die höchsten E n zymaktivitäten, der Zusatz von Rapsöl bewirkt jedoch einen Aktivitätsrückgang. 500-ml-Rundkolben mit glatter Wandung ergeben geringere Aktivitätsausbeuten als 100-ml-Kulturkolben. E i n e Ölzugabe führt in keinem Falle zu einer nennenswerten Steigerung der E n z y m a k t i v i t ä t . I m Hinblick auf den Oleffekt zeigen 500-ml-Rundkolben mit 4 Schikanen (500-ml E - K o l b e n ) ähnliche Ergebnisse wie die Rührfermentor e n : Die Zugabe von Rapsöl führt zu einer deutlichen Erhöhung der Proteaseausschüttung. Die Ergebnisse lassen den Schluß zu, daß die durch die Schikanen bewirkte erhöhte Turbulenz die Belüftung intensiviert und damit einen wesentlichen Einfluß auf den Oleffekt ausübt. Zur weiteren Klärung dieses Problems sind Versuche in Schüttelkultur mit 500-mlRundkolben, die eine unterschiedliche Anzahl bzw. Tiefe von Schikanen besitzen, durchgeführt worden. Die Auswirkungen auf die Proteaseausschüttung sind in Abbildung 4, auf die Biomasseentwicklung in Abbildung 5 dargestellt. Die Resultate bestätigen die Vermutung, daß die Belüftungsintensität sowohl den öleffekt als auch Wachstum und Proteasebildung stark beeinflußt. Aus Abbildung 4 geht hervor, daß mit zunehmender Anzahl und Tiefe der Schikanen die Proteasebildung gemindert wird. Dieser Einfluß tritt jedoch bei den Kontrollvarianten stärker als bei den Varianten mit ölzusatz in Erscheinung. Während bei den Rundkolben mit glatter W a n d die

Sporenkonserve Glasampulle mit lurns-Heilerde VC lngertempemtur Vorkulturl MQWl-Agar. 56 °C, 20SM.

ca. 2x 109Sporen/ml i

Sporensuspension 10 m! Tween80-Lösg-[0ß1%] pro Röhrchen 0,S% Inokuium VorkultiirK 201MYP-Medium 50 X, 560 ty min 201/min, kStd.

1%

Inokuium

5% Inokuium

Arbeitskultur

500 ml Rundkolben m. Einstichen

A b b . 1.

V e r s u c h s a b l a u f in verschiedenen Kulturgefäßen

2-1- Fermentar 30-1-Fermentar

soiMMO. MOWR 0,1% Rapsöl 50°C 210 U/min ca. ifSStd.

« MQWO. MSWB 0,2%Rapsö! SO'C 500Uf min 601/ min ca. ttStd.

201 MQWIO. MOWZ 0,2% Rapsöl 50 "C 370 U/min 101/min ca. 22Std.

Variante I

Variante U

Variante HL

A b b . 2 . 3 V a r i a n t e n v o n K u l t u r k o l b e n ( v . r . n . 1.: 1 0 0 m l S t e i l w a n d k o l b e n m i t 3 0 m l M e d i u m , 500-ml-Rundkolben mit 50 ml Medium, 500-ml-Rundkolben mit 4 Schikanen und 50 ml Medium) 43

Z. Allg. Mikrobiol., Bd. 23, H. 10

638

A . LEUCHTENBERGER u n d H . RUTTLOFF

TE/ml

Fermentor

Kultur-

Rund-

Einstich-

flaschen

kalben

Aolben

Fermentor

Abb. 3. Wirkung von Rapsölzusätzen auf die Proteasebildung in verschiedenen Kulturgefäßen (MQW I-Medium, 21 Std., 0,1% Rapsölzusatz)

£

Kolbenform Zahl der Schikanen

Ä

—

—

2

2

4

Tiefe der Schikanen

—

—

0,5

0,5

0,5

0,5

Öl-Zusatz

—

0,1

—

0,1

—

0,1

4

*

1

1

cm

0,1

%

—

TEjml 737

800 700

6 lf9

600

Ski

500

U83

MO 295

300

383 /

ZOO

z

150

100

055

n

•

1

2

3

4

-

5

6

7

8

Varianten

Abb. 4. Einfluß der Belüftungsintensität auf die Proteasebildung (500 ml Rundkolben mit und ohne Schikanen, MQW I-Medium, 19 Std., 0,1% Rapsölzusatz)

639

Wirkung von Ölen und Fettsäuren auf T. vulgaris. III. Protein mg/ml 7 "

o o

o

o o o o o o o 1

Z.

3

4

5

6

7

8

Varianten Abb. 5. Wirkung der Belüftungsintensität auf die Biomasseentwicklung (Bedingungen wie Abb. 4)

Olzugabe zu einem deutlichen Aktivitätsrückgang gegenüber der Kontrolle f ü h r t , vermindert sich bei der Kolbenform mit Schikanen der Unterschied zwischen Kontrollu n d Ölvariante. Mit weiterer Zunahme der Belüftung liegt schließlich die Proteaseaktivität der Ölvariante deutlich über der entsprechenden Kontrolle. Die gleiche Tendenz zeigt sich hinsichtlich des Einflusses der Belüftung auf die Biomasseentwicklung (Abb. 5). E s wird festgestellt, daß das W a c h s t u m in der Kontrolle mit zunehmender Belüftung stark gehemmt und bei der Variante mit der größten Turbulenz das Mycel fast völlig lysiert. Die Zugabe von Rapsöl wirkt diesem Lyseprozeß entgegen, was sich u. a. in einer erhöhten Proteasebildung auswirkt. Eür weitere Versuche werden daher nur noch 500-ml-Rundkolben mit 4 x 0,5 cm tiefen Schikanen verwendet. Stammaterial Welchen Einfluß das f ü r die Ölversuche verwendete Stammaterial besitzt, wird mit Sporenkonserven unterschiedlicher Leistung geprüft (Tab. 1). E s zeigt sich, daß 0 , 1 % Rapsölzusatz bei allen getesteten Stammkonserven eine Steigerung der Proteasebildung herbeiführt. Die Höhe des Zuwachses ist jedoch verschieden. Ein Zusammenhang zwischen der absoluten Aktivitätshöhe u n d dem Steigerungsgrad k a n n aus den E r gebnissen nicht abgeleitet werden. E s wird jedoch bei der mikroskopischen Kontrolle der K u l t u r beobachtet, daß die verschiedenen Stämme zum Abbautermin einen unterschiedlichen Grad an Mycellyse aufweisen. Auf Grund dieses Hinweises werden weitere Konserven auf ihr Lyseverhalten in 500-ml-E-Kolben und dessen Auswirkung auf die Proteaseaktivität untersucht (Abb. 6). E s wird festgestellt, daß die einzelnen S t ä m m e eine unterschiedliche Lyseneigung unter den erhöhten Turbulenzbedingungen der Einstichkolben zeigen. Diese Mycellyse ist im komplexen löslichen Medium MQW V sowohl mikroskopisch als auch mittels Proteinbestimmung nachweisbar. Zwischen der Intensität der Mycellyse und der gebildeten proteolytischen Aktivität besteht ein Zusammenhang. S t ä m m e mit geringer Lyseneigung sind zu einer höheren Proteasebildung befähigt als solche mit starkem lytischen Effekt. 43*

640

A . L e u c h t e n b e r g e r und H . R u t t l o f f

Tabelle 1 Wirkung von Rapsöl auf die Proteasebildung durch Stämme mit unterschiedlichem Leistungsvermögen in 500-ml-E-Kolben (MQW I-Medium, 50 °C, 0,1% Rapsöl, p H 7,0) K — Sporenkonserve, ö l — Ölvariante, % — Leistung der ö l Variante bezogen auf 100% der Kontrolle Proteaseaktivität (TE/ml) verschiedener Sporenkonserven

Kulturdauer (Std.)

K 112 K

15 18 22

100 135 200

K 219

K 125

Öl 115 175 260

115 130 130

K

Öl

K

120 165 220

220 270 300

183 164 136

Lysegrad ( mikroskopische

Kulturdauer KÔ21

16

—

19

—

22

-t-

280 375 420

K31

KW

—

*

— —

+

+

# *

-H-

+

-H-

-H-

•tt-

•Hf

Lysegrad: - keine, + schwache Biomasse (Protein)

219 194 169

K

Öl

295 320 285

335 425 233

114 133 82

variante KS

K438

Öl

Beobachtung )

Stamm

(Std.)

128 193 248

K 305

K509

+

# mittlere, -Hf starke Lyse

nach 22 Stunden

mg/ml

0,5

Oß

S

0,1 TS/m! 800 _ 700

500

K521

K138

. *

Proteaseaktivität

I

KS

K31

u n KW

K509

Stamm

( TE/ml )

r

wo 300 200

100 ' ' • i ' ' i ' ' i i ' i'i 1619 22 16 1922 16 1922 161922 161922 16 19 22Std. K521 Km KS K31 KW K509 Stamm Abb. 6. Beziehung zwischen Lyseverhalten und Proteasebildung verschiedener Stämme in 500 ml-E-Kolben (MQW V-Medium, 50 °C, 22 Std., ohne Rapsöl)

Wirkung von ölen und Fettsäuren auf T. vulgaris.

641

III.

Medienzusammensetzung Von wesentlicher Bedeutung für die Ölwirkung ist die Medienzusammensetzung (Abb. 7). Vergleichsansätze in 2-1-Fermentoren unter Verwendung des nährstoffärmeren Grundmediums MQW I und des nährstoffreicheren Mediums MQW I I mit und ohne Ölzusatz zeigen, daß die durch Rapsöl verursachte stimulierende Wirkung auf die Proteasebildung im MQW I-Medium größer ist (um ca. 1 4 0 % ) als im MQW II-Medium (um ca. 8 0 % ) . Wmi m 3

300

OZ

200

700 16

19

22

W SM.

Abb. 7. Wirkung von Rapsöl auf die Proteasebildung in Medien mit unterschiedlichem Nährstoffgehalt (2-l-Fermentor, MQW I- und MQW II-Medium, 22 Std., 50 °C, 0 , 2 % Rapsölzusatz, 60 l Luft/Std., 500 U/min) 1 - MQW 1 , 2 - MQW I I , 3 - MQW I + 0 , 2 % Rapsöl, 4 - MQW I I + 0 , 2 % Rapsöl

Diskussion Nach den erzielten Befunden wird die Wirkung von Rapsöl auf das Wachstum von T. vulgaris und dessen Proteasesynthese durch die untersuchten Parameter in unterschiedlichem Ausmaß beeinflußt. So zeigt sich gemäß Abbildung 3 und 4, daß die in einem Fermentationsgefäß vorliegenden Belüftungs- und Turbulenz Verhältnisse ausschlaggebend für die Ölwirkung sind. Während in glattwandigen Kulturflaschen Öl keinen oder sogar einen hemmenden Effekt auf die Proteaseproduktion ausübt, wirkt es in Kulturflaschen mit Schikanen (Erhöhung der Turbulenz) aktivitätsfördernd. E s ist denkbar, daß einige Autoren infolge Verwendung ungeeigneter Kulturgefäße negat i v e B e f u n d e e r z i e l t h a b e n (ISMAILOVA U. LOGINÜVA 1 9 7 5 , ALFORD et al.

1971).

Über den Einfluß von Schikanen in Kulturflaschen auf den 0 2 -Übergang im Medium l i e g e n U n t e r s u c h u n g s e r g e b n i s s e v o n YAMADA et al. ( 1 9 7 8 ) v o r . S i e f i n d e n , d a ß d i e 0 2 -

Übergangsrate bei Erlenmeyerkolben mit 3 Schikanen bestimmter Größe etwa lOfa h höher ist als in glattwandigen Gefäßen. Die Kolben sind in dieser Hinsicht mit Riihrfermentoren unter normalen Arbeitsbedingungen vergleichbar. Aus der Tatsache, daß nur in Einstichkolben und in Rührfermentoren mit einer hohen Ö 2 -Übergangsrate ein stimulierender Effekt von Öl auf die Proteasesynthese bei T. vulgaris erzielt wird, ist einmal mehr der Einfluß von Öl auf die 0 2 -Versorgung der Kultur abzuleiten. In Übereinstimmung mit bereits im Rührfermentor erhaltenen Befunden (LEUCHTENBERGER et al. 1979) weist Abbildung 4 aus, daß mit zunehmdener Belüftungsintensität die absolute Höhe der Proteaseaktivität zurückgeht. Dieser Einfluß ist bei den Versuchen ohne Ölzusatz wesentlich stärker ausgeprägt als mit Öl. Gemäß Abbildung 3 wird demonstriert, daß dies insbesondere auf die Beeinflussung der Biomassebildung

642

A . LEUCHTENBERGER u n d H . RTJTTLOFF

zurückzuführen ist, die bei intensiver Luftzufuhr durch Mycellyse stark gehemmt wird. Über den negativen Einfluß einer zu starken Belüftung auf Wachstum und Synthese verschiedener Enzyme berichten auch andere Autoren. ARAVINA et al. (1976) finden bei Aspergillus terricola maximales Wachstum und gute Proteasesynthese bei einer Sauerstoffzufuhr von 0,38 bis 0,86 g/Std. je 1 Medium. Sowohl eine zu geringe als auch eine zu intensive Belüftung (0,18 g 0 2 /l • Std. bzw. 1,07 g 0 2 /l • Std.) führen zu einem drastischen Aktivitätsrückgang. Die Biomassebildung wird insbesondere bei zu hoher Belüftung gehemmt, und es erfolgt eine starke Abnahme der Biomassemenge durch Mycellyse. Die Zugabe von Sonnenblumenöl wirkt diesen negativen Erscheinungen entgegen. G A L Y N K I N et al. (1978) beobachten bei Schüttelkulturversuchen in 750-mlErlenmeyerkolben eine optimale Synthese des proteolytisch aktiven Enzyms Hygrolytin durch Actinomyces hygroscopicus unter Verwendung eines komplexen Mediums mit 1% Zahnwalöl bei einer Mediummenge von 150 ml. Höhere (200 ml) als auch geringere Kolbenfüllungen (50 ml) haben einen Aktivitätsrückgang zur Folge. Einen regulativen Einfluß von Sauerstoff auf die Enzymsynthese vermuten auch W Y S S U. E T T L I N G E R (1981). Die Bildung von Tyrosinase durch Streptomyces glaucescens ist umgekehrt proportional der Konzentration von Gelöst-0 2 im Medium. Bei hoher 0 2 Konzentration beträgt die Enzymaktivität nur einen Bruchteil derjenigen, wie sie unter niedriger 0 2 -Zuführung erreicht wird. Eine Hemmung der Asparaginaseproduktion bei Escherichia coli durch intensive Belüftung in 500 ml Kulturflaschen mit Schikanen gibt N E T R V A L (1980) an. In einem 2%igen Maisquellwassermedium geht die Enzymaktivität zurück, sobald eine 0 2 -Absorptionsrate von mehr als 0,22 mmol 0 2 /l • min überschritten wird. Bei 1,25 mmol 0 2 /l • min erhält dieser Autor nur noch ca. 5 % der Maximalausbeute. Nach Untersuchungsergebnissen von P A C A et al. (1978) bewirkt die Zugabe oberflächenaktiver Substanzen (z. B. Pottwalöl) zu einem Fermentationsmedium einen Rückgang der 0 2 -Absorptionsgeschwindigkeit aus der gasförmigen in die flüssige Phase um ca. 50%. Es wird angenommen, daß dies auf einer Senkung der Strömungsgeschwindigkeit in der Umgebung der Luftblasen, einer Dämpfung der Ausbildung von Wirbeln sowie einer Erniedrigung der Oberflächenspannung beruht. Bei der Prüfung der Rapsölwirkung auf die Proteasebildung unterschiedlich aktiver Stammvarianten kann eine Korrelation zwischen der Steigerungsrate und der absoluten Aktivitätshöhe nicht festgestellt werden (Tab. 1). Möglicherweise wird dieser Zusammenhang infolge des unterschiedlichen Lyseverhaltens der verschiedenen Stämme nicht erkennbar (gemäß Abb. 6 ist die Proteasebildung von solchem Stamm-Material geringer, das in Einstichkolben stärker zur Mycellyse neigt). Daraus resultiert, daß bei der Stamm-Auswahl für Fermentationen im Rührfermentor das Lyseverhalten überprüft werden muß. Autolytische Vorgänge sind bei Bakterien, insbesondere aber bei Actinomyceten, weit verbreitet ( G O L O W I N A et al. 1 9 7 3 , O K A Z A K I 1 9 7 2 , S T O L P U. S T A R R 1 9 6 5 , B E R N HARD 1 9 8 0 ) . Sie werden durch das komplexe Wirken verschiedener zellwandabbauender Enzyme verursacht. Nach G H Y U S E N U. SHOCKMAN ( 1 9 7 3 ) sowie G L A S E R ( 1 9 7 3 ) treten diese in allen Wachstumsphasen auf. Sie wirken unter physiologischen Bedingungen bei auf- und abbauenden Prozessen mit und besitzen unter normalen Bedingungen eine relativ niedrige Aktivität. Unter extremen Bedingungen (z. B. Temperatur- oder pHÄnderung, 0 2 -Zufuhr) kann jedoch ihre Synthese wesentlich angeregt werden. Es ist anzunehmen, daß die bei 0 2 -t)berangebot auftretende verstärkte Mycellyse bei T. vulgaris auf eine derartige Enzymaktivierung zurückzuführen ist. Eine deutliche Beziehung besteht auch zwischen dem Nährstoffgehalt des Kulturmediums und der Ölwirkung. Gemäß Abbildung 7 ist der aktivitätsfördernde Effekt

W i r k u n g v o n Ölen u n d F e t t s ä u r e n auf T. vulgaris.

III.

643

v o n R a p s ö l in n ä h r s t o f f a r m e n M e d i e n s t ä r k e r als in n ä h r s t o f f r e i c h e n , w a s die A n n a h m e b e s t ä r k t , d a ß Öl e n t w e d e r s e l b s t als C-Quelle g e n u t z t w i r d o d e r die M e t a b o l i s i e r u n g a n d e r e r N ä h r s t o f f k o m p o n e n t e n b e g ü n s t i g t . Ü b e r die W i r k u n g v o n Ol als C-Quelle g i b t es z a h l r e i c h e L i t e r a t u r h i n w e i s e (LEUCHTENBERGEE U. R U T T L O F F 1 9 7 9 ) ; auf diesb e z ü g l i c h e U n t e r s u c h u n g e n m i t T. vulgaris w i r d s p ä t e r n o c h e i n g e g a n g e n . Ü b e r e i n e n Z u s a m m e n h a n g zwischen der Medienzusammensetzung u n d der Ölwirkung ä u ß e r n s i c h a u c h Y A M A M O T O et al. ( 1 9 6 4 ) . D i e s e A u t o r e n f i n d e n , d a ß d i e G l u c o a m y l a s e p r o d u k t i o n d u r c h Endomyces spec. v o n R a p s ö l (in A b h ä n g i g k e i t v o m G e h a l t a n W e i z e n k l e i e ) b e e i n f l u ß t w i r d , u n d sie e r k l ä r e n d i e s m i t e i n e r V e r ä n d e r u n g d e r V i s k o s i t ä t i m M e d i u m sowie einer Beeinflussung des W a c h s t u m s .

Literatur ALFOED, J . A., SMITH, J . L. a n d LILLY, H . D., 1971. Relationship of microb : al a c t i v i t y to changes in lipids of food. J . appl. Bacteriol., 34, 133 — 146. A R A V I N A , L . A . , PONOMAREVA, V . D . , T E R E S T U N , J . M . ,

KASATKINA, J . D .

and

GREKOVA, V .

K.,

1976. Conditions of cultivation of t h e m u t a n t of Aspergillus terricola producing p r o t e o l y t i c enzyme. Microbiologija (Mosk.), 45, 770 — 776. BERNHARD, R., 1980. F- u n d E - B e r i c h t „ G e w i n n u n g u n d A n w e n d u n g von T h e r m i t a s e " , u n v e r öffentlicht. F R A N Z K E , C . , G Ö B E L , R . , K R O L L , J . , L I E B S , P . , R I T T E R , G . u n d S C H U L T Z E , M . , 1 9 7 9 . Ü b e r die V e r ä n d e r u n g des F e t t e s bei der K u l t i v i e r u n g v o n Endomycopsis bispora. N a h r u n g , 23, 2 8 3 — 2 8 7 . G A L Y N K I N , V . A . , E F I M O V A , T . P . , GREKOVA, V . K . u n d KOROLEVA, T . A . , 1 9 7 8 . E i n f l u ß der Kultivierungsbedingungen auf die F e t t v e r w e r t u n g d u r c h den P r o d u z e n t e n v o n H y g r o l y t i n . K h i m - . F a r m . Zh., 1 2 , 1 1 6 - 1 1 9 . G H Y U S E N , J . M . a n d SHOCKMAN, G . I)., 1 9 7 3 . Biosynthesis of peptidoglycan. I n : L. L E V I E (Ed.) Bacterial Membranes a n d Walls. Marcel Dekker Inc. New Y o r k . G L A S E R , L . , 1 9 7 3 . Bacterial cell surface polysaccharides. A n n . R e v . Biochem., 4 2 , 9 1 . GOLOVINA, J . G . ,

GUZHOVA, E . P . ,

BOGDANOVA, T . J .

and

LOGINOVA, L . G . ,

1973. L y t i c

enzymes

p r o d u c e d by thermophilic actinomycete Micromonospora vulgaris P A II-4. Mikrobiologija, 42, 6 2 0 - 6 2 7 . ISMAILOVA, D. Y . a n d LOGINOVA, L . G . , 1 9 7 5 . E f f e c t of some substances on t h e cellulase s y n t h e s i s of t h e t h e r m o t o l e r a n t f u n g u s Aspergillus terreus 17 P . P r i k l a d n a j a Biochim. i. Microbiol., 5, 676-681. KLINGENBERG, P . , ZICKLER, F . , LEUCHTENBERGER, A . u n d RUTTLOFF, H . , 1979. G e w i n n u n g

und

Charakterisierung von P r o t e a s e n aus Thermoactinomyces vulgaris. I I . Selektion v o n leistungsgesteigerten S t ä m m e n . Z . Allg. Mikrobiol., 19, 1 7 — 2 5 . K O B A Y A S H I , H . a n d S U Z U K I , H . , 1 9 7 2 . Studies on t h e decomposition of raffinose b y a-galactosidase of mold. I I I . S t i m u l a t o r y effect of f a t t y acids on t h e enzyme production. J . F e r m e n t a t i o n Technol., 50, 8 3 5 - 8 4 3 . L E U C H T E N B E R G E R , A., K L I N G E N B E R G , P . u n d R U T T L O F F , H . , 1 9 7 9 . Gewinnung u n d Charakterisier u n g von P r o t e a s e n aus Thermoactinomyces vulgaris. I I I . U n t e r s u c h u n g e n zur P r o t e a s e b i l d u n g in einer kleintechnischen Versuchsanlage. Z. Allg. Mikrobiol., 19, 27 — 35. L E U C H T E N B E R G E R , A. u n d R U T T L O F F , H . , 1 9 7 9 . W i r k u n g von Ölen u n d F e t t s ä u r e n auf Wachst u m u n d E n z y m b i l d u n g bei Thermoactinomyces vulgaris. I . E i n f l u ß von Ölen u n d F e t t s ä u r e n auf den Stoffwechsel von Mikroorganismen (Literaturüberblick). Z. Allg. Mikrobiol., 19, 609 bis 627.

A., S C H A F F N E R , P . u n d R U T T L O F F , H . , 1 9 8 1 . W i r k u n g von Ölen u n d F e t t s ä u ren auf W a c h s t u m u n d E n z y m b i l d u n g bei Thermoactinomyces vulgaris. I I . Methodik der Wachst u m s m e s s u n g in einer S u b m e r s k u l t u r in Gegenwart von ö l . Z. Allg. Mikrobiol., 21, 671—678. NETRVAL, J . , 1980. E f f e c t s of aeration a n d of corn steep concentration on L-asparaginase production in Escherichia coli. E u r o p . J . Appl. Microbiol., 9, 185 — 187. O K A Z A K I , H . , 1 9 7 2 . On cell wall lytic activity produced b y thermophilic actinomyces. I I I . Some properties of t h e components of t h e lytic enzyme produced b y Micropolyspora spec. J . F e r m e n t a t i o n Technol., 50, 5 8 0 - 5 9 1 . LEUCHTENBERGER,

PACA, J . , ETTLER, P . a n d GREGR, V., 1978. E f f e c t of v i s c o s i t y a n d s u r f a c e a c t i v e a g e n t s o n t h e

t r a n s f e r of oxygen in a pilot-scale fermentor. K v a s n y P r u m y s l , 24, 181 — 184.

SCHIERBAUM, F . , R I C H T E R , M . , AUGUSTAT, S . u n d RADOSTA, S . , 1 9 7 7 . H e r s t e l l u n g ,

Eigenschaften

u n d A n w e n d u n g gelbildender S t ä r k e h y d r o l y s e p r o d u k t e . D t . Lebensm.-Rdsch., 73, 390—394.

644

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STOLF, H . a n d STARR, M . P . , 1965. B a c t e r i o l y s i s . A n n . R e v . Microbiol., 19, 79.

WYSS, M. and ETTLINGER, L., 1981. Oxygen as a regulator of tyrosinase formation in Streptomyces glaucescens. Experentia, 37, 1232. YAKOVLEVA, M. B . , L E U C H T E N B E R G E R , A., LOGINOVA, L . G. and R U T T L O F F , H . , 1 9 8 2 . Effect of vegetable oils and surfactants on the growth and synthesis of proteases by pigmented and pigmentless strains of Thermoactinomyces vulgaris. Mikrobiologija, 51, 70 — 76. Y A M A D A , S . , W A D A , M . and C H I B A T A , J., 1 9 7 8 . Oxygen transfer in shaken flask cultures and t h e conversion of sorbitol to sorbose by Acetobacter suboxydans. J . Fermentation Technol., 56, 20-28.

T., H A T T O K I , F . and T A K A T S U , H . , 1 9 6 4 . The effect of lipids on amylolytic enzyme production by Endomyces spec. Bull. Inst. chem. Res. Kyoto, 4 2 , 2 5 2 — 2 6 9 .

YAMAMOTO,

Anschrift: Dr. A. L E U C H T E N B E R G E R Zentralinstitut für Ernährung der AdW DDR-1505 Bergholz-Rehbrücke, Arthur-Scheunert-Allee 114/116

Zeitschrift für Allgemeine Mikrobiologie

23

1983

10

645 — 651

(* Bereich Zellbiologie und Regulation, 2 Bereich Biochemie der Sektion Biowissenschaften der Karl-Marx-Universität, Leipzig, DDR)

Intracytoplasmic membrane induction by hexadecane in Acinetobacter calcoaceticus H . MÜLLER1, A. NAUMANN2, R . CLAUS2 a n d H . - P .

KLEBER2

(Eingegangen am 11. 4. 1983) Acinetobacter calcoaceticus 69-V is capable of utilizing ra-alkanes as the sole carbon source. By conventional and high voltage electron microscopy the fine-structure of these bacteria grown on hexadecane (with or without addition of DL-carnitine), succinate or yeast extract was analysed. A. calcoaceticus grown on hexadecane exhibited intracytoplasmic unit membrane aggregates in contrast to controls grown on the nonhydrocarbon substrates. Probably these aggregates are formed by the cytoplasmic membrane. Most of these membranes are surrounding and penetrating hexadecane inclusions. Ultrastructurally no essential difference caused by addition of carnitine could be seen. The results suggest that the membrane aggregates are the sites of enzymes which are required for the utilization of hexadecane. T h e cell envelope complex of G r a m - n e g a t i v e bacteria is composed of an undulating outer membrane, an electron-dense peptidoglycan layer, and a cytoplasmic membrane. C o m p l e x intracellular membrane s y s t e m s h a v e been described in specialized groups of microorganisms such as the autotrophic-nitrifying bacteria (WALLACE and NICHOLS 1969), and

the

HAGEN

methane-oxidizing 1972),

the

bacteria

(DAVIES

nitrogen-fixing

Azotobacter

and

WHITTENBURY (OPPENHEIM

and

1970,

DE BOER

MARCUS

1970,

PATE et al. 1973), and the photosynthetic Thiorhodaceae and Athiorhodaceae (NIETH and DREWS 1974). Intrac3'toplasmic hydrocarbon inclusions (KENNEDY et al. 1975) and intracytoplasmic membranes (KENNEDY and FINNERTY 1975) were demonstrated in the alkane-oxidizing microorganism Acinetobacter sp. H 0 1 - N . Acinetobacter calcoaceticus is capable of assimilation of w-alkanes greater than 11 carbons in length (KLEBER et al. 1973). Addition of L-carnitine to the hexadecane medium stimulates the g r o w t h of A. calcoaceticus (KLEBER et al. 1981). T h e present report extends the information on formation and structure of intracytoplasmic membranes of p r o k a r y o t e s during g r o w t h on hexadecane as the sole carbon and energy source b y an ultrastructural analysis of A. calcoaceticus. Materials

and

methods

Organism and culture conditions: Acinetobacter calcoaceticus 69-V (KLEBER et al. 1973) was cultured aerobically at 30 °C in a minimal medium as described previously ( K L E B E R and AURICH 1973). Hexadecane was added at a concentration of 10 g/1, succinate at a concentration of 20 g/1. In some experiments the hexadecane medium contained also DL-carnitine (0.5 g/1). For comparison some studies have been made on A. calcoaceticus 69-V cultivated continuously on a minimal salt medium developed for chemostate cultivation of this bacteria (GLAUS, R., unpublished) with a hydrocarbon mixture ranging from CJ2—C18 or yeast extract as the sole c a r b o n s o u r c e (CLAUS et al.

1982).

Determination of growth: Growth of cells was followed by measuring A 600 nm (SPEKOL, VEB

CARL ZEISS, J e n a ) .

Isolation of hexadecane inclusions: The inclusions were prepared as described by SCOTT and ( 1 9 7 6 ) , homogenized and extracted with benzene. The components of the clear benzene solution were detected by capillary gas chromatography using a 25 m OV1 quartz capillary

FINNERTY

c o l u m n (NAUMANN 1 9 8 3 ) .

Zeitschrift für Allgemeine Mikrobiologie

23

1983

10

645 — 651

(* Bereich Zellbiologie und Regulation, 2 Bereich Biochemie der Sektion Biowissenschaften der Karl-Marx-Universität, Leipzig, DDR)

Intracytoplasmic membrane induction by hexadecane in Acinetobacter calcoaceticus H . MÜLLER1, A. NAUMANN2, R . CLAUS2 a n d H . - P .

KLEBER2

(Eingegangen am 11. 4. 1983) Acinetobacter calcoaceticus 69-V is capable of utilizing ra-alkanes as the sole carbon source. By conventional and high voltage electron microscopy the fine-structure of these bacteria grown on hexadecane (with or without addition of DL-carnitine), succinate or yeast extract was analysed. A. calcoaceticus grown on hexadecane exhibited intracytoplasmic unit membrane aggregates in contrast to controls grown on the nonhydrocarbon substrates. Probably these aggregates are formed by the cytoplasmic membrane. Most of these membranes are surrounding and penetrating hexadecane inclusions. Ultrastructurally no essential difference caused by addition of carnitine could be seen. The results suggest that the membrane aggregates are the sites of enzymes which are required for the utilization of hexadecane. T h e cell envelope complex of G r a m - n e g a t i v e bacteria is composed of an undulating outer membrane, an electron-dense peptidoglycan layer, and a cytoplasmic membrane. C o m p l e x intracellular membrane s y s t e m s h a v e been described in specialized groups of microorganisms such as the autotrophic-nitrifying bacteria (WALLACE and NICHOLS 1969), and

the

HAGEN

methane-oxidizing 1972),

the

bacteria

(DAVIES

nitrogen-fixing

Azotobacter

and

WHITTENBURY (OPPENHEIM

and

1970,

DE BOER

MARCUS

1970,

PATE et al. 1973), and the photosynthetic Thiorhodaceae and Athiorhodaceae (NIETH and DREWS 1974). Intrac3'toplasmic hydrocarbon inclusions (KENNEDY et al. 1975) and intracytoplasmic membranes (KENNEDY and FINNERTY 1975) were demonstrated in the alkane-oxidizing microorganism Acinetobacter sp. H 0 1 - N . Acinetobacter calcoaceticus is capable of assimilation of w-alkanes greater than 11 carbons in length (KLEBER et al. 1973). Addition of L-carnitine to the hexadecane medium stimulates the g r o w t h of A. calcoaceticus (KLEBER et al. 1981). T h e present report extends the information on formation and structure of intracytoplasmic membranes of p r o k a r y o t e s during g r o w t h on hexadecane as the sole carbon and energy source b y an ultrastructural analysis of A. calcoaceticus. Materials

and

methods

Organism and culture conditions: Acinetobacter calcoaceticus 69-V (KLEBER et al. 1973) was cultured aerobically at 30 °C in a minimal medium as described previously ( K L E B E R and AURICH 1973). Hexadecane was added at a concentration of 10 g/1, succinate at a concentration of 20 g/1. In some experiments the hexadecane medium contained also DL-carnitine (0.5 g/1). For comparison some studies have been made on A. calcoaceticus 69-V cultivated continuously on a minimal salt medium developed for chemostate cultivation of this bacteria (GLAUS, R., unpublished) with a hydrocarbon mixture ranging from CJ2—C18 or yeast extract as the sole c a r b o n s o u r c e (CLAUS et al.

1982).

Determination of growth: Growth of cells was followed by measuring A 600 nm (SPEKOL, VEB

CARL ZEISS, J e n a ) .

Isolation of hexadecane inclusions: The inclusions were prepared as described by SCOTT and ( 1 9 7 6 ) , homogenized and extracted with benzene. The components of the clear benzene solution were detected by capillary gas chromatography using a 25 m OV1 quartz capillary

FINNERTY

c o l u m n (NAUMANN 1 9 8 3 ) .

646

H . MÜLLER et al.

Electron microscopy: a) Chemical fixation. First prefixation: Immediately after cultivation 40 — 60 ml cell suspension of every sample was mixed with 250 g/1 freshly distilled glutaraldehyde in 0.2 mol/1 sodium phosphate buffer, p H 7.2 (final concentration 30 — 40 g/1 glutaraldehyde). Second prefixation: After centrifugation at 6000 X g for 15 min at 4 °C in the first prefixation medium the pellets were prefixed again with 60 g/1 glutaraldehyde in 0.1 mol/1 sodium phosphate buffer, pH 7.2 (60 min at room temp.). The samples were rinsed with 0.1 mol/1 sodium phosphate buffer, pH 7.2, and postfixed 90 min at room temperature with a solution containing 10 g/1 0 s 0 4 + 10 g/1 K 2 Cr 2 0 7 , p H 7.2 (WOHLFARTH-BOTTERMANN 1957). After washing in 0.1 mol/1 sodium phosphate buffer, p H 7.2, the cells were dehydrated by graded series of acetone-water mixtures (700 g/1 acetone contained 10 g/1 phosphotungstic acid and 5 g/1 uranyl acetate, WOHLFARTH-BOTTERMANN 1957), and embedded in Durcupan (FLUKA). Ultrathin sections for conventional electron microscopy and thick sections (about 0.5 — 1 jim) for high voltage electron microscopy were cut on an Ultrotome I I I (LKB, Sweden). Thin sections were stained with lead citrate (VENABLE a n d COGGESHALL 1 9 6 5 ) a n d e x a m i n e d i n a TESLA B S 5 0 0 e l e c t r o n m i c r o s c o p e ( C S S R ) o p e r a t -

ing at 60 kV. Thick sections have been stained with lead citrate followed by uranyl acetate (for every 20 min each side) and examined at 1000 kV in the JEM-1000 (JEOL, Japan) of the Institut für Festkörperphysik und Elektronenmikroskopie, Halle, of the Academy of Sciences of the GDR. b) Cryofixation: In order to increase the freezing rate, very low-mass (13 mg) specially shaped copper platelets were used. A gold grid (2.3 mm/400 mesh) was dipped into the bacterial suspension, placed between two copper platelets, and frozen by the propane-jet technique (MÜLLER et al. 1980a). Freeze-substitution: The substitution procedure was carried out according to MÜLLER et al. (1980b). Thin sections were stained with lead citrate and uranyl acetate and examined at 100 kV in a PHILIPS E M 3 0 1 .

Results In contrast to the controls grown on succinate (Fig. 1) or yeast extract Acinetobacter calcoaceticus cultivated on hexadecane showed inclusions (Fig. 2, 3, 4, 7). According to the findings by gas chromatography these inclusions are hexadecane pools (NAUMANKT 1983). In contrast to the results in Acinetobacter sp. H 0 1 - N (SCOTT and F I N N E R T Y 1976) the hexadecane pools in A. calcoaceticus are surrounded by aggregates of unit membranes, and usually these membranes are penetrating the hexadecane pools (Fig. 4, 5, 6, 7). With the aid of the high voltage electron microscope in thick sections (about 1 fim) the bacterial cells were visible in their whole depth. I n the peripheral cell areas hexadecane inclusions of different size and of different electron density are seen (Fig. 3). Higher magnification and thinner sections (about 0.5 (j.m) showed t h a t the denser inclusions are rich in membranes. Neighbouring membrane aggregates of different size often form enlarged membrane areas (Fig. 8). All of these membrane differentiations seem to be in continuity with the cytoplasmic membrane. The membranes of the aggregates have the same thickness of about 6.5 nm as the cytoplasmic membrane. Possibly the membrane aggregates are formed b y repeated looping of the cytoplasmic membrane. In the nonhydrocarbongrown bacteria membrane aggregates could not be observed. Here and there in controls and in alkane-grown bacteria the cytoplasmic membrane appeared to reach the cell surface. The hexadecane-grown cells often showed a membrane 'layer' consisting of small membrane loops close lieing together in place of the normal single cytoplasmic membrane. I n hexadecane-grown bacteria of lag phase with or without addition of carnitine already small numerous membrane aggregates (averaging 80 nm in diam.) connected with the cytoplasmic membrane were present. Bacteria grown exponentially on hexadecane with or without carnitine exhibited inclusions averaging 180 n m in diam. During the late exponential and stationary growth phase the size of the inclusions increased scarcely. Ultrastructurally no essential difference caused b y addition of carnitine could

Intracytoplasmic membrane induction in A.

calcoaceticus

Fig. 1—3. Acinetobacter calcoaceticus grown on succinate (Fig. 1), on hexadecane with (Fig. 2), on hexadecane without carnitine (Fig. 3), exponential growth phase, chemical Fig. 3. High voltage electron micrograph taken at 1000 kV using a thick section (1 H Hexadecane inclusion, M Mesosomal-like structure. Fig. 1. x 5 4 0 0 0 , Fig. 2. x 0 0 0 0 0 . X 34000

647

carnitine fixation. ij.m). Fig. 3. x

he seen. After isolation of the cell envelope complex of hexadecane-grown cells of A . calcoaceticus (Auricii et al. 1977) membrane aggregates were still associated with the cytoplasmic membrane. The results concerning all the mentioned membrane structures were identical under both fixation conditions, chemical and crvofixation (Fig. 4 , 5, 7).

The membrane aggregates differed distinctly from the mesosomal-like structures. Such structures were present in succinate-grown controls (Fig. 1), also in acetategrown cells of A. calcoaceticus (Auricii etal. 1977, Fig. 2c), but seldom in hexadecanegrown bacteria. Bacteria grown on hexadecane (exponential phase of growth) and than (after thoroughly washing with 0.1 mol/1 potassium phosphate buffer, pH 7.4) incubated 1 h in the minimal medium without carbon source showed some fewer inclusions, and membrane aggregates were still present. If the incubation time without hexadecane had been prolonged till 3 h in most of the bacteria the number and size of inclusions and membrane envelopes were much reduced.

Discussion M a k u l a and F i n n e r t y

( 1 9 7 0 ) d e m o n s t r a t e d a d o u b l i n g of t h e p h o s p h o l i p i d s i n

hydrocarbon-grown bacteria. A comparison with the nonhydrocarbon-grown controls

H . MULLER et

648

al.

Fig. 4 — 6. Acinetobacter calcoaceticus grown on hexadecane. Fig. 4. and 6. Chemical fixation, Fig. 5. Cryofixation. Fig. 6. Partially lysed cell. A Unit membrane aggregates, C Cytoplasmic membrane, H Hexadecane inclusions. Fig. 4. x 158000, Fig. 5. x 160000, Fig. 6. X 150000

showed that the formation of specific intracytoplasmic membrane aggregates in Acinetobacter calcoaceticus was induced by the hydrocarbon medium. I.e., concomitant with the formation of membrane aggregates hexadecane was resorbed, accumulated in inclusions, and incorporated into the cell metabolism. The close contact between the hydrocarbon pools and the membrane aggregates on the one side and the results on modifications of the enzyme system of A. calcoaceticus during w-alkane-assimilation ( T A U C H E R T et al. 1 9 7 5 , A U R I C H et al. 1 9 7 7 , A S P E R G E R et al. 1 9 7 8 ) on the other side indicate t h a t the membrane aggregates are the sites of enzymes required for the utilization of the hexadecane. This assumption is supported by the findings that the membrane-bound NADP-dependent aldehyde dehydrogenase in A. calcoaceticus ( A U R I C I I et al. 1 9 7 7 ) is localized at the surface of small vesicles containing hydrocarbons ( Y O R I S E K et al.

1982).

The presence or absence of a surrounding membrane is the criterium for dividing the cell inclusions of prokaryotes into two major groups ( S H I V E L Y 1 9 7 4 ) . The membranes surrounding most inclusions of prokaryotes (for instance sulfur granules) are 2 to 4 nm thick and composed entirely of protein, i.e., the membranes are of the nonunit type ( S H I V E L Y 1 9 7 4 ) . T h e surrounding membranes of hexadecane inclusions in Acine-

Intracytoplasmic membrane induction in A.

calcoaceticus

649

Fig. 7. —8. Acinetobacter calcoaceticus grown on hexadecane. Fig. 7. Cryofixation, arrow: unit membrane. Fig. 8. Chemical fixation. A Enlarged areas of unit membrane aggregates. Fig. 7. X 1 3 5 0 0 0 , Fig. 8. X

104000

Electron micrographs of Fig. 5 and 7 provided by BRUNO HUMBEL, Zürich

tobacter sp. H01-N appeared as monolayers, but they proved lipid-rich structures containing polypeptides ( S C O T T and F I N N E R T Y 1 9 7 6 ) . Besides, in some cells of the strain Acinetobacter sp. H01-N these authors found intracellular membrane aggregates which appeared as lamellar sheaths extending across the cytoplasm. Such aggregates composed of some or many unit membranes were present in alkane- and also in alk-1ene-grown cells of the strain Acinetobacter sp. H 0 1 - N ( K E N N E D Y and F I N N E R T Y 1 9 7 5 ) . These membrane aggregates showed local contact or attachment to the hydroca.rbon pools but they did not surround or penetrate them. A continuity of these unit membranes with the cytoplasmic membrane was suggested by the authors. K Ä P P E L I and F I N N E R T Y ( 1 9 7 9 ) demonstrated an extracellular vesicular component that binds hexadecane in the growth medium of hexadecane-grown Acinetobacter. The authors suggest that these phospholipid- and lipopolysaccharid-rich particles and other extracellular factors are involved in the transport of alkanes and possibly in the interaction with the cell surface. Under the conditions of continuous cultivation of A. calcoaceticun on alkanes CLAUS et al. ( 1 9 8 2 ) succeeded isolating of above described vesicles. The peculiarity of unit membranes of A. calcoaceticus to form membrane aggregates in contact with hexadecane pools indicated their significance for resorption, accumulation, and utilization of the hydrocarbon substrate.

650

H . M Ü L L E R et

al.

K E N N E D Y et al. ( 1 9 7 5 ) d e s c r i b e d m e s o s o m a l - l i k e s t r u c t u r e s i n N B Y E ( n u t r i e n t b r o t h - y e a s t e x t r a c t ) - g r o w n c e l l s of t h e s t r a i n Acinetobacter sp. H O L - N . E B E R S O L D et al. ( 1 9 8 1 ) u s e d c r y o f i x a t i o n c o m b i n e d w i t h f r e e z e - s u b s t i t u t i o n a n d d e m o n s t r a t e d t h a t m e s o s o m e s of G r a m - p o s i t i v e b a c t e r i a a r e a r t i f a c t s a r i s i n g d u r i n g c h e m i c a l f i x a t i o n . P o s s i b l y t h e m e s o s o m a l - l i k e s t r u c t u r e s of A. calcoaceticus are also method-dependent artifacts. A. calcoaceticus is c a p a b l e of u t i l i z i n g D L - c a r n i t i n e a s t h e s o l e c a r b o n s o u r c e . T h e u t i l i z a t i o n of t h i s c o m p o u n d a n d t h e g r o w t h c o r r e l a t e d w i t h t h e c l e a v a g e of t h e C - N b o u n d a n d t h e r e b y w i t h t h e f o r m a t i o n of t r i m e t h y l a m i n e (KLEBER et al. 1 9 7 7 ) . B y a d d i t i o n of D L - c a r n i t i n e t o t h e h e x a d e c a n e m e d i u m t h e l a g p h a s e is r e d u c e d ( K L E BER et al. 1 9 8 1 ) . T h e c a u s e of t h e e f f e c t of c a r n i t i n e is n o t y e t c l e a r . U l t r a s t r u c t u r a l l y n o e s s e n t i a l d i f f e r e n c e c a u s e d b y a d d i t i o n of c a r n i t i n e c o u l d b e s e e n . T h e o b s e r v e d s t i m u l a t o r y e f f e c t o n t h e g r o w t h of A. calcoaceticus probably depends o n t h e i n f l u e n c e of c a r n i t i n e o n s u b s t r a t e m e t a b o l i s m . E s p e c i a l l y a n a c t i v a t i o n of f a t t y acid catabolism has to be considered.

A

cknowledgements

W e are grateful t o Prof. Dr. H . BETHGE, head of t h e I n s t i t u t f ü r F e s t k ö r p e r p h y s i k u n d Elektron e n m i k r o s k o p i e der A k a d e m i e der Wissenschaften der D D R , Halle-, for t h e possibility to use t h e h i g h voltage electron microscope J E M - 1 0 0 0 . We t h a n k B R U N O H U M B E L , I n s t i t u t e of Cell Biology of t h e Swiss Federal I n s t i t u t e of Technology for performing t h e ' c r y o m e t h o d s a n d for providing a p a r t of t h e electron micrographs (Fig. 5 a n d 7). F o r performing gas c h r o m a t o g r a p h y we t h a n k R . M Ü L L E R , a n d for technical assistance S . M E H N E R T , C. S C H N E I D E R , E . S I E B E R T , I . S E I F E R T .

References B., K L E B E R , H . - P . u n d A U R I C H , H . , 1 9 7 8 . O x y d a t i o n langkettiger. n-Alkane d u r c h zellfreie E x t r a k t e von Acinetobacter calcoaceticus. Identifizierung u n d B e s t i m m u n g von ra-Tetradekansäure d u r c h I n k u b a t i o n m i t n - T e t r a d e k a n . Wiss. Z. Karl-Marx Univ. Leipzig, M a t h . - N a t . R., 27, 3 - 1 5 . A U R I C H , H . , SORGER, H . u n d M Ü L L E R , H . , 1977. Isolierung u n d Charakterisierung der Zellgrenzschichten v o n Acinetobacter calcoaceticus. Z. allg. Mikrobiol., 17, 333 — 338. ASPERGER, 0 . , FUTTIG, A . , BEHRENS,

CLAUS, R . ,

KÄPPELI, 0 . ,

MÜLLER, M . ,

HUMBEL, B .

and

FIECHTER, A.,

1982.

infrastructure

of

Acinetobacter calcoaceticus 69 V in relation t o hexadecane u p t a k e . J . Cell Biol, (in press). D A V I E S , S. L . a n d W H I T T E N B U R Y , R . , 1970. Fine s t r u c t u r e of m e t h a n e a n d other h y d r o c a r b o n utilizing bacteria. J . gen. Microbiol., 61, 227—232. DE BOER, W . E . a n d HAGEN, W., 1972. Observations on t h e fine s t r u c t u r e of a methane-utilizing bacterium. Antonie v a n Leeuwenhoek J . Microbiol. Serol., 38, 33 — 47. E B E R S O L D , H . R . , C O R D I E R , J . L . a n d L Ü T H Y , P . , 1 9 8 1 . Bacterial mesosomes: Method d e p e n d e n t artifacts. Arch. Microbiol., 130, 1 9 - 2 2 . K Ä P P E L I , O . a n d F I N N E R T Y , W . R . , 1 9 7 9 . P a r t i t i o n of alkane b y an extracellular vesicle derived f r o m hexadecane-grown Acinetobacter. J . Bacteriol., 140, 707 — 712. K E N N E D Y , R . S . , F I N N E R T Y , W . R . , S U D A R S A N A N , K . a n d Y O U N G , R . A., 1 9 7 5 . Microbial assimilation of hydrocarbons. I . The f i n e - s t r u c t u r e of a h y d r o c a r b o n oxidizing Acinetobacter sp. Arch. Microbiol., 102, 7 5 - 8 3 . KENNEDY, R . S. a n d FINNERTY, W . R., 1975. Microbial assimilation of hydrocarbons. I I . I n tracytoplasmic m e m b r a n e induction in Acinetobacter sp. Arch. Microbiol., 102, 85 — 90. K L E B E R , H . - P . , S C H O P P , W . u n d A U R I C H , H., 1 9 7 3 . V e r w e r t u n g von n-Alkanen d u r c h einen S t a m m von Acinetobacter calcoaceticus. Z. allg. Mikrobiol., 13, 445 — 447. K L E B E R , H . - P . u n d A U R I C H , H . , 1 9 7 3 . E i n f l u ß von »-Alkanen auf die Synthese der E n z y m e des Glyoxylatzyklus in Acinetobacter calcoaceticus. Z . allg. Microbiol., 13, 4 7 3 — 4 8 0 . K L E B E R , H . - P . , S E I M , H . , A U R I C H , H . u n d S T R A C K , E . , 1 9 7 7 . V e r w e r t u n g von T r i m e t h y l a m m o n i u m v e r b i n d u n g e n d u r c h Acinetobacter calcoaceticus. Arch. Microbiol., 112, 2 0 1 — 2 0 6 . K L E B E R , H.-P., C L A U S , R . , S E I M , H . u n d STRACK, E., 1 9 8 1 . Verfahren z u m Z ü c h t e n von Bakterien. W P C 1 2 K / 2 3 4 3 1 7 (Wirtsch.-Patent).

I n t r a c y t o p l a s m i c m e m b r a n e induction in A.

calcoaceticus

651

a n d F I N N E R T Y , W . B . , 1 9 7 0 . Microbial assimilation of hydrocarbons. Identification of phospholipids. J . Bacteriol., 103, 348 — 355. MÜLLER, M., MEISTER, N. a n d MOOR, H . , 1980a. Freezing in a propane jet a n d its application in freeze-fracturing. Mikroskopie, 86, 129 — 140. M Ü L L E R , M., M A R T I , T . a n d R R I T Z , S., 1980b. I m p r o v e d structural preservation b y freeze-subs t i t u t i o n . I n : Electron Microscopy 1980, Seventh E u r o p e a n Congress on Electron Microscopy F o u n d a t i o n , Leiden (BREDEROO, P . a n d DE PRIESTER, W., Eds.), Vol. 2, pp. 720 — 721. NAUMANN, A., 1983. Untersuchungen zum n-Alkan oxydierenden System aus Acinetobacter calcoaceticus. Dissertation A, Karl-Marx-Univ. Leipzig, M a t h . - N a t . F a k . XTETII, K . F . a n d DREWS, G., 1974. The protein p a t t e r n s of intracytoplasmic membranes a n d reaction center particles isolated from Bhodopseudomonas capsulata. Arch. Mikrobiol., 96, 161 t o 174. O P P E N H E I M , J . a n d M A R C U S , L . , 1 9 7 0 . Correlation of u l t r a s t r u c t u r e in Azotobacter vinelandii with nitrogen source for growth. J . Bacteriol., 101, 2 8 6 — 2 9 1 . PATE, J . L., SHAK, V. K . a n d BRILL, W. J . , 1973. I n t e r n a l m e m b r a n e control in Azotobacter vinelandii. J . Bacteriol., 114, 1346 — 1350. SCOTT, C. C . L. a n d F I N N E R T Y , W . R., 1 9 7 6 . Characterization of intracytoplasmic hydrocarbon inclusions f r o m t h e hydrocarbon-oxidizing Acinetobacter species H 0 1 - N . J . Bacteriol., 127, MAKULA, R . A.

481-489.

SHIVELY, J . M., 1974. Inclusion bodies of prokaryotes. Ann. Rev. Microbiol., 28, 167 — 187. T A U C H E R T , H., R O Y , M., S C H O P P , W . u n d A U R I C H , H . , 1 9 7 5 . Pyridinnucleotidunabhängige Oxydation von längerkettigen aliphatischen Alkoholen durch ein E n z y m aus Ac. calcoaceticus. Z. allg. Mikrobiol., 15, 4 5 7 - 4 6 0 . V E N A B L E , J . H . a n d COGGESHALL, R . A., 1 9 6 5 . A simplified lead citrate stain for use in electron microscopy. J . Cell. Biol., 25, 4 0 7 - 4 0 8 . V O R I S E K , J . , SORGER, H . , A U R I C H , H . a n d L O J D A , Z . , 1 9 8 2 . Ultracytochemical staining of aldehyde dehydrogenase activity in Acinetobacter calcoaceticus cultivated on M-alkanes. Symposium histochemicum K a r l o v y Vary 2 4 . - 2 6 . 3. 1982 (Abstract). W A L L A C E , W . a n d N I C H O L S , D. J . D., 1 9 6 9 . The biochemistry of nitrifying microorganisms. Biol. Rev., 44, 3 5 9 - 3 9 1 . WOHLFARTH-BOTTERMANN, K . E . ,

1 9 5 7 . Die K o n t r a s t i e r u n g tierischer Zellen u n d Gewebe im R a h m e n ihrer elektronenmikroskopischen U n t e r s u c h u n g an u l t r a d ü n n e n Schnitten. N a t u r wiss., 4 4 , 2 8 7 — 2 8 8 .

Mailing address: Doz. Dr. H I L D E G A R D M Ü L L E R Sektion Biowissenschaften der Karl-Marx-Universität Bereich Zellbiologie u. Regulation DDR-7010 Leipzig, Talstr. 33

Zeitschrift f ü r Allgemeine Mikrobiologie

10

23

1983

653-660

(Akademie der Wissenschaften der D D R , F o r s c h u n g s z e n t r u m f ü r Molekularbiologie u n d Medizin, Z e n t r a l i n s t i t u t f ü r Molekularbiologie, Berlin-Buch, D i r e k t o r : Prof. Dr. W . Z S C H I E S C H E )

Isolierung und Rekonstitution des Alkan-Monooxygenase-Systems der Hefe Lodderomyces elongisporus1) W . - H . SCHUNCK, P . R I E G E , H . H O S ECK u n d H . - G . MÜLLEK

(Eingegangen

am 3.

3.1983)

I t is generally assumed t h a t t h e f i r s t step of alkane degradation in several y e a s t strains a n d b a c t e r i a is catalyzed b y a monooxygenase system containing cytochrome P-450 as t h e t e r m i n a l oxidase a n d a N A D P H - c y t o c h r o m e P-450 reductase as t h e electron transfer component. To prove this hypothesis b o t h proteins were purified to homogeneity f r o m t h e microsomal f r a c t i o n of t h e alkane-assimilating yeast L. elongisporus a n d r e c o n s t i t u t e d in vitro. T h e cytochrome P-450, having a specific content of 12 — 17 nmoles/mg protein t u r n e d o u t to b e a typical m e m b e r of this class of hemoproteins with respect t o t h e molecular weight (53 500) a n d spectral properties. T h e reductase c o m p o n e n t with a molecular weight of 79000 contains F A D a n d F M N as prosthetic groups. The recombination of t h e hemoprotein a n d t h e flavoprotein results in a n enzyme system which catalyzes t h e monoterminal h y d r o x y l a t i o n of hexadecane. T h e a c t i v i t y of t h e reconstituted enzyme system is r e m a r k a b l y s t i m u l a t e d b y nonionic detergents, suggesting t h e r e q u i r e m e n t of a n additional lipid factor. T h e possible i m p o r t a n c e of o t h e r electron transfer systems a n d of s u b s t r a t e t r a n s p o r t is discussed with respect t o t h e f u n c t i o n of t h e alkane monooxygenase system u n d e r in vivo conditions. Ziel d e r U n t e r s u c h u n g e n w a r d i e A u f k l ä r u n g d e r N a t u r d e s E n z y m s y s t e m s , d a s i n d e r H e f e L. elongisporus d e n 1. S c h r i t t d e s A l k a n a b b a u s k a t a l y s i e r t . D a s e x p e r i mentelle H e r a n g e h e n wurde dabei vor allem durch die beiden folgenden B e f u n d e bestimmt: a) d e r H e f e s t a m m v e r f ü g t ü b e r w i e g e n d ü b e r e i n e n m o n o t e r m i n a l e n W e g d e s A l k a n abbaus. b) w-Alkane induzieren die B i l d u n g v o n C y t P - 4 5 0 , das bereits in d e n unzerstörten Hefezellen a n h a n d seines charakteristischen, CO-Differenzspektrums nachweisbar i s t (SCHUNCK et al. 1 9 7 8 , MAUERSBERGER et al. 1 9 8 1 ) . D i e E i g e n s c h a f t C y t P - 4 5 0 bei K u l t i v i e r u n g auf » - A l k a n e n z u bilden, teilt der Lodderomyces-Stamm mit m i n d e s t e n s 8 weiteren H e f e s t ä m m e n sowie einer Reihe v o n B a k t e r i e n ( T a b . 1). Bereits die ersten N a c h w e i s e dieses H ä m o p r o t e i n s in a l k a n v e r w e r t e n d e n Mikroo r g a n i s m e n (CARDINI U. JURTSHUK 1 9 6 8 , 1 9 7 0 , GALLO et al. 1 9 7 1 , LEBEAULT et al. 1 9 7 1 , GALLO et al. 1 9 7 3 ) f ü h r t e n z u d e r A n n a h m e , d a ß C y t P - 4 5 0 i n e i n i g e n M i k r o o r g a n i s m e n d e n 1. S c h r i t t d e s A l k a n a b b a u s k a t a l y s i e r t . T a b e l l e 2 soll v e r a n s c h a u l i c h e n auf welche Ergebnisse sich diese H y p o t h e s e s t ü t z e n kann. F o l g t m a n der kritischen B e w e r t u n g der a u f g e f ü h r t e n K r i t e r i e n (COOPER et al. 1 9 7 9 , BURKE 1 9 8 1 ) , s o g a b e s n e b e n e i n e r V i e l z a h l i n d i r e k t e r H i n w e i s e ( P u n k t e 1 b i s 8 i n T a b . 2) l a n g e Z e i t k e i n e n e i n d e u t i g e n e x p e r i m e n t e l l e n N a c h w e i s ( P u n k t e 9 b i s 11 i n T a b . 2) f ü r d i e p o s t u l i e r t e Hervorgegangen aus einem Vortrag, gehalten auf dem Leipziger Biotechnologie-Symposium, Sept. 1982; vgl. auch M Ü L L E R et al. (1983) A b k ü r z u n g : Cytochrom — Cyt 44

Z. Allg. Mikrobiol., B d . 23, H. 10

Zeitschrift f ü r Allgemeine Mikrobiologie

10

23

1983

653-660

(Akademie der Wissenschaften der D D R , F o r s c h u n g s z e n t r u m f ü r Molekularbiologie u n d Medizin, Z e n t r a l i n s t i t u t f ü r Molekularbiologie, Berlin-Buch, D i r e k t o r : Prof. Dr. W . Z S C H I E S C H E )

Isolierung und Rekonstitution des Alkan-Monooxygenase-Systems der Hefe Lodderomyces elongisporus1) W . - H . SCHUNCK, P . R I E G E , H . H O S ECK u n d H . - G . MÜLLEK

(Eingegangen

am 3.

3.1983)

I t is generally assumed t h a t t h e f i r s t step of alkane degradation in several y e a s t strains a n d b a c t e r i a is catalyzed b y a monooxygenase system containing cytochrome P-450 as t h e t e r m i n a l oxidase a n d a N A D P H - c y t o c h r o m e P-450 reductase as t h e electron transfer component. To prove this hypothesis b o t h proteins were purified to homogeneity f r o m t h e microsomal f r a c t i o n of t h e alkane-assimilating yeast L. elongisporus a n d r e c o n s t i t u t e d in vitro. T h e cytochrome P-450, having a specific content of 12 — 17 nmoles/mg protein t u r n e d o u t to b e a typical m e m b e r of this class of hemoproteins with respect t o t h e molecular weight (53 500) a n d spectral properties. T h e reductase c o m p o n e n t with a molecular weight of 79000 contains F A D a n d F M N as prosthetic groups. The recombination of t h e hemoprotein a n d t h e flavoprotein results in a n enzyme system which catalyzes t h e monoterminal h y d r o x y l a t i o n of hexadecane. T h e a c t i v i t y of t h e reconstituted enzyme system is r e m a r k a b l y s t i m u l a t e d b y nonionic detergents, suggesting t h e r e q u i r e m e n t of a n additional lipid factor. T h e possible i m p o r t a n c e of o t h e r electron transfer systems a n d of s u b s t r a t e t r a n s p o r t is discussed with respect t o t h e f u n c t i o n of t h e alkane monooxygenase system u n d e r in vivo conditions. Ziel d e r U n t e r s u c h u n g e n w a r d i e A u f k l ä r u n g d e r N a t u r d e s E n z y m s y s t e m s , d a s i n d e r H e f e L. elongisporus d e n 1. S c h r i t t d e s A l k a n a b b a u s k a t a l y s i e r t . D a s e x p e r i mentelle H e r a n g e h e n wurde dabei vor allem durch die beiden folgenden B e f u n d e bestimmt: a) d e r H e f e s t a m m v e r f ü g t ü b e r w i e g e n d ü b e r e i n e n m o n o t e r m i n a l e n W e g d e s A l k a n abbaus. b) w-Alkane induzieren die B i l d u n g v o n C y t P - 4 5 0 , das bereits in d e n unzerstörten Hefezellen a n h a n d seines charakteristischen, CO-Differenzspektrums nachweisbar i s t (SCHUNCK et al. 1 9 7 8 , MAUERSBERGER et al. 1 9 8 1 ) . D i e E i g e n s c h a f t C y t P - 4 5 0 bei K u l t i v i e r u n g auf » - A l k a n e n z u bilden, teilt der Lodderomyces-Stamm mit m i n d e s t e n s 8 weiteren H e f e s t ä m m e n sowie einer Reihe v o n B a k t e r i e n ( T a b . 1). Bereits die ersten N a c h w e i s e dieses H ä m o p r o t e i n s in a l k a n v e r w e r t e n d e n Mikroo r g a n i s m e n (CARDINI U. JURTSHUK 1 9 6 8 , 1 9 7 0 , GALLO et al. 1 9 7 1 , LEBEAULT et al. 1 9 7 1 , GALLO et al. 1 9 7 3 ) f ü h r t e n z u d e r A n n a h m e , d a ß C y t P - 4 5 0 i n e i n i g e n M i k r o o r g a n i s m e n d e n 1. S c h r i t t d e s A l k a n a b b a u s k a t a l y s i e r t . T a b e l l e 2 soll v e r a n s c h a u l i c h e n auf welche Ergebnisse sich diese H y p o t h e s e s t ü t z e n kann. F o l g t m a n der kritischen B e w e r t u n g der a u f g e f ü h r t e n K r i t e r i e n (COOPER et al. 1 9 7 9 , BURKE 1 9 8 1 ) , s o g a b e s n e b e n e i n e r V i e l z a h l i n d i r e k t e r H i n w e i s e ( P u n k t e 1 b i s 8 i n T a b . 2) l a n g e Z e i t k e i n e n e i n d e u t i g e n e x p e r i m e n t e l l e n N a c h w e i s ( P u n k t e 9 b i s 11 i n T a b . 2) f ü r d i e p o s t u l i e r t e Hervorgegangen aus einem Vortrag, gehalten auf dem Leipziger Biotechnologie-Symposium, Sept. 1982; vgl. auch M Ü L L E R et al. (1983) A b k ü r z u n g : Cytochrom — Cyt 44

Z. Allg. Mikrobiol., B d . 23, H. 10

654

W . - H . S C H U N C K et

al.

Tabelle 1 Cytochrome P-450 in alkanverwertenden Mikroorganismen Mikroorganismus Acinetobacter spp. Corynebacterium sp. Candida tropicalis

Candida

guilliermondii

Candida lipolytica Candida pulcherima Candida maltosa Torulopsis sp. Torulopsis Candida Saccharomycopsis lipolytica Lodderomyces

elongisporus

Literatur A S P E R G E R et al. ( 1 9 8 1 ) CARDINI u . JURTSHUK (1968, G A L L O et al. ( 1 9 7 1 ) L E B E A U L T et al. ( 1 9 7 1 ) D Ü P P E L et al. ( 1 9 7 3 ) G A L L O et al. ( 1 9 7 3 ) B E R T R A N D et al. ( 1 9 7 9 ) T A K A G I et al. ( 1 9 8 0 ) G M Ü N D E R et al. ( 1 9 8 1 ) T I T T E L B A C H et al. ( 1 9 7 6 ) M A U E R S B E R G E R et al. ( 1 9 8 0 ) I L C H E N K O et al. ( 1 9 8 0 ) T A K A G I et al. ( 1 9 8 0 ) T A K A G I et al. ( 1 9 8 0 ) H E I N Z et al. ( 1 9 7 0 ) I L C H E N K O et al. ( 1 9 8 0 ) DÉLAISSÉ u . N Y N S (1974) D É L A I S S É et al. ( 1 9 8 1 ) S C H U N C K et al. ( 1 9 7 8 ) M A U E R S B E R G E R et al. ( 1 9 8 1 ) R I E G E et al. ( 1 9 8 1 )

1970)

Tabelle 2 Allgemeine Kriterien für die enzymatische Funktion von Cyt P-450-Systemen (zusammengestellt aus COOPER et al. ( 1 9 7 9 ) und B U R K E ( 1 9 8 1 ) ) und bisherige Ergebnisse zur Rolle von Cyt P-450 bei der Alkarioxydation durch Mikroorganismen Kriterien 1. Induktion durch Substrat 2. Parallele intrazelluläre Verteilung von Cyt P-450 und Enzymaktivität 3. Das Substrat induziert charakteristische spektrale Veränderungen 4. Stöchiometrie einer Monooxygenasereaktion 5. NAD(P)H und 0 2 als Cosubstrate

6.

Geringe CN~-Empfindlichkeit

7. Starke Hemmung durch CO

8. Partielle Reinigung und Rekonstitution des Enzymsystems 9. Photochemisches Wirkungsspektrum mit Amax = 450 nm 10. Hemmung der enzymatischen Reaktion durch Cyt P-450 spezifische Antikörper 11. Rekonstitution des aktiven Enzymsystems aus den hochgereinigten Proteinkomponenten

entsprechende Ergebnisse bei alkanoxidierenden Mikroorganismen vgl. Tab. 1 G A L L O et al. (1973) D É L A I S S É et al. (1981) M Ü L L E R et al. (1970) G M Ü N D E R (1979) M A N S U Y et al. (1980) S C H U N C K et al. (1978) G A L L O et al. (1971) L E B E A U L T et al. (1971) D Ü P P E L et al. (1973) S C H U N C K et al. (1978) D Ü P P E L et al. (1973) S C H U N C K et al. (1978) G A L L O et al. (1971) L E B E A U L T et al. (1971) G A L L O et al. (1973) C A R D I N I u. J U R T S H U K (1970) D Ü P P E L et al. (1973) B E R T R A N D et al. (1979)

R I E G E et HONECK

al. (1981) et al. (1982)

Alkan-Monooxygenase-System aus Lodderomyces

655

enzymatische Funktion des Cyt P-450 im Alkanstoffwechsel. Versuche zur Rekonstitution eines aktiven Enzymsystems wurden erstmalig für das Cyt P-450-System aus Candida tropicalis ( D Ü P P E L et al. 1 9 7 3 , B E R T R A N D et al. 1 9 7 9 ) , allerdings mit einer nur geringfügig gereinigten Cyt P-450-Komponente, durchgeführt. Material

und

Methoden

Die Kultivierung des Hefestammes Lodderomyces elongisporus EH 15 D (Institut für Technische Chemie, Leipzig) auf einem »i-Alkangemisch (C n — C19) (MATJERSBERGER et al. 1981) und die Reinigung von Cyt P-450 ( R I E G E et al. 1981, S C H U N C K et al. 1983) und der NADPH-Cyt P-450 Reduktase ( H O N E C K et al. 1982) aus der mikrosomalen Membranfraktion erfolgte wie in früheren Arbeiten beschrieben. Der Standardansatz (Totalvolumen 1 ml) zur Rekonstitution des Alkan-Monooxygenase-Systems enthielt: Cyt P-450 (0,24 nMol), NADPH-Cyt P-450 Reduktase (1,2 ¡¿Mol Cyt c/min), Präwozell W-ON 100 (0,02%) sowie Hexadecan (100 nMol zugegeben in 10 ¡J-1 Äthanol) und NADPH (200 nMol) in 200 MM Kaliumphosphatpuffer pH 7,25. Die durch die Hexadecan-Zugabe ausgelöste NADPH-Oxydation wurde bei 340 nm (e = 6,3 mM - 1 • cm"') spektralphotometrisch verfolgt. Das bei Verwendung von [1-14C] Hexadekan (3 x 106 DPM/100 nMol) gebildete la C-Hexadecanol wurde nach Stoppen der Reaktion mit 0,5 ml 8%iger Schwefelsäure, Extraktion mit Heptan (nach D O L E 1956) und Trennung über Aluminiumoxid (nach GHOLSON et al. 1963) durch Szintillationsmessung quantitativ bestimmt. Ergebnisse

und

Diskussion

Reinigung und E i g e n s c h a f t e n von Cyt P-450 und seiner N A D P H a b h ä n g i g e n R e d u k t a s e a u s L. elongisporus In L. elongisporus sind sowohl Cyt P-450 als auch seine NADPH-abhängige Reduktase membrangebunden. Sie können nach mechanischem Zellaufschluß innerhalb einer mikrosomalen Fraktion gewonnen werden, die die N A D P H - und 0 2 -abhängige (durch CO-hemmbare) Oxydation von Hexadecan bis zur Palmitinsäure katalysiert 2+ ( S C H U N C K et al. 1978). Die Ca -sedimentierte Membranfraktion enthält 0,3—0,4nMol Cyt P-450/mg Protein und eine Reduktaseaktivität von 0,6 —0,9 ¡j.mol Cyt c/min X mg Protein. Abbildung 1 gibt einen Überblick über die angewandten Proteinreinigungsverfahren. Die Reinigung des Cyt P-450 war im wesentlichen durch einen ChromatographieCa 2+ -sedimentierte | mikrosmonale Fraktion | Solubilisierung Solubilisierung mit Desoxycholat mit Cholat DEAE-Lphacell (KCl-Gradient) Hydroxylapatit (Phosphat-Gradient) Calciumphosphat-Gel

| 8-NH 2 -Octyl-Sepharose4B (Elution mit Tween 20) Calciumphosphat-Gel

gereinigte gereinigtes NADPH-Cyt c Reduktase Cytochrome P-450 Abb. 1. Schematische Darstellung der Verfahren zur Reinigung des Cyt P-450 ( R I E G E et al. (1981)) und der NADPH-Cyt P-450 (Cyt c)-Reduktase ( H O N E C K et al. (1982)) aus der mikrosomalen Fraktion von L. elongisporus 44»

656

W . - H . SCHUNCK et al.

Schritt an 8-Amino-octyl-Sepharose 4B möglich. Durchschnittlich wurde eine 30 bis 50fache Anreicherung des Hämoproteins auf einen spezifischen Gehalt von 12 bis 17 nMol/mg Protein erreicht, die Ausbeuten lagen zwischen 20 und 30%. Die relativ gute Stabilität des Cyt P-450 während der Präparation wurde vor allem durch den Zusatz von EDTA, Dithioerythritol und Glycerol erreicht. Die Reduktase wurde im Anschluß an eine Solubilisierung mit Desoxycholat durch Chromatographie an DEAE-Sephacell und Hydroxylapatit gereinigt, wobei die Elution des jeweils zunächst adsorbierten Enzyms mit Hilfe eines KCl- bzw. Phosphatgradienten erfolgte. Diese Methode gestattet eine etwa lOOfache Anreicherung der Reduktase bei Ausbeuten zwischen 30 und 50%. Die sehr guten Ausbeuten wurden vor allem durch den Zusatz von FAD und FMN zu allen Präparationspuffern erreicht. Die Chromatographie an Calciumphosphat-Gel diente bei beiden Proteinen zur Detergensentfernung. Beide Proteine konnten in elektrophoretisch reiner Form gewonnen werden; das in der Na-Dodecylsulfat-Polyacrylamid-Gelelektrophorese ermittelte Molekulargewicht des Cyt P-450 liegt bei 53500, das der Reduktase bei 79000. Die prosthetischen Gruppen — Häm beim Cyt P-450 und FAD/FMN bei der Reduktase — verleihen den beiden gereinigten Proteinen charakteristische spektrale Eigenschaften. Das Soretmaximum der oxidierten Form des Cyt P-450 liegt bei 417 nm, das der Dithionit-reduzierten Form bei 412 nm; die Bindung von CO an die reduzierte Form führt zu der für alle Cyt P-450-Proteine charakteristischen starken Rotverschiebung des Soretmaximums auf annähernd 450 (genau 447 nm). Die gereinigte Reduktase zeigt dagegen ein für Flavoproteine typisches Absorptionsspektrum mit Maxima bei 453 und 380 nm. R e k o n s t i t u t i o n des A l k a n - M o n o o x y g e n a s e - S y s t e m s Abbildung 2 zeigt, daß das ursprünglich als NADPH-Cyt c Reduktase gereinigte Flavoprotein in der Lage ist, Elektronen vom NADPH auf Cyt P-450 zu übertragen. Bemerkenswert war bei diesen Versuchen, daß eine annähernd vollständige Reduktion des Cyt P-450 (gemessen an der Umwandlung der oxidierten Form in den reduzierten + CO-Komplex) erst durch Zugabe von Hexadecan sowie geringer Konzentrationen an Triton X 100 bzw. Präwozell W-ON 100 erreicht werden konnte. Unter den so optimierten Bedingungen für die Wechselwirkung von Reduktase und Cyt P-450 wurde die Umsetzung von 14 C-Hexadecan überprüft. Es zeigte sich, daß weder Cyt P-450 noch die Reduktase allein die Alkanoxydation katalysieren, erst ihre Rekombination führt zur Ausbildung eines aktiven Enzymsystems ( R I E G E et al. 1981, H O N E C K et al. 1982). Die Aktivität des rekonstituierten Enzymsystems konnte nicht nur durch die quantitative Erfassung des radioaktiven Hydroxylierungsproduktes, sondern auch spektrophotometrisch anhand der NADPH-Oxydation gemessen werden, die durch die Zugabe des Alkansubstrates ausgelöst wird (Abb. 3). Dabei lag die Geschwindigkeit der NADPH-Oxydation im allgemeinen 2 bis 3mal höher als die der Bildung des radioaktiven Produktes (Tab. 3). Die erhaltenen Sättigungskurven (Abb. 3) bei Variation einer Proteinkomponente sprechen für die Ausbildung eines definierten, stöchiometrischen Enzymkomplexes — mit großer Wahrscheinlichkeit stellt ein 1:1Komplex von Cyt P-450 und Reduktase die aktive Form des Enzymsystems dar. Die stimulierende Wirkung von nichtionischen Detergenzien weist auf eine funktionelle Bedeutung von Lipiden für die Wechselwirkung der ursprünglich membrangebundenen Proteinkomponenten hin.

Alkan-Monooxydgenase-System aus Lodderomyces

657

Wellenlänge (nm) Abb. 2. Nachweis der enzymatischen Reduktion des gereinigten Cyt P-450 anhand der Ausbildung des reduzierten + CO-Komplexes. Die Meßküvetten enthielten in 200 mM KaliumphosphatPuffer, p H 7,25, Cyt P-450(0,18nMol/ml),gereinigte Reduktase (0,7 |xMol Cyt c/min ml), Hexadecan (0,16 mM) und NADPH (0,4 mM) sowie folgende Detergenszusätze: 0,008% Präwozell W-ON100 (• • • •).0,032% Präwozell W-ON100 ( ), 0,04% Triton X100 ( ) bzw. keinen Detergenszusatz ( —). Die Ansätze wurden vor und nach der NADPHZugabe 30 s mit CO durchströmt. Die Reverenzküvetten enthielten jeweils die gleichen Komponenten mit Ausnahme des Cyt P-450. Tabelle 3 Eigenschaften der Komponenten und Aktivität des rekonstituierten Alkan-Monooxygenase-Systems aus L. elongisporus Komponente Cyt P-450 Reduktase

prosthetische Gruppe Eisen-Protoporphyrin IX FAD/FMN 1:1

Molekulargewicht

spezif. Gehalt bzw. Aktivität

53500 79000

12 —171) 60-702) 4 - 63) 8 —124)

reionstituiertes System (Cyt P-450 + Reduktase) r

2 ) nMol/mg Protein; ) |j.Mol Cyt c/min • mg Protein; ) nMol 1-Hexadecanol/min • n Mol Cyt P-450; 4 + ) nMol NADP /min • nMol Cyt P-450 3

Als Produkt der v o m rekonstituierten E n z y m s y s t e m katalysierten U m s e t z u n g v o n H e x a d e c a n wurde 1-Hexadecanol identifiziert (wird an anderer Stelle veröffentlicht). Die Reaktion verläuft dabei offensichtlich nach der in Gl. 1 a wiedergegebenen Bruttogleichung. Die im Vergleich zur Alkanumsetzung höhere N A D P H - O x y d a t i o n könnte, wie bei einigen anderen Cyt P-450-Systemen bekannt (IMAI 1981), durch eine E n t kopplungsreaktion nach Gl. l b hervorgerufen werden. R - C H 3 + 0 2 + N A D P H + H+ -

R-CH2OH + H 2 0 + NADP+

0 2 + N A D P H + H + - H202 + NADP+

(Gl. l a ) (Gl. l b )

Zusammenfassend kann festgestellt werden, daß durch die Reinigung von Cyt P-450 und seiner N A D P H - a b h ä n g i g e n R e d u k t a s e aus L. elongisporus und die R e konstitution dieser beiden Proteine zu einem aktiven Alkan-Monooxygenase-System,

658

W . - H . SCHUNCK et al.

100

300 500 700 Cyt.P-450 (p Mo/)

100 200 300 WO 500 Hexodecan(p.M)

100 200 300 Detergens (n Mo!)

1 2 Reduktase (juMo/ red. Cyt.c-min')

0

20 40 60 80 100 120 NADPH(jiM)

Abb. 3. Rekonstitution des Alkan-Monooxygenase-Systems aus L. elongisporus, gemessen an der Geschwindigkeit der durch Hexadecan ausgelösten NADPH-Oxydation. Bei jeweils fester Konzentration an allen anderen Komponenten (vgl. Material und Methoden) wurden die Menge bzw. Konzentration an Cyt P-450 (A), Reduktase (B) und Detergens (C: "o"o • = Präwozell W-ON 100, — A —A — = Triton X 100) sowie von Hexadecan (D) und NADPH (E) variiert die postulierte enzymyatische P u n k t i o n des Cyt P - 4 5 0 direkt unter in wiro-Bedingungen nachgewiesen werden konnte. Geht man auf Grund dieser Ergebnisse davon aus, daß das Cyt P - 4 5 0 - S y s t e m unter in w'i'o-Bedingungen tatsächlich den 1. S c h r i t t des Alkanabbaus katalysiert, so erscheint seine in wiro-Aktivität als um etwa 2 Größenordnungen zu gering, um die bekannte Wachstumsgeschwindigkeit der Hefezellen auf w-Alkanen zu gewährleisten. Ähnliche Diskrepanzen treten offensichtlich bei fast allen bisher beschriebenen alkanoxidierenden E n z y m e n in Verbindung mit dem Zellaufschluß auf. Als geschwindigkeitslimitierende Schritte können diskutiert werden: die Substrat-Bindung, der Elektronentransfer und die Produktdissoziation. Versuche zur Steigerung der A k t i v i t ä t des rekonstituierten Alkan-Monooxygenase-Systems durch eine Komplettierung mit zusätzlichen K o m p o n e n t e n könnten daher interessante Einblicke z. B . in die Prozesse des intrazellulären Substrat- und Produkttransportes ermöglichen.

Literatur ASPEROER, O., NAUMANN, A .

and

KLEBER, H. P.,

1981.

Occurrence

o f c y t o c h r o m e P - 4 5 0 in

Acinetobacter strains after growth on rc-hexadecane. FEMS Microbiol. Letters, 11, 309—312.

BERTRAND, J . C., GILEWICZ, M., BAZIN, H . , ZACEK, M. a n d AZOLAY, E . ,

1979. Partial

purifica-

tion of cytochrome P-450 of Candida tropicalis and reconstitution of hydroxylase activity. FEBS Letters, 105, 1 4 3 - 1 4 6 .

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BURKE, M. D., 1981. Cytochrome P-450: A pharmaceutical necessity or a biochemical curiosity. Biochem. Pharmacol., 30, 181 — 187. C A R D I N I , G . and J U R T S H U K , P . , 1 9 6 8 . Cytochrome P - 4 5 0 involvement in the oxidation of »-octane by cell-free extracts of Corynebacterium sp. strain 7 E IC. J . biol. Chemistry, 243, 6 0 7 1 — 6 0 7 2 . CARDINI, G. and JURTSHUK, P., 1970. The enzymatic hydroxylation of re-octane by Corynebacterium sp. strain 7E 1C. J . biol. Chemistry, 245, 2789—2796. COOPER, D . Y . ,

SCHLEYER, H . ,

LEVIN, S. S.,

EISENHARDT, R . H . ,

NOVACK, B . G .

and

ROSEN-

THAL, O., 1979. A réévaluation of the role of cytochrome P-450 as the terminal oxidase in hepatic microsomal mixed function oxidase cytalyzed reactions. Drug Metabol. Rev., 10, 153 — 185. D É L A I S S É , J . M . and N Y N S , E . J . , 1974. Detection of cytochrome P-450 in subcellular fractions of Endomycopsis lipolytica grown on w-hexadecane. Arch, internat, physiol. Biochem., 82, 179. D É L A I S S É , J . M . , M A R T I N , P . , V E R I I E Y E N - B O U V Y , M . F . and N Y N S , E . J . , 1 9 8 1 . Subcellular distribution of enzymes in the yeast Saccharomycopsis lipolytica, grown on n-hexadecane, with special reference to the co-hydroxylase. Biochim. biophysica Acta, 6 7 6 , 7 7 — 9 0 . D O L E , V . P . , 1 9 5 6 . A relation between non-esterified f a t t y acids in plasma and the metabolism of glucose. J . Clin. Invest., 35, -150 — 154. D Ü P P E L , W . , L E B E A U L T , J . M . and COON, M . J . , 1973. Properties of a yeast cytochrome P-450 containing enzyme system which catalyzes the hydroxylation of f a t t y acids, alkanes and drugs. Europ. J . Biochem., 36, 5 8 3 - 5 9 2 . G A L L O , M., B E R T R A N D , J . C. et A Z O U L A Y , E . , 1971. Participation du cytochrome P-450 dans 1' oxydation des alcanes chez Candida tropicalis. F E B S Letters, 1 9 , 45—49. G A L L O , M . , B E R T R A N D , J . C . , R O C H E , B. and A Z O U L A Y , E . , 1 9 7 3 . Alkane oxidation in Candida tropicalis. Biochim. biophysica Acta, 296, 6 2 4 — 6 3 8 . GHOLSON, R . K . , BAPTIST, J . N . a n d COON, M. J . , 1963. H y d r o c a r b o n o x i d a t i o n b y a b a c t e r i a l

enzyme system. I I . Cofactor requirements for octanol formation from octane. Biochemistry, 2, 1155-1159. GMÜNDER, F. K., 1979. Die Assimilation von Hexadekan durch Candida tropicalis. Diss. E T H Nr. 6427. G M Ü N D E R , F . K . , K Ä P P E L I , 0 . and F I E C H T E R , A., 1981. Chemostat studies on the hexadecane assimilation b y t h e yeast Candida tropicalis. I I . Regulation of cytochromes and enzymes. Europ. J . Appl. Microbiol. Biotechnol., 12, 135 — 142. HEINZ, E., TULLOCH, A. D. and SPENCER, J . F. T., 1970. Hydroxylation of oleic acid by cell-free extracts of a species of Torulopsis. Biochim. biophysica Acta, 202, 49—55. H O N E C K , H . , SCHTJNCK, W . - H . , R I E G E , P . and M Ü L L E R , H . - G . , 1982. The cytochrome P-450 alkane monooxygenase system çf the yeast Lodderomyces elongisporus: Purification and some properties of t h e NADPH-cytochrome P-450 reductase. Biochem. Biophys. Res. Commun., 106, 1 3 1 8 - 1 3 2 4 . I L C H E N K O , A . P . , M A U E R S B E R G E R , S . , MATYASHOVA, R . N . , a n d LOSINOV, A . B . , 1 9 8 0 . I n d u c t i o n of cytochrome P - 4 5 0 in the course of yeast growth on different substrates. Mikrobiologia, 49, 452-458.

IMAI, Y., 1981. The roles of cytochrome &5 in reconstituted monooxygenase systems containing various forms of hepatic microsomal cytochrome P-450. J . Biochem., 89, 351 — 362. LEBEAULT, J . M., LODE, E. T. and COON, M. J., 1971. F a t t y acid and hydrocarbon hydroxylation in yeast: Role of cytochrome P-450 in Candida tropicalis. Biochem. Biophys. Res. Commun., 42, 4 1 3 - 4 1 9 . M A N S U Y , D., C A R L I E R , M . , B E R T R A N D , J . C . and A Z O U L A Y , E., 1980. Spectral characterization of cytochrome P-450 of a strain of Candida tropicalis grown on tetradecane. Europ. J . Biochem., 109, 1 0 3 - 1 0 8 . M A U E R S B E R G E R , S . , MATYASHOVA, R . N . , M Ü L L E R , H . - G . a n d LOSINOV,, A . B . , 1 9 8 0 . I n f l u e n c e

of

the growth substrate and the oxygen concentration in the medium on the cytochrome P-450 content in Candida guilliermondii. Europ. J . Appl. Microbiol. Biotechnol., 9, 285—294. M A U E R S B E R G E R , S . , S C H U N C K , W.-H., and M Ü L L E R , H.-G., 1981. The induction of cytochrome P-450 in Lodderomyces elongisporus. Z. Allg. Mikrobiol., 21, 313—321. M Ü L L E R , H . - G . , S C H U N C K , W . - H . , R I E G E , P . and H O N E C K , H . , 1 9 7 9 . The alkane-hydroxylating enzyme system of the yea.-t Candida guilliermondii. Acta biol. med. german., 38, 345 — 349. M Ü L L E R , H.-G., M A U E R S B E R G E R , S . , S C H U N C K , W.-H. und W I E D M A N N , B., 1983. Enzym-Induktion in der Hefe Lodderomyces in Gegenwart von ra-Alkanen. Z. Allg. Mikrobiol., 23, 589 — 593. R I E G E , P . , S C H U N C K , W . - H . , H O N E C K , H . and M Ü L L E R , H . - G . , 1 9 8 1 . Cytochrome P - 4 5 0 from Lodderomyces elongisporus: I t s purification and some properties of the highly purified protein. Biochem. Biophys. Res. Commun., 98, 527—534. S C H U N C K , W . - H . , R I E G E , P . , B L A S I G , R . , H O N E C K , H . and M Ü L L E R , H . - G . , 1 9 7 8 . Cytochrome P-450 and alkane hydroxylation activity in Candida guilliermondii. Acta biol. med. german., 37,

K 3 - K 7 .

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Schunck, W.-H., Riege, P., M ü l l e e , H.-G. and Schelee, W., 1983. Purification and some molecular properties of cytochrome P-450 from the alkane-assimilating yeast Lodderomyees elongisporus (russ.). Biochimija, 48, 518—526. Takagi, M., Moriya, K. and Yano, K., 1980. Induction of cytochrome P-450 in petroleum-assimilating yeast. 1. Selection of a strain and basic characterization of cytochrome P-450 induction in the strain. Cell. Molecul. Biol., 25, 363—369. Takagi, M., Kawamura, H., Moriya, K. and Yano, K., 1980. Biochemical studies on cytochrome P-450 induction in Candida cells grown on n-alkane and its derivatives. Proc. 5th Internat. Yeast. Sump., 417-421. T i t t e l b a c h , M., Rohde, H.-G. und Weide, H., 1976. Nachweis eines CO-bindenden Hämoproteins in Candida guilliermondii, Stamm H 17, nach Kultur auf n-Alkanen. Z. Allg. Mikrobiol., 16, 155-156. Anschrift: W.-H. Schunck Zentralinstitut für Molekularbiologie der AdW der DDR, Bereich Angewandte Enzymologie DDR-1115 Berlin-Buch, Robert-Rössle-Str. 10

Zeitschrift f ü r Allgemeine Mikrobiologie

23

1983

10

661-668

(Akademie der Wissenschaften der D D R , F o r s c h u n g s z e n t r u m f ü r Molekularbiologie u n d Medizin, Z e n t r a l i n s t i t u t f ü r Mikrobiologie u n d experimentelle Therapie, J e n a , D i r e k t o r : Prof. D r . U . TAUBENECK)

Methylosin A und B, Pigmente aus Methylosinus

trichosporium

D . G . STBAUSS u n d U . BERGEB

(Eingegangen

am 11.

4.1983)

Screening for m e t h a n o t r o p h i c bacteria led t o t h e isolation of a Methylosinus trichosporium strain which was characterized b y t h e ability t o produce a b r i g h t red-violet pigment, which was f o u n d t o be nearly insoluble in water. This communication r e p o r t s on t h e f o r m a t i o n , isolation, a n d purification of t h e p i g m e n t a n d its separation i n t o t h e components methylosin A a n d B. B y comparison of physico-chemical p a r a m e t e r s we identified t h e p i g m e n t as a prodiginine-type c o m p o u n d . T h e chemical constitution of methylosin B was established. To our knowledge this is t h e f i r s t observation of prodiginine-type compounds t o be metabolites of m e t h a n o t r o p h i c bacteria. Bei den obligat methanotrophen Bakterien werden ausgeprägte Pigmentierungen für die Vertreter v o n zwei G a t t u n g e n beschrieben, als R o s a bis Orange bei Methylomonas u n d G e l b b i s B r a u n b e i Methylobacter (WHITTENBUBY et al. 1 9 7 0 ) . D i e S t ä m m e d e r ü b r i g e n G a t t u n g e n Methylococcus, Methylosinus u n d Methylocystis weisen keine oder lediglich s c h w a c h gelbliche, bräunliche oder rosa F ä r b u n g e n auf, die o f t erst in alten K u l t u r e n erscheinen. D i e s e s B i l d v o n der Verbreitung der P i g m e n t b i l d u n g in dieser Organismengruppe zeigte sich a u c h im Ergebnis eigener Anreicherungs- u n d Isolationsarbeiten, die v o n M a t e r i a l a u s u n t e r s c h i e d l i c h e n H a b i t a t e n a u s g i n g e n u n d zu e i n e r V i e l f a l t v o n I s o l a t e n führten, unter d e n e n alle bisher beschriebenen G a t t u n g e n m e t h a n o t r o p h e r B a k t e r i e n vertreten sind. U n g e w ö h n l i c h war dabei die Isolierung eines S t a m m e s v o n Methylosinus, der sich durch die B i l d u n g eines l e u c h t e n d roten P i g m e n t e s auszeichnet. I n der vorliegenden Arbeit wird über die Gewinnung, Isolierung u n d Charakterisierung des roten Farbstoffs, den Vergleich mit anderen Bakterienpigmenten sowie über die R e i n i g u n g des P i g m e n t e s u n d die T r e n n u n g in seine K o m p o n e n t e n berichtet. A u ß e r d e m wird die K o n s t i t u t i o n der H a u p t k o m p o n e n t e ermittelt.

Material

und

Methoden

Organismus: Der pigmentierte B a k t e r i e n s t a m m S M 6 w u r d e über eine Anreicherungskultur aus d e m Wasser eines s t a r k eutrophierten Flusses (Saale, Mittellauf, Brücke Maua, Kreis J e n a ) isoliert. Medium u n d K u l t u r b e d i n g u n g e n : Zur K u l t i v i e r u n g des Organismus dient ein Mineralmedium, zusammengestellt n a c h HEYER, J. (unveröffentlicht): NH 4 C10,5 g/1 a q u a dest., M g S 0 4 • 7 H „ 0 0,1 g/1, N a „ H P 0 4 • 2 H , 0 0,7 g/1, K H 2 P 0 4 0,3 g/1, CaCl 2 • 6 H „ 0 0,01 g/1, F e S 0 4 • 7 H 2 0 0,005 g/1, ZnS0 4 "- 7 H 2 0 0,44 mg/1, C u S 0 4 • 5 H 2 0 0,20 mg/1, M n S 0 4 • 4 H 2 0 0,20 mg/1, (NH 4 ),Mo0 4 • 2 H 2 0 0,10 mg/1, H 3 B 0 3 0,10 mg/1, CoCl2 • 6 H 2 0 0,08 mg/1. ( p H 7,0—7,4; Sterilisation im Autoklaven). Die K u l t u r erfolgt bei 30 °C u n t e r einer Gasphase v o n 20 vol.% Methan, 5 vol.% C 0 2 u n d 75 vol.% L u f t . Man b r i n g t die K u l t u r e n in E x s i k k a t o r e n u n t e r oder v e r w e n d e t K u l t u r g e f ä ß e m i t gasdicht abschließbaren Aufsätzen, die f ü r S c h ü t t e l k u l t u r e n geeignet sind. Zur Pigmentgewinn u n g erfolgt die Zellernte n a c h 10—20 Tagen K u l t u r d a u e r aus flüssigen R u h e k u l t u r e n d u r c h Zentrifugation (6000 X g, 20 min).

Zeitschrift f ü r Allgemeine Mikrobiologie

23

1983

10

661-668

(Akademie der Wissenschaften der D D R , F o r s c h u n g s z e n t r u m f ü r Molekularbiologie u n d Medizin, Z e n t r a l i n s t i t u t f ü r Mikrobiologie u n d experimentelle Therapie, J e n a , D i r e k t o r : Prof. D r . U . TAUBENECK)

Methylosin A und B, Pigmente aus Methylosinus

trichosporium

D . G . STBAUSS u n d U . BERGEB

(Eingegangen

am 11.

4.1983)

Screening for m e t h a n o t r o p h i c bacteria led t o t h e isolation of a Methylosinus trichosporium strain which was characterized b y t h e ability t o produce a b r i g h t red-violet pigment, which was f o u n d t o be nearly insoluble in water. This communication r e p o r t s on t h e f o r m a t i o n , isolation, a n d purification of t h e p i g m e n t a n d its separation i n t o t h e components methylosin A a n d B. B y comparison of physico-chemical p a r a m e t e r s we identified t h e p i g m e n t as a prodiginine-type c o m p o u n d . T h e chemical constitution of methylosin B was established. To our knowledge this is t h e f i r s t observation of prodiginine-type compounds t o be metabolites of m e t h a n o t r o p h i c bacteria. Bei den obligat methanotrophen Bakterien werden ausgeprägte Pigmentierungen für die Vertreter v o n zwei G a t t u n g e n beschrieben, als R o s a bis Orange bei Methylomonas u n d G e l b b i s B r a u n b e i Methylobacter (WHITTENBUBY et al. 1 9 7 0 ) . D i e S t ä m m e d e r ü b r i g e n G a t t u n g e n Methylococcus, Methylosinus u n d Methylocystis weisen keine oder lediglich s c h w a c h gelbliche, bräunliche oder rosa F ä r b u n g e n auf, die o f t erst in alten K u l t u r e n erscheinen. D i e s e s B i l d v o n der Verbreitung der P i g m e n t b i l d u n g in dieser Organismengruppe zeigte sich a u c h im Ergebnis eigener Anreicherungs- u n d Isolationsarbeiten, die v o n M a t e r i a l a u s u n t e r s c h i e d l i c h e n H a b i t a t e n a u s g i n g e n u n d zu e i n e r V i e l f a l t v o n I s o l a t e n führten, unter d e n e n alle bisher beschriebenen G a t t u n g e n m e t h a n o t r o p h e r B a k t e r i e n vertreten sind. U n g e w ö h n l i c h war dabei die Isolierung eines S t a m m e s v o n Methylosinus, der sich durch die B i l d u n g eines l e u c h t e n d roten P i g m e n t e s auszeichnet. I n der vorliegenden Arbeit wird über die Gewinnung, Isolierung u n d Charakterisierung des roten Farbstoffs, den Vergleich mit anderen Bakterienpigmenten sowie über die R e i n i g u n g des P i g m e n t e s u n d die T r e n n u n g in seine K o m p o n e n t e n berichtet. A u ß e r d e m wird die K o n s t i t u t i o n der H a u p t k o m p o n e n t e ermittelt.

Material

und

Methoden

Organismus: Der pigmentierte B a k t e r i e n s t a m m S M 6 w u r d e über eine Anreicherungskultur aus d e m Wasser eines s t a r k eutrophierten Flusses (Saale, Mittellauf, Brücke Maua, Kreis J e n a ) isoliert. Medium u n d K u l t u r b e d i n g u n g e n : Zur K u l t i v i e r u n g des Organismus dient ein Mineralmedium, zusammengestellt n a c h HEYER, J. (unveröffentlicht): NH 4 C10,5 g/1 a q u a dest., M g S 0 4 • 7 H „ 0 0,1 g/1, N a „ H P 0 4 • 2 H , 0 0,7 g/1, K H 2 P 0 4 0,3 g/1, CaCl 2 • 6 H „ 0 0,01 g/1, F e S 0 4 • 7 H 2 0 0,005 g/1, ZnS0 4 "- 7 H 2 0 0,44 mg/1, C u S 0 4 • 5 H 2 0 0,20 mg/1, M n S 0 4 • 4 H 2 0 0,20 mg/1, (NH 4 ),Mo0 4 • 2 H 2 0 0,10 mg/1, H 3 B 0 3 0,10 mg/1, CoCl2 • 6 H 2 0 0,08 mg/1. ( p H 7,0—7,4; Sterilisation im Autoklaven). Die K u l t u r erfolgt bei 30 °C u n t e r einer Gasphase v o n 20 vol.% Methan, 5 vol.% C 0 2 u n d 75 vol.% L u f t . Man b r i n g t die K u l t u r e n in E x s i k k a t o r e n u n t e r oder v e r w e n d e t K u l t u r g e f ä ß e m i t gasdicht abschließbaren Aufsätzen, die f ü r S c h ü t t e l k u l t u r e n geeignet sind. Zur Pigmentgewinn u n g erfolgt die Zellernte n a c h 10—20 Tagen K u l t u r d a u e r aus flüssigen R u h e k u l t u r e n d u r c h Zentrifugation (6000 X g, 20 min).

662

D . G . STKAUSS u n d U . BERGES

Isolierung des Pigments Methylosin: Das Zentrifugat und der Uberstand der Bakterienkultur wurden zur Zersetzung schleimiger Produkte mit Schwefelsäure auf einen pH-Wert unter 2 angesäuert und die Biomasse von der flüssigen Phase durch Filtration getrennt. Das Filtrat von 150 ml wurde nacheinander mit 75 ml und 50 ml ra-Butanol extrahiert. Die intensiv violettrot gefärbten Extrakte wurden vereinigt und im Vakuum auf ein Endvolumen von 10 ml konzentriert. Die abfiltrierte Biomasse von 15 g (Frischgewicht) wurde dreimal mit je 30 ml Methanol extrahiert. Die Methanolextrakte wurden vereinigt und unter schonenden Bedingungen im Vakuum bis auf ein Endvolumen von 15 ml konzentriert. Das Konzentrat wurde mit Butanol (50 ml) versetzt und die erhaltene Lösung im Vakuum auf 10 ml konzentriert. Die Butanolkonzentrate aus der Filtratund Biomasse-Extraktion wurden vereinigt, durch Waschen mit wenig 0,1 N Natriumbicarbonatlösung von restlichen Säurespuren befreit und im Vakuum zur Trockne eingeengt. Obwohl der Farbwechsel des Methylosins in wäßrigen Lösungen reversibel ist, verstärkte sich zunehmend der Eindruck, daß gleichzeitig auch eine Zersetzung des Farbstoffs erfolgt. Es wurde daher in den weiteren Untersuchungen darauf verzichtet, den Anteil des Pigmentes aus dem angesäuerten Kulturfiltrat zu gewinnen, und dieses nur noch aus der Zellmasse isoliert. Dazu wurden die Zellen von der Kulturlösung abzentrifugiert, in wenig Wasser suspendiert, erneut abzentrifugiert und in Methanol eingetragen. 50 g frische, feuchte Zellmasse wurden mit 75 ml Methanol dreimal nacheinander unter Erhitzen bis auf 40—45 °C behandelt, dabei ging der überwiegende Anteil des relativ festsitzenden Farbstoffs in das Methanol über. Die Methanolextrakte wurden vereinigt, getrocknet und im Vakuum bei einer Temperatur unter 20 °C zur Trockne eingeengt. Der Rückstand betrug 175 mg. Reinigung und Trennung des Methylosins: Das Rohprodukt wurde in wasserfreiem Chloroform gelöst und an einem Dextran-Gel (Sephadex LH 20, PHARMACIA, Schweden) säulenchromatographisch in eine rein blaue Fraktion A und eine intensiv rote Fraktion B getrennt. Dazu wurden 150 mg Rohprodukt in 50 ml Chloroform gelöst, die Lösung auf ein Gelbett von 50 cm Länge und 2 cm Durchmesser aufgetragen. Anschließend wurde mit reinem Chloroform eluiert. Die blaue Komponente bildete die 1. Zone und die rote die 2. Beide wurden getrennt aufgefangen und die Eluate zur Trockne gebracht. Der Trockenrückstand der blauen Komponente betrug 7,2 mg, der der roten 31,7 mg.

Ergebnisse Charakterisierung des Organismus Der untersuchte Stamm wächst obligat methanotroph, d. h. unter obligater Verwertung von Methan als alleiniger Kohlenstoff- und Energiequelle. Der Organismus bildet auf festem Medium nach 7- bis 12tägiger Kulturdauer Kolonien mit 0,5—3 m m Durchmesser, die farblos, schwach rot bis blutrot gefärbt sein können. Der Farbstoff diffundiert nicht ins Agarmedium. Die Zellen sind gramnegative, bewegliche, gerade oder schwach gebogene, manchmal leicht birnenförmige Stäbchen (0,7 — 1,0 X 2,5—4,5 [i.m), die Exosporen und Rosetten bilden. Mit dieser Zellmorphologie wird der von uns isolierte Organismus als Methylosinus trichosporium charakterisiert, der sich von den bisher beschriebenen Vertretern dieser Art jedoch durch die Bildung des roten Pigments unterscheidet. Diese Art der Familie Methylomonadaceae ist seit W H I T T E N B U R Y et al. ( 1 9 7 0 ) beschrieben, jedoch nicht in den Approved Lists of Bacterial Names (SKERMAN et al. 1980) enthalten. Kulturbedingungen und Pigmentierung In Ruhekultur mit flüssigem Medium bildet sich eine rote Wachstumshaut, die aus Aggregaten von rosettenförmig angeordneten Zellen besteht. Mit Schüttelkulturen läßt sich eine größere Zellmasse erzeugen als mit Standkulturen, jedoch bleibt dabei die Pigmentbildung fast völlig aus. Wird eine Schüttelkultur als Ruhekultur (nach Erneuerung der Gasphase) weiterbebrütet, so kommt es wiederum zur Ausbildung einer pigmentierten Oberflächenhaut.

Pigmente aus Methylosinus trichosporium

663

Zur Gewinnung von pigmentierten Zellen in Massenkultur waren Ruhekulturen in Kulturgefäßen (z. B. Fernbachkolben) mit flüssigem Medium in flacher Schicht mit großer Oberfläche am geeignetsten, da sich hier die Wachstumshaut an einer großen Kontaktfläche zur Gasphase hin ausbilden kann. Der rote Farbstoff ist in Wasser nahezu unlöslich. Die geringe Rotfärbung der Kulturlösung beruht auf partikulär und an Schleimstoffe gebundenem Farbstoff. Mikroskopisch zeigt sich eine Anreicherung des roten Pigments in alten Zellen, in Resten abgestorbener Zellen und in der interzellularen Schleimsubstanz der Rosetten. Durch Induzieren eines Sauerstoffmangels für die Kultur ließ sich die Pigmentierung nicht beeinflussen. Biologische A k t i v i t ä t des P i g m e n t s Beim Testen von Methanol-, Äthanol-, n-Butanol- und Acetonextrakten der pigmentierten Zellmasse wurden im Agar-Diffusionstest gegen die folgende Keimauswahl keine antimikrobiellen Wirksamkeiten festgestellt: Bacillus sublilis Escherichia coli Pseudomonas aeruginosa Pseudomonas spec. Proteus vulgaris Micrococcus flavus Alkaligenes faecalis Comamonas terrigena Staphylococcus aureus Mycobacterium smegmatis Saccharomyces cerevisiae Kloeckera brevis Pénicillium notatum

ATCC 6633 SG 458 und C 600 SG 137 B 7 SG 2 ATCC 10240 ATCC 8750 ATCC 8461 SG 511 SG 987 JH 1 JH 2 J P 36

In den mikrobiologischen Modelltests auf potentiell carcinostatische Aktivität (Prophagen-Induktions-Test GADO et al. 1966; BIP-Test FLECK 1968) erwies sich das Pigment als unwirksam. Chemische C h a r a k t e r i s i e r u n g des Methylosins Methylosin ist ein roter, in konzentrierter Form blauroter Farbstoff mit Indikatoreigenschaften. Seine Lösungen in organischer Phase sind in saurem Milieu rot, in alkalischem Milieu gelb gefärbt. Diese Farbreaktion is reversibel, jedoch nicht unbegrenzt stabil. Ein Vergleich der UV/Vis-Absorption des Methylosins mit dem beobachteten braunen Zersetzungsprodukt zeigt, daß letzteres ein von der Ausgangssubstanz erheblich abweichendes Spektrum besitzt, was auf eine weitgehende Änderung des chromophoren Systems schließen läßt. Die Absorptionsmaxima des Methylosins liegen bei 243 und 536 nm in saurem Milieu, das längerwellige Maximum wird beim Alkalisieren hypsochrom verschoben und liegt dann bei 470 nm. Die Art des Färb wechseis, die Lage der Absorptionsmaxima und der relativ hohe Stickstoffgehalt der Substanz (ca. 12%) ließen die Annahme zu, daß es sich beim Methylosin um Prodigiosin oder ein Derivat davon handelt, was bestätigt werden konnte. Für die Prodigiosin-ähnlichen Pigmente gibt es ein Bezeichnungssystem nach dem Vorschlag von GEHBER (1969). Danach wird der Name Prodigiosin für den ersten bekannten Vertreter der Gruppe, für 2-Methyl-3-pentyl-prodiginin, reserviert und

664

D . G . STRAUSS u n d U . B E R G E R

„Prodiginin" als Trivialname für den gleichbleibenden aromatischen Teil der Prodigiosin-Struktur dieser Pigmente verwendet. Indem wir uns dem anschließen, sind die von uns gefundenen Farbstoffe auf Grund ihres spektralen Verhaltens als ProdigininDerivate zu bezeichnen. C h a r a k t e r i s t i k des M e t h y l o s i n A Methylosin A ist eine in organischen Lösungsmitteln intensiv blaue, in festem Zustand graublaue Substanz. Sie konnte bisher nicht zur Kristallisation gebracht werden. Methylosin A löst sich in praktisch allen organischen Lösungsmitteln, einschließlich Petroläther, mit blauer Farbe, hat Indikatoreigenschaften und zeigt unter dem Einfluß von Alkalien eine gelbe Farbe, welche auf Zusatz von Säuren wieder nach blau umschlägt. Der Farbwechsel ist zwar reversibel, doch deuten sich Zersetzungserscheinungen an. Wenn der Medienwechsel mehrmals wiederholt wird, treten in zunehmendem Maße braune Produkte auf, die keine Indikatoreigenschaften mehr besitzen. Das UV-Spektrum und IR-Spektrum von Methylosin A ist mit dem von Methylosin B weitgehend identisch (Abb. 1, 2 und 3). Der gefundene Molpeak mit per Masse 729 war für die Bestimmung der Bruttoformel zu schwach. £ C

1,0 -

o -0,1 moo

' moo

1

' 25000

1

35000

30000

1 20000

1 , 15000cm'1

Abb. 1. UV/Vis-Spektrum von Methylosin A und B in 0,1 N salzsaurem Methanol

t

e

8

10

12

n

16

IS

20 22

24

26

28

30

C h a r a k t e r i s t i k des M e t h y l o s i n

32

34

36

38 WxWOcm'7

B

Methylosin B ist ein in allen geprüften organischen Lösungsmitteln gut lösliches Produkt und löst sich darin mit intensiv roter Farbe. Im festen Zustand ist Methylosin B blaurot, zeigt keine Fluoreszenz, hat Indikatoreigenschaften, welche sich im Farbwechsel sauer/neutral rot und alkalisch gelb äußern (Abb. 4).

665

Pigmente aus Methylosinus trichosporium if

100

6

8

10

12

»

16

18

20

22

fr

26

2$

30

32

3k

36

38

Wx 0 10

100cm''

20

L—i

i

25 20

15

n

i i 10 9

i 8

1 7

1 6

' 5

' 1,5

'

1 *

3,5

-90 —hoo 2,5)im

1—

3

Abb. 3. IR-Spektrum von Methylosin B in K B r

Dieser Farbwechsel ist reversibel, ohne daß Zersetzungserscheinungen wie bei Methylosin A zu beobachten sind. In K B r gibt (B) ein gut ausgeprägtes IR-Spektrum (Abb. 3) mit den Banden bei 3055, 2955, 2930, 2870, 2850, 1730, 1630, 1600, 1575, 1540, 1510, 1475, 1460, 1440, 1375, 1270, 1260, 1080, 1020, 955, 840, 740 und 685 cm" 1 , welches sich nur wenig von dem des Methylosin A unterscheidet. Methylosin B besteht aus C, H, N und 0 , hat schwach basische Eigenschaften und bildet mit Säuren Salze, welche aber bisher nicht formelrein erhalten werden konnten (ca. 1,15 mol HCl/3 N). Die Summenformel und das Molekulargewicht wurden massenspektrometrisch bestimmt. C 22 H 29 N 3 0; MG 351. Für die angegebene Summenformel wurde berechnet 351,2310 und 351,2330 gefunden. Der Basispeak wurde zu 266,1282 bestimmt. E r entspricht einem Fragment C 16 H 16 N 3 0, dessen berechnete Molmasse 266,1293 beträgt. Aus beiden Daten ergibt sich ein Spaltstück von der Größe C6H13, welches eine aliphatische Seitenkette des Methylosin B darstellt, und das Fragment mit der Massenzahl 266 repräsentiert somit den Chromophor des Moleküls. Die Prodiginine zeigen im Massenspektrum ein charakteristisches Fragmentierungsmuster, indem sowohl vom Grundmolekül als auch nach Abspaltung der aliphatischen respektive alicyclischen Seitenkette vom verbleibenden Fragment ein doppelt geladenes Ion gebildet wird, was zu gebrochenen Massenzahlen führt (Tab. 1). Diese werden nun auch im Massenspektrum des Methylosin B gefunden, und zwar für die Molmasse 351 das doppelt geladene Ion mit der Molmasse 175,5 sowie das Fragment £ 18500s 540,5 nm

Komponente B im sauren Methanol ( 0,1 n HCl)

0,5

0 75000

20000

15000

cm' 1

Abb. 4. Vis-Spektrum von Methylosin B, Lage des längstwelligen Maximums in Abhängigkeit vom pH-Wert des Milieus

666

D . G . STKAUSS u n d U . B E R G E R

Tabelle 1 Molmassenpeaks und Fragmentpeaks der Prodiginine substance 2-Methyl-3-pentyl-I (Prodogiosin) 8-Desmethoxy-8-hydroxy2-methyl-3-pentyl-I(Norprodigiosin) 2-Methyl-3-heptyl-I (UB2B2) 2-Undecyl-I 7'-Butyl-2,3-cycloheptyl-I 3'-Ethyl-3,4-cyclopropyl-I

M+

M++

M+-side chain

M ++ -side chain

323

161,5

267/266(—C4H9)

133,5/133

309

154,5

253/252(—C4H9)

126,5/126

351

175,5

267/266(—C6H13)

133,5/133

393 391 321

196,5 195,5 160,5

253/252( —C10H21) 348(-C3H7) 306/305-(CH 3 )

126,5/126 153/152,5

ohne Seitenkette mit der Molmasse 2 6 7 / 2 6 6 sowie dessen doppelt geladenes Ion mit der Molmasse 133,5/133. Der Fragmentpeak mit der Masse 266 stellt den Basispeak dar, für seine Masse wurde eine Bruttoformel von C l g H 1 6 N ermittelt. Wie bereits erwähnt, existieren von den Prodigininen aliphatisch und alicyclisch substituierte Derivate. Letztere unterscheiden sich von den ersteren deutlich im Protonenresonanzspektrum dadurch, daß ein Signal für das Proton in C-3 Position auftritt, welches in ersterem fehlt, und dessen Signal bei 5,7 ppm liegt (Tab. 2). Das Fehlen dieses Signals im Methylosin B weist darauf hin, daß die Komponente B zur Gruppe der Prodigiosine gehört, was außerdem durch das Signal für die olefinisch Tabelle 2 Protonenresonanzsignale der olefinischen Substituenten in Abhängigkeit vom Substitutionsmuster (RI-R4) substance

RI

(ppm)

2-Methyl-3-pentyl-I (Prodigiosin) 3'-Bthyl-3,4-cyclopropyl-l (Pigment 2) 7'-Butyl-2,3-cycloheptyl-I 2-MethyI-3-heptyl-I (UB2B2) 2-Undecyl-I

CH3 (1,8 s) CH3 (2,0 s) CH2 CH3 (2,02 s) CH2

9'-Ethyl-2,4-cyclononyl-I 2.14-cycloalkyl-I

CH2 CH2

olefinic substituents R3 R2 (ppm) (ppm) CH2 (2,35 m) CH, (2,35 IN) CH2 CH, (2,35 m) H (5,77) H H

characteristic signal of the I-substitution pattern by Thesis 1965 OCH3

H

R-| -R^=H

References

R4 (ppm)

H (6,4 s) CH

H (6,7) H

H H (6,4 s) H

H H

D . G . STRAUSS,

H

GERBER (1969)

CH2 H

Varían N.M.R.spectra catalogue G E R B E R et al.

(1979)

unpubl.

H CH2

G . C . RODGERS

and

R.

H.

R3

H : Prodiginine

(1)

(N.N. Gerber; Appt. Microbiol.

1Í ,1-3

(1969)

WILLIAMS,

Ph. D.

Pigmente aus Methylosinus trichosporium.

667

gebundene CH 3 -Gruppe unterstützt wird, da diese in allen übrigen bisher bekannten Prodigininen fehlt. Auf Grund der chemisch-physikalischen Daten konnte ein Vergleich mit den für Prodigiosin publizierten Angaben durchgeführt werden. Danach besteht für Methylosin A und B eine weitgehende Übereinstimmung mit den von W I L L I A M S et al. ( 1 9 5 6 ) publizierten UV-Daten für Prodigiosin und im IR-Spektrum mit den Angaben von G R E E N et al. ( 1 9 5 6 ) für die blaue und rote Traktion des Prodigiosins. Methylosin B unterscheidet sich aber vom Prodigiosin in seinem Molekulargewicht und von den durch W R E E D E U. R O T H H A A S ( 1 9 3 4 ) publizierten und von D ' A O U S T und G E R B E R ( 1 9 7 4 ) diskutierten Strukturformeln für Prodigiosin durch seine um zwei —CH 2 -Gruppen längere Seitenkette und ist deshalb als 2-Methyl-3-heptyl-prodiginin zu bezeichnen, ursprüngliche Arbeitsbezeichnung siehe Tabelle 1 UB2B2. Diskussion Prodigiosin ist ursprünglich als charakteristisches rotes Pigment von Serratia marcescens ( W I L L I A M S U. H E A R N 1 9 6 7 ) beschrieben worden; inzwischen hat sich jedoch gezeigt, daß die Bildung von Prodigiosin oder Prodigiosin-ähnlichen Pigmenten nicht auf diese Gattung beschränkt ist, sondern auch bei anderen Mikroorganismen vorkommt. Es gibt eine ganze Reihe von Literatvirmitteilungen über das Vorkommen der leuchtend roten, lipophilen, Prodigiosin-ähnlichen Pigmente mit neuen und ungewöhnlichen Strukturen bei Vertretern einiger Actinomycetengattungen (Streptomyces spec. ( G E R B E R U. L E C H E V A L I E R 1 9 7 6 , W A S S E R M A N et al. 1 9 6 9 ) ; Actinomadura madurae, A. pelletiert ( G E R B E R 1 9 6 9 , 1 9 7 1 , 1 9 7 3 ) ; Streptoverticillium rubrireticuli ( G E R B E R U. S T A H L Y 1 9 7 5 ) ) , bei verschiedenen marinen Bakterien (Alteromonas rubra ( G A U T H I E R 1 9 7 6 , G E R B E R U. G A U T H I E R 1 9 7 9 ) ; Vibrio psychroereythreus ( D ' A O U S T U. G E R B E R 1 9 7 4 ) ; Pseudomonas magnesiorubra ( G A N D H I et al. 1 9 7 3 , G E R B E R 1 9 7 5 ) und andere marine Pseudomonas-Arten ( G H A N D I et al. 1 9 7 6 ) ; mesophiles gramnegatives marines Bakterium ( L E W I S U. C O R P E 1 9 6 4 ) sowie einem nicht identifizierten roten Abwasserbakterium ( G E R B E R U. G A U T H I E R 1 9 7 9 , G E R B E R 1 9 7 5 ) . Unsere Identifizierung von Prodigiosin-ähnlichen Pigmenten bei Methylosinus trichosporium SM 6 erweitert erneut die Kenntnis über das Vorkommen dieser Verbindungsklasse und unterstreicht, daß diese Pigmente unter Mikroorganismen unterschiedlicher systematischer Stellung und bei Bewohnern verschiedener Ökosysteme verbreitet sind. Die physiologische Bedeutung der Pigmente ist unbekannt. Mit dem Auffinden dieser Farbstoffe wird das Vorkommen derartiger Verbindungen als Metabolite in der Gruppe der methanotrophen Bakterien erstmalig nachgewiesen. Es bleibt ungeklärt, inwieweit es sich bei diesem Pigment von Methylosinus trichosporium lediglich um ein Alterungsprodukt der Zellen handelt oder um einen Bestandteil der Haftsubstanz bei der Rosettenbildung. Bereits untersuchte Pigmente von methanotrophen Bakterien sind die rosa, braunen und gelben Farbstoffe bei Methylomonas methanica, die von L E A D B E T T E R und F O S T E R ( 1 9 5 8 ) als Karotinoide identifiziert wurden, und die von B U D O H O S K I et al. ( 1 9 7 8 ) bei Methylomonas-Stämmen nachgewiesenen Pteridine mit Fluoreszenzeigenschaften. Der Unterschied zu dem von uns isolierten Pigment ist offensichtlich. Literatur BUDOHOSKI, L . , MICHALIK, J . a n d RACZYNSKA-BOJANOWSKA, K . , 1978. F l u o r e s c e n t p i g m e n t s i n

the newly isolated methylotrophs: Pseudomonas J 16 and Methylomonas P 11. Acta Microbiol. Pol., 27, 257-266.

668

D . G . STRAUSS u n d U . BERGER

and G E R B E R , N . ST., 1 9 7 4 . Isolation and purification of prodigiosin from Vibrio psychroerythreus. J . Bacteriol., 118, 756—757. F L E C K , W . , 1 9 6 8 . Eine neue mikrobiologische Screening-Methode für die Suche nach potentiellen Carcinostatica und Virostatica mit Wirkung im Nukleinsäure-Stoffwechsel. Z. Allg. Mikrobiol.,

D'AOUST, J . Y .

8,

139-144.

I., SAVTSCIIENKO, G . and H O R V A T H , I . , 1 9 6 6 . Agardiffusion method for the screening of anticancer substances by phage induction. Acta Microbiol. Acad. Sei. Hung., 12, 363. G A N D H I , N . M., N A Z A R E T H , J . , D I V E K A R , P . V . , K O H L , H . and D E SOUZA, N . J . , 1 9 7 3 . Magnesidin, a novel magnesium-containing antibiotic. J . Antib., 2 6 , 7 9 7 — 7 9 8 . G A U T H I E R , M. J., 1976. Alteromonas rubra sp. nov., a new marine antibiotic-producing bacterium. Int. J . Syst. Bacteriol., 26, 459-466. G H A N D I , N. M., P A T E L L , J . R., G H A N D I , J., DE SOUZA, N. J., and K O H L , H., 1976. Prodigiosin metabolites of a marine Pseudomonas species. Mar. Biol., 34, 223—227. G E R B E R , N. N., 1969. Prodigiosin-like Pigments from Actinomadura (Nocardia) pelletieri and Actinomadura madurae. Appl. Microbiol., 18, 1 — 3. G E R B E R ^ N. N., 1971. Prodigiosin-like pigments from Actinomadura (Nocardia) pelletieri. J. Antib., 24, 636. G E R B E R , N. N., 1973. Minor prodiginine pigments from Actinomadura madurae and Actinomadura pelletieri. J . Heterocycl. Chem., 10, 925—929. G E R B E R , N. N., 1975. Prodigiosin-like pigments. Crit. Rev. Microbiol., 3, 469—485. G E R B E R , N. N. and S T A H L Y , D. P., 1975. Prodiginine (prodigiosin-like) pigments from Streptoverticillium rubrireticuli, an organism that causes pink staining of polyvinyl chloride. Appl. Microbiol., 30, 807-810. G E R B E R , N. N. and L E C H E V A L I E R , M. P., 1 9 7 6 . Prodiginine (prodigiosin-like) pigments from Streptomyces and other aerobic actinomycetes. Canad. J . Microbiol., 2 2 , 6 5 8 — 6 6 7 . G E R B E R , N. N. and G A U T H I E R , M. J., 1979. New prodigiosin-like pigment from Alteromonas rubra. Appl. environm. Microbiol., 37, 1176 — 1179. G R E E N , J . A . , R A P P O P O R T , D . A . and W I L L I A M S , R . P . , 1 9 5 6 . Studies on pigmentation of Ser. marcescens. J . Bacteriol., 72, 483—487. L E A D B E T T E R , E . R . and F O S T E R , J . W . , 1 9 5 8 . Studies on some methane-utilizing bacteria. Arch. Mikrobiol., 3 0 , 9 1 - 1 1 8 . L E W I S , S. M. and C O R P E , W. A., 1964. Prodigiosin-producing bacteria from marine sources. Appl. Microbiol., 12, 1 3 - 1 7 . S K E R M A N , V. B. D., Mc G O W A N , V. and S N E A T H , P. H. A. (ed.), 1980. Approved Lists of Bacterial Names. Int. J . Syst. Bacteriol., 30, 225—420. W A S S E R M A N , H. H., R O D G E R S , G . C . and K E I T H , D . D . , 1969. Metacyloprodigiosin, a tripyrrole pigment from Streptomyces longisporus ruber. J . Amer. Chem. Soc., 91, 1263 — 1264. W H I T T E N B U R Y , R., P H I L L I P S , K. C. and W I L K I N S O N , J. F., 1970. Enrichment, isolation and some properties of methane-utilizing bacteria. J . gen. Microbiol., 61, 205 —218. W I L L I A M S , R. P. and H E A R N , W . R., 1 9 6 7 . Prodigiosin. I n : Antibiotics, 2 , Biosynthesis ( D . G O T T L I E B and P. D . S H A W ) . Springer-Verlag, Inc., New York, 4 1 0 — 4 3 2 , 4 4 9 . W I L L I A M S , R . P . , G R E E N , J . A. and R A P P O P O R T , D . A., 1 9 5 6 . Studies on pigmentation of Ser. marcescens. J . Bacteriol., 71, 115 — 120. W R E D E , F. und R O T H H A A S , A., 1934. Über das Prodigiosin, den roten Farbstoff des B. prodigiosus. Hoppe-Seylers Z. physiol. Chem., 226, 9 5 - 1 0 7 . GADO,

Anschrift: Dr. sc. D. G . STRAUSS Zentralinstitut für Mikrobiologie und experimentelle Therapie der AdW DDR-6900 Jena, Beutenbergstraße 11

RICHARD CAMPBELL

Mikrobielle Ökologie Übersetzung aus dem Englischen (Wissenschaftliche Taschenbücher, Reihe Biologie)

1981. 243 Seiten — 55 Abbildungen — 24 Tabellen — kl. 8° — 12,50 M Bestell-Nr. 7628487 (7272)

Der Autor gibt in diesem Taschenbuch eine Einführung in die Ökologie der Mikroorganismen. Nach der Darstellung der Grundkonzeptionen der Ökologie und der Methoden der Mikrobenökologie werden die mikrobiologischen Umsetzungen des Kohlenstoffs, Stickstoffs, Schwefels, Phosphors, Eisens und Siliziums sowie die Nutzung des mikrobiellen Abbaus dieser Elemente und ihrer Verbindungen behandelt.

Bitte richten Sie Ihre Bestellungen an eine Buchhandlung

AKADEMIE-VERLAG DDR-1086 Berlin, Leipziger Str. 3 - 4

Z E I T S C H R I F T FÜR ALLGEMEINE MIKROBIOLOGIE VOLUME 23

1983

N U M B E R 10

CONTENTS Formation of additional contacts of chromosome with membrane in the process of DNA repair synthesis in bacterial cells Production of oxalic acid by some fungi infected tubers Ultrastructural characterization of core structures and paracrystalline inclusion bodies in L-form cells of streptomycetes Effect of oils and fatty acids on growth and enzyme production of Thermoactinomyces

vulgaris.

III.

Influence of culture vessels, strains, and medium composition Intracytoplasmic membrane induction by hexadecane in Acinetobacter

calcoaceticus

V . G . BEZLEPKIN, Y U . Y U . MALINOVSKY AND A . I . GAZIEV

607

0 . FABOYA, T . IKOTUN AND 0 . S . FATOKI

621

J . GUMPERT

625

A . LEUCHTENBEBGEB AND H . R U T T LOFF 635

H . MÜLLER, A . NAUMANN, R . CLAUS AND H . - P . KLEBER 645

Isolation and reconstitution of the alkane monooxygenase system of the yeast Lodderomycea elongi-

W . - H . SCHUNCK, P . RIEGE, H . H O NECK AND H . - G . MÜLLER 653

Methylosins A and B, pigments from Methylosinua

D . G . STBAUSS a n d U . BERGER

sporiis

trichosporium

661

Z E I T S C H R I F T FÜR ALLGEMEINE MIKROBIOLOGIE VOLUME 23

1983

N U M B E R 10

CONTENTS Formation of additional contacts of chromosome with membrane in the process of DNA repair synthesis in bacterial cells Production of oxalic acid by some fungi infected tubers Ultrastructural characterization of core structures and paracrystalline inclusion bodies in L-form cells of streptomycetes Effect of oils and fatty acids on growth and enzyme production of Thermoactinomyces

vulgaris.

III.

Influence of culture vessels, strains, and medium composition Intracytoplasmic membrane induction by hexadecane in Acinetobacter

calcoaceticus

V . G . BEZLEPKIN, Y U . Y U . MALINOVSKY AND A . I . GAZIEV

607

0 . FABOYA, T . IKOTUN AND 0 . S . FATOKI

621

J . GUMPERT

625

A . LEUCHTENBEBGEB AND H . R U T T LOFF 635

H . MÜLLER, A . NAUMANN, R . CLAUS AND H . - P . KLEBER 645

Isolation and reconstitution of the alkane monooxygenase system of the yeast Lodderomycea elongi-

W . - H . SCHUNCK, P . RIEGE, H . H O NECK AND H . - G . MÜLLER 653

Methylosins A and B, pigments from Methylosinua

D . G . STBAUSS a n d U . BERGER

sporiis

trichosporium

661