Zeitschrift für Allgemeine Mikrobiologie: Volume 24, Heft 2 [Reprint 2021 ed.] 9783112565780, 9783112565773

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ZEITSCHRIFT FÜR ALLGEMEINE MIKRQ-BIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS VOLUME 24 • 1984 • NUMBER 2

AKADEMIE-VERLAG • BERLIN ISSN 0044-2208

Z. allg. Mikrobiol., Berlin 24 (1984), 2, 65-136

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 Englisch 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 "eí 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.: B E R T H E L I N , J., B E L G Y , G. and M A G N E , R., 1977. Some aspects of the mechanism of solubilization and insolubilization of uranium from granites by heterotrophic microorganisms. In: Bacterial Leaching Conference (W. S C H W A R T Z , Editor), pp. 261 to 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.: K A P P E L I , O . , M Ü L L E R , M . and F I E C H T E R , A . , 1978. Chemical and structural alterations at the cell surface of Candida tropicalis, induced by hydrocarbon substrate. J . Bacterid., 133, 952—958. 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 fiir 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 MIKRO- BIOLOGIE

HERAUSGEGEBEN VON

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

UNTER D E R CHEFREDAKTION VON

W. Schwartz, Braunschweig

AN INTERNATIONAL JOURNAL ON

und

MORPHOLOGY, PHYSIOLOGY, GENETICS,

U. Taubeneck, Jena

AND ECOLOGY OF MICROORGANISMS UNTER 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

U. May, Jena

VOLUME 2 4 • 1984 • N U M B E R 2

AKADEMIE-VERLAG • BERLIN

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Zeitschrift für allgemeine Mikrobiologie 2 4 (1984) 2, 67 — 75

(Sektion Biologie der Ernst-Moritz-Arndt-Universtität Greifswald, W B Molekularbiologie)

Charakterisierung von drei aromatischen Aminotransferasen aus Candida maltosa R . BODE u n d D . BIRNBAUM

(Eingegangen

am 8.

7.1983)

T o characterize the aromatic aminotransferase activity cell free extracts of Candida maltosa were chromatographed on DEAE-cellulose, P A A G electrophorese, and gel filtration, respectively. Three active fractions could be separated: AAT I was constitutively synthesized, A A T I I appears t o be regulated by the general control of amino acid biosynthesis, a result which suggests its probable role in phenylalanine/tyrosine biosynthesis, and A A T I I I was inducible by tryptophan, phenylalanine, and/or tyrosine. Gel filtration analyses indicate t h a t aromatic aminotransferases have molecular weights of 73,000, 85,000, and 105,000, respectively. All three enzymes showed overlapping specificities, each capable of transamination with phenylpyruvate, 4-hydroxyphenylpyruvate, prephenate, or indolepyruvate and the corresponding amino acids, but they were slowly active to glutamate and 2-oxoglutarate. The apparent MICHAELIS constants were determined and the possibilities of the enzyme reactions in the organism were discussed.

Die Aminotransferasen stellen zumeist Enzyme dar, die sich durch eine breite Substratspezifität auszeichnen. Auf Grund der dadurch bedingten überlappenden Funktionen ist es häufig schwierig, sie in bestimmte biochemische Wege einzuordnen. Hinzu kommt noch, daß sie durch ihre Eigenschaft, die Enzymreaktion reversibel gestalten zu können, in der Lage sind, sowohl synthetische als auch katabole Prozesse zu katalysieren ( J E N S E N U. CALHOUN 1 9 8 1 ) . Für die Transaminierung der aromatischen Aminosäuren läßt sich eine Vielzahl dieser unterschiedlichen Möglichkeiten aufzeigen, die in den verschiedenen Pro- und Eukaryoten realisiert werden. Neben Organismen, die für die Transaminierung von Tryptophan, Tyrosin und Phenylalanin nur ein Enzym besitzen (SPEEDIE et al. 1 9 7 5 , B E S O H L E et al. 1 9 8 2 ) , gibt es solche mit spezifischen Enzymen für einzelne Reaktionen ( T E U S C H E R 1 9 7 0 , B O D E U. BIRNBAUM 1 9 7 8 b, B Y N G et al. 1 9 8 1 ) bzw. mehrere Enzyme für gleiche Sequenzen (MAVRIDES U. COMERTON 1 9 7 8 , R U B I N U. J E N S E N 1 9 7 9 , P A R I S U. MAGASANIK 1 9 8 1 ) und andere mit einem über die drei aromatischen Aminosäuren hinausgehenden Substratspektrum ( P O W E L L U. MORRISON 1 9 7 8 , F A Z E L U. J E N S E N 1 9 7 9 , K O I D E et al.

1980).

Über die Syntheseregulation der aromatischen Aminotransferasen liegen relativ wenige Ergebnisse vor. K R A D O L F E R et al. ( 1 9 8 2 ) konnten für Saccharomyces cerevisiae die Existenz von zwei aromatischen Aminotransferasen zeigen, von denen eine durch die aromatischen Aminosäuren induziert werden kann. Eine Regulierbarkeit der Synthese dieser Enzyme konnte auch in Bakterien nachgewiesen werden (COLLIER u . KOHLHAW 1 9 7 2 , P A T E L et al.

1978).

Unsere Untersuchungen der Transaminierung aromatischer Aminosäuren in Candida maltosa zeigten, daß diese Aktivität durch die allgemeine Kontrolle der Aminosäurebiosynthese reguliert werden kann (BODE et al. 1983). Das Anliegen der vorliegenden Arbeit ist es, die bei diesem Organismus gefundenen drei unterschiedlichen aromatischen Aminotransferasen vorzustellen und Aussagen über deren Funktion, Regulierbarkeit und Substratspezifität zu treffen, s*

Zeitschrift für allgemeine Mikrobiologie 2 4 (1984) 2, 67 — 75

(Sektion Biologie der Ernst-Moritz-Arndt-Universtität Greifswald, W B Molekularbiologie)

Charakterisierung von drei aromatischen Aminotransferasen aus Candida maltosa R . BODE u n d D . BIRNBAUM

(Eingegangen

am 8.

7.1983)

T o characterize the aromatic aminotransferase activity cell free extracts of Candida maltosa were chromatographed on DEAE-cellulose, P A A G electrophorese, and gel filtration, respectively. Three active fractions could be separated: AAT I was constitutively synthesized, A A T I I appears t o be regulated by the general control of amino acid biosynthesis, a result which suggests its probable role in phenylalanine/tyrosine biosynthesis, and A A T I I I was inducible by tryptophan, phenylalanine, and/or tyrosine. Gel filtration analyses indicate t h a t aromatic aminotransferases have molecular weights of 73,000, 85,000, and 105,000, respectively. All three enzymes showed overlapping specificities, each capable of transamination with phenylpyruvate, 4-hydroxyphenylpyruvate, prephenate, or indolepyruvate and the corresponding amino acids, but they were slowly active to glutamate and 2-oxoglutarate. The apparent MICHAELIS constants were determined and the possibilities of the enzyme reactions in the organism were discussed.

Die Aminotransferasen stellen zumeist Enzyme dar, die sich durch eine breite Substratspezifität auszeichnen. Auf Grund der dadurch bedingten überlappenden Funktionen ist es häufig schwierig, sie in bestimmte biochemische Wege einzuordnen. Hinzu kommt noch, daß sie durch ihre Eigenschaft, die Enzymreaktion reversibel gestalten zu können, in der Lage sind, sowohl synthetische als auch katabole Prozesse zu katalysieren ( J E N S E N U. CALHOUN 1 9 8 1 ) . Für die Transaminierung der aromatischen Aminosäuren läßt sich eine Vielzahl dieser unterschiedlichen Möglichkeiten aufzeigen, die in den verschiedenen Pro- und Eukaryoten realisiert werden. Neben Organismen, die für die Transaminierung von Tryptophan, Tyrosin und Phenylalanin nur ein Enzym besitzen (SPEEDIE et al. 1 9 7 5 , B E S O H L E et al. 1 9 8 2 ) , gibt es solche mit spezifischen Enzymen für einzelne Reaktionen ( T E U S C H E R 1 9 7 0 , B O D E U. BIRNBAUM 1 9 7 8 b, B Y N G et al. 1 9 8 1 ) bzw. mehrere Enzyme für gleiche Sequenzen (MAVRIDES U. COMERTON 1 9 7 8 , R U B I N U. J E N S E N 1 9 7 9 , P A R I S U. MAGASANIK 1 9 8 1 ) und andere mit einem über die drei aromatischen Aminosäuren hinausgehenden Substratspektrum ( P O W E L L U. MORRISON 1 9 7 8 , F A Z E L U. J E N S E N 1 9 7 9 , K O I D E et al.

1980).

Über die Syntheseregulation der aromatischen Aminotransferasen liegen relativ wenige Ergebnisse vor. K R A D O L F E R et al. ( 1 9 8 2 ) konnten für Saccharomyces cerevisiae die Existenz von zwei aromatischen Aminotransferasen zeigen, von denen eine durch die aromatischen Aminosäuren induziert werden kann. Eine Regulierbarkeit der Synthese dieser Enzyme konnte auch in Bakterien nachgewiesen werden (COLLIER u . KOHLHAW 1 9 7 2 , P A T E L et al.

1978).

Unsere Untersuchungen der Transaminierung aromatischer Aminosäuren in Candida maltosa zeigten, daß diese Aktivität durch die allgemeine Kontrolle der Aminosäurebiosynthese reguliert werden kann (BODE et al. 1983). Das Anliegen der vorliegenden Arbeit ist es, die bei diesem Organismus gefundenen drei unterschiedlichen aromatischen Aminotransferasen vorzustellen und Aussagen über deren Funktion, Regulierbarkeit und Substratspezifität zu treffen, s*

68

R . BODE u n d D . BIRNBAUM

Material und Methoden Stämme und Wachstumsbedingungen: Für die Untersuchungen wurde der Hefestamm Candida maltosa E H 15 verwendet, den uns freundlicherweise das Institut für Technische Chemie der AdW der DDR zur Verfügung stellte. Die aus ihm erzeugten Doppelmutanten arg-2 lys-1 und met-6 arg-10 wurden von der FA Hefegenetik der BMA-Universität bereitgestellt. Die Anzucht der Hefe erfolgte als Schüttelkultur bei 30 °C für 16 h. Als Nährlösung wurde das Minimalmedium (MM) nach T A N A K A et al. (1967) mit 1% Glucose und 1 mg/1 Biotin verwendet. Zur Herstellung des Tryptophan-Minimalmediums (TrpMM) wurde die Konzentration des Ammoniums durch 10 MM L-Tryptophan ersetzt. Gewinnung des Enzymextraktes: Der Aufschluß der Hefezellen erfolgte mit Hilfe einer XPresse in 0,1 M Tris-HCl (pH 8,6). Das Homogenat wurde bei 20000 g 20 min zentrifugiert und der Überstand zur Ionenaustauschchromatographie und Disk-Gelelektrophorese eingesetzt. Chromatographie mit DEAE-Cellulose: Der Enzymextrakt (etwa 100 mg Protein) wurde an eine DEAE-Cellulose-Säule (2 X 20 cm) adsorbiert, die mit 0,1 M Tris-HCl (pH 8,6) äquilibriert worden war. Die Proteinelution erfolgte mit 300 ml eines linearen 0—0,5 M KCl-Gradienten. Bei einer Durchflußgeschwindigkeit von 30 ml/h wurden 4-ml-Fraktionen gesammelt. Die aktiven Fraktionen wurden bis zu ihrer Verwendung bei —25 °C gelagert. Polyacrylamid-Gelelektrophorese: Die Präparation der Polyacrylamidgele und die Durchführung der disk-elektrophoretischen Trennung der Proteine erfolgte nach MAURER ( 1 9 6 8 ) . Der Nachweis von aromatischen Aminotransferase-Aktivitäten wurde durch 30 min Inkubation der Gele in 0,1 M Tris-HCl (pH 8,6), 10 MM L-Tryptophan, 10 MM 2-Oxoglutarat und 0,1 MM Pyridoxal5-phosphat durchgeführt. Danach wurden die Gele in eine 20 MM FeCl 3 -Lösung in 35%iger Perchlorsäure übergeführt und nach 20 min die rotgefärbten Banden densitometrisch ausgewertet. Molekulargewichtsbestimmung: Zur Bestimmung des Molekulargewichtes der nach DEAECellulose-Chromatographie gewonnenen Aminotransferasen diente Sephadex G-200 (2 X 50 cm). E s wurde mit 0,1 M Tris-HCl (pH 8,6) äquilibriert und Fraktionen zu 0,8 ml gesammelt. Als Eichproteine dienten Katalase, Alkohol-DHG, Rinderserumalbumin und Ovalbumin. Enzymbestimmungen: Die Aminotransferase-Aktivitäten wurden durch die Konzentrationsmessung der gebildeten oder verbrauchten aromatischen Ketosäuren bestimmt. Der Umsatz von Phenylpyruvat und 4-Hydroxyphenylpyruvat wurde spektrophotometrisch nach COLLIER u. KOHLHAW (1972) verfolgt. Indolpyruvat wurde kolorimetrisch nach B O D E U. B I R N B A U M (1978b) bestimmt. Der Reaktionsansatz (1 ml) wurde 10 min bei 37 °C inkubiert und enthielt neben dem Aminogruppenacceptor und -donator in den im Ergebnisteil angegebenen Konzentrationen noch 0,1 MM Pyridoxal-5-phosphat und 0,1 M Tris-HCl (pH 8,6). Proteinbestimmung: Die Proteinkonzentration der Enzymextrakte wurde nach LOWRY et al. (1951) bestimmt. Rinderserumalbumin diente als Standardprotein. Substratgewinnung: Prephenat wurde nach der Methode von GIBSON (1964) und Arogenat nach B O D E u. B I R N B A U M (1979) aus Chorisminsäure gewonnen. Die mikrobielle Synthese von Chorisminsäure erfolgte nach B O D E U. B I R N B A U M (1978a). Die Herkunft der anderen Substrate war kommerziell.

Ergebnisse Mit H i l f e v o n D E A E - C e l l u l o s e k o n n t e n im zellfreien E x t r a k t v o n C. maltosa drei aromatische A m i n o t r a n s f e r a s e n (AAT) identifiziert werden (Abb. 1). D i e A b l ö s u n g v o m I o n e n a u s t a u s c h e r erfolgte mit einer K C l - K o n z e n t r a t i o n v o n 0,15, 0 , 2 0 bzw. 0,26 M. D i e A u s p r ä g u n g des Elutionsprofils war u n a b h ä n g i g d a v o n , welche der arom a t i s c h e n A m i n o s ä u r e n als A m i n o g r u p p e n d o n a t o r bzw. welche K e t o s ä u r e (2-Oxoglutarat, P r e p h e n a t ) eingesetzt worden war. D i e drei isolierten A A T unterscheiden sich a u c h sehr deutlich in ihrem Molekulargewicht. Mit H i l f e v o n S e p h a d e x G - 2 0 0 k o n n t e für die A A T I 7 3 0 0 0 ± 3000, für die A A T I I 8 5 0 0 0 ± 5 0 0 0 und für die A A T I I I 1 0 5 0 0 0 + 5 0 0 0 ermittelt werden ( A b b . 2 ) . D i e P A A G - E l e k t r o p h o r e s e ließ sich ebenfalls z u s a m m e n mit d e m beschriebenen N a c h w e i s v e r f a h r e n b e n u t z e n , u m die T r a n s a m i n a s e - A k t i v i t ä t a u f z u t r e n n e n (Abb. 3). Mit Hilfe dieser M e t h o d e gelang es auch nachzuweisen, daß die E n z y m k o n z e n t r a t i o n e n der A A T durch die A n z u c h t b e d i n g u n g e n variiert w e r d e n k ö n n e n . I n Gegenwart v o n Amitrol, das die allgemeine K o n t r o l l e in dieser H e f e auslösen k a n n ( B O D E et al. 1 9 8 3 ) ,

69

Aromatische Aminotransferasen aus C. maltosa

0.5

20

40

60

Fraktion Abb. 1. Fraktionierung aromatischer Aminotransferase-Aktivitäten nach DEAE-Cellulose-Chromatographie. Die Bestimmung erfolgte in 0,1 M Tris-HCl (pH 8,6) und 0,1 mii PLP mit folgenden Ansätzen: ( • ) , 5 mM L-Tryptophan, 5 mM 2-Oxoglutarat; (o), 5 mM L-Tyrosin, 5 mM 2-Oxoglutarat; (A), 5 mM L-Phenylalanin, 5 mji 2-Oxoglutarat; 5 mM L-Tryptophan, 0,1 mM Prephenat

vergrößert sich der P e a k der AAT I I , während im Beisein von L-Tryptophan eine Z u n a h m e der A k t i v i t ä t der AAT I I I zu beobachten war. U m q u a n t i t a t i v e Aussagen über die V e r ä n d e r u n g der E n z y m k o n z e n t r a t i o n e n der AAT n a c h unterschiedlichen Anzuchtbedingungen treffen zu können, wurden die H e f e e x t r a k t e mit Hilfe der DEAE-Cellulose a u f g e t r e n n t u n d die höchsten Aktivitätswerte bestimmt. Wie aus Tabelle 1 zu e n t n e h m e n ist, spiegeln sich die Ergebnisse, die mit der PAAG-Elektrophorese gewonnen wurden waren auch nach der T r e n n u n g im Ionenaustauscher wider. Die AAT I I I war dereprimierbar durch die aromatischen Aminosäuren. Diese können entweder einzeln, in K o m b i n a t i o n oder als alleinige Stickstoffquelle dem A n z u c h t m e d i u m beigegeben werden. Die Dereprimierung, die dabei erreicht wurde, betrug etwa das 4fache. Die Aktivitätswerte der AAT I als a u c h der AAT I I wurden bei der Anzucht mit den aromatischen Aminosäuren nicht v e r ä n d e r t . Die AAT I I ließ sich durch eine Aminosäurelimitierung dereprimieren. Beim W i l d s t a m m wurden dabei die Aminosäureantagonisten Amitrol u n d Äthionin eingesetzt, während bei den verwendeten D o p p e l m u t a n t e n das W a c h s t u m durch unterschiedliche Konzentrationen der benötigten Aminosäure limitiert wurde. Beide Möglichkeiten f ü h r t e n zu einer E r h ö h u n g der A k t i v i t ä t der AAT I I u m das 2-3fache. E i n e Veränderung der AAT I u n d auch der AAT I I I war u n t e r diesen Bedingungen nicht zu beobachten. N a c h diesen Ergebnissen stellt die AAT I eine k o n s t i t u t i v e

70

R . B O D E u n d D . BIRNBAUM

Mr • t04 Abb. 2. Bestimmung des Molekulargewichtes der aromatischen Aminotransferasen mittels Sephadex G-200. Als Eichproteine dienten Ovalbumin (o), Rinderserumalbumin (•), Alkohol-DHG ( • ) und Katalase (A)

Abb. 3. Densitogramm der disk-elektrophoretischen Trennung der aromatischen Aminotransferasen. Die Anzucht der Hefe erfolgte in MM (A), MM + 0,5 m x Amitrol (B) bzw. in MM + 5 IHM LTryptophan (C)

Aromatische Aminotransferasen aus C. maltosa

71

Tabelle 1 Spezifische Aktivitäten (nkat/mg Protein) der aromatischen Aminotransferasen nach unterschiedlichen Anzuchtbedingungen. Die Trennung erfolgte durch DEAE-Cellulose; es sind die Fraktionen mit den höchsten Aktivitätswerten angegeben. Die Bestimmung erfolgte mit 5 m l L-Tryptophan, 5 nM Oxoglutarat, 0,1 mM PLP in 0,1 m Tris-HCl (pH 8,6) Anzuchtbedingung

TG

AAT

(g/I)

I

II

HI

MM MM + 0,5 mM Amitrol MM + 1 , 0 mM DL-Äthionin MM + 5,0 mM L-Tryptophan MM 4- 5,0 mM L-Phenylalanin MM + 5,0 mM L-Tyrosin MM + 5,0 mM L-Trp/L-Phe/L-Tyr TrpMM

4,9 2,0 2,5 5,0 4,7 5,2 5,2 3,7

0,35 0,33 0,30 0,37 0,35 0,30 0,32 0,35

2,34 3,98 4,15 2,20 2,41 2,22 2,18 2,25

0,75 0,67 0,60 2,35 2,85 2,24 2,92 3,21

MM + 0,1 mM L-Arg/L-Lys MM + 1,0 mM L-Arg/L-Lys

0,7 3,5

0,30 0,32

5,45 3,55

0,54 0,64

MM + 0,1 mM L-Met/L-Arg MM + 1 , 0 mM L-Met/L-Arg

1,0 3,9

0,32 0,32

4,98 3,68

0,58 0,75

Wildstamm

arg-2

lys-1

met-6

arg-10

Aminotransferase dar, während sich die AAT I I und die AAT I I I durch unterschiedliche Regulationsmechanismen dereprimieren lassen. Alle drei Enzyme können eine Reihe von Aminogruppenacceptoren mit unterschiedlicher Aktivität aminieren (Tab. 2). Von den geprüften Ketosäuren wurden vor allem Phenylpyruvat, 4-Hydroxyphenylpyruvat, Prephenat und Indolpyruvat umgesetzt. Geringe Aktivitäten waren mit Oxoglutarat und Oxalacetat zu verzeichnen, während P y r u v a t und Ketoisovalerat nicht als Acceptor genutzt werden konnten. Eine ähnliche Spezifität der AAT konnte auch bei der Verwendung der aromatischen Aminosäuren als Aminogruppendonator festgestellt werden. Wie aus Tabelle 3 hervorgeht, waren die drei AAT aber auch in der Lage weitere Aminosäuren umzusetzen. So konnten neben Tryptophan, dessen Umsatzrate am größten war, Phenylalanin, Tyrosin und Arogensäure auch Histidin, Methionin, Leucin und Glutaminsäure als Donator genutzt werden. Der prozentuale Umsatz der aromatischen Aminosäuren durch die drei AAT ist miteinander vergleichbar, während die Aktivität der AAT I gegenüber Histidin, Methionin, Leucin und Glutaminsäure deutlich größer als die der AAT I I oder AAT I I I war. Die drei isolierten Transaminasen waren weiterhin nicht in der Lage, das D-Isomer der verwendeten Aminosäuren umzusetzen. Tabelle 2 Aminogruppenacceptor-Spezifität der aromatischen Aminotransferasen. Als Bezugspunkt wurde der Umsatz mit Oxoglutarat verwendet Acceptor (0,5 mM)

Donator (5 mM)

AAT I

II

III

2-Oxoglutarat Oxalacetat Pyruvat 2-Ketoisovalerat Phenylpyruvat 4-Hydroxyphenylpyruvat Prephenat Indolpyruvat

L-Trp L-Trp L-Trp L-Trp L-Trp L-Trp L-Trp L-Phe

100 50 0 0 380 290 410 470

100 150 0 0 950 890 850 1100

100 220 0 0 1780 1150 1690 1560

72

R . BODE u n d D . BIRNBAUM

Tabelle 3 Aminogruppendonator-Spezifität der aromatischen Aminotransferasen. Als Bezugspunkt wurde der Umsatz mit L-Tryptophan bzw. L-Phenylalanin verwendet Donator

Acceptor

(5 mu) L-Tryptophan D-Tryptophan L-Tyrosin D-Tyrosin L-Phenylalanin D-Phenylalanin L-Phenylalanin L-Tyrosin L-Arogenat L-Histidin L-Leucin L-Methionin L-Glutaminsäure L-Asparaginsäure alle weiteren proteinogenen L-Aminosäuren

5 MM 2-Oxoglutarat 2-0xogIutarat 2-Oxoglutarat 2-0xoglutarat 2-Oxoglutarat 2-Oxoglutarat 0,5 M M Indolpyruvat Indolpyruvat Indolpyruvat Indolpyruvat Indolpyruvat Indolpyruvat Indolpyruvat Indolpyruvat

AAT I

II

III

100 0 59 0 69 0

100 0 50 0 55 0

100 0 58 0 64 0

100 86 94 63 50 58 24 0

100 91 100 21 11 1 4 0

100 90 97 30 8 1 4 0

0

0

0

Indolpyruvat

Zur weiteren Präzisierung der Substratspezifität der AAT wurden die MICHAELISKonstanten für die verwendeten Amino- und Ketosäuren bestimmt. Dabei wurde die Intercept-Darstellung der doppelt reziproken Werte benutzt, da die AAT-katalysierten Reaktionen dem nicht-sequentiellen T y p entsprachen. Die -iC m -Werte der AAT I und der AAT I I I sind einander ähnlich, während die Werte für die A A T I I deutlich größer sind (Tab. 4). F ü r Phenylalanin, Tyrosin und Arogenat ist bei allen drei AAT eine bessere Spezifität gegenüber den anderen benutzten Aminosäuren zu erkennen. Das Gleiche trifft für die aromatischen Ketosäuren zu. F ü r diese konnten relativ geringe MiCHAELis-Konstanten ermittelt werden, die 1—2 Zehnerpotenzen kleiner waren als z. B . die Werte für Oxoglutarat. Tabelle 4 K m -Werte (mi) der aromatischen Aminotransferasen. Zur Bestimmung wurde die Intercept-Darstellung der doppelt reziproken Werte benutzt Substrat

AAT 1

L-Tryptophan L-Phenylalanin L-Tyrosin L-Arogenat L-Histidin L-Leucin L-Methionin L-Glutaminsäure 2-Oxoglutarat Oxalacetat Phenylpyruvat 4-Hydroxyphenylpyruvat Prephenat Indolpyruvat Pyridoxal-5-phosphat

2,5 0,44 0,40 0,45 5,0 4,0 5,0 7,0 5,0 3,3 0,08 0,05 0,08 0,06 0,0010

II 3,3 1,1 2,0 1,8 9,0 9,0 — —

10,0 10,0 0,25 0,20 0,30 0,25 0,0005

III 2,0 0,45 0,50 0,50 3,3 5,0 — —

2,0 3,0 0,11 0,05 0,09 0,05 0,0005

73

Aromatische Aminotransferasen aus C. maltosa

Bei höheren Konzentrationen der aromatischen Ketosäuren konnte eine Substratinhibition der AAT festgestellt werden. Wie aus Tabelle 5 hervorgeht, waren besonders die AAT I und die AAT I I empfindlich. Eine Inhibition der AAT I I war dagegen erst bei hohen Konzentrationen von Phenylpyruvat, 4-Hydroxyphenylpyruvat und Prephenat festzustellen; eine geringere Inhibitorkonstante konnte für Indolpyruvat bestimmt werden. Sowohl Oxoglutarat als auch Oxalacetat waren nicht in der Lage, die AAT-Reaktion bis zu einer geprüften Konzentration von 50 mM zu hemmen. Tabelle 5 Substratinhibition (KI in MM) der aromatischen Aminotransferasen Substrat

Phenylpyruvat 4-Hydroxylphenylpyruvat Prephenat Indolpyruvat

AAT I

II

III

0,5 0,4 0,4 0,3

15 10 15 1,5

0,5 0,3 0,4 0,3

Diskussion I n C. maltosa konnten durch chromatographische Methoden drei unterschiedliche aromatische Aminotransferasen getrennt und charakterisiert werden. Die Aktivität der AAT I in der Zelle war relativ gering und deren Enzymsynthese konnte nicht beeinflußt werden, so daß wir einen konstitutiven Charakter dieses Enzyms annehmen. Die AAT I war auch in der Lage mit einer relativ hohen Aktivität Histidin und einige aliphatische Aminosäuren umzusetzen. Auf Grund dieser Spezifität ist es möglich, daß diese AAT in vivo am Metabolismus dieser Aminosäuren beteiligt ist. Die AAT I I besitzt nach Anzucht in MM die Hauptmenge der Transaminaseaktivit ä t und ließ sich durch Aminosäure-Mangelbedingungen dereprimieren. Die mRNASynthese der AAT I I unterliegt demnach der allgemeinen Kontrolle der Aminosäurebiosynthese. Da es sich bei diesem Regulationsmechanismus um eine Dereprimierung von anabolen Aminosäureenzymen handelt, postulieren wir, daß die AAT I I die Aminotransferase der Phenylalanin/Tyrosin-Biosynthese darstellt. Von dieser Transkriptionsregulation werden bei C. maltosa eine Reihe von Enzymen kontrolliert (BODE et cd. 1 9 8 3 ) .

Die AAT I I I ließ sich durch die Anzucht mit aromatischen Aminosäuren induzieren. Ein geringer Level war aber auch bei der Anzucht in MM zu beobachten, dessen Synthese sich wahrscheinlich auf die Anwesenheit des freien Zellpools der aromatischen Aminosäuren von 5,4 mMol/kg TG (BODE et al. 1983) zurückführen läßt. Die Aufgabe der AAT I I I in vivo kann demnach als katabole Funktion bei der Gewinnung des Stickstoffs aus den aromatischen Aminosäuren determiniert werden. Dieser erste Schritt des Aminosäureabbaus f ü h r t bei Hefen über weitere Sequenzen zum entsprechenden Alkohol bzw. der Säure (RAINBOW 1970, ROSAZZA et al. 1973). In Sacch. cerevisiae konnte eine ähnliche Situation wie in C. maltosa beobachtet werden. KRADOLFER et al. (1982) fanden bei diesem Organismus zwei AAT, von denen ebenfalls eine durch die aromatischen Aminosäuren induzierbar war. Von den vier AAT mit überlappenden Spezifitäten in Pseudomonas aeruginosa konnte ebenfalls eine als synthetische und eine als katabole AAT charakterisiert werden; die Synthese der beiden anderen Transaminasen war nicht reguliert (PATEL et al. 1978). Die Transaminierungsreaktion des Prephenats zum Arogenat wurde zum ersten Male von STENMARK et al. (1974) bei Blaualgen beschrieben und führte zur Entdeckung

74

R . BODE u n d D . BIRNBAUM

einer veränderten Phenylalanin/Tyrosin-Biosynthese. Auch in der Hefe Hansenula henricii konnte dieser Arogenat-Weg nachgewiesen werden (BODE U. B I R N B A U M 1979). Die aromatischen Aminotransferasen von C. maltosa setzten sowohl Prephenat als auch Arogenat mit einer ähnlichen Spezifität und Geschwindigkeit um wie die anderen aromatischen Amino- bzw. Ketosäuren. Die Existenz der Synthese von Phenylalanin bzw. Tyrosin via Arogenat wird bei dieser Hefe zur Zeit untersucht. Die MiCHAELis-Konstanten der drei isolierten Transaminasen weisen eine deutliche Spezifität gegenüber Phenylpyruvat, 4-Hydroxyphenylpyruvat, Prephenat und Indolpyruvat als auch gegenüber Tyrosin, Phenylalanin und Arogenat aus. Die KmWerte für Tryptophan waren dagegen 2—5 mal größer als für die drei letzteren. Ähnliche Verhältnisse konnten auch bei einigen anderen Organismen festgestellt werden (POWELL U. M O R R I S O N 1978, K R A D O L F E R et al. 1982). Die geringen Umsatzraten von Glutaminsäure, Oxoglutarat sowie von Oxalacetat und die relativ großen MiCHAELis-Konstanten für diese Substrate lassen die Frage aufkommen, inwieweit diese Aminogruppenacceptoren bzw. der Donator bei der in two-Transaminierung aromatischer Aminosäuren in dieser Hefe überhaupt genutzt werden können. Es erscheint eher möglich, daß das Tryptophan den Aminogruppendonator für die Synthese von Phenylalanin, Arogenat und Tyrosin darstellt. Dadurch würde eine direkte Verknüpfung der AAT-katalysierten katabolen Reaktion des Tryptophans zum Indolpyruvat mit dem Anabolismus der anderen aromatischen Aminosäuren erfolgen. Neben der Aktivierung der Chorismat-Mutase (MELO RAMOS u. BODE unveröff.) käme dem Tryptophan in C. maltosa dadurch eine weitere regulatorische Funktion innerhalb der Phenylalanin/Tyrosin-Biosynthese zu. Diese Vermutung muß in vivo noch überprüft werden, ist aber nicht auszuschließen, da auch in anderen Organismen Aminotransferasen mit ähnlichem Wirkungsmuster gefunden wurden (SUKANYA U. V A I D Y A N A T H A N 1964, K O I D E et al. 1980).

Literatur BESOHLE, H . G . , SÜSSMUTH, R . u n d LINGENS, F . , 1 9 8 2 . E i g e n s c h a f t e n v o n a r o m a t i s c h e n

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säure-Aminotransferasen aus zwei Chloramphenicol-resistenten Flavobakterien. HoppeSeyler's Z. Phyiol. Chem., 863, 1365 — 1375. BODE, R. und BIRNBAUM, D., 1978a. Die Enzyme der Biosynthese aromatischer Aminosäuren bei Hansenula henricii: Charakterisierung der Anthranilat-Synthase. Biochem. Physiol. Pflanzen, 172, 2 4 5 - 2 5 3 . BODE, R. und BIRNBAUM, D. 1978 b. Die Enzyme der Biosynthese aromatischer Aminosäuren bei Hansenula henricii: Die Aminotransferasen. Biochem. Physiol. Pflanzen, 178, 44—50. BODE, R. und BIRNBAUM, D., 1979. Die Enzyme der Biosynthese aromatischer Aminosäuren bei Hansenula henricii: Nachweis und Charakterisierung der Enzyme des Pretyrosin-Weges. Z. allg. Mikrobiol., 19, 83—88. B O D E , R . , CASPER, P. und K U N Z E , G . , 1983. Auslösung einer allgemeinen Kontrolle der Aminosäurebiosynthese bei Candida spec. E H 15/D durch Amitrol. Biochem. Physiol. Pflanzen, 178, 457-468. BYNG, G. S.,

WHITAKER, R . J . ,

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a n d JENSEN, R . A.,

1981.

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acid pathway branches at L-arogenate in Euglena gracilis. Mol. Cell. Biol., 1, 426—438. COLLIER, R. H. and KOHLHAW, G., 1972. Nonidentity of the aspartate and the aromatic aminotransferase components of transaminase A in Escherichia coli. J . Bacteriol., 112, 365—371. FAZEL, A. M. and JENSEN, R. A., 1979. Aromatic aminotransferases in coryneform bacteria. J . Bacteriol., 140, 580—587. GIBSON, F., 1964. Chorismic acid: purification and some chemical and physical studies. Biochem. J . , 90, 2 5 6 - 2 6 1 . J E N S E N , R. A. and CALHOUN, D . H . , 1 9 8 1 . Intracellular roles of microbial aminotransferases: overlap enzymes across different biochemical pathways. CRC Crit. Rev. Microbiol., 8 , 2 2 9 — 2 6 6 . KOIDE, Y . ,

HONMA, M . a n d

SHIMOMURA, T . ,

1980. L-tryptophan-A-ketoisocaproate

ferase from Pseudomonas sp.. Agric. Biol. Chem., 44, 2013—2019.

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P., N I E D E R B E R G E R , P. and H Ü T T E R , R., 1 9 8 2 . Tryptophan degradation in Saccharomyces cerevisiae: characterization of two aromatic aminotransferases. Arch. Microbiol., 133,

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and R A N D A L L , R . J . , 1 9 5 1 . Protein measurements with the Folin phenol reagent. J . Biol. Chem., 193, 265—275. M A U R E R , H. R., 1968. Disk-Elektrophorese. Theorie und Praxis der diskontinuierlichen Polyacrylamidgel-Elektrophorese. Walter de Gruyter & Co., Berlin. M A V R I D E S , C . and COMERTON, B., 1 9 7 8 . Aminotransferases for aromatic amino acids and aspartate in Bacillus subtilis. Biochim. Biophys. Acta, 524, 60 —37. P A R I S , C. G . and M A G A S A N I K , B., 1 9 8 1 . Purification and properties of aromatic amino acid aminotransferase from Klebsiella aerogenes. J . Bacteriol., 145, 2 6 6 — 2 7 1 . P A T E L , N . , S T E N M A R K - C O X , S . L. and J E N S E N , R. A . , 1 9 7 8 . Enzymological basis of reluctant auxotrophy for phenylalanine and tyrosine in Pseudomonas aeruginosa. J . Biol. Chem., 253, L O W K Y , 0 . H . , ROSEBROUGH, N . J . , F A R R , A . L .

2972—2978. P O W E L L , J . T.

and MORRISON, F . J., 1 9 7 8 . The purification and properties of the aspartate trans aminotransferase from Escherichia coli. Eur. J . Biochem., 87, 391—400. R A I N B O W , C., 1970. Brewer's yeast. I n : The yeast 3, 179 — 199 ( R O S E , A. H . and H A R R I S O N , J . S., Editors). Academic Press Inc., New York. ROSAZZA, J . P . , J U H L , R . and D A V I S , P . , 1 9 7 3 . Tryptophol formation by Zygosaccharomyces priorianus. Appl. Microbiol., 26, 98 — 105. R U B I N , J . L. and J E N S E N , R . A., 1979. Enzymology of L-tyrosine biosynthesis in mung bean (Vigna radiata [L.] WICZEK). Plant Physiol., 64, 727—734. S P E E D I E , M. K., H O R N M A N N , U. and F L O S S , H. G., 1975. Isolation and characterization of tryptophan transaminase and indolepyruvate C-methyltransferse. Enzymes involved in indolmycin biosynthesis in Streptomyces griseus. J . Biol. Chem., 250, 7819—7825. S T E N M A R K , S . L . , P I E R S O N , D . L . , G L O V E R , G . I . and J E N S E N , R . A . , 1 9 7 4 . Blue-green bacteria synthesize L-tyrosine by the pretyrosine pathway. Nature, 2 4 7 , 2 9 0 — 2 9 2 . S U K A N Y A , N . K . and V A I D Y A N A T H A N , C . S . , 1 9 6 4 . Aminotransferases of Agrobacterium tumefaciens. Transamination between tryptophan and phenylpyruvate. Biochem. J., 92, 5 9 4 — 5 9 8 . T A N A K A , A . , O H I S H I , N . and F U K U I , S., 1967. Studies on the formation of vitamins and their function in hydrocarbon fermentation. Production of vitamin B e by Candida albicans in hydrocarbon medium. J . Ferment. Technol., 45, 617—623. T E U S C H E R , E., 1970. Tryptophanstoffwechsel und Alkaloidbildung beim Mutterkornstamm SD 58 in saprophytischer Submerskultur. I I I . Tryptophan-Transaminase. Z. allg. Mikrobiol., 10, 137-146. Anschrift: Doz. Dr. R. BODE, Ernst-Moritz-Arndt-Universität Greifswald, Sektion Biologie, WB Molekularbiologie, DDR-2200 Greifswald, Fr.-Ludwig-Jahn-Str. 15 a

Zeitschrift f ü r Allgemeine Mikrobiologie 24 (1984) 2, 76

Buchbesprechung (Editor), The Population Dynamics of Infectious Diseases: Theory and Applications. 368 S., 135 Abb., 104 Tab. London-New York 1982. Chapman and Hall. £ 17.50. R.M.ANDERSON

Die vorliegende Monographie enthält populationsdynamische Modelluntersuchungen ausgewählter infektiöser Krankheitserreger (Viren, Bakterien, Protozoen und vor allem Enthelminthen), die sich auf ein umfangreiches epidemiologisches Beobachtungsmaterial stützen. Die mathematischen Modelle stehen weniger im Vordergrund der Untersuchungen als die Diskussion der biologischen Bedingungen, die jeweils zu den vorgestellten Modellen führen. Ausführlich werden die epidemiologischen Konzepte und Prinzipien behandelt, die sich aus den Modellstudien ableiten, sowie diejenigen Aspekte, die f ü r die Aufstellung von Krankheitskontrollprogrammen von Wichtigkeit sein könnten. Obwohl sich die Autoren in erster Linie an Epidemiologen, Mediziner, P a r a sitologen und Ökologen richten, wird die Hoffnung ausgesprochen, auch bei Mathematikern Interesse f ü r diese Thematik zu wecken. Die Kapitel und ihre Autoren: R. M. ANDERSON: Direkte Übertragung viraler und bakterieller Infektionen des Menschen; Populationsdynamik und Kontrolle von Haken- und Spulwurminfektionen; Tollwut des Fuchses. B. CVJETANOVIC: Dynamik bakterieller Infektionen. A . K E Y M E R : Bandwurminfektionen. J . L. A R O N , R . M . M A Y : Populationsdynamik der Malaria. A. D . B A R B O U R : Mathematisches Modell zur Bilharziose. K . D I E T Z : Populationsdynamik der Onchozerkose. R . A . W I L S O N , G . S M I T H , M . R . T H O M A S : Mathematisches Modell zur Fasciolosis. D. J . BRADLEY: Epidemiologische Modelle — Theorie und Wirklichkeit. Das Buch enthält eine umfangreiche Literaturübersicht (28 Seiten). G . MÜLLER ( J e n a )

and N. R . M O R R I S (Editors), Microtubules in Microorganisms. 145 Abb., 10 Tab. New York-Basel 1982. Marcel Dekker. SFr. 156.

P . CAPPUCCINELLI

XIII +

410 S.,

Die vorliegende Ausgabe präsentiert auf der Basis eines Symposiums 1981 in Porto Conte/Sardinien das junge, sich rasch entwickelnde Gebiet des Cytoskeletts in eukaryotischen Mikroorganismen. F ü r sein mikrotubuläres Teilsystem sind in 23 Kapiteln Synthese, Gewinnung und Konservativität des Tubulins, die Assemblierung von Mikrotubuli (MT) in vivo und in vitro, ihre Nucleation an MT-Organisationszentren (MTOC), Struktur, Organisation und Organisationsmodi von MT-Systemen, Funktion der MT für Zellstruktur, den Chromosomentransport und Zellwandsynthese, die Beeinflussung von MT und MT-Funktion durch MT-Gifte sowie die Isolierung, Struktur, Zusammensetzung und Funktion von MTOC, die Gewinnung und Charakterisierung von Mutanten mit geänderten MT-Eigenschaften und -Funktionen, der Evolution der Meiose und den versuchten Nachweis von Tubulin in einem Prokaryoten (Acholeplasma) beschrieben. Die Untersuchungen wurden vorzugsweise an Vertretern der Pilze, Algen und Protozoen ausgeführt. Der skizzierte Inhalt ist über ca. 850 Stichworte leicht und vollständig zugänglich. Die Beiträge sind von hervorragenden Fachleuten verfaßt und repräsentieren den gegenwärtigen Wissensstand auf den jeweiligen Gebieten. Es wird ein ausgezeichneter Überblick geliefert, der zugleich die z. Z. bestehenden Grenzen unseres Verständnisses der mit MT verbundenen bzw. an MT gebundenen Phänomene aufzeigt und insbesondere das molekulare Verständnis zellulärer Funktionen der MT betrifft. Das vorliegende Buch ist unbedingt empfehlenswert für alle, die sich mit der zellbiologischen Bedeutung der MT befassen bzw. sich darüber informieren wollen und die MT-abhängigen Eigenschaften von eukaryotischen Mikroorganismen verstehen und praxisrelevante Aspekte eingeschlossen, gezielt beeinflussen wollen. E . UNGER ( J e n a )

Z e i t s c h r i f t f ü r allgemeine Mikrobiologie 24 (1984) 2, 77 — 84

(Akademie der Wissenschaften der D D R , I n s t i t u t f ü r technische Chemie, Leipzig, D i r e k t o r : P r o f . Dr. M. RINGPFEIL)

Dissimilation of methanol in Acetobacter sp. MB 58 M . W . GBÜNDIG a n d N . V . DORONINA1)

(Eingegangen

am 28.

7.1983)

I n t h e f a c u l t a t i v e m e t h y l o t r o p h Acetobacter sp. M B 58, which uses t h e hexulose-6-phosphate p a t h w a y , t h e linear a n d cyclic dissimilation of m e t h a n o l was investigated b y using different growth substrates. Methanol a n d f o r m a l d e h y d e were oxydized in t h e presence of phenazine m e t h o s u l f a t e only in methanol-grown cells. A p a r t i c u l a t e dichlorphenol indophenol-dependent f o r m a t e dehydrogenase h a v i n g a p H - o p t i m u m of 5.0 was d e t e c t e d anaerobically. F o r m a t e oxidation was n o t controlled b y m e t h a n o l . T h u s in Acetobacter sp. M B 58 a complete linear dissimilatory sequence is operative. During growth on m e t h a n o l cyclic dissimilation is possible in Acetobacter sp. M B 58 like in other obligate methylotrophs. P r o b l e m s in measuring hexulose-6-phosphate s y n t h a s e b y using t h e coupled optical assay were solved b y adding partially purified hexulose-6-phosphate isomerase f r o m Pseudomonas W 6 t o t h e t e s t system. A new finding in m e t h y l o t r o p h y was m a d e : I n Acetobacter sp. M B 58 hexulose-6p h o s p h a t e isomerase is controlled b y m e t h a n o l a n d hexulose-6-phosphate s y n t h a s e is n o t . Finally, in Acetobacter sp. M B 58 t h e dissimilatory enzyme steps, which are epigenetically controlled b y m e t h a n o l , include phenazine m e t h o s u l p h a t e - d e p e n d e n t oxidation of m e t h a n o l a n d f o r m a l d e h y d e a n d hexulose-6-phosphate isomerase. T h e f a c u l t a t i v e m e t h y l o t r o p h Acetobacter sp. M B 5 8 a s s i m i l a t e s m e t h a n o l v i a t h e F B P 2 ) - v a r i a n t of t h e H u P - p a t h w a y (MIETHE 1 9 7 7 , B A B E L a n d M U L L E R 1 9 7 7 ) . T h e T C A - c y c l e s e e m s to be i n c o m p l e t e , w h i c h is a characteristic in obligate m e t h a n o l assimilating bacteria (BABEL a n d HOFMANN 1 9 7 7 ) . I n obligate m e t h y l o t r o p h s energy is g e n e r a t e d i n t h e s o - c a l l e d d i s s i m i l a t o r y H u P - c y c l e i n t h e f o r m of r e d u c t i o n e q u i valents. In general, N A D ( P ) + - l i n k e d formate dehydrogenase could not be detected, suggesting t h a t t h e linear dissimilatory sequence d o e s not operate. H o w e v e r , STEUDEL et al. ( 1 9 8 0 ) w e r e a b l e t o m e a s u r e p o l a r o g r a p h i c a l l y f o r m a t e o x i d i z i n g a c t i v i t y i n i n t a c t c e l l s of Acetobacter sp. M B 5 8 . I n t h e p r e s e n t p a p e r w e d e m o n s t r a t e t h e e x i s t e n c e of a f o r m a t e - o x i d i z i n g e n z y m e in vitro a n d s t u d y t h e r e g u l a t i o n of d i s s i m i l a t o r y e n z y m e s .

Materials and methods

E n z y m e s a n d biochemicals: All enzymes a n d biochemicals were obtained f r o m SIGMA Chemical Co. (St. Luis, Mo., U.S.A.) exept ribose-5-phosphate, NAD+, N A D H , N A D P + , a n d N A D P H which

1

) A c a d e m y of Sciences of t h e U.S.S.R., I n s t i t u t e of Biochemistry a n d Physiology of Microorganisms, P u c h i n o on Oka, Moscow Region ) A b b r e v i a t i o n s : D C I P = dichlorphenol indophenol; F A D = flavine adenine dinucleotide; F B P = f r u c t o s e - l , 6 - b i s p h o s p h a t e ; F M N = f l a v i n e mononucleotide; G 6 P D H = glucose-6-phosphate d e h y d r o g e n a s e ; G S H = glutathion, r e d u c e d ; H u P = hexulose-6-phosphate; H u P I = hexulose-6p h o s p h a t e isomerase; H u P S = hexulose-6-phosphate s y n t h a s e ; 6 P G D H = 6-phosphogluconate dehydrogenase; PMS = phenazine m e t h o s u l p h a t e ; TCA = tricarboxylic acid 2

Z e i t s c h r i f t f ü r allgemeine Mikrobiologie 24 (1984) 2, 77 — 84

(Akademie der Wissenschaften der D D R , I n s t i t u t f ü r technische Chemie, Leipzig, D i r e k t o r : P r o f . Dr. M. RINGPFEIL)

Dissimilation of methanol in Acetobacter sp. MB 58 M . W . GBÜNDIG a n d N . V . DORONINA1)

(Eingegangen

am 28.

7.1983)

I n t h e f a c u l t a t i v e m e t h y l o t r o p h Acetobacter sp. M B 58, which uses t h e hexulose-6-phosphate p a t h w a y , t h e linear a n d cyclic dissimilation of m e t h a n o l was investigated b y using different growth substrates. Methanol a n d f o r m a l d e h y d e were oxydized in t h e presence of phenazine m e t h o s u l f a t e only in methanol-grown cells. A p a r t i c u l a t e dichlorphenol indophenol-dependent f o r m a t e dehydrogenase h a v i n g a p H - o p t i m u m of 5.0 was d e t e c t e d anaerobically. F o r m a t e oxidation was n o t controlled b y m e t h a n o l . T h u s in Acetobacter sp. M B 58 a complete linear dissimilatory sequence is operative. During growth on m e t h a n o l cyclic dissimilation is possible in Acetobacter sp. M B 58 like in other obligate methylotrophs. P r o b l e m s in measuring hexulose-6-phosphate s y n t h a s e b y using t h e coupled optical assay were solved b y adding partially purified hexulose-6-phosphate isomerase f r o m Pseudomonas W 6 t o t h e t e s t system. A new finding in m e t h y l o t r o p h y was m a d e : I n Acetobacter sp. M B 58 hexulose-6p h o s p h a t e isomerase is controlled b y m e t h a n o l a n d hexulose-6-phosphate s y n t h a s e is n o t . Finally, in Acetobacter sp. M B 58 t h e dissimilatory enzyme steps, which are epigenetically controlled b y m e t h a n o l , include phenazine m e t h o s u l p h a t e - d e p e n d e n t oxidation of m e t h a n o l a n d f o r m a l d e h y d e a n d hexulose-6-phosphate isomerase. T h e f a c u l t a t i v e m e t h y l o t r o p h Acetobacter sp. M B 5 8 a s s i m i l a t e s m e t h a n o l v i a t h e F B P 2 ) - v a r i a n t of t h e H u P - p a t h w a y (MIETHE 1 9 7 7 , B A B E L a n d M U L L E R 1 9 7 7 ) . T h e T C A - c y c l e s e e m s to be i n c o m p l e t e , w h i c h is a characteristic in obligate m e t h a n o l assimilating bacteria (BABEL a n d HOFMANN 1 9 7 7 ) . I n obligate m e t h y l o t r o p h s energy is g e n e r a t e d i n t h e s o - c a l l e d d i s s i m i l a t o r y H u P - c y c l e i n t h e f o r m of r e d u c t i o n e q u i valents. In general, N A D ( P ) + - l i n k e d formate dehydrogenase could not be detected, suggesting t h a t t h e linear dissimilatory sequence d o e s not operate. H o w e v e r , STEUDEL et al. ( 1 9 8 0 ) w e r e a b l e t o m e a s u r e p o l a r o g r a p h i c a l l y f o r m a t e o x i d i z i n g a c t i v i t y i n i n t a c t c e l l s of Acetobacter sp. M B 5 8 . I n t h e p r e s e n t p a p e r w e d e m o n s t r a t e t h e e x i s t e n c e of a f o r m a t e - o x i d i z i n g e n z y m e in vitro a n d s t u d y t h e r e g u l a t i o n of d i s s i m i l a t o r y e n z y m e s .

Materials and methods

E n z y m e s a n d biochemicals: All enzymes a n d biochemicals were obtained f r o m SIGMA Chemical Co. (St. Luis, Mo., U.S.A.) exept ribose-5-phosphate, NAD+, N A D H , N A D P + , a n d N A D P H which

1

) A c a d e m y of Sciences of t h e U.S.S.R., I n s t i t u t e of Biochemistry a n d Physiology of Microorganisms, P u c h i n o on Oka, Moscow Region ) A b b r e v i a t i o n s : D C I P = dichlorphenol indophenol; F A D = flavine adenine dinucleotide; F B P = f r u c t o s e - l , 6 - b i s p h o s p h a t e ; F M N = f l a v i n e mononucleotide; G 6 P D H = glucose-6-phosphate d e h y d r o g e n a s e ; G S H = glutathion, r e d u c e d ; H u P = hexulose-6-phosphate; H u P I = hexulose-6p h o s p h a t e isomerase; H u P S = hexulose-6-phosphate s y n t h a s e ; 6 P G D H = 6-phosphogluconate dehydrogenase; PMS = phenazine m e t h o s u l p h a t e ; TCA = tricarboxylic acid 2

78

M . W . GRUNDIG a n d N . V . DORONINA

came from REANAL (Budapest, Hungary). 14 C-formaldehyde came from V/O IZOTOP (U.S.S.R.) and Aquasol from NEN (New England Nuclear Ltd., U. K.). Preparation of crude sonic extracts: Methanol, 0.5% (v/v), glucose, 0.5% (w/v) and glycerol, 0.8% (v/v) were used as growth substrates. Acetobacter sp. MB 58 was grown and harvested in the mid-exponential growth phase as described by STEUDEL et al. (1980). Biomass was washed twice with 50 MM potassium phosphate buffer p H 7.5 and stored a t —20 °C. The suspensions (1 g wet weight per 5 ml buffer) were sonicated at 20 kHz and 0 °C with a MSE ultrasonifier using four 30-s pulses interupted by one-min cooling periods. The extracts were centrifuged at 30000 xg for 40 min at + 2 °C. The supernatant was used immediately. To determine NAD+ /GSH-dependent formaldehyde dehydrogenase, NADH-oxidase-free supernatant (200000 xg, 90 min, + 2 °C) was used. Formate dehydrogenase activity was measured in 30000 xg-pellets (90 min, + 2 °C). The pellets were homogenized in the same volume of oxygene-free potassium phosphate/citric acid buffer, p H 5.0. All buffers were prepared by bubbling argon through them. Frozen biomass was thawed under argon stream. The procedure of homogenizing was carried out under the same conditions. The obtained suspension was kept closed in argon atmosphere. Enzyme assays: All assays were performed with crude sonic extracts at 30 °C. The spectrophotometry assays were performed with a UV/ VIS spectrophotometer using 2 ml reaction volumes. Methanol dehydrogenase (PMS): The reaction mixture contained Tris-HCl buffer, p H 9.0, 100 MM; PMS, 0.5 MM; DCIP, 0.05 MM; NH4C1, 1 5 m M ; K C N , 1 MM and extract. The reaction was started with 15 MM methanol or 10 MM formaldehyde. Formaldehyde dehydrogenases (NAD+/GSH and NADP+) were assayed according to BABEL a n d MOTHES ( 1 9 7 8 ) .

Formate dehydrogenase (DCIP) was assayed anaerobically by using syringes for filling the cuvette side arm through which argon was blown continuously. Argon pressure was used for adding suspension and substrate to the cuvette. The cuvette already contained oxygen-free buffer and DCIP. During the reaction argon was blown through the cuvette, but not through the reaction mixture. Methylenblue, methylviologene, benzylviologene, ferricyanide, FMN, FAD, NAD+ and NADP+ did not act as electron acceptors. No formate-dependent oxidase activity could be obtained from the membrane fraction. Additions of EDTA and MgCl2 did not result in higher activities, as d e s c r i b e d e l s e w h e r e (MULLER et al. 1978. EGEROV et al. 1 9 7 9 , NIEKUS et al. 1980). T h e u s e of S H -

group-protecting agents such as dithioerytritol or mercaptoethanol (LEONHARDT and ANDREESEN 1 9 7 7 , EGEROV et al. 1979, NIEKUS et al. 1 9 8 0 , SCHATJER a n d FERRY 1982) r e s u l t e d i n e n d o g e n o u s

reaction with DCIP. Finally the reaction mixture contained potassium phosphate/citric acid buffer, p H 5.0; DCIP, 0.1 MM and suspension. The reaction was started by adding 30 MM sodium formate. The measurements were carried out at 520 nm. For calculation of specific activities the equation e = 0.96 X 103 1 • M - 1 • c m - 1 was used. Specific activities, obtained from suspensions which were not prepared under anaerobic conditions were 30 to 50 precent lower. Suspensions prepared with press disruption (X-press, 30000 kg per cm2) did not result in higher values. Hexulose-6-phosphate synthase was assayed by three methods, a) The radiochemical assay was used according to LAWRENCE et al. (1970) by measuring pentosephosphate-dependent fixation of " C - fo r m a 1 de h y de into sugar phosphates. A reaction mixture (0.4 ml) contained 0.2 ml, potassium phosphate buffer, p H 7.0, 50 MM; MgCl2, 0.005 MM and ribose-5-phosphate, 0.002 MM was incubated for 5 min. Then 0.019 MM (2.5 N Ci) 14 C-formaldehyde was added. After 1 min of incubation the reaction was started by adding the extract (0.05 ml). The mixture was allowed to stand for another 5 min and stopped by adding 1.5 ml icecold absolute ethanol. Sugar phosphates were then precipitated with 0.1 ml 5 % barium acetate. The mixture was kept in ice for 10 min, filtered through CF/A WHATMAN glasfiber filter and washed once with 1 ml ice-cold absolute ethanol. The filter was dried for 20 min at 110 °C. Radioactivity was measured in a liquid scintillation counter (Intertechnique, France) in 10 ml Aquasol. b) The spectrophotometrical assay was carried out according to DAHL et al. (1972). The mixture contained potassium phosphate buffer, p H 7.0, 100 mM; MgCl2, 4 mM; NADP+, 0.25 MM; ribose5-phosphate, 5 mM; glucosephosphate isomerase, 3.5 U ; glucose-6-phosphate dehydrogenase, 9 U and extract. The reaction was started by adding 5 MM formaldehyde. c) In a second spectrophotometric assay the reaction mixture described above was used but partially purified H u P I from Pseudomonas W6, 800 (ig was added. Glucose-6-phosphate dehydrogenases and 6-phosphogluconate dehydrogenases were assayed according to KORNBERG and HORECKER (1955). The reaction mixture contained Tris-HCl buffer, p H 7.0, 50 MM; MgCl2, 5MM; glucose-6-phosphate or 6-phosphogluconate, 5 MM and NAD(P)+, 0 . 8 MM.

Dissimilation of methanol in Acetobacter sp.

79

NAD(P)H-oxidases were determined in 30000 xg-supernatant. The reaction mixture contained potassium phosphate buffer, p H 8.0, 200 mM and NAD(P)H, 0.25 mM. The reaction was started b y adding the extract. Partial purification of H u P I from Pseudomonas W6 was performed according t o L E V E R I H G et al. (1981). Pseudomonas W6, an obligate methylotroph, was cultivated a t 30 °C in shaking flasks in a mineral salt medium as described for cultivation of Acetobacter sp. MB 58 b u t with 310 mg/ml phosphate and 1 % (v/v) methanol. The cells were harvested in the mid-exponential growth phase, washed twice with potassium phosphate buffer, 50 mM, p H 7.0 containing 5 mM E D T A a n d then resuspended in t h e same buffer to a concentration of 0.1 g wet weight per ml. 150 ml of this suspension were sonicated using 5 one-min pulses interupted by 30-sec, cooling periods. The extract was centrifuged a t 30000 xg for 40 min a t + 2 °C. The supernatant was subjected to ammonium sulfate precipitation between 50 and 90% saturation. Then the protein was precipitated by centrifugation, dissolved in 20 ml buffer containing 1 mM E D T A and dialysed against 2 1 of t h e same buffer. The dialysed enzyme solution (10 mg protein/ml) was free of any H u P S activity. Protein determination was carried out by the method of S C H A C T E R L E and P O L L O C K ( 1 9 7 3 ) and b y t h e method of B R A D F O R D ( 1 9 7 6 ) .

Results Linear

dissimilatory

sequence

of

methanol

Methanol- and formaldehyde-oxidizing activities could be measured with P M S only i n m e t h a n o l - g r o w n Acetobacter sp. M B 58 b u t n o t in n o n m e t h y l o t r o p h i c a l l y g r o w n cells (Table 1). N A D + / G S H - a n d N A D P + - d e p e n d e n t o x i d a t i o n of f o r m a l d e h y d e w a s a l w a y s p r e s e n t a t l o w l e v e l s i n d e p e n d e n t l y of t h e g r o w t h s u b s t r a t e (Table 1). Table 1 Enzyme activities [nM • m i n - 1 • m g - 1 protein] of t h e linear dissimilatory sequence in Acetobacter sp. MB 58 as revealed b y different growth substrates Growth substrate

E n z y m e activities Methanol oxidation PMS Formaldehyde oxidation PMS NAD+/GSH NADP+ F o r m a t e oxidation DCIP NADH-oxidation NADPH-oxidation

Methanol

Glucose

Glycerol

237

—

—

—

—

150 24 13

29 12

55 12

193 6

156 6

179 18

—

—

—

— = not detectable DCIP-linked formate-oxidizing activity was only detectable under anaerobic c o n d i t i o n s . N o r e a c t i o n t o o k p l a c e w i t h o t h e r e l e c t r o n a c c e p t o r s (see Materials a n d Methods). The reaction could be inhibited b y cyanide. The highest specific activities w e r e o b t a i n e d a t p H 5.0. F o r m a t e - o x i d i z i n g a c t i v i t y w a s o n l y d e t e c t a b l e in t h e membrane fraction. In methanol- and nonmethylotrophically grown Acetobacter sp. M B 5 8 similar l e v e l s of D C I P - d e p e n d e n t a c t i v i t y were f o u n d (Table 1). Cyclic

dissimilation

of

formaldehyde

M e t h a n o l - a n d g l u c o s e - g r o w n Aacetobacter sp. M B 5 8 p o s s e s s t h e s a m e l e v e l s of N A D + - a n d N A D P + - d e p e n d e n t G 6 P D H . I n c o n t r a s t , g l y c e r o l - g r o w n cells e x i b i t less

80

M. W . GRÜNDIG a n d N . V. DORONINA

Table 2 E n z y m e activities [nM • m i n - 1 • m g _ 1 protein] of the cyclic dissimilation of formaldehyde in Acetobacter sp. M B 58 as revealed b y different growth substrates Growth substrate

Enzyme Hexulose-6-phosphate s y n t h a s e Radiochemical assay Spectrophotometrical assay Glucose-6-phosphate dehydrogenase NAD+ NADP+ 6-Phosphogluconate dehydrogenase NAD+ NADP+

Methanol

Glucose

Glycerol

735 707

722 267

893 285

99 770

85 683

60 356

625 148

854 171

771 158

activity. The NADP+-dependent G6PDH-activities are in all cases about twice as high as the NAD+-dependent ones (Table 2). N A D + - d e p e n d e n t 6PGDH-activities are about four times higher t h a n the N A D P + - d e p e n d e n t ones. H u P S and H u P I are amphibolic enzymes. They are involved both in the dissimilatory as in the assimilatory sequence of bacteria with methylotrophic nutrition. Assaying H u P S with the spectrophotometric method in nonmethylotrophically grown Acetobacter sp. MB 58 always resulted in much lower values t h a n those measured with the direct radioactive method. Only in methanol-grown Acetobacter sp. MB 58 the two assays gave similar specific activities. Our hypothesis t h a t this is due to much lower levels of H u P I was based on the results of L E V E E I N G et al. ( 1 9 8 1 ) . Therefore, we repeated the spectrophotometric HuPS-assay b y adding partially purified H u P I from Pseudomonas W6. The results obtained are shown in Table 3. Table 3 Influence of H u P I on the results obtained w i t h the spectrophotometric m e t h o d for assaying H u P S in Acetobacter sp. M B 58 grown on different substrates [nM • m i n - 1 • m g - 1 protein] Growth substrate

without H u P I with H u P I

Methanol

Glucose

Glycerol

668 824

118 834

80 693

Using the " i m p r o v e d " spectrophotometric HuPS-assay, we obtained activities which were nearly identical with those found with the radiochemical method (compare Table 2). The use of ribulose-5-phosphate instead of ribose-5-phosphate led to the same results.

Discussion The methanol-oxidizing enzyme of Acetobacter sp. MB 58 must be similar to the methanol dehydrogenases of other methylotrophs with regard to PMS-dependence, a m m o n i u m requirement and the p H - o p t i m u m of 9 or more ( A N T H O N Y and Z A T M A N 1 9 7 2 , 1 9 7 8 ,

D i s s i m i l a t i o n o f m e t h a n o l in Acetobacter M E H T A 1 9 7 3 , S P E R L et al.

81

sp.

1 9 7 4 , H A R D E R a n d ATTWOOD 1 9 7 5 , WADZINSKI a n d R I B B O N S

1 9 7 5 , G O L D B E R G 1 9 7 5 , Y A M A N A K A a n d MATSUMOTO 1 9 7 7 , 1 9 7 9 , B A M F O R T H a n d Q u A Y L E 1 9 7 8 , W O L F a n d H A N S O N 1 9 7 8 , D U I N E et al.

and KOMAGATA 1 9 8 1 , O H T A et al. 1 9 8 1 , inducible (or derepressible) by methanol 1 9 7 7 , L Y N C H et al.

GOSH

1978, BELLION a n d W u

and

(DUNSTAN

1978,

URAKAMI

This enzyme is also O ' C O N N O R and H A N S O N

QUALE 1981).

et al.

1972,

1980).

Several enzymes are capable of oxidizing formaldehyde ( S T I R L I N G and D A L T O N 1978) including both the NAD(P)+-dependent and dye-linked ones. It is assumed that, in organisms which lack the NAD(P) + -dependent dehydrogenase, formaldehyde is oxidized by either methanol dehydrogenase ( A N T H O N Y and Z A T M A N 1 9 6 4 , J O H N S O N and Q U A Y L E 1 9 6 4 , P A T E L a n d H O A R E 1 9 7 1 , M E H T A 1 9 7 3 , S P E R L et al. 1 9 7 4 ) , which can oxidize formaldehyde in vitro, or by the NH 4 -independent dye-linked formaldehyde dehydrogenases ( J O H N S O N and Q U A L E 1 9 6 4 , M E H T A 1 9 7 5 , M A R I S O N and A T T W O O D + 1 9 8 0 ) . However, in Acetobacter sp. MB 5 8 NAD(P) -dependent formaldehyde-oxidizing activities are negligibly low. But formaldehyde is oxidized well in the presence of PMS. At the present time the results suggest that in Acetobacter sp. MB 58 formaldehyde is oxidized by the methanol-oxidizing enzyme. This reaction requires ammonium and has a pH-optimum of 9.0, as is the case for methanol oxidation. Independently of this PMS-linked formaldehyde oxidation is induced (or derepressed) by methanol, too. The results suggest that Acetobacter sp. MB 58 possesses a "special" formateoxidizing enzyme because of the low pH-optimum (5.0) of the reaction. Common formate dehydrogenases were measured at a pH-range from 7 to 8.5 ( J O H N S O N and Q U A Y L E 1964, P A T E L and H O A R E 1971, L E S T E R and D E Moss 1971, A N D R E E S E N and L J U N G D A H L 1 9 7 1 , STIEGLITZ a n d MATELES 1 9 7 3 , LEONHARDT a n d A N D R E E S E N M Ü L L E R et al.

1 9 7 8 , F R I E D R I C H et al.

1977,

1 9 7 9 , B A B E L a n d M O T H E S 1 9 8 0 , LOGINOVA et

al.

1981, S C H A U E R and F E R R Y 1982). Other methylotrophs may also possess formateoxidizing enzymes, which can use artificial electron acceptors under anaerobic conditions, as indicated by the results of D I J K H U I Z E N et al. (1979) who used Pseudomonas oxalaticus OX1. We think, therefore, that in Acetobacter sp. MB 58 a complete linear dissimilatory sequence is operative. As is the case if other methylotrophs that use the HuP-pathway Acetobacter sp. M B 5 8 can oxidize CJ-carbon to C 0 2 by an additional cyclic sequence via G 6 P D H and 6 P G D H . In our strain G 6 P D H can generate more N A D P H and 6 P G D H more NADH. This distribution was also found in the methylotrophs Pseudomonas C ( B E N - B A S S A T and G O L D B E R G 1 9 7 7 ) , Pseudomonas oleovorans (LOGINOVA and + T R O T S E N K O 1 9 7 7 ) and in Acetobacter suboxydans ( S T E U D E L et al. 1 9 8 0 ) . NAD -dependent 6PDH is even lower compared with the other and is not controlled by methanol, though in Acetobacter suboxydans NADP+-dependent G 6 P D H and 6 P G D H are both controlled by methanol. (In all other strains mentioned NADP + -dependent + 6 P G D H is much lower than NAD -dependent activity.) HuPS was measured in methanol- and nonmethylotrophically grown Acetobacter sp. MB 58. In glucose- and glycerol-grown cells, however, HuPS showed lower activities with the spectrophotometric method than with the direct radiochemical assay. L E V E R I N G et al. ( 1 9 8 1 ) reported on the same discrepancy. They could not detect HuPS with the spectrophotometric method in extracts of Arthrobacter P I because H u P I was absent. On the basis of our data we conclude that H u P I was much lower in nonmethylotrophically grown than in methanol-grown Acetobacter sp. MB 58. For this reason we used partially purified H u P I from another methylotroph (Pseudomonas W6) in the spectrophotometric HuPS-assay. With this "improved" assay, we obtained nearly the same specific activities of HuPS from methanol- both from glucose- and 6

Z. allg. Mikrobiol., Bd. 24, H. 2

82

M . W . G R Ü N D I G a n d N . V . DORONINA

g l y c e r o l - g r o w n cells. T h i s m e a n s t h a t i n Acetobacter sp. M B 5 8 H u P S is c o n s t i t u t i v e a n d H u P I is e p i g e n e t i c a l l y controlled b y m e t h a n o l . I n c o n t r a s t t o our results, H u P S is r e g u l a t e d b y m e t h a n o l in Pseudomonas C (BENB A S S A T a n d G O L D B E R G 1 9 7 7 ) a n d i n Pseudomonas oleovorans (LOGINOVA and TKOTS E N K O 1 9 7 7 ) or b y m e t h y l a m i n e in Arthrobacter P I ( L E V E E I N G et al. 1 9 8 1 ) . S T E U D E L et al. ( 1 9 8 0 ) , u s i n g t h e c o m m o n s p e c t r o p h o t o m e t r i c a s s a y , f o u n d t h a t H u P S is c o n trolled b y m e t h a n o l i n Acetobacter suboxydans. Considering our r e s u l t s it c o u l d b e p o s s i b l e t h a t c o n s t i t u t i v i t y of H u P S a n d control of H u P I b y m e t h a n o l are c o m m o n to methanol-utilizing Acetobacter. F i n a l l y , in t h e linear d i s s i m i l a t o r y s e q u e n c e of Acetobacter sp. M B 5 8 P M S - l i n k e d o x i d a t i o n of m e t h a n o l a n d f o r m a l d e h y d e a n d t h e r a t h e r a m p h i b o l i c o p e r a t i n g H u P I are e p i g e n e t i c a l l y c o n t r o l l e d b y m e t h a n o l . Acknowledgements The main p a r t of this work was done in the laboratory of Dr. Y. A. T R O T S RNKO (Puchino, U.S.S.R.). We are grateful to him for his support and helpful advice. We also t h a n k Prof. Dr. W. B A B E L for stimulating discussion and for his help in preparing this manuscript. References ANDRESSEN, J . R. and LJUNGAHL, L. G., 1974. Nicotinamide adenine dinuleotide phosphatedependent formate dehydrogenase from Clostridium thermoacetium: Purification and properties. J . Bacteriol., 120, 6 — 14. ANTHONY, C. and ZATJVIAN, L. J., 1964. The microbial oxidation of methanol: Methanol oxidizing enzyme of Pseudomonas sp. M 27. Biochem. J . , 92, 614—621. BABEL, W. und HOFMANN, K. H., 1977. Regulation des P y r u v a t - und a-Ketoglutarat-Dehydrogenase-Komplexes eines fakultativ methylotrophen Bakteriums. Z. allg. Mikrobiol., 17, 403 bis 406. BABEL, W. und MOTHES, G., 1978. Dissimilatorische Sequenzen in methylotrophen Bakterien. Z. allg. Mikrobiol., 18, 17—26. BABEL, W. und MOTHES, G., 1980. Rolle der Formiat-Dehydrogenase in „Serinweg-Bakterien". Z. allg. Mikrobiol., 20, 167 — 175. BABEL, W. und MÜLLER, R., 1977. Kinetik der Hexulose-6-phosphat Synthase methylotropher Bakterien in vitro und in situ. Z. allg. Mikrobiol., 17, 175 —182. B A M F O R T H , C . W. and Q U A Y L E , J . R., 1 9 7 8 . The dye-linked alcoholdehydrogenase of Rhodopseudomonas acidophila. Biochem. J . , 169, 677—686. B E N - B A S S A T , A. and G O L D B E R G , I . , 1 9 7 7 . Oxidation of Cj-eompounds in Pseudomonas C . Biochim. biophysica Acta, 497, 5 8 6 — 5 9 7 . BELLION, E. and W u , G. T.-S., 1978. Alcohol dehydrogenase from a facultative methylotrophic bacterium. J . Bacteriol., 185, 251—258. BRADFORD, M. M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248—254. D A H L , J . S., M E H T A , R . J . and H O A R E , D . S., 1 9 7 2 . New obligate methylotroph. J . Bacteriol., 109, 9 1 6 - 9 2 1 . D I J K H U I Z E N , L . C., TIMMERMAN, J . W . C. a n d H A R D E R , W . , 1 9 7 9 . A p y r i d i n e

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oxalaticus 0 X 1 . F E M S Microand

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dehydrogenase from Hyphomicrobium X. Biochim. biophysica Acta, 524, 277—287. D U N S T A N , P . M., A N T H O N Y , C . and D R A B B L E , W . , 1972. Microbial metabolism of C X and C A compounds. The role of glyoxylate, glycolate and acetate in the growth of Pseudomonas AM 1 on ethanol and on Cj compounds. Biochem. J . , 128, 101 —115.

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a n d Q U A Y L E , J . R . , 1 9 8 1 . Purification a n d properties of t h e methanol dehydrogenase f r o m Methylophilus methylotrophus. Biochem. J . , 199, 145—250. HARDER, W . and ATTWOOD, M., 1975. Oxidation of organic Cx compounds b y Hyphomicrobium spp. Antonie van Leeuwenhoek, 41, 421—429. J O H N S O N , P . A. a n d Q U A Y L E , J . R . , 1 9 6 4 . Microbial growth on Cj-compounds. 6 . Oxidation of methanol, formaldehyde a n d formate by methanol-grown Pseudomonas AM 1. Biochem. J . GOSH, R .

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281-290.

A. and H O R E C K E R , B . L . , 1 9 5 5 . GIucose-6-phosphate dehydrogenase. I n : Methods in Enzymology, Vol. I, (COLOWICK, S . P . and K A P L A N , N . O . , Editors). Academic Press Inc. Publishers, New York, 323. LAWRENCE, A., KEMP, M. and QUAYLE, J . R., 1970. Synthesis of cell constituents by methanegrown Methanomonas methanoxidans. Biochem. J . , 116, 631—639. LEONHARDT, U. a n d ANDREESEN, J . R., 1977. Some properties of formate dehydrogenase, accumulation a n d incorporation of 1 8 6 W-tungsten into protein of Clostridium formiaceticum. Arch. Microbiol., 115, 277—284. LESTER, R . L. and DE MOSS, J . A., 1971. Effects of molybdate and selenite on formate and n i t r a t e metabolism of Escherichia coli. J . Bacteriol., 105, 10Ö6 —1014. L E V E R I N G , P . R . , V A N D I J K E N , J . P . , V E E N H U I S , M. and H A R D E R , W . , 1 9 8 1 . Arthrobacter PI, a fast growing versatile methylotroph with amine oxidase as a key enzyme in the metabolism of m e t h y l a t e d amines. Arch. Microbiol., 129, 7 2 — 8 0 . LOGINOVA, N. V. a n d TROTSENKO, Y. A., 1977. Metabolism of methanol in Pseudomonas oleovorans. Mikrobiologia, 46, 2 1 0 — 2 1 6 . LOGINOVA, N . V . , GOVORHUKINA, N . I . a n d T R O T S E N K O , Y . A . , 1 9 8 1 . Metabolism of the obligate methylotroph Methylophilus methanolovorus. Mikrobiologia, 50, 3 0 5 — 3 1 0 . KORNBERG,

LYNCH, M . J . , WOPAT, A . E . a n d O'CONNOR, M . L . , 1980. C h a r a c t e r i z a t i o n of t w o n e w f a c u l t a t i v e

methylotrophs. Appl. Environ. Microbiol., 40, 4 0 0 — 4 0 7 . MARISON, I. W. and ATTWOOD, M. M., 1980. Partial purification a n d characterization of a dyelinked formaldehyde dehydrogenase f r o m Hyphomicrobium X. J . gen. Microbiol., 117, 305—313. MEHTA, R . J . , 1973. Studies on methanol-oxidizing bacteria.' I I . Purification and properties of methanol dehydrogenase from Pseudomonas R J 1 . Antonie van Leeuwenhoek, 39, 303—312. M E H T A , R . J . , 1 9 7 5 . A novel inducible formaldehyde dehydrogenase of Pseudomonas R J 1 . Antonie v a n Leeuwenhoek, 41, 8 9 — 9 5 . MIETHE, D., 1977. P h . D. Thesis, Academy of Sciences of t h e G.D.R. MÜLLER, U . , WILLNOW, P . , RUSCHIG, U . a n d HÖPNER, T . , 1978. F o r m a t e d e h y d r o g e n a s e

Pseudomonas

oxalaticus.

from

Europ. J . Biochem., 83, 485—498.

N I E K U S , H . G . D . , VAN D O O R N , E . ,

D E V R I E S , W . a n d STOUTHAMER, A . H . , 1 9 8 0 . A e r o b i c

growth

of Campylobacter sputorum, subspecies bubulus with formate. J . gen. Microbiol., 118, 419—428. O ' C O N N O R , M. L . , and H A N S O N , R . S., 1 9 7 7 . E n z y m e regulation in Methylobacterium organophilum. J . gen. Microbiol., 101, 3 2 7 - 3 3 2 . O H T A , S . , F U J I T A , T . and T O B A R I , J . , 1 9 8 1 . Methanol dehydrogenase of Methylomonas J : Purification, crystallization, and some properties. J . Biochem., 90, 2 0 5 — 2 1 3 . PATEL, R . N. and HOARE, D. S., 1971. Physiological studies of methane- and methanol-oxidizing bacteria: Oxidation of Cj- compounds by Methylococcus capsulatus. J . Bacteriol., 107, 187 — 192. PATEL, R . N . , BOSE, H . R . , MANDY, W . J . a n d HOARE, D . S., 1972. P h y s i o l o g i c a l s t u d i e s of m e t h a -

n e - a n d methanol-oxidizing bacteria: Comparism of primary alcohol dehydrogenase from Methylococcus capsulatus (Texas strain) and Pseudomonas species M 27. J . Bacteriol., 110, 570 — 577. PATEL, R. N., H o u , C. T. a n d FELIX, A., 1978. Microbial oxidation of m e t h a n e a n d m e t h a n o l : Crystallization of holo- a n d apo-methanol dehydrogenase f r o m Methylomonas methanica. J . Bacteriol., 133, 6 4 1 - 6 4 9 . S C H A C T E R L E , J . R . a n d P O L L O C K , R . L . , 1 9 7 3 . A simplified method for q u a n t i t a t i v e assay of small a m o u n t s of protein in biological material. Anal. Biochem., 51, 654—655. SCHAUER, N. L. a n d FERRY, J . G., 1982. Properties of formate dehydrogenase in Methanobacterium formicicum. J . Bacteriol., 150, 1—7. SPERL, G. T . , FORREST, H . S. a n d GIBSON, D . T . , 1974. S u b s t r a t e s p e c i f i t y of p u r i f i e d p r i m a r y

alcohol dehydrogenase from methanol-oxidizing bacteria. J . Bacteriol., 118, 541—550. u n d B A B E L , W . , 1 9 8 0 . Bakterium MB 5 8 , ein methylotrophes „Essigsäurebakterium". Z. allg. Mikrobiol., 20, 663—672. STIEGLITZ, B. a n d MATELES, R . I., 1973. Methanol metabolism in Pseudomonas C. J . Bacteriol., 114, 390—398. S T I R L I N G , D. I . a n d S ALTON, H . , 1 9 7 8 . Purification a n d properties of a NAD(P)+-linked formaldehyde dehydrogenase f r o m Methylococcus capsulatus ( B A T H ) . J . gen. Microbiol., 107, 1 9 — 2 9 . U R A K A M I , T . and KOMAGATA, K . , 1981. Eleetrophoretic comparism of enzymes in the gram negative methanol-utilizing bacteria. J . gen. appl. Microbiol., 27, 381—403. STEUDEL, A . , MIETHE, D .

84

M . W . GKUNDIG a n d N . V . DOBONINA

WADZINSKI, A. M. and RIBBONS, D. W., 1975. Oxidation of Cj-compounds by particulate fractions from Methylococcus capsulatus : Properties of methanol oxidase and methanol dehydrogenase. J . Bacteriol., 112, 1 3 6 4 - 1 3 7 4 .

WOLF, H. J . and HANSON, R. S., 1978. Alcohol dehydrogenase from Methylobacterium organophilium. Appl. environ. Microbiol., 36, 105 — 114. YAMANAKA, K. and MATSTTMOTO, K., 1977. Purification and crystallization and properties of primary alcohol dehydrogenase from methanol-oxidizing Pseudomonas sp. J\s 2941. Agric. biol. Chemistry, 41, 4 6 7 - 4 7 5 . YAMANAKA, K. and MATSTTMOTO, K., 1979. Isolation of facultative methanol-oxidizing bacteria which produce electrophoretically different methanol dehydrogenases. Agric. biol. Chemistry, 43, 1 — 7 . Mailing adress : M. W. GRUNDIG, Institut fur technische Chemie der AdW, DDR 7050 Leipzig, Permoserstr. 15

Zeitschrift für allgemeine Mikrobiologie 24 (1984) 2, 8 5 - 9 2

( I n s t i t u t für I m p f s t o f f e Dessau der A k a d e m i e der Landwirtschaftswissenschaft der D D R , Direktor: O b . - V e t . - R a t Prof. Dr. sc. D . URBANECK)

Eine neue Methode zur Darstellung der Kapsel v o n Bordetella bronchiseptica im Elektronenmikroskop U . KLUDAS j u n . u n d W . RUDOLPH

(Eingegangen

am 27. 5.

1983)

A n e w a n d rapid working m e t h o d is presented for electronmicroscopic preparation of capsules from g r a m n e g a t i v e bacteria, e. g. Bordetella bronchiseptica and Pasteurella multocida. The a d v a n t a g e of the n e w technique is the availability of t h e results w i t h i n 30 m i n after starting t h e preparation. The staining of the capsule b y alcian blue has to be done as a first s t e p together w i t h glutarald e h y d e f i x a t i o n , before staining the bacterial cell w i t h phosphotungstic acid. The n e w staining technic also reveals structural details of t h e capsule. The described procedure was f o u n d t o be useful in controlling the d e v e l o p m e n t of the bacterial capsule depending on culture media for propagation and maintenance of the a b o v e m e n t i o n e d bacteria.

Zahlreiche gramnegative Bakterien haben als äußere Begrenzung auf der Zellwand eine Kapsel. F ü r das Zelleben ist sie nur scheinbar bedeutungslos, denn sie schützt vor Phagozytose, v e r s t ä r k t e m Befall durch Bakteriophagen u n d einem raschen Austrocknen der Zelle ( K Ö H L E R U. MOCHMANN 1 9 8 0 ) . Die Kapsel der Species Bordetella bronchiseptica besteht überwiegend aus Polysacchariden. Sie gehören zu den K-Antigenen und sind hitzelabil. Bekapselte Zellen bilden S, O und H-Antigene aus. Unbekapselte Zellen sind nur Träger der O-Antigene ( N A K A S E 1 9 5 7 ) . Die Fähigkeit zur Ausbildung von Kapseln ist in jeder Bakterienzelle genetisch verankert ( B R I X T O N 1 9 6 5 ) . Die Anzahl der Bakterien eines Stammes, die eine Kapsel ausbilden, wird durch die Kultivierungsbedingungen beeinflußt. B. bronchiseptica bildet in vivo Kapseln aus, die in vitro nach wenigen Passagen verlorengehen. Dieser Kapselverlust ist reversibel, solange keine Schädigung der genetischen Anlagen erfolgte. Eine häufige Passagierung f ü h r t allerdings zu einem irreversiblen Verlust der Kapsel ( N A K A S E 1 9 5 7 ) . YOKOMIZO und SHIMIZU ( 1 9 7 9 ) k o n n t e n an B. bronchiseptica durch spezifische Antiseren diese Kapselrückbildung nachweisen, die sich in drei P h a s e n unterteilen läßt (Abb. 1). Phase I

Kapsel+

Phase I

Phase M

Kapsel*/-

Kapsel -

CD — O CD

Abb. 1. Darstellung des Kapselverlustes v o n B. bronchiseptica nur kugelförmige Mikroorganismen ausgebildet

(NAKASE 1957). I n der P h a s e I I I werden

Zeitschrift für allgemeine Mikrobiologie 24 (1984) 2, 8 5 - 9 2

( I n s t i t u t für I m p f s t o f f e Dessau der A k a d e m i e der Landwirtschaftswissenschaft der D D R , Direktor: O b . - V e t . - R a t Prof. Dr. sc. D . URBANECK)

Eine neue Methode zur Darstellung der Kapsel v o n Bordetella bronchiseptica im Elektronenmikroskop U . KLUDAS j u n . u n d W . RUDOLPH

(Eingegangen

am 27. 5.

1983)

A n e w a n d rapid working m e t h o d is presented for electronmicroscopic preparation of capsules from g r a m n e g a t i v e bacteria, e. g. Bordetella bronchiseptica and Pasteurella multocida. The a d v a n t a g e of the n e w technique is the availability of t h e results w i t h i n 30 m i n after starting t h e preparation. The staining of the capsule b y alcian blue has to be done as a first s t e p together w i t h glutarald e h y d e f i x a t i o n , before staining the bacterial cell w i t h phosphotungstic acid. The n e w staining technic also reveals structural details of t h e capsule. The described procedure was f o u n d t o be useful in controlling the d e v e l o p m e n t of the bacterial capsule depending on culture media for propagation and maintenance of the a b o v e m e n t i o n e d bacteria.

Zahlreiche gramnegative Bakterien haben als äußere Begrenzung auf der Zellwand eine Kapsel. F ü r das Zelleben ist sie nur scheinbar bedeutungslos, denn sie schützt vor Phagozytose, v e r s t ä r k t e m Befall durch Bakteriophagen u n d einem raschen Austrocknen der Zelle ( K Ö H L E R U. MOCHMANN 1 9 8 0 ) . Die Kapsel der Species Bordetella bronchiseptica besteht überwiegend aus Polysacchariden. Sie gehören zu den K-Antigenen und sind hitzelabil. Bekapselte Zellen bilden S, O und H-Antigene aus. Unbekapselte Zellen sind nur Träger der O-Antigene ( N A K A S E 1 9 5 7 ) . Die Fähigkeit zur Ausbildung von Kapseln ist in jeder Bakterienzelle genetisch verankert ( B R I X T O N 1 9 6 5 ) . Die Anzahl der Bakterien eines Stammes, die eine Kapsel ausbilden, wird durch die Kultivierungsbedingungen beeinflußt. B. bronchiseptica bildet in vivo Kapseln aus, die in vitro nach wenigen Passagen verlorengehen. Dieser Kapselverlust ist reversibel, solange keine Schädigung der genetischen Anlagen erfolgte. Eine häufige Passagierung f ü h r t allerdings zu einem irreversiblen Verlust der Kapsel ( N A K A S E 1 9 5 7 ) . YOKOMIZO und SHIMIZU ( 1 9 7 9 ) k o n n t e n an B. bronchiseptica durch spezifische Antiseren diese Kapselrückbildung nachweisen, die sich in drei P h a s e n unterteilen läßt (Abb. 1). Phase I

Kapsel+

Phase I

Phase M

Kapsel*/-

Kapsel -

CD — O CD

Abb. 1. Darstellung des Kapselverlustes v o n B. bronchiseptica nur kugelförmige Mikroorganismen ausgebildet

(NAKASE 1957). I n der P h a s e I I I werden

86

U . KLUDAS j u n . u n d W . RUDOLPH

Die Kapsel, die für das Bakterium offensichtlich eine gewisse Schutzfunktion ausübt, ist damit gleichzeitig ein bedeutender Virulenzfaktor. Das wird deutlich aus der Tatsache, daß die bekapselten Keime der Phase I virulent sind, während die kapsellosen der Phase I I I avirulent sind. Die Phase I I ist eine Zwischenphase (GOODNOW 1980).

Obwohl die für die protektive Immunität verantwortlichen Antigene von B. bronchiseptica in der Zellwand lokalisiert sind ( K R Ü G E R 1 9 8 2 ) , ist nur bei den Phase I-Bakterien mit dem Vorhandensein des kompletten Antigenspektrums zu rechnen. Für die Impfstoffproduktion müssen deshalb bekapselte Keime eingesetzt werden. Die schon erwähnte Möglichkeit des Kapselverlustes unter verschiedenen Kultivierungsbedingungen erfordert deshalb eine ständige Kontrolle der Kapselbildung während der Kultivierung von B. bronchiseptica für die Impfstoffproduktion. Die Darstellung der Bakterienkapsel im Lichtmikroskop ist nach dem Tusche-Verfahren ( W E I D E U . A U R I C H 1 9 8 0 ) oder der Kapselfärbung nach M A N E V A L ( S C H R Ö D E R 1977) möglich. Diese beiden Methoden lassen keine Aussage über den Grad der Zerstörung der Kapsel, die Kapselstruktur und sehr kleine Kapselreste zu, weil das Auflösungsvermögen des Lichtmikroskopes dafür nicht ausreicht. Für die elektronenmikroskopische Darstellung von Bakterienkapseln gibt es prinzipiell zwei Möglichkeiten, nämlich die Herstellung von Ultradünnschnitten oder die Untersuchung ganzer Keime bei Verwendung verschiedener Kontrastierungsverfahren. Die Kontrolle der Kapselausbildung während des Kultivierungsprozesses erfordert eine schnelle Verfügbarkeit der Ergebnisse. Damit scheidet die Dünnschnittechnik aus. Von den Kontrastierungsverfahren erschien uns die Negativkontrastierung als die geeigneteste Methode. Die Standardmethode mit Phosphorwolframsäure (PWS) ( R E I M E R 1 9 6 7 ) ist zwar einfach und schnell durchführbar, brachte aber keine befriedigende Darstellung der Kapsel von B. bronchiseptica. Ziel dieser Untersuchungen war es deshalb, ein Kontrastierungsverfahren zu finden, das bei vertretbarem Arbeitsaufwand hinreichende Informationen über die Kapselausbildung von B. bronchiseptica in sehr kurzer Zeit, d. h. innerhalb von etwa 30 min, liefert. Material und Methoden D i e Untersuchungen wurden a m B. bronchiseptica S t a m m S 3634 des B I V P o t s d a m vorgenomm e n ' ) . Der S t a m m war zu B e g i n n unserer U n t e r s u c h u n g e n kein Frischisolat. D i e K u l t i v i e r u n g u n d Passagierung der Mikroorganismen erfolgte in einem flüssigen Spezialnährmedium im ANKUMF e r m e n t o r (KLUDAS et al. in Vorbereitung). D i e K e i m e wurden m i t sterilen P i p e t t e n aus d e m N ä h r m e d i u m e n t n o m m e n u n d auf die m i t F o r m v a r beschichteten N e t z b l e n d e n aufgetragen. D i e E n t f e r n u n g der Flüssigkeit erfolgte entweder durch Lufttrocknung der freiliegenden N e t z b l e n d e n •oder durch Diffusion in 2%iges Agarosegel, auf das die N e t z b l e n d e n vor der Beschickung m i t Bakterienkultur aufgelegt worden waren. D i e Fixierung erfolgte entweder m i t 0 , 4 % i g e m Formalin oder m i t 2 , 5 % i g e m Glutaraldehyd. F ü r die Negativkontrastierung wurden P W S , Osmiumsäure oder B l e i a c e t a t a n g e w a n d t , die K o n t r a s t i e r u n g der Kapselpolysaccharide gelang m i t Alcianblau. D i e Untersuchung der Präparate wurde in einem TESLA B S 500 Elektronenmikroskop vorgekommen.

Ergebnisse und Diskussion •Trocknung Als ein wichtiger Präparationsschritt erwies sich bereits die Entfernung der Kulturilüssigkeit nach Aufbringen der Bordetella-Suspension auf die beschichteten Netzä

) Für die Überlassung des S t a m m e s d a n k e n wir Herrn Dr. KÖHLER v o m B I V P o t s d a m .

Darstellung der Kapsel von

Bordetella

87

blenden. Mit Rücksicht auf die Zielstellung unserer Untersuchungen ist dies nur durch Lufttrocknung oder Agarosediffusion möglich, da alle anderen Methoden einen grösseren Aufwand an Zeit erfordern. Beide Methoden unterscheiden sich in der Strukturerhaltung der Bakterien wesentlich. Die Oberflächenspannung des Wassers auf die Mikroorganismen, die bei einer Partikelgröße von 1 [J.m Durchmesser ca. 1,4 kg/mm 2 beträgt (REIMER 1967), kann nicht vermieden werden. Darüber hinaus sind die Mikroorganismen während der Lufttrocknung einer starken Änderung des osmotischen Druckes und des pH-Wertes ausgesetzt. Die Zellwände von B. bronchiseptica (gramnegative Bakterien) sind dieser Belastung, im Gegensatz zu den Zellwänden grampositiver Bakterien, nicht gewachsen. Es kommt zur Dehnung der Zellwand. In der elektronenmikroskopischen Abbildung erscheint die Zellwand dann nicht mehr als straffe Membran, sondern als gefalteter, unregelmäßiger ca. 100 nm breiter Saum, der nach außen durch eine scharfe Linie begrenzt wird (Abb. 2).

Abb. 2. A) B. bronchiseptica, Entwässerung durch Lufttrocknung. Z = Zellwand K = Kapselrest B) Schematische Darstellung der Zellwandveränderung durch die Oberflächenspannung der Flüssigkeit

88

U . KLUDAS jun. u n d W . RUDOLPH

Durch das Aufbringen der Netzblenden auf Agarosegel dringt die Flüssigkeit in. die Agarose ein. D a b e i kann den Bakterien die Flüssigkeit nicht vollständig entzogen werden, weil sich zwischen der Diffusion der Nährlösung in das Agarosegel und der Wasserverdunstung aus dem Gel sehr bald ein Gleichgewicht einstellt. Dieser Prozess dauert, abhängig vom Alter des Agarosegels, etwa 1 5 — 2 0 min. B i s zur unmittelbar sich anschließenden Fixierung behalten die B a k t e r i e n eine gewisse Restfeuchte, die, begünstigt durch nahezu konstanten osmotischen D r u c k und p H - W e r t , eine gute Strukturerhaltung sichert (Abb. 3). Bakterienkapseln sind aber gegenüber äußerer Krafteinwirkung sehr empfindlich. Auch während der relativ schonenden Entwässerung durch Agarosediffusion k o m m t es daher zur Verschiebung der Kapsel. Dicke Kapselschichten ( > 2 0 0 nm) werden

^ Netzblende Abb. 3. A) B. bronchiseptica, 200 nm starke Kapsel. Entwässerung durch Agarosediffusion B) Schematische Darstellung der Kapselveränderung bei einer Stärke von 200 nm

Darstellung der Kapsel von Bordetella

89

d u r c h die O b e r f l ä c h e n s p a n n u n g gleichmäßig zur Seite gedrängt, was im E l e k t r o n e n m i k r o s k o p als R i n g e r k e n n b a r ist (Abb. 3). D e m g e g e n ü b e r gleiten d ü n n e r e K a p s e l s c h i c h t e n ( < 1 0 0 u m ) n a c h einer Seite a b (Abb. 4). E n t s p r e c h e n d dieser Verschiebung wird die K a p s e l im E l e k t r o n e n m i k r o s k o p s t ä r k e r dargestellt als sie in Wirklichkeit ist.

Abb. 4. A) B. brochiseptica, 100 nm starke Kapsel. Entwässerung durch Agarosediffusion. K R = Kapselrest B) Schematische Darstellung der Kapselveränderung bei einer Stärke von 100 nm

Fixierung Die F i x i e r u n g der B a k t e r i e n w u r d e m i t 0,4%iger Eormalinlösung oder 2,5%iger G l u t a r a l d e h y d l ö s u n g versucht. E s war n u r G l u t a r a l d e h y d geeignet. Die fixierende W i r k u n g l ä ß t sich d u r c h die Vernetzung der A m i n o g r u p p e n ben a c h b a r t e r P o l y p e p t i d k e t t e n oder der H y d r o x y l g r u p p e n der Polysaccharide erklären.

90

U . KLUDAS j u n . u n d W . RUDOLPH

Kontrastierung Die Kontrastierung der Bakterien wurde mit verschiedenen Schwermetallverbindungen, wie PWS, Osmiumsäure und Bleiacetat, versucht. Diese hüllen die Mikroorganismen in eine elektronendichte amorphe Schicht ein, oder bilden schwerlösliche Salze mit Proteinen und Polysacchariden. Die so mit Schwermetallverbindungen behandelten Bakterien haben eine bis zu 3mal größere Dichte gegenüber unbehandelten Objekten. Da die Kapsel mit den oben genannten Schwermetallen nicht darstellbar war, griffen wir einen Hinweis von S T E E D M A N ( 1 9 5 0 ) auf, der in der Histochemie zum Nachweis saurer Mucopolysaccharide Alcianblau 8 GS verwendet. Alcianblau ist einKupferphthalocyaninkomplex. Der wasserlösliche Farbstoff bildet mit den Polysaccharidanionen der Kapsel ( C A S S I T Y et dl. 1 9 7 8 ) wasserunlösliche elektronendichte Präzipitate. Eine Verstärkung dieser Reaktion wird durch Zusatz von Elektrolyten (z. B. NaCl und HCl) erreicht ( L U P P A 1 9 7 7 ) . Der Kontrast der Bakterienzelle wird durch die Reaktion der Anionenkomplexe der P W S [(PW 3 O 10 ) 4 ] _3 mit den positiv geladenen NH 3 -Gruppen im Protein der Zellwand erzielt. Dabei werden schwerlösliche elektronendichte PWS-Salze gebildet. Beim Einsatz von P W S vor Alcianblau reagieren die Anionen der P W S nicht nur mit den NH 3 -Gruppen der Zellwand, sondern auch mit den OH-Gruppen der Kapsel-Polysaccharide. Es k o m m t zu einer Veresterung der Polysaccharide. Der Phosphorwolfram-Zuckerester ist auf Grund seiner geringen Elektronendichte im Elektronenmikroskop aber nicht sichtbar. Nach der Veresterung mit P W S können die Kapselpolysaccharide nicht mehr mit Alcianblau reagieren, so daß die Kapselkontrastierung mit Alcianblau vor der Kontrastierung mit P W S durchgeführt werden muß. Da aber bereits durch die Fixierung die f ü r die Alcianblaukontrastierung notwendigen Hydroxylgruppen vernetzt werden, ist es erforderlich, die Alcianblaufärbung gleichzeitig mit der Fixierung vorzunehmen (Abb. 5).

NH3 -W30K,

-w3o10 3NH,

P =

n„3

W3010

NH3

- W o

R -OH R — OH

+

R-OH Polysaccharid

H -

W301o .

H -

W3010

H

-W30W

P = W3010

PWS

-W3010

schwer lösliches Salz elektronendichl

PWS-Anion

Kation der Zellwand

_w 3 O w —P=W 3 O w

-H,0

Zuckerest er

*• Atcianblau

keine Reaktion

löslich, nicht elektronendicht

Abb. 5. Reaktionsunterschiede von PWS gegenüber dem Protein der Zellwand und dem Kapselpolysaccharid

Elektronenmikroskopische

Darstellung

Die Kapsel ist im Elektronenmikroskop als hellgrauer Saum ohne scharfe äußere Begrenzung erkennbar (Abb. 3). I n stark gerührten oder geschüttelten Bakterien-

Darstellung der Kapsel von Bordetella

91

kulturen kann ein Abreißen der Kapsel von der Bakterienzelle beobachtet werden. Die abgetrennten Polysaccharide bilden im Medium mehr oder weniger große Ablagerungen (Abb. 4). Die elektronenmikroskopische Darstellung im Negativkontrast von B. bronchiseptica ermöglicht es, im Gegensatz zur Lichtmikroskopie, strukturelle Veränderungen an der Kapsel bzw. ihren Verlust unter in mtro-Bedingungen schneller und mit einer größeren Genauigkeit zu erfassen. Damit ist die elektronenmikroskopische Kapseldarstellung von B. bronchiseptica eine sehr wirksame Prüfmethode bei der Entwicklung von Stammhaitungs- und Fermentationsmedien ( K L U D A S et al., in Vorbereitung). Bisher führen wir den Kapselnachweis entsprechend der von uns aufgezeigten Methode nur für B. bronchiseptica und an Pasteurella multocida durch (Abb. 6). Wir nehmen an, daß der Nachweis auch bei anderen gramnegativen Bakterien möglich ist. Folgende Vorraussetzungen müssen erfüllt sein: 1. Die Entwässerung der Mikroorganismen muß nach der Agarosediffusion erfolgen. 2. Die Alcianblau-Reaktion mit der Kapsel muß vor der PWS-Kontrastrierung und gleichzeitig mit der Fixierung erfolgen.

Abb. 6. Kapseldarstellung an Pasteurella

multocida

Für die wertvollen Hinweise am Anfang unserer Arbeiten danken wir Herrn Dr. BAETSCH von der Ernst-Moritz-Arndt Universität Greifswald, Sektion Biologie, Abt. Elektronenmikroskopie

Literatur D. A . , G R E I S E N , H. A . , and A P P E L , M . J. 1977. Bacteriological variation among Bordetella bronchiseptica isolates from dogs and other species. J. Clinical Microbiol., 5/4, 471—480.

BEMIS,

92

U . RLUDAS j u n . u n d W . RUDOLPH

BRIXTON, C. C., 1965. The structure, function, synthesis and genetic control of bacterial pili molecular modul for DNA and RNA transport in gram-negative bacteria. Trans. New Yor. Acad. Sei., 27, 1003-1054. CASSITY, T . R . , KOLODZIEJ, B . J . a n d PFISTER, R . M . , 1978. U l t r a s t r u c t u r e

of t h e c a p s u l e

of

Bacillus megaterium ATCC 19213. Microbios, 21, 153 — 160. GOODNOW, A., 1980. Biology of Bordetella bronchiseptica. Microbiol. Rev., 44, 722—738. KÖHLER, W. undMocHMANN, H. R., 1980. Grundriß der Medizinischen Mikrobiologie. VEB Gustav Fischer Verlag Jena, 5. Auflage. LUPPA, H., 1977. Histochemie. Akademie Verlag Berlin. NAKASE, Y., 1957. Studies on Hemophilus bronchisepticus. I I . Phase variation of H. bronchisepticus. Kitasato, Arch, of Exp. Med. Vol. XXX/3-4, 7 3 - 7 8 . REIMER, L., 1967. Elektronenmikroskopische Untersuchungs- und Präparationsmethoden. Springer Verlag Berlin-Heidelberg-New York. SCHRÖDER, H., 1977. Mikrobiologisches Praktikum. Volk und Wissen Berlin. STEEDMAX, H. F., 1950. Alcian blue 8 GS: A new stain for mucin. Quart. J . Micr. Sei..91,447—479. WEIDE, H. und AUBICH, H., 1979. Allgemeine Mikrobiologie. VEB Gustav Fischer Verlag Jena. YOKOMIZO, Y. and SHIMIZU, T., 1979. Adherence of B. bronchiseptica to swine nasal epithelial cells and its possible role in virulence. Res. vet. Sei., 27, 15—21. Anschrift : Dipl.-Chem. U. KLUDAS, Institut für Impfstoffe der Akademie der Landwirtschaftswissenschaften, DDR 4500 Dessau, Jahnstr. 8

Zeitschrift f ü r allgemeine Mikrobiologie 24 (1984) 2, 9 3 - 1 0 0

(Akademie der Wissenschaften der D D R , Forschungszentrum f ü r Molekularbiologie und Medizin, Zentralinstitut f ü r Mikrobiologie und Experimentelle Therapie, Jena, Direktor: Prof. Dr. U. TAUBENECK)

Alternative life cycles in Thermoactinomyces

vulgaris

SIGRID KRETSCHMER

(Eingegangen

am 26. 6. 1983)

The differentiation of Thermoactinomyces vulgaris in liquid medium was studied morphologically. Sporogenesis could be initiated in strain 0 4 if growing mycelia were transferred into nitrogen free medium. After the shift down about 30% of the hyphae lysed. The remaining gave rise to new hyphae in which after about 4.5 hours sporulation started. 4 hours later t h e spores have been completed. Concerning kinetics as well as morphology this life cycle was identical to the SSMcycle (secondary substrate mycelium-cycle) observed during cultivation on CD-agar, b u t was different to the AM-cycle (aerial mycelium-cycle) which ran on CSL-agar. The complex d a t a now available allow to describe completely t h e differentiation behaviour of T. vulgaris, which is characterized by the existence of 2 alternative life cycles. Comparison of both revealed t h a t they partly consist of similar steps. B u t the SSM-cycle in addition contains a secondary growth phase which is initiated about 1 hour after t h e end of primary growth. The 3 phenotypically different mycelia occurring in T. vulgaris (primary, aerial and secondary substrate mycelium) were comparatively characterized. The SSM is the most highly differentiated mycelium because of alteration of gene expression already some hours before onset of sporulation and the formation of emergences in which the spores develop.

Under unlimited growth conditions Thermoactinomyces vulgaris form undifferentiated primary mycelium (PM). Upon exhaustion of nutrients in agar media growth of PM ceases and alternatively 2 kinds of secondary, sporogenic mycelia develop. Depending on the composition of the agar either aerial mycelium (AM) or secondary substrate mycelium (SSM) is formed. Kinetics as well as manner of differentiation of both mycelia have been described ( K R E T S C H M E R 1978a, 1982, 1984). Concerning differentiation behaviour of actinomycetes in liquid medium only few and mostly contradictory results have been published. Thus, it is unknown if there are homologies or at least some functional similarities during development on agar and in liquid medium ( K A L A K O U T S K I I 1 9 7 6 ) . For T. vulgaris it is known that sporulation may occur in liquid media. But there, neither the kinetics nor morphology of the whole life cycle has been analyzed. The behaviour of T. vulgaris G 4 during long-term cultivation in a stirring fermentor has been studied in detail ( K R E T S C H M E R et al. 1982). But under the conditions favouring the production of extracellular proteases nearly no spores were formed. The present study was undertaken to find conditions allowing sporulation during cultivation in liquid medium. There, the differentiation behaviour had to be analyzed mainly by use of the electron microscope. Afterwards, all known life cycles observed with T. vulgaris are compared. Further, the 3 phenotypically different mycelia of T. vulgaris (PM, AM, SSM) are comparatively characterized.

Materials and methods The strains used were Thermoactinomyces vulgaris G4 and 1227 (the latter was kindly supplied by D. A. HOPWOOD). Methods for production, storage and activation of spores, which were always used as inoculum, were described by STROHBACH and KRETSCHMER ( 1 9 7 7 ) . The medium used for

Zeitschrift f ü r allgemeine Mikrobiologie 24 (1984) 2, 9 3 - 1 0 0

(Akademie der Wissenschaften der D D R , Forschungszentrum f ü r Molekularbiologie und Medizin, Zentralinstitut f ü r Mikrobiologie und Experimentelle Therapie, Jena, Direktor: Prof. Dr. U. TAUBENECK)

Alternative life cycles in Thermoactinomyces

vulgaris

SIGRID KRETSCHMER

(Eingegangen

am 26. 6. 1983)

The differentiation of Thermoactinomyces vulgaris in liquid medium was studied morphologically. Sporogenesis could be initiated in strain 0 4 if growing mycelia were transferred into nitrogen free medium. After the shift down about 30% of the hyphae lysed. The remaining gave rise to new hyphae in which after about 4.5 hours sporulation started. 4 hours later t h e spores have been completed. Concerning kinetics as well as morphology this life cycle was identical to the SSMcycle (secondary substrate mycelium-cycle) observed during cultivation on CD-agar, b u t was different to the AM-cycle (aerial mycelium-cycle) which ran on CSL-agar. The complex d a t a now available allow to describe completely t h e differentiation behaviour of T. vulgaris, which is characterized by the existence of 2 alternative life cycles. Comparison of both revealed t h a t they partly consist of similar steps. B u t the SSM-cycle in addition contains a secondary growth phase which is initiated about 1 hour after t h e end of primary growth. The 3 phenotypically different mycelia occurring in T. vulgaris (primary, aerial and secondary substrate mycelium) were comparatively characterized. The SSM is the most highly differentiated mycelium because of alteration of gene expression already some hours before onset of sporulation and the formation of emergences in which the spores develop.

Under unlimited growth conditions Thermoactinomyces vulgaris form undifferentiated primary mycelium (PM). Upon exhaustion of nutrients in agar media growth of PM ceases and alternatively 2 kinds of secondary, sporogenic mycelia develop. Depending on the composition of the agar either aerial mycelium (AM) or secondary substrate mycelium (SSM) is formed. Kinetics as well as manner of differentiation of both mycelia have been described ( K R E T S C H M E R 1978a, 1982, 1984). Concerning differentiation behaviour of actinomycetes in liquid medium only few and mostly contradictory results have been published. Thus, it is unknown if there are homologies or at least some functional similarities during development on agar and in liquid medium ( K A L A K O U T S K I I 1 9 7 6 ) . For T. vulgaris it is known that sporulation may occur in liquid media. But there, neither the kinetics nor morphology of the whole life cycle has been analyzed. The behaviour of T. vulgaris G 4 during long-term cultivation in a stirring fermentor has been studied in detail ( K R E T S C H M E R et al. 1982). But under the conditions favouring the production of extracellular proteases nearly no spores were formed. The present study was undertaken to find conditions allowing sporulation during cultivation in liquid medium. There, the differentiation behaviour had to be analyzed mainly by use of the electron microscope. Afterwards, all known life cycles observed with T. vulgaris are compared. Further, the 3 phenotypically different mycelia of T. vulgaris (PM, AM, SSM) are comparatively characterized.

Materials and methods The strains used were Thermoactinomyces vulgaris G4 and 1227 (the latter was kindly supplied by D. A. HOPWOOD). Methods for production, storage and activation of spores, which were always used as inoculum, were described by STROHBACH and KRETSCHMER ( 1 9 7 7 ) . The medium used for

94

SIGRID KRETSCHMER

liquid cultivation contained: sucrose 10 g, Casamino acids ( D I F C O ) 6 g, NaCl 0 . 5 g, K 2 H P 0 4 • 1 g Mg S0 4 • 7 H 2 0 0.3 g, CaC03 O.Olg, FeS0 3 • 7 H 2 00.01 g per liter, pH was 7.0. Growth occurred in flasks which were shaken in a 50° C-water bath. For studying the ultrastructure of developing mycelia samples taken were fixed with glutaraldehyde and embedded according to K E L L E N B E R G E R et al. (1958).Ultrathin sections of the mycelia were studied with an electron microscope Type SEM 3-2 (VEB Werk fur Fernsehelektronik, Berlin-Oberschoneweide). Formation and sporulation of aerial mycelium (AM) occurred on CSL-agar (KRETSCHMER 1984} and of secondary substrate mycelium (SSM) on CD-agar (KRETSCHMER 1982). Results Sporulation of strain G 4 in liquid medium could be induced b y transferring exponentially growing mycelium into medium lacking the sole nitrogen source casamino acids. W i t h strain 1227 appropriate conditions for sporulation have not been found. After the shift to nitrogen starvation the behaviour of strain G 4 was followed (Fig. 1 and 2). T h e mycelia having been grown for 5 doublings (doubling time 34 min) were ultrastructurally sound and intact. After 1.5 hours about 3 0 % of the hyphae showed destruction and were about lysing. T h e remaining hyphae showed increased frequency of cross walls and often carried branches, which subsequently elongated. 4.5 hours after the shift the mostly unseptated branches frequently formed cross walls. Then the individual cells formed each an emergence, in which lateron an endospore developed. T h e kinetics of cross wall formation during the transition to nitrogen starvation is represented in Fig. 3. Finally, the average length of cells carrying a sporulation emergence was 0.8 [j,m. T h e distribution of the ultrastructural different hyphae in subsequent samples allows to reconstruct the manner of differentiation of T. vulgaris G 4. Upon exhaustion of nitrogen immediately a considerable number of hyphae — probably the most oldest — underwent autolysis. T h e surviving hyphae increased their frequency of cross wall formation, which generally is an indicator of retardation of growth. B u t the lysing cells delivered nutrients, which allowed resumption of growth, which mainly occurred by outgrowth of branches. T h e continuation of growth after nitrogen limitation was also documented by proving synthesis of D N A . F o r several hours after the shift down 3 H-Thymidine was found to be incorporated specifically into the D N A of the mycelia ( S T R O H B A C H unpubl.). T h e newly formed branches initially did not show cross walls. B u t at about the 4 t h hour, when the nutrients available were obviously exhausted, the frequency of septation increased and sporogenesis was initiated. T h e formation of emergences at about the 4 . 5 t h hour was the first sign of initiation of sporogenesis, since they were never formed by non sporulating hyphae. T h e spore septum always appeared a t the tip of the emergence (Fig. 2). T h e forespores were completed in average a t the 6 t h hour and most spores had attained refractility b y t h e 8 . 5 t h hour after the shift. Concerning morphology as well as kinetics of differentiation T. vulgaris G 4 cultivated in the liquid medium behaved similarly as if developing on CD-agar ( K R E T S C H M E R 1978a), and to strain 1227 also cultivated on CD-agar ( K R E T S C H M E R 1982). Consequently, the life cycle observed in liquid medium was the SSM-cycle. Regarding the complex differentiation behaviour of T. vulgaris, it finally has turned out that 3 phenotypically different mycelia m a y occur. F o r comparison main traits of each were listed within T a b l e 1. Discussion I n differentiating actinomycetes, according to the appearance 2 kinds of mycelia are distinguished, the substrate and the aerial mycelium (AM). These terms are used syn-

Fig. 1. E a r l y developmental stages of a liquid culture of T. vulgaris G4 after a shift to nitrogen starvation. A t the indicated times after t h e shift (hours) samples were t a k e n for embedding. Bar = 0.5 ¡¿m

96

SIGRID K R E T S C H M E R

Fig. 2. Later development stages of a liquid culture of T. vulgaris after a shift to nitrogen starvation. At the indicated times after the shift (hours) samples were taken for embedding. Bar = 0.5 [im

Alternative life cycles in T. vulgaris

A 1

B 1

97

C 1

D i

E 1

Hours after shift down Fig. 3. Kinetics of cross wall formation of T. vulgaris after transition to nitrogen starvation. With each sample of the submerse culture form at least 60 longitudinal sections the average distance between neighbouring cross walls was determined. Morphological characteristics of the samples: A: about 3 0 % of the hyphae were destructed, the remaining intact hyphae often bear short branches. B : intact hyphae prevailed. C: hyphae were multiseptated and often showed emergences. D: different stages of forespore development were seen within the emergences. E : the spores had attained refractility.

onymous with the terms: undifferentiated, primary mycelium (PM) and secondary mycelium (SM) which is able to sporulate (KALAKOUTSKII 1976). This classification has also been applied to T. vulgaris. The genus Thermoactinomyces has been described to form PM and SM, both bearing single spores (BALDACCI and Locci 1966, Locci 1971). According to our results this description is incorrect, since PM is not sporogenic. Instead, there are 2 phenotypically different kinds of sporogenic, secondary mycelium. Besides the AM in T. vulgaris the SSM is formed, which is clearly distinguishable from PM by altered gene expression (Table 1), taking place some hours before onset of sporulation. The occurrence of 2 kinds of secondary mycelia indicates that at the end of the primary growth the metabolism may alternatively switch to 2 different pathways (Fig. 4). In the case of AM-formation metabolism directly turns to sporogenesis. In the other case growth is stopped only reversibly, and the shift down initiates alteration of gene expression to overcome deprivations, for example by production of extracellular proteases (KRETSCHMER et al. 1982). The mycelium growing thereafter is the SSM. The decision, which one of the sporogenic mycelia is formed, depends on the circumstances under which primary growth ceases. AM developed densely and sporulated well on CSL-agar (pH 7.2), while SSM prevailed on CD-agar (pH 6.2) where the also present AM did not sporulate (KRETSCHMER 1982,1984). Because of the complex composition of the CSL-agar it is difficult to decide which limiting substance may be responsible for AM-formation. A candidate may be soluble phosphor, since CSL-agar contains only small amounts in contrast to the CD-medium which contains 0 . 4 % soluble phosphate. The formation of SSM is dependent on nitrogen starvation, as is shown with liquid culture as well as with CD-agar, which contains only 0 . 3 % nitrogenous compounds. Comparison of the life sequences which alternatively may run in T. vulgaris shows that they partly consists of identical steps (Fig. 4). Functional similarity is exhibited during sporogenesis, and seems to be present already 1 hour before initiation of sporulation. Sporulating AM differs from sporulating SSM only by its hydrophobic cell 7

Z. allg. M i k r o b i o l . , B d . 2 4 , H . 2

98

SIGRID K R E T S C H M E R

Table 1. Comparative characterization of the 3 phenotypically different mycelia occurring in T. vulgaris PM (primary mycelium)

SSM (secondary substrate mycelium)

AM (aerial mycelium)

occurrence

on solid and in liquid medium

on solid and in liquid medium

on solid medium facing air

initiation

by outgrowth of spores or a shift up

after overcoming a lag phase induced by substrate limitation 1 ), 3)

directly after limitation of nutrients 4 )

kinetics of growth

exponential2)

linear1)

transition from exponential to stationary growth phase4)

width of hyphae

0.3—0.4 (im

0.3—0.4 |im

0.3—0.6 (im

average frequency of cross walls

1/10 (im

gradually decreasing to 1/0.8 (im 1/0.75 [im

cell wall surface

normal

normal

hydrophobic in wild type strains

frequency of branching

1/20 - 4 0 (im1), 2)

1/6.7 (im1)

low, probably similar to PM 4 )

—

-

no

after termination of secondary growth

shortly after termination of primary growth

within emergences of the hyphal cells

mostly within emergence-less cells

tendency to cell seperation initiation of sporulation

site of endospore formation

+

production of bacteriophages upon infection

+3), 4)

-3)

-4)

formation of prophage carrier spores

- 3 ), 4)

+3)

+ 4)

expression of competence

- 3 ), 4)

+3)

-4)

—

+5)

?

production of extracellular proteases 1) 2)

KRETSCHMER 1 9 7 8 a KRETSCHMER 1 9 7 8 B

3) 4)

KRETSCHMER 1 9 8 2 KRETSCHMER 1 9 8 4

6)

K R E T S C H M E R et al.

1982

Alternative life cycles in T.

AM

I

1

1

if

2

3

I

I

1

J/.

U

pM

SSM

0

99

vulgaris

1

L 1

6

J

1

8

1

1

10

1

1

12

1

1

U

Hours of incubation Fig. 4. Comparison of the two alternative life cycles observed with T. vulgaris. events the following stages are correlated: Stage

germination primary growth lag of growth secondary growth septation formation of forespores maturation of the spores

According to similar

AM-cycle

SSM-cycle

(KRETSCHMEE 1 9 8 4 )

(KRETSCHMER 1 9 8 2 )

1 2

I II L III IV V VI

3 4 5

wall and the lack of emergences ( K R E T S C H M E E 1 9 8 4 ) . Concerning kinetics of the life cycle as well as morphology of sporulation AM resembles bacilli. On the contrary, the SSMlife cycle is more complex, since it consists of primary growth, secondary growth and sporulation. As was observed earlier, the growing SSM of T. vulgaris 1261 expresses competence for genetic transformation ( K R E T S C H M E R 1982). I n Bacillus subtilis 168 the competent state appears during a specific, resting presporulation stage ( K R E T S C H M E R 1973). With both organisms competence is expressed within a stage intermediate between vegetative growth and sporulation. Initiation of sporogenesis destroys competence of both. According to the similarity of the mode of endospore formation with both organism an evolutionary relationship is assumed. Then, in an evolutionary aspect, the competent presporulation stage of B. subtilis 168 may be a precursor of the growing SSM of T. vulgaris. Acknowledgement The skillful technical assistance of Mrs.

GERDA ELSKE

is gratefully acknowledged.

References BALDACCI, E. and Locci, R., 1966. A tentative arragement of the genera in Actinomyeetales. Giorn. Microbiol., 14, 131 — 139. KALAKOUTSKII, L. V. and AGRE, N. S., 1976. Comparative aspects of development and differentiation in Actinomycetes. Bacteriol. Rev., 40, 469—524. 7*

100

SIGRID KRETSCHMER

E., R Y T E B , A. and S E C H A U D , J . , 1 9 5 8 . Electron microscope study of DNAcontaining plasm. I I . Vegetative and phage DNA as compared with normal bacterial nucleoids in different physiological states. J . Biophys. Biochem. Cytol., 4, 671—678. KRETSCHMER, S., 1973. Sporenbildung in wachsenden Bacillus suitilis-Kalturen und ihre Korrelation zur Kompetenz für genetische Transformation. Z. allg. Mikrobiol., 12, 221—232. KRETSCHMER, S., 1978 a. Transition of Thermoactinomyces substrate mycelium from growth to sporulation. Z. allg. Mikrobiol., 18, 6 1 3 — 6 1 6 . KRETSCHMER, S., 1978 b. Kinetics of vegetative growth of Thermoactinomyces vulgaris. Z. allg. Mikrobiol., 18, 7 0 1 - 7 1 1 . KRETSCHMER, S., 1982. Alteration of interaction with virulent bacteriophage T a l during differentiation of Thermoactinomyces vulgaris. Z. allg. Mikrobiol., 22, 6 2 9 — 6 3 7 . KRETSCHMER, S., 1984. Characterization of aerial mycelium of Thermoactinomyces vulgaris. Z. allg. Mikrobiol., 2 4 , 1 0 1 - 1 1 1 . KELLENBERGER,

K R E T S C H M E R , S . , K Ö R N E R , D . , STROHBACH, G . , K L I N G E N B E R G , P . , JACOB, H . - E . , G U M P E R T , J . a n d R U T T L O F F , H . , 1 9 8 2 . Physiologische und zellbiologische Charakterisierung des Proteasebildners

Thermoactinomyces vulgaris während langzeitiger Kultivierung im Rührfermentor. Z. allg. Mikrobiol., 22, 6 9 3 - 7 0 3 . Locci, R., 1971. On the spore formation process in Actinomycetes. IV. Examination by scanning electron microscopy of the genera Thermoactinomyces, Actinobifida and Thermomonospora. Rev. P a t . Veg., 4 (Suppl.), 6 3 - 8 0 . STROHBACH, G. und KRETSCHMER, S., 1977. Einbau von Thymidin in die DNS von Actinomyceten. I. Der Einbau exogenen Thymidinsin die DNS von Thermoactinomyces vulgaris. Z. allg. Mikrobiol., 17, 5 5 9 - 5 6 8 .

Mailing address: Dr. S. KRETSCHMER, Zentralinstitut f ü r Mikrobiologie und experimentelle Therapie der AdW, D D R 6900 Jena, Beutenbergstr. 11

Zeitschrift f ü r allgemeine Mikrobiologie 24 (1984) 2, 1 0 1 - 1 1 1

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

Chaiacterization of aerial mycelium of Thermoactinomyces

vulgaris

SIGRID K R E T S C H M E R

(Eingegangen am 26. 4. 1983) After growth of primary mycelium (PM) for 6.5 hours on CSL-agar with hydrophobic aerial mycelium (AM) appeared. The development of AM of the Thermoactinomyces vulgaris strains 1227 and 1261 was studied by light and electron microscope. Immediately after its appearance the AM was represented by long unbranched hyphae, consisting of cells about 1 um in length. After 2.5 hours within most of them a forespore has been developed. Formation of hyphal emergences as observed with secondary substrate mycelium (SSM) did not precede forespore formation in AM. The developing spores attained refractility when the AM was 5 hours old. Since a prolonged period of AM-growth was not observed and sporulation was initiated already 1 hour after appearance of the AM it must be concluded that AM is formed by conversion of PM. Obviously, this conversion is induced by a nutritional shift down and takes place during residual growth while the culture passes to the stationary growth phase. When subsequent stages of the AM-life cycle of T. vulgaris were infected with the virulent bacteriophage T a l , only the PM was found to propagate the phage. In infected AM the sporulation went on unaffectedly. Infection during the first 2 hours of AM-development resulted in the formation of prophage-carrier spores. Afterwards, when the forespores were completed, infection of AM was abortive. Competence for genetic transformation — found to be expressed in SSM — was absent in AM. From the strains 1227 and 1261 bald mutants were isolated. Under conditions favouring AMformation morphogenesis as well as mode of interaction with phage T a l and transforming DNA was similar to the parent strains. Their AM only failed to develop the hydrophobic appearance and had a lower frequency of sporulation. W h e n Thermoactinomyces vulgaris was c u l t i v a t e d o n different agar m e d i a w e o b s e r v e d t h a t at the e n d of the primary growth 2 kinds of secondary, sporogenic m y c e l i a appeared. U s i n g conditions favourable for genetic transformation (HOPWOOD a n d WRIGHT 1972) aerial m y c e l i u m (AM) as well as secondary substrate m y c e l i u m (SSM) were formed, but o n l y the latter sporulated (KRETSCHMER 1982). Manner as well as kinetics of d i f f e r e n t i a t i o n of SSM o n the CD-agar m e d i u m h a v e b e e n a n a l y z e d (KRETSCHMER 1 9 7 8 a , 1982). H i t h e r t o the d e v e l o p m e n t of AM has not b e e n studied. I t is o n l y k n o w n that it is able to form endospores (MACH a n d FUTTERLIEB 1966, CROSS 1968, L o c c i 1971, HENSSEN et al. 1981). B u t the q u e s t i o n is still o p e n w e t h e r b o t h sporogenic m y c e l i a of T. vulgaris b e h a v e similarly w i t h respect to kinetics and morp h o l o g y of differentiation. T h e a i m of this paper is to a n a l y z e the d e v e l o p m e n t of AM under appropriate c o n d i t i o n s a n d to describe its m a i n features.

Materials and methods Bacterial and bacteriophage strains: The strains used were Thermoactinomyces vulgaris 1227 and its m u t a n t derivative 1261 nic-1 str-1 thi-3 (both kindly supplied by D. A. HOPWOOD). From these strains spontaneously appearing bald mutants were isolated, called 1227 bid and 1261 bid. In a previous paper the strain 1261 bid was called 1261 amy- (KRETSCHMER 1982). But later we got aware t h a t this m u t a n t is able to form AM, which only lacks the white, hydrophobic appearance.

Zeitschrift f ü r allgemeine Mikrobiologie 24 (1984) 2, 1 0 1 - 1 1 1

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

Chaiacterization of aerial mycelium of Thermoactinomyces

vulgaris

SIGRID K R E T S C H M E R

(Eingegangen am 26. 4. 1983) After growth of primary mycelium (PM) for 6.5 hours on CSL-agar with hydrophobic aerial mycelium (AM) appeared. The development of AM of the Thermoactinomyces vulgaris strains 1227 and 1261 was studied by light and electron microscope. Immediately after its appearance the AM was represented by long unbranched hyphae, consisting of cells about 1 um in length. After 2.5 hours within most of them a forespore has been developed. Formation of hyphal emergences as observed with secondary substrate mycelium (SSM) did not precede forespore formation in AM. The developing spores attained refractility when the AM was 5 hours old. Since a prolonged period of AM-growth was not observed and sporulation was initiated already 1 hour after appearance of the AM it must be concluded that AM is formed by conversion of PM. Obviously, this conversion is induced by a nutritional shift down and takes place during residual growth while the culture passes to the stationary growth phase. When subsequent stages of the AM-life cycle of T. vulgaris were infected with the virulent bacteriophage T a l , only the PM was found to propagate the phage. In infected AM the sporulation went on unaffectedly. Infection during the first 2 hours of AM-development resulted in the formation of prophage-carrier spores. Afterwards, when the forespores were completed, infection of AM was abortive. Competence for genetic transformation — found to be expressed in SSM — was absent in AM. From the strains 1227 and 1261 bald mutants were isolated. Under conditions favouring AMformation morphogenesis as well as mode of interaction with phage T a l and transforming DNA was similar to the parent strains. Their AM only failed to develop the hydrophobic appearance and had a lower frequency of sporulation. W h e n Thermoactinomyces vulgaris was c u l t i v a t e d o n different agar m e d i a w e o b s e r v e d t h a t at the e n d of the primary growth 2 kinds of secondary, sporogenic m y c e l i a appeared. U s i n g conditions favourable for genetic transformation (HOPWOOD a n d WRIGHT 1972) aerial m y c e l i u m (AM) as well as secondary substrate m y c e l i u m (SSM) were formed, but o n l y the latter sporulated (KRETSCHMER 1982). Manner as well as kinetics of d i f f e r e n t i a t i o n of SSM o n the CD-agar m e d i u m h a v e b e e n a n a l y z e d (KRETSCHMER 1 9 7 8 a , 1982). H i t h e r t o the d e v e l o p m e n t of AM has not b e e n studied. I t is o n l y k n o w n that it is able to form endospores (MACH a n d FUTTERLIEB 1966, CROSS 1968, L o c c i 1971, HENSSEN et al. 1981). B u t the q u e s t i o n is still o p e n w e t h e r b o t h sporogenic m y c e l i a of T. vulgaris b e h a v e similarly w i t h respect to kinetics and morp h o l o g y of differentiation. T h e a i m of this paper is to a n a l y z e the d e v e l o p m e n t of AM under appropriate c o n d i t i o n s a n d to describe its m a i n features.

Materials and methods Bacterial and bacteriophage strains: The strains used were Thermoactinomyces vulgaris 1227 and its m u t a n t derivative 1261 nic-1 str-1 thi-3 (both kindly supplied by D. A. HOPWOOD). From these strains spontaneously appearing bald mutants were isolated, called 1227 bid and 1261 bid. In a previous paper the strain 1261 bid was called 1261 amy- (KRETSCHMER 1982). But later we got aware t h a t this m u t a n t is able to form AM, which only lacks the white, hydrophobic appearance.

102

SIGRID KRETSCHMER

Therefore, the term amy- is replaced by the term bid. The 4 strains mentioned are competent for genetic transformation. The virulent bacteriophage employed was phage T a l which along with its DNA has been described by S A K F E R T et al. (1979). There the methods for production of phage lysates (titre about 1 • 1010 phages/ml) are also mentioned. Media and experimental conditions: Methods for production, storage and activation of T. vulgaris spores, which always were used as inoculum, were described by S T R O H B A C H and K R E T SCHMER (1977). The complete development and behaviour of AM was studied during cultivation on CSL-agar. The CSL-agar consisted of: corn step liquor (50% dry weight) 0.5%, casamino acids vitamin free (Dirco) 0.3%, sucrose 1.5%, corn starch 0.5%, N a N 0 3 0.1%, NaCl 0.25%, KC1 0.0025%, CaCl2 0.025%, FeS0 4 • 7 H 2 0 0.0005%, magnesium glycerophosphate 0.025%, agar 2%, aqua dest. The p H was adjusted to 7.2. Incomplete differentiation of AM occurred on CDagar, p H 6.2 (KRETSCHMER 1982). Incubation temperature was 52 °C. The development of AM was followed either by removing samples from agar cultures or directly with microcultures. To maintain lucidness of the microcolonies it was necessary to dilute the CSL-agar 1:4 with water-agar, otherwise a dense layer of primary mycelium (PM) would appear before AM-formation is initiated. The preparation fo the microculture has been described earlier ( K R E T S C H M E R 1978b). For taking series of fotographs a site was chosen where between agar and cover slip there was a layer of air. The AM was discernible from the PM resp. SSM by bright appearance, if oblique lighting was used. For electron microscopical studies mycelium was scraped from the CSL-agar plates, fixed for 18 hours in glutaraldehyde and embedded (KELLENBERGER et al. 1958). Ultrathin sections of hyphae were studied with the electron microscope Type SEM 3—2 (VEB Werk fur Fernsehelektronik, Berlin-Oberschoneweide). As similarly was done with SSM (KRETSCHMER 1982) the abilities of AM to produce phages upon infection and to undergo genetic transformation were tested. At different developmental stages either 0.1 ml suspension of bacteriophage T a l or 25 ;xg 1227 DNA were spread onto plates. The transforming DNA was prepared by E. S A R F E R T according to M A R M U R ( 1 9 6 1 ) . The transformation process was stopped after 1 hour by spreading 20 jig DNase/plate. -The spores were harvested about 40 hours later and tested for the frequency of transformation of the raic-marker. In the case of treatment with phage T a l the degree of mycelial lysis caused by phage production and the frequency of formation of prophage-carrier spores (phage trapping) were determined. The latter were represented by spores which gave rise to plaques upon outgrowth. The spores have been harvested after about 40 hours of incubation and were heated for 15 min at 90 °C before testing to destroy free phages.

Results Morphogenesis

of aerial mycelium

(AM)

AM was only formed where mycelium extended into air. This fact makes direct microscopical observation difficult. Nevertheless, conditions have been found which allowed selective observation of the AM (Fig. 1). At the 7thhour of incubation chains of bright spots appeared simultaneously on primary hyphae. They either grew out to short branches or merged, thus giving rise to longer AM-hyphae, which soon break into rods. In the microculture about 1.5 hours after initiation of AM-formation its development ceased. Prolonged growth and sporulation of AM was not observed. Lack of sporulation of AM was also observed during growth on CD-agar plates, where AM appeared 6.5 hours after inoculation. Independend of age this nonsporulating AM was heterogenous with respect to the frequency of septation. The aerial hyphae consisted of 1 to several ¡xm long cells, which tended to separate (Fig. 2 A-C). Well sporulating AM was obtained if development occurred on CSL-agar plates. Macroscopically, the white hydrophobic AM appeared on the PM at the 6.5 th hour of incubation. One hour later the agar was covered by a dense layer of AM. Microscopically several steps of morphological development could be distinguished (Fig. 3). At the time when AM appeared the rarely septated primary hyphae were replaced by hyphae being multiply septated. These hyphae were straight, unbranched and consisted of about 1 ¡xm long cells. 2.5 hours after initiation of AM-formation within

103

Characterization of aerial mycelium of T. vulgaris

most cells a forespore became visible. The forespore was formed within the cylindricalshaped cells and remained there until maturation of the spore (Fig. 4). Refractility of the spores was attained 11.5 hours after inoculation. After initiation of sporogenesis AM becomes very sensitive to handling and lysed quickly when placed under a cover glass. Therefore, the AM had to be fixed before observation by submerging in a solution of 1% formaldehyde. The activity of cell wall lytic enzymes in sporulating AM was also evidenced by the curling of the hyphae and their tendency to separate into single cells. The life cycle of the bald mutants of the strains 1227 and 1261 was studied too. With respect to kinetics as well as morphology of differentiation no major differences were observed. But mostly the frequency of sporulation was reduced, sometimes to about 10% (Fig. 2D). Interaction of differentiating AM with bacteriophage Tal and transforming

DNA

In the course of differentiation on CSL-agar plates 3 modes of interaction with the virulent phage T a l were observed (Fig. 5 a and b). Until appearance of AM the lawn of PM became lysed upon phage production. Afterwards, the mycelium attained resistance against phage infection and sporulation went on unaffectedly. When the AM was older than 2.5 hours the infection was abortive. But when phages were spread during the first 2.5 hours of AM-development the prophage became locked into the future spore in a heat-resistant state. Upon outgrowth of these prophage-carrier spores progeny phages were released. The peak of formation of carrier spores, i.e. of phage trapping occurred 1 hour after initiation of AM-formation, at the 7.5 th hour of incubation. With strains 1261 bid often a smaller second peak was found between the 11 th and 12 th hour. The frequency of formation of carrier spores was dependent on the titre of the phage suspension used. The pattern of interaction with phage T a l was similar within the 4 strains used, except that with strain 1227 the frequency of phage trapping was 10 times less than with the others (Fig. 5a and b). In order to study the expression of competence for genetic transformation during differentiation on CSL-agar the mycelia of strains 1261 and 1261 bid were exposed for hourly intervals to 1227-DNA. A small peak of competence appeared with both strains 2 hours after initiation of AM-formation (Fig. 5a and b).

Discussion Concerning the mode of formation of AM it seems to be proven that aerial hyphae arise and grow away from primary hyphae by branching (Locci 1971, Cross and Unsw o r t h 1977). Our direct observation of microcolonies seems — at least partly — to support this assumption. But the appearance of only short, non-sporulating aerial hyphae indicates that the conditions of the microculture are unfavourable for the development of AM. But in the series of microfotographs (Fig. 1) also another mode of AM-formation can be observed, which consists in a conversion of primary hyphae to aerial hyphae by alteration of cell wall composition. We assume that the dense AM on CSL-agar plates is formed by hyphal conversion. This assumption is mainly based on the facts that branching was observed only seldom and that sporulation in the AM started already 1 hour after its appearance. At the time when AM appears the hyphae are multiply septated. The increase of cross wall formation generally is an indicator of retardation of growth. From the kinetics of differentiation of AM it can

104

SIGRID RRETSCHMER

Fig. 1. Development of aerial mycelium by a microcolony of T. vulgaris. Diluted CSL-agar was inoculated with 1227-spores. Pictures were t a k e n a t intervals of 30 min f r o m t h e 7 t h t o t h e 8.5 t h hour of incubation (1—4). Oblique lighting, magnification 160:1. F o r b e t t e r orientation 3 withe m a r k s were placed above aereas of well visibly development of aerial h y p h a e

Characterization of aerial mycelium of T. vulgaris

105

106

SIGRID KRETSCHMER

Fig. 2. Morphology of non and poorly sporulating aerial mycelium of T. vulgaris. Strain 1261 was grown on CD-agar plates for 11 (A), 15 (B) and 11 (C) hours. Strain 1227 bid was grown on CSL-agar plates for 12 hours (D)

Characterization of aerial mycelium of T. vulgaris

107

Fig. 3. Main developmental stages of T. vulgaris 1227 during 6. —11. hour of incubation on CSL-agar plates. Phase contrast microscopy of samples removed from the mycelial lawn. The pictures represent following stages: 6: primary mycelium, 7.5: septated AM, 9: septated AM at different stages of forespore formation. 11: forespores are mostly matured to refractile endospores. Bar = 10 ¡xm

108

SIGRID KRETSCHMER

F i g . 4. I n f r a s t r u c t u r e o f spore f o r m a t i o n in AM o f T. vulgaris

1261. S a m p l e s were t a k e n a f t e r i n c u b a t i o n

on C S L - a g a r plates for 8 . 5 (A), 9 ( B ) , 11 (C) a n d 1 1 . 5 (D) hours. I n A a forespore is seen within t h e u p p e r m o s t cell. B a r = 0 . 5 ¡xm

be concluded that on one hand limitation of nutrients induces the hyphal conversion, and on the other hand, the change to hydrophobic appearance itself prevents further uptake of nutrients, thus favouring initiation of sporogenesis. The latter fact would explain why in the bald mutants the frequency of sporulation is largely reduced (Fig. 2D). In contrast to the AM, where a prolonged period of growth was not observed, the formation of SSM by branched outgrowth of secondary, sporogenic hyphae from P M has been observed directly ( K R E T S C H M E R 1978a). There, secondary growth lasted about 3.5 hours, before sporulation was initiated. The similarity of development of the parent strains and the bald mutants indicates that the latter also form AM, which is clearly distinguishable from SSM. That means, the alteration of the composition of the cell wall is not the main indicator for AM. A detailed comparison of the 3 phenotypically different mycelia observed with T. vulgaris will be given separately ( K R E T S C H M E R 1 9 8 4 ) . Hence sporulation is initiated the differentiation seems to run similarly within AM and SSM, except that in SSM emergences are formed which are the sites for spore formation ( K R E T S C H M E R 1 9 8 4 ) . Studying the interaction with bacteriophages and transforming DNA has proved to be a suitable way to characterize developmental stages of T. vulgaris beyond morphological description ( K R E T S C H M E R 1 9 8 2 . ) . As with SSM the aquaintance of resistance against phage infection indicates the moment when the primary growth ceases. The AM behaves similarly to sporulating SSM and bacilli. There, as long as the forespore has not been completed the injection of phage-DNA results in the formation of prophage-carrier spores ( G O L D B E R G and GOLLAKOTA 1 9 6 1 , TAKAHASHI 1 9 6 4 , Y E H L E and Doi 1 9 6 7 , K R E T S C H M E R 1 9 8 2 ) . Afterwards, the infection with phages is abortive, since the phage DNA is not able to enter the forespore.

109

Characterization of aerial mycelium of T. vulgaris

(V'o) ßuiddajj

aßoqdp

dsaj (x) uoyom/a/suajf jo Aouanbajj

*

•2 S s g

bcä 13 TO^ 1QJ ~ bo ® C3 ( g Js 60 cä 5 1 "ft (») uo/pnpojd eßoqd Aq pssnoj

sisAi

(v 'oj ßuiddoj) dßüqd jo dsaj(x) uoijDLujopuojf. jo

Aoudnbajj

•S j •tí o

cS ®

-+-

-+-

ft . X e 'S tí 3 "2 ,0 8 3 . o cä 60 cä ft O cä h ft tí o ®

BR322)

A, B, C: A B 2463 ( p B R 3 2 2 ) , D, E , F : A B 1157 ( p B R 3 2 2 ) , A, D : 2 (ig/ml Tc, B, E : 10 (ig/ml Tc, C: 70 ¡ig/ml Tc, F : 170 fxg/ml Tc

zu überprüfen, sind Stämme mit definierten Oligomeren konstruiert worden. Aus C600 recBC (andere recA+-Stämme sind ebenso geeignet) wurde die Plasmid-DNS isoliert, ohne daß dieser Stamm zuvor höheren Tc-Konzentrationen als 10 [ig/ml ausgesetzt worden ist. Nach elektrophoretischer Auftrennung sind die Oligomere (Monobis Tetramer) aus dem Agarosegel elektrophoretisch eluiert und in die recA "-Mutante H B 101 transformiert worden. Plasmid-Oligomere können in recA-Mutanten relativ stabil erhalten werden (vgl. P O T T E B U. D K E S S L E R 1 9 7 7 ) . In den vier H B 101-Transformanten, die die Mono-, Di-, Tri- oder Tetramere von pBR322 enthalten, sind gleiche Plasmid-DNS-Mengen bestimmt worden. Darüber hinaus erreicht die /?-Lactamaseaktivität, die sich proportional zur Gendosis verhält (UHLIN U. NORDSTEÖM 1 9 7 7 ) , bei den untersuchten Stämmen ein gleiches Niveau (Abb. 4). Ebenso zeigten sich keine deutlichen Unterschiede in der Tc-Resistenz (Abb. 5), so daß sich der PlasmidDNS-Gehalt mit zunehmender Oligomerisierung nicht erhöht.

Abb. 4. Relativer Plasmidgehalt • und /J-Lactamaseaktivität bei H B 101 ( p B R 3 2 2 , Mono-, Tri- bzw. Tetramer) in der späten logarithmischen Wachstumsphase im M9-Medium. Die angegebenen Beträge sind Mittelwerte aus jeweils 4 voneinander unabhängigen Bestimmungen. Die Schwankungsbreiten sind über jeder Säule angegeben

Bildung von Oligomeren des Plasmids pBR322

Abb. 5. Tc-Resistenz von HB 101 (pBR322, bei Abb. 2

123

Mono-, Di-, Tri- bzw. Tetramer) Versuchsbedingungen wie

Diskussion Zirkuläre Plasmidoligomere, bei denen die Monomereinheiten in Tandem-Stellung kovalent miteinander verbunden sind, werden bei verschiednenen E. coii-Plasmiden (GOEBEL U. HELLNSKI 1968, HELINSKI u . CLEWELL 1971, LURQUIN U. KADO 1977)

und in Bacillus subtilis (CANOSI et dl. 1978) gefunden. Die genetischen Voraussetzungen, die für die Bildung und die Auflösung von Plasmidoligomeren in der E. coli-Zelle notwendig sind, wurden in den letzten Jahren intensiv untersucht, wobei gezeigt wurde, daß das recA-Genprodukt entscheidend beteiligt ist (POTTER U. DRESSLER 1977, FISHEL et al. 1981, JAMES et al. 1982).

Weitaus weniger gut bekannt ist, welche äußeren Einflüsse den Anteil unterschiedlicher Plasmid-Oligomere an der gesamten Plasmid-DNS bestimmen. In der vorliegenden Arbeit wurde gezeigt, daß mit steigenden Tc-Konzentrationen größere Oligomere (bis zu Tetramere) des Plasmids pBR322 dominant werden, während die Anteile der Mono-, Di- und z.T. auch derTrimere unter die Nachweisgrenze der Agarosegelelektrophorese sinken (WIEHLE 1983). RecA~-Mutanten können unter gleichen Bedingungen keine Oligomere bilden, weshalb wir einen Zusammenhang mit der geringeren Tc-Resistenz (vgl. Abb. 2) vermuteten. BASTLN (1981) hatte rekombinante Derivate von pBR322, die Insertionen im Promoterbereich des Tc r -Gens trugen, in XI776 (recA+) eingeführt und die nur schwach Tc-resistenten Transformanten mit Tcselektiert. Nach Isolation und erneuter Transformation der Plasmid-DNS in HB 101 fand BASTIN mehr als 5 0 % Transformanten, die Oligomere (bis Hexamere) enthielten und eine höhere Tc-Resistenz zeigten. Die Ergebnisse unserer Vorversuche sowie die von BASTIN (1981) ließen die Vermutung zu, daß Zellen mit Plasmid-Oligomeren einen insgesamt höheren Plasmid-DNS-Gehalt aufweisen. Diese Fragestellung wurde 9*

124

W . WIEHLE, M . HECKER, B . REICHSTEIN u n d F . MACH

durch Transformation von H B 101 mit Mono-, Di-,Tri- sowie Tetrameren überprüft. Dabei zeigten die Klone des Stammes H B 101 mit definierten Oligomeren (Monobis Tetramer) von pBR322 mit zunehmender Oligomerisierung weder einen signifikant höheren Plasmid-DNS-Gehalt noch eine Zunahme von /5-Lactamaseaktivität und Tc-Resistenz. Warum sich bei hohen Tc-Konzentrationen dennoch Zellen mit Oligomeren anreichern, sollte Gegenstand weiterer Untersuchungen sein.

Literatur BASTIN, M., 1981. Molecular cloning in plasmid pBR322 giving altered expression of the tetracyclin resistance gene. J. Gen. Microbiol., 123, 187 — 191. BIRNBOIM, H. C. and DOLY, J., 1980. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res., 7, 1513 — 1523. BOLIVAR, F . ,

RODRIGUEZ, R . L . ,

GREENE, P . J . ,

BETLACH, M . C.,

HEYNECKER, H . L . ,

BOYER,

H. W., CROSA, J . H. and FALKOW, S., 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene, 2, 95 — 113. BOYER, H. W. and ROULLAND-DCSSOIX, R., 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol., 41, 459—472. CANOSI, U., MORELLI, G. and TRAUTXER, A. T., 1978. The relationship between molecular structure and transformation efficiencv of some Staphylococcus aureus plasmids isolated form Bacillus subtilis. Mol. gen. Genet., 166, 259—267.

CORNELIS, P . , DIGNEFFE, C., WILLEMOT, K . a n d COLSON, C.,

1981. P u r i f i c a t i o n

of

Escherichia

coli amplifiable plasmids by high salt sepharose chromatography. Plasmid, 5, 221—223. ECKHARDT, T., 1978. A rapid method for the identification of plasmid DNA in bacteria. Plasmid I, 584-588. FISHEL, R. A., JAMES, A. A. and KOLODNER, R., 1981. recA-independent general genetic recombination of plasmids. Nature, 294, 184—186. GOEBEL, W. and HELINSKI, D. R., 1968. Generation of higher multiple circular DNA forms in bacteria. Proc. Natl. Acad. Sci. USA, 61, 1406-1413. HELINSKI, D . R . a n d CLEWELL, D . B., 1971. Circular D N A . A n n . R e v . Biochem., 40, 899—942.

HINTERMANN, G . , FISCHER, H . - M . , CRAMERI, R . a n d

HTTTTER, R . ,

1981. S i m p l e p r o c e d u r e

for

distinguishing CCC,OC and L-forms of plasmid DNA by agarose gel electrophoresis. Plasmid, 5, 371-373. HOWARD-FLANDERS, P. and THERIOT, L., 1966. Mutants of Escherichia coli K12 defective in DNA repair and in genetic recombination. Genetics, 53, 1137 — 1150.

JAMES, A. A., MORRISON, P . T. a n d KOLODNER, R . , 1982. Genetic r e c o m b i n a t i o n of bacterial

plasmid DNA. J . Biol., 160, 411—430. KLEINSCHMIDT, A. K., 1968. Monolayer techniques in electron microscopy of nucleic acids molecules Methods in Enzymology, 12 B, 361—377. LURQUIN, P. F. and KADO, C. I., 1977. Escherichia coli plasmid pBR313 insertion into plant protoplast and into their nuclei. Molec. gen. Genet., 154, 113 — 121.

MARKO, M. A., CHIPPERFIELD, P . a n d BIRNBOIM, H . C., 1982. A procedure for t h e large scale

isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal. Biochem., 121, 382—387. MILLER, J . H., 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, p. 431. PERRET, C. J., 1954. Jodometric assay of penicillinase. Nature (London), 174, 1012 — 1013. POTTER, H. and DRESSLER, D., 1977. On the mechanism of genetic recombination: The maturation of recombination intermediates. Proc. Natl. Acad. Sci., USA, 74, 4168—4172. UHLIN, B. E. and NORDSTRÖM, K., 1977. R-plasmid gene dosage effects in Escherichia coli K12: copy mutants of the if-plasmid Rldrdl9. Plasmid, 1, 1—7. WIEHLE, W., HECKER, M. u n d MACH, F . , 1982. U n t e r s u c h u n g e n zur E r h a l t u n g des Plasmids

pBR322 in diskontinuierlicher Kultur von Escherichia coli K12 C600. Z. allg. Mikrobiol., 22, 273-277. WIEHLE, W., 1983. Untersuchungen zur Replikation und Expression des Plasmids pBR327 in verschiedenen Escherichia coZi-Stämmen. Dissertation A, Greifswald. Anschrift: Prof. Dr. F. MACH Sektion Biologie der Ernst-Moritz-Arndt-Universität, DDR 2200 Greifswald, Jahnstr. 15

Z e i t s c h r i f t f ü r allgemeine Mikrobiologie 24 (1984) 2, 1 2 5 - 1 2 7

Kurze

Originalmitteilung

(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 u r Molekularbiologie u n d Medizin, Z e n t r a l i n s t i t u t f u r Mikrobiologie u n d experimentelle Therapie, J e n a , D i r e k t o r : Prof. Dr. U . TAUBENECK)

Use of nystatin for random spore selection in the yeast Saccharomycopsis lipolytica G . BARTH a n d H .

(Eingegangen

am 2.

WEBER

9.1983)

N y s t a t i n was used t o develop a new m e t h o d to select spores of t h e yeast Saccharomycopsis lipolytica. A t low concentrations n y s t a t i n killed preferently growing cells of this yeast. A t high concent r a t i o n s nongrowing cells were affected as well. I n contrast, spores were n o t sensitive to n y s t a t i n action. This differential response t o t h e antibiotic suggested its use t o select spores f r o m s p o r u l a t e d y e a s t cultures. S e p a r a t i o n of s p o r e s f r o m v e g e t a t i v e c e l l s is a n i m p o r t a n t p r e r e q u i s i t e f o r m a n y m e t h o d s of g e n e t i c a n a l y s i s i n y e a s t . T h e r e f o r e , s e v e r a l p r o c e d u r e s h a v e b e e n d e v e l o p e d t o i s o l a t e s p o r e s f r o m Saccharomyces (Sacch.) cerevisiae ( r e v i e w s s. S H E R M A N 1 9 7 5 , M O R T I M E R a n d H A W T H O R H E 1 9 7 5 ) . S p o r e s of t h e y e a s t Saccharomycopsis (8.) lipolytica» h a v e b e e n s e p a r a t e d b y t h e p a r a f f i n oil m e t h o d ( E M E I S a n d G U T Z 1 9 5 8 , G A I L L A R D I N et al. 1 9 7 3 ) a n d c a n a v a n i n e r e s i s t a n c e m e t h o d ( S H E R M A N a n d R O M A N 1 9 6 3 , B A S S E L etal. 1 9 7 1 ) . Besides these methods both ether vapour (DAWES and H A R D I E 1 9 7 4 ) a n d a c e t o n e v a p o u r (EGEL 1 9 7 7 ) h a v e b e e n u s e d f o r t h e i s o l a t i o n of s p o r e s f r o m S. lipolytica (BARTH 1980). I n t h i s p a p e r w e d e s c r i b e a s i m p l e m e t h o d w h i c h m a k e s u s e of n y s t a t i n t o selects p o r e s f r o m S. lipolytica. Strains a n d materials: 8. lipolytica strains used t h r o u g h o u t t h e experiments h a v e been described previously (BARTH a n d W E B E R 1 9 8 3 , 1 9 8 4 ) . N y s t a t i n was obtained f r o m t h e D e p a r t m e n t of Antibiotic Chemistry of t h e Central I n s t i t u t e for Microbiology a n d E x p e r i m e n t a l T h e r a p y , J e n a . Media: Minimal m e d i u m (MMT) and complete m e d i u m (YEP-G) are described previously (BARTH a n d K U N K E L 1 9 7 9 ) . The composition of t h e sporulation m e d i u m (CSM) will b e p u b l i s h e d e l s e w h e r e (BARTH a n d WEBER 1984).

M e t h o d s : N y s t a t i n was suspended in absolute ethanol a t concentrations of 1 m g or 2 mg p e r ml. The suspension was always freshly prepared a n d immediately applied to t h e culture. E t h a n o l concentrations u p to three percent did n o t significantly affect t h e growth of cells in glucose containing m e d i u m . S p o r u l a t i o n : The m e t h o d used to induce sporulation will be described elsewhere (BARTH a n d WEBER, in preparation).

R a n d o m spore selection: Sporulated strains were suspended in Y E P - G a n d shaken for 2 h o u r s t o a c t i v a t e vegetative cells. This time was t o short to induce germination of spores. T h e p H was t h e n a d j u s t e d to 4.5 to 5.0 b y t h e addition of HC1 a n d n y s t a t i n solution was a d d e d t o t h e desired concentration. These cultures were shaken a n o t h e r 1.5 hours, centrifuged, a n d washed twice with w a t e r . Subsequently cells were resuspended in helicase solution (200 mg/7 ml a q u a dest.) a n d i n c u b a t e d for one more hour. The cells were t h e n sedimented again, washed twice w i t h w a t e r a n d resuspended in a q u a dest. A f t e r sonication (BRANSON sonicator, 5 X 1 min) suspensions w e r e spreaded on Y E P - G plates a n d i n c u b a t e d a t 28 °C. Two d a y s later Y E P - G plates were replicap l a t e d o n t o a p p r o p r i a t e minimal m e d i u m plates a n d CSM plates. Colonies of diploid' cells w e r e coloured brownish a f t e r sporulation in c o n t r a s t t o t h e white unsporulated colonies. T h e f r e q u e n c y of s u r v i v a l a f t e r n y s t a t i n t r e a t m e n t w a s e s t i m a t e d f o r s e v e r a l h a p l o i d a n d d i p l o i d s t r a i n s of S. lipolytica. Nystatin strongly inactivates growing haploid

Z e i t s c h r i f t f ü r allgemeine Mikrobiologie 24 (1984) 2, 1 2 5 - 1 2 7

Kurze

Originalmitteilung

(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 u r Molekularbiologie u n d Medizin, Z e n t r a l i n s t i t u t f u r Mikrobiologie u n d experimentelle Therapie, J e n a , D i r e k t o r : Prof. Dr. U . TAUBENECK)

Use of nystatin for random spore selection in the yeast Saccharomycopsis lipolytica G . BARTH a n d H .

(Eingegangen

am 2.

WEBER

9.1983)

N y s t a t i n was used t o develop a new m e t h o d to select spores of t h e yeast Saccharomycopsis lipolytica. A t low concentrations n y s t a t i n killed preferently growing cells of this yeast. A t high concent r a t i o n s nongrowing cells were affected as well. I n contrast, spores were n o t sensitive to n y s t a t i n action. This differential response t o t h e antibiotic suggested its use t o select spores f r o m s p o r u l a t e d y e a s t cultures. S e p a r a t i o n of s p o r e s f r o m v e g e t a t i v e c e l l s is a n i m p o r t a n t p r e r e q u i s i t e f o r m a n y m e t h o d s of g e n e t i c a n a l y s i s i n y e a s t . T h e r e f o r e , s e v e r a l p r o c e d u r e s h a v e b e e n d e v e l o p e d t o i s o l a t e s p o r e s f r o m Saccharomyces (Sacch.) cerevisiae ( r e v i e w s s. S H E R M A N 1 9 7 5 , M O R T I M E R a n d H A W T H O R H E 1 9 7 5 ) . S p o r e s of t h e y e a s t Saccharomycopsis (8.) lipolytica» h a v e b e e n s e p a r a t e d b y t h e p a r a f f i n oil m e t h o d ( E M E I S a n d G U T Z 1 9 5 8 , G A I L L A R D I N et al. 1 9 7 3 ) a n d c a n a v a n i n e r e s i s t a n c e m e t h o d ( S H E R M A N a n d R O M A N 1 9 6 3 , B A S S E L etal. 1 9 7 1 ) . Besides these methods both ether vapour (DAWES and H A R D I E 1 9 7 4 ) a n d a c e t o n e v a p o u r (EGEL 1 9 7 7 ) h a v e b e e n u s e d f o r t h e i s o l a t i o n of s p o r e s f r o m S. lipolytica (BARTH 1980). I n t h i s p a p e r w e d e s c r i b e a s i m p l e m e t h o d w h i c h m a k e s u s e of n y s t a t i n t o selects p o r e s f r o m S. lipolytica. Strains a n d materials: 8. lipolytica strains used t h r o u g h o u t t h e experiments h a v e been described previously (BARTH a n d W E B E R 1 9 8 3 , 1 9 8 4 ) . N y s t a t i n was obtained f r o m t h e D e p a r t m e n t of Antibiotic Chemistry of t h e Central I n s t i t u t e for Microbiology a n d E x p e r i m e n t a l T h e r a p y , J e n a . Media: Minimal m e d i u m (MMT) and complete m e d i u m (YEP-G) are described previously (BARTH a n d K U N K E L 1 9 7 9 ) . The composition of t h e sporulation m e d i u m (CSM) will b e p u b l i s h e d e l s e w h e r e (BARTH a n d WEBER 1984).

M e t h o d s : N y s t a t i n was suspended in absolute ethanol a t concentrations of 1 m g or 2 mg p e r ml. The suspension was always freshly prepared a n d immediately applied to t h e culture. E t h a n o l concentrations u p to three percent did n o t significantly affect t h e growth of cells in glucose containing m e d i u m . S p o r u l a t i o n : The m e t h o d used to induce sporulation will be described elsewhere (BARTH a n d WEBER, in preparation).

R a n d o m spore selection: Sporulated strains were suspended in Y E P - G a n d shaken for 2 h o u r s t o a c t i v a t e vegetative cells. This time was t o short to induce germination of spores. T h e p H was t h e n a d j u s t e d to 4.5 to 5.0 b y t h e addition of HC1 a n d n y s t a t i n solution was a d d e d t o t h e desired concentration. These cultures were shaken a n o t h e r 1.5 hours, centrifuged, a n d washed twice with w a t e r . Subsequently cells were resuspended in helicase solution (200 mg/7 ml a q u a dest.) a n d i n c u b a t e d for one more hour. The cells were t h e n sedimented again, washed twice w i t h w a t e r a n d resuspended in a q u a dest. A f t e r sonication (BRANSON sonicator, 5 X 1 min) suspensions w e r e spreaded on Y E P - G plates a n d i n c u b a t e d a t 28 °C. Two d a y s later Y E P - G plates were replicap l a t e d o n t o a p p r o p r i a t e minimal m e d i u m plates a n d CSM plates. Colonies of diploid' cells w e r e coloured brownish a f t e r sporulation in c o n t r a s t t o t h e white unsporulated colonies. T h e f r e q u e n c y of s u r v i v a l a f t e r n y s t a t i n t r e a t m e n t w a s e s t i m a t e d f o r s e v e r a l h a p l o i d a n d d i p l o i d s t r a i n s of S. lipolytica. Nystatin strongly inactivates growing haploid

126

G. BABTH and H. WEBER

Table 1 Inactivation of S. lipolytica cells by nystatin (10 ng/ml) after cultivation in nonsupplemented MMT + 1 % glucose for 6 hours a) Inactivation of growing cells of haploid (H 194, H 195, H 222) and diploid strains (Bl, B215)

b) Inactivation of nongrowing cells of auxotrophic mutant strains

Strains

Frequency of survival

Strains

Frequency of survival

H H H B B

5 8 6 8 9

H 194-15 H 195-5 H 222-20

5 X 10- 1 3 X 10" 1 3 X 10- 1

194 195 222 1 215

X X X x X

10-" 10- 4 lO"4 10- 4 10- 4

Table 2 Inactivation of nongrowing cells of S. lipolytica by different concentrations of nystatin. Non growing cells from stationary growth phase were used Survival of cells after treatment with different concentration of nystatin

H 195-5 B 157 B 215

10 (ig/ml

20 jig/ml

40 ng/ml

3 X 10" 1 6 X 10- 1 5 X 10" 1

2 x 10- 2 3 X 10~ 2 1 X 10- 2

6 X 10- 3 8 X 10~ 3 3 X 10" 3

and diploid cells of S. lipolytica (Table la) but showed little effect on nongrowing cells like auxotrophic mutants when incubated in nonsupplemented minimal medium (Table lb). Therefore, nystatin was useful for enrichment of S. lipolytica mutants

b y special procedures (GAILLABDIN et al. 1 9 7 3 , BAKTH 1 9 8 0 , BABTH a n d W E B E R 1 9 8 3 ) .

Nystatin inactivates nongrowing haploid and diploid cells of S. lipolytica stronger at higher concentrations than at lower concentrations (Table 2). These data suggested the use of nystatin to kill selectively vegetative cells in sporulated cultures in order to select ascospores of this yeast. Spores were expected not to be killed since they were protected by the ascus wall and their own thick spore wall as well as the material covering spores (WEBER 1979, BARTH and WEBER, in preparation). Therefore, the procedure for random spore selection described in the methods and materials sections was elaborated. This method has been applied to several sporulated diploid cultures. The portion of diploid cells among the survivers was easily detectable by brownish colour of sporulated colonies on CSM. In all cases tested so far no or only one to two diploid cells were observed among five thousand surviving cells per strain investigated. No discriminating effects by nystatin were observed among spore isolates using several auxotrophic markers or mating type alleles A and B (Table 3).

Our data show that nystatin may successfully be used for spore selection and for random spore analysis in S. lipolytica. SAMSONOVA and BOTTCHER (1980) used nystatin to select genetic segregants in Pichia guilliermondii strains. The authors discussed the possibilities that nystatin either induced haploidization or, what seemed more likely to us, that it selectively killed vegetative cells thus leading to the selection of spores in this yeast, too.

Selection of spores by nystatin

127

Table 3 Segregation pattern among survivied cells after nystatin treatment of two sporulated diploid strains a) Segregation pattern of auxotrophic markers Random spore type

Diploid strain

Genotype

B 108

ade, LYS+/ADE+, lys

P

= 0.25 0.80 >

P

1150

NP

1174

> 0.50

b) Segregation pattern of mating type alleles Diploid strain B 86 X2 =

Mating type of random spores

Genotype

A/B 0.12 0.80 >

P >

A

B

71

67

0.50

References BARTH, G., 1980. Untersuchungen zur genetischen Bearbeitbarkeit der alkanverwertenden Hefe Saccharomycopsis lipolytica unter Beachtung von Mutanten mit Defekten im Trikarbonsäurezyklus und Glyoxylatweg. Dissertation. Akademie der Wissenschaften der DDR. BARTH, G. and K Ü N K E L , W . , 1 9 7 9 . Alcohol dehydrogenase in yeast. I I . NAD+ and NADP+ dependent alcohol dehydrogenases in Saccharomycopsis lipolytica. Z. allg. Mikrobiol., 19, 381 to 390. BARTH,

G. and W E B E R , H . 1 9 8 3 . Genetic studies on the yeast Saccharomycopsis lipolytica. Inactivation and mutagensis. Z. allg. Mikrobiol., 23, 147 —157. BASSEL, J . , W A R F E L , J . and MORTIMER, R . 1 9 7 1 . Complementation and genetic recombination in Candida lipolytica. J . Bacteriol., 108, 609—611. D A W E S , I. W. and H A R D I E , I . D . , 1974. Selective killing of vegetative cells in sporulated yeast culture by exposure to diethyl ether. Mol. Gen. Genet., 131, 281—289. EGEL, R., 1977. Selective spore survival during replica plating of fission yeast. Arch. Microbiol., 112, 1 0 9 - 1 1 0 . E M E I S , C . C . und GÜTZ, H., 1958. Eine einfache Technik zur Massenisolation von Hefesporen. Z. Naturforsch., 10, 6 4 7 - 6 5 0 . GAILLARDIN, C . M., CHAROY, V . and HESLOT, H . , 1973. A study of copulation and meiotic segregation in Candida lipolytica. Arch. Mikrobiol., 92, 48—57. MORTIMER, R . K . and HAWTHORNE, D . C., 1 9 7 5 . Genetic mapping in yeast. I n : Methods in Cell Biology (ed. by D. M. PRESCOTT), Vol. X I , 221—233. Academic Press New York. SAMSONOVA, J . A. und BÖTTCHER, F . , 1 9 8 0 . Genetische Segregation durch Nystatin in Hefen. Wiss. Z. EMAU Greifswald, X X I X , 6 5 - 6 6 . SHERMAN, F . , 1 9 7 5 . Use of micromanipulators in yeast studies. I N : Methods in Cell Biology (ed. by D. M. PRESCOTT), Vol. X I , 189 — 199. Academic Press New York. SHERMAN, F . and ROMAN, H . , 1963. Evidence for two types of allelic recombination in yeast. Genetics, 48, 2 5 5 - 2 6 1 . W E B E R , H . , 1 9 7 9 . Substructural studies on sporulation of Saccharomycopsis lipolytica. Z . allg. Mikrobiol., 19, 1 8 3 - 2 9 7 .

Mailing address: Dr. G. BARTH, Zentralinstitut für Mikrobiologie und experimentelle Therapie der AdW, D D R 6900 Jena, Beutenbergstr. 11

Zeitschrift f ü r allgemeine Mikrobiologie 24 (1984) 2, 128

Buchbesprechungen P. S K U L A C H E V and P. C . H I N K L E (Editors), Chemiosmotic Proton Circuits in Biological Membranes. X V I I I + 633 S., Abb. 93, Tab. 15. London-Amsterdam-Don Mills, Ontario-SydneyTokyo 1981. Addison-Wesley Publishing Company Inc. $ 29.50. V.

Das vorliegende Buch ist dem Nobelpreisträger P E T E R M I T C H E L L anläßlich seines 60. Geburtstages gewidmet. In 38 Beiträgen wird ein Überblick über Protonen-Transport-Reaktionen an membrangebundenen Enzymen gegeben. Ausgangspunkt 20 J a h r e intensiver Untersuchungen dieser Reaktionsmechanismen war die von M I T C H E L L im J a h r e 1961 aufgestellte chemiosmotische Hypothese der Energieerhaltung bei der oxidativen und photosynthetischen Phosphorylierung. Neben A T P tritt bei diesen membrangekoppelten Reaktionen der Protonengradient quer zur Membran als zweite Energiequelle auf. I n 21 bzw. 16. Orginal- bzw. Übersichtsarbeiten werden Reaktionen diskutiert, die der Erzeugung bzw. dem Abbau des Protonentransmembranpotentials dienen. Diese Gliederung erleichtert dem interessierten Biologen und Biochemiker die Einarbeitung in das wohl faszinierendste Gebiet der Bioenergetik lebender Zellen. Dabei stehen folgende biologische Systeme im Vordergrund: die 3 Komplexe der mitochondrialen Atmungskette; Transhydrogenase; Photoelektrontransport ; Bakteriorhodopsin; H, K ATP-ase des Magens; mitochondriale und bakterielle ATP-Synthetase {I\F l j ); A T P Transporter; bakterielles Transportsystem; Flageila. Ein umfangreiches Literaturverzeichnis von insgesamt 1635 Zitaten ist den Orginalarbeiten nachgestellt. Der das Buch abschließende Beitrag des Jubilars über vektorielle Liganden-Leitungsmechanismen in der Biochemie eröffnet auch dem Biophysiker Ansätze zur Entwicklung einer molekularmechanischen Theorie lokaler biochemischer Prozesse, die die Beschreibung des Metabolismus und des Stofftransportes einbezieht. P . MÜHLIG ( J e n a )

N. S T R A T H E R N , E. W. J O N E S and J . R. B R O A C H (Editors), The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, Monograph I I B . X + 680 S., 79 Abb., 71 Tab. Cold Spring H a r b o r 1982. Cold Spring Harbor Laboratory. $90.00.

J.

Den Autoren ist es sehr zu danken, daß sie den erfolgreichen Versuch unternommen haben, in zwei Bänden die wichtigsten Daten aus den sehr schnell voranschreitenden genetischen, biochemischen und physiologischen Untersuchungen an der Hefe Saccharomyces zusammenfassend darzustellen. I n dem nunmehr vorliegenden zweiten Band wird eine sehr gute Übersicht der wichtigsten Kenntnisse des Metabolismus und der Genexpression in dieser Hefe gegeben. Dabei wird insbesondere deutlich, welche große Bedeutung der Kombination der klassischen genetischen Untersuchungsmethodik, der Gentechnologie und der Enzymologie zukommt. Wertvoll sind auch die Ergänzungen der genetischen K a r t e sowie die Zusammenstellung der biochemischen Marker der Hefezellorganelle. Dieses Buch ist nicht nur allen Wissenschaftlern, die auf diesem Gebiet tätig sind, zu empfehlen, sondern auch all denen, die sich über diese Forschung eine Übersicht verschaffen wollen. Außerdem ist es als Nachschlagewerk zu Fragen der Molekularbiologie eukaryotischer Zellen geeignet. G . BARTH ( J e n a )

Zeitschrift für allgemeine Mikrobiologie 24 (1984) 2, 1 2 9 - 1 3 2

Kurze

Originalmitteilung

(Laboratorio de Microbiologia, Institute Gulbenkian de Ciencia, Apartado 14, 2781 Oeiras Codex, Portugal)

Temperature relations of yield, growth and thermal death in the y e a s t Hansenula

polymorpha

C. CABEQA-SILVA a n d A . MADEIKA-LOPES

(Eingegangen

am 23.

8.1983)

The temperature profile of thermal death and growth was studied in a strain of the methylotrophic yeast Hansenula polymorpha, together with the temperature dependence of the growth yield coefficients on glucose. The profile was typically dissociative with a maximum temperature for growth around 48 °C, above which the yield was found to decline sharply.

Previous studies concerning the dependence on temperature of the growth yield and of the specific rates of growth and thermal death led to the distinction between two types of temperature profiles among yeasts (For a review see VAN U D E N 1 9 8 4 ) . In the so-called associatively-profiled yeasts, which include species of the genera Saccharomyces, Candida and Torulopsis, thermal death concurred with exponential growth in the supra-optimal temperature range of growth (VAN U D E N et al. 1 9 6 8 , VAN U D E N and

MADEIRA-LOPES

LOPES

1974,

1970,

OLIVEIBA-BAPTISTA

SIMOES-MENDES

el al.

1978,

and

VAN U D E N

MADEIRA-LOPES

and

]971,

MADEIRA-

VAN U D E N

1979,

et al. 1 9 8 2 , S I M O E S - M E N D E S and M A D E I R A - L O P E S 1 9 8 3 ) , whereas in the dissociatively-profiled yeasts, such as Cryptococcus neoformans, Lipomyces kononenkoae and psychrophilic species of the genus Candida, no biologically significant interaction was found of thermal death with exponential growth (VAN U D E N et al. 1 9 6 8 ,

LEMOS-CAROLINO

MADEIRA-LOPES

a n d VAN U D E N 1 9 7 9 , V I D A L - L E I R I A a n d

L O P E S a n d VAN U D E N and MADEIRA-LOPES

VAN U D E N 1 9 8 0 ,

1 9 8 2 , S P E N C E R - M A R T I N S a n d VAN U D E N 1 9 8 2 , 1 9 8 3 , MADEIRA-LOPES a n d CABEQA-SILVA

MADEIRA-

SIMOES-MENDES

1984).

The current interest in the methylotrophic yeast Hansenula polymorpha (see for example EGLI and FIECHTER 1981) prompted us to study its temperature profile. We used a mineral medium with vitamins and 0.3% (w/v) glucose and methods described earlier (VAN UDEN et al. 1968, VAN UDEN and

MADEIRA-LOPES

1970,

SPENCER-MARTINS

a n d VAN

UDEN

1982).

The

type-strain

(CBS 4732), kept in our laboratory under IGC 4025, was isolated from soil irrigated with waste water from distilleries (WICKERHAM 1970). Its maximum temperature for growth, the highest found among yeasts lies in the neighbourhood of 48 °C (VAN UDEN et al. 1968).

Three distinct temperature zones were detected with respect to growth of Hansenula polymorpha-. (1) At temperatures below 46 °C balanced exponential growth was almost immediately established, the initial specific growth rate being constant as long as nutrients were available (Fig. 1-A); (2) at temperatures between 46 °C and 47 °C balanced exponential growth was preceded by unbalanced mass growth and zero cell number growth (Fig. 1-B); (3) at temperatures between 47 °C and 48 °C balanced exponential growth was preceded by unbalanced mass growth and exponential cell death (Fig. 1-C). When the specific rates of balanced exponential growth were plotted together with the specific thermal death rates, obtained with populations pre-incubated for 48 hours

Zeitschrift für allgemeine Mikrobiologie 24 (1984) 2, 1 2 9 - 1 3 2

Kurze

Originalmitteilung

(Laboratorio de Microbiologia, Institute Gulbenkian de Ciencia, Apartado 14, 2781 Oeiras Codex, Portugal)

Temperature relations of yield, growth and thermal death in the y e a s t Hansenula

polymorpha

C. CABEQA-SILVA a n d A . MADEIKA-LOPES

(Eingegangen

am 23.

8.1983)

The temperature profile of thermal death and growth was studied in a strain of the methylotrophic yeast Hansenula polymorpha, together with the temperature dependence of the growth yield coefficients on glucose. The profile was typically dissociative with a maximum temperature for growth around 48 °C, above which the yield was found to decline sharply.

Previous studies concerning the dependence on temperature of the growth yield and of the specific rates of growth and thermal death led to the distinction between two types of temperature profiles among yeasts (For a review see VAN U D E N 1 9 8 4 ) . In the so-called associatively-profiled yeasts, which include species of the genera Saccharomyces, Candida and Torulopsis, thermal death concurred with exponential growth in the supra-optimal temperature range of growth (VAN U D E N et al. 1 9 6 8 , VAN U D E N and

MADEIRA-LOPES

LOPES

1974,

1970,

OLIVEIBA-BAPTISTA

SIMOES-MENDES

el al.

1978,

and

VAN U D E N

MADEIRA-LOPES

and

]971,

MADEIRA-

VAN U D E N

1979,

et al. 1 9 8 2 , S I M O E S - M E N D E S and M A D E I R A - L O P E S 1 9 8 3 ) , whereas in the dissociatively-profiled yeasts, such as Cryptococcus neoformans, Lipomyces kononenkoae and psychrophilic species of the genus Candida, no biologically significant interaction was found of thermal death with exponential growth (VAN U D E N et al. 1 9 6 8 ,

LEMOS-CAROLINO

MADEIRA-LOPES

a n d VAN U D E N 1 9 7 9 , V I D A L - L E I R I A a n d

L O P E S a n d VAN U D E N and MADEIRA-LOPES

VAN U D E N 1 9 8 0 ,

1 9 8 2 , S P E N C E R - M A R T I N S a n d VAN U D E N 1 9 8 2 , 1 9 8 3 , MADEIRA-LOPES a n d CABEQA-SILVA

MADEIRA-

SIMOES-MENDES

1984).

The current interest in the methylotrophic yeast Hansenula polymorpha (see for example EGLI and FIECHTER 1981) prompted us to study its temperature profile. We used a mineral medium with vitamins and 0.3% (w/v) glucose and methods described earlier (VAN UDEN et al. 1968, VAN UDEN and

MADEIRA-LOPES

1970,

SPENCER-MARTINS

a n d VAN

UDEN

1982).

The

type-strain

(CBS 4732), kept in our laboratory under IGC 4025, was isolated from soil irrigated with waste water from distilleries (WICKERHAM 1970). Its maximum temperature for growth, the highest found among yeasts lies in the neighbourhood of 48 °C (VAN UDEN et al. 1968).

Three distinct temperature zones were detected with respect to growth of Hansenula polymorpha-. (1) At temperatures below 46 °C balanced exponential growth was almost immediately established, the initial specific growth rate being constant as long as nutrients were available (Fig. 1-A); (2) at temperatures between 46 °C and 47 °C balanced exponential growth was preceded by unbalanced mass growth and zero cell number growth (Fig. 1-B); (3) at temperatures between 47 °C and 48 °C balanced exponential growth was preceded by unbalanced mass growth and exponential cell death (Fig. 1-C). When the specific rates of balanced exponential growth were plotted together with the specific thermal death rates, obtained with populations pre-incubated for 48 hours

130

C. CABEGA-SILVA and A . MADEIRA-LOPES

.5 .1 . 0 o .05

i

-

rff

9

• • 0

2

4

6

24 26 28 30

Time (hj Fig. 1. Colony counts and optical densities obtained at 44.9 °C (A), 46.8 °C (B) and 47.9 °C (C) with Hansenula polymorpha in liquid mineral medium with glucose

at 47.5 °C (in order to overcome transient cell death) a typically dissociative temperature profile emerged (Fig. 2): the AERHENIUS plot of growth displayed only branch in the supraoptimal temperature range while the extrapolated AEEHENIUS plot of death intersected the AEEHENIUS plot of growth at a biologically non-significant value. The behaviour of the growth yield coefficients on glucose (Fig. 3), which did not significantly vary from 25 °C until almost 48 °C but sharply decreased down to zero 50 o

10 a a V.

5-

o, u

,

«> CL 10

.5 56 56 5« 52 50 48 «6 44 42 40 36 36 34 32 30(°C ) I L_ I I _L I L_ -1—_J I , I I L. I I J i 300 305 310 315 325 320 330 T-l xlO5

IK'1}

Fig. 2. Temperature profile of the specific growth ( o ) and thermal death ( A ) rates of Hansenula polymorpha

Temperature profile of Hansenula polymorpha

131

therefrom, was qualitatively identical with those of Lipomyces MARTINS

and

VAN U D E N

1982)

and

Crytococcus

neoformans

kononenkoae (SPENCER(MADEIRA-LOPES

and

VAN UDEN 1982). While in yeasts with an associative profile the yield on glucose declines above the optimum temperature for growth (SPENCER-MARTINS and VAN UDEN 1982), probably due to dissipation of glucose by non-viable cells (VAN UDEN and MADEIRA-LOPES 1976), dissociative yeasts appear to be "thermotolerant" (PozMOGOVA 1978, KVASNIKOV and ISAKOVA 1978) with respect to yield. In such yeasts non-viable cells are not formed during exponential growth at supraoptimal temperatures which m a y explain the stability of the yield on carbon.

6 3 "Si O 5 ¿,0 45 Temperature I'C) Fig. 3. Temperature dependence of the growth yield of Hansenula polymorpha in mineral medium with 0.3% glucose (w/v)

Acknowledgements We wish to record our thanks to Prof. N.

VAN U D E N

for encouragement and helpful discussion.

References T. and F I E C H T E R , A., 1981. Theoretical analysis of media used in the growth of yeast on methanol. J . Gen. Microbiol., 123, 365—369. K V A S N I K O V , E. I. and ISAKOVA, D. M., 1978. The Physiology of Thermotolerant Microorganisms (in Russian). Yerlag „ N A U K A " , Moscow. L E M O S - C A R O L I N O , M . , M A D E I R A - L O P E S , A . and VAN U D E N , N . 1982. The temperature profile of the pathogenic yeast Candida albicans. Z. allg. Mikrobiol., 22, 705—709. M A D E I R A - L O P E S , A . , 1 9 7 4 . Inheritance of the maximum temperature for growth in Saccharomyces cerevisiae. Cienc. Biol. (Portugal), 1 , 8 9 — 9 2 . M A D E I R A - L O P E S , A . and C A B E ^ A - S I L V A , C . , 1984. The dependence on temperature of thermal death, growth and yield of Candida tropicalis. Z. allg. Mikrobiol., 24, 133 — 135. M A D E I R A - L O P E S , A . and VAN U D E N , N . , 1 9 7 9 . Thermal association and dissociation in thermosensitive mutants of Saccharomyces cerevisiae. Z. allg. Mikrobiol., 19, 303—305. M A D E I R A - L O P E S , A. and VAN U D E N , N., 1982. The temperature profile of Cryptococcus neoformans. Sabouraudia, 20, 331—334. O L I V E I E A - B A P T I S T A , A . and VAN U D E N , JST., 1 9 7 1 . Occurrence of two maximum temperatures for growth in yeast. Z. allg. Mikrobiol., 11, 5 9 — 6 1 . EGLI,

132

C . CABE9A-SILVA a n d A . MADEIRA-LOPES

POZMOGOVA, I . N., 1978. T h e effect of above-optimal t e m p e r a t u r e s on bacteria a n d y e a s t s (in Russian). I n v e s t í a Akademii N a u k SSR, Seria Biologitscheskaia, no. 4, 588—597. S I M O E S - M E N D E S , B. a n d M A D E I R A - L O P E S , A . , 1 9 8 3 . H e a t - i n d u c e d respiratory-deficiency a n d t h e t e m p e r a t u r e profile of yeasts. Ciénc. Biol. (Portugal), 8, 1—8. SIMOES-MENDES, B . , MADEIRA-LOPES, A . a n d VAN UDEN, N . , 1978. K i n e t i c s of p e t i t e m u t a t i o n

a n d t h e r m a l d e a t h in Saccharomyces cerevisiae a t superoptimal t e m p e r a t u r e s . Z. allg. Mikrobiol., 18, 2 7 5 - 2 7 9 . SPENCER-MARTINS, I . a n d VAN UDEN, N., 1982. T h e t e m p e r a t u r e profile of growth, d e a t h a n d yield of t h e starch-converting yeast Lipomyces kononenkoae. Z. allg. Mikrobiol., 22, 503—505. VAN UDEN, N., 1984. T e m p e r a t u r e profiles of yeasts. Adv. Microbiol. Physiol., 25, in press. VAN U D E N , N., A B R A N C H E S , P . a n d C A B E Z A - S I L V A , C . , 1968. T e m p e r a t u r e f u n c t i o n s of t h e r m a l d e a t h in yeasts a n d their relation to t h e m a x i m u m t e m p e r a t u r e for growth. Arch. Mikrobiol., 61, 3 8 1 - 3 9 3 . VAN UDEN, N. a n d MADEIRA-LOPES, A., 1970. Concurrent exponential growth a n d d e a t h of cell populations of Saccharomyces cerevisiae a t superoptimal growth t e m p e r a t u r e s . Z. allg. Mikrobiol., 10, 5 1 5 - 5 2 6 . VAN U D E N , N . a n d M A D E I R A - L O P E S , A . , 1 9 7 6 . Yield a n d m a i n t e n a n c e relations of yeast g r o w t h in t h e chemostat a t superoptimal t e m p e r a t u r e s . Biotechnol. Bioeng., 18, 791—804. V I D A L - L E I R I A , M. a n d VAN U D E N , N . , 1980. T h e r m a l dissociation in t h e psychrophilic yeast Candida curiosa. Z. allg. Mikrobiol., 20, 471—474. W I C K E R H A M , L . J . , 1 9 7 0 . Genus Hansenula H. et P . S Y D O W . I n : L O D D E R (ed.). T h e Yeasts, a T a x o n o m y S t u d y . X o r t h - H o l l a n d Publishing Company, p p . 2 2 6 — 3 1 5 . Mailing address: Dr. A.MADEIRA-LOPES, I n s t i t u t o Gulbenkian de Ciencia, A p a r t a d o 14, 2781 Oeiras-Codex, P o r t u g a l

Zeitschrift für allgemeine Mikrobiologie 2 4 (1984) 2, 1 3 3 - 1 3 5

Kurze

Originalmitteilung

{Laboratorio de Microbiologia, Institute Gulbenkian de Ciencia, Apartado 14, 2781 Oeiras Codex, Portugal)

The dependence on temperature of thermal death, growth and yield of Candida tropicalis A . MADEIRA-LOPES a n d C. CABEQA-SILVA

(Eingegangen

am, 23.

8.1983)

The temperature profile of thermal death and growth of a strain of the yeast Candida tropicalis was found to be dissociative. The yield coefficients on glucose did not significantly v a r y from 28 °C up to 39 °C, the maximum temperature for growth.

The combined graphical representation of the ARRHENIUS plot of the specific thermal death rates of a yeast species, together with the ARRHENIUS plot of its specific growth rates, either exhibits a conjunction of the two plots (associative profile), which was shown to indicate a concurrence of death with growth in the supraoptimal temperature range (VAN U D E N and MADEIRA-LOPES 1 9 7 0 ) , or displays separation of the plots, excluding such a concurrence: dissociative profile (MADEIRA-LOPES and VAN U D E N 1 9 7 9 , VIDAL-LEIRIA and VAN U D E N 1 9 8 0 ) . These findings of two types of response of yeasts to temperature have recently been reinforced by the additional study of temperature dependence of growth yield coefficients (SPENCER-MARTINS and VAN U D E N 1 9 8 2 , VAN U D E N 1 9 8 4 ) . Here we report on the temperature profile of Candida tropicalis, a yeast both of medical and industrial importance (ZIOBRO 1 9 7 9 , YAMADA etal. 1 9 8 0 ) . W e utilized the type-strain of Candida tropicalis (CBS 94), kept in our laboratory under IGC 3097, formerly isolated by CASTELLANI from human material (VAN UDEN and BUCKLEY 1970) and the methods described elsewhere (VAN UDEN et al. 1968, VAN UDEN and MADEIRA-LOPES 1970, SPENCEK-MARTINS and VAN UDEN 1982).

At temperatures from 27.9 °C up to 38.G °C, sustained balanced exponential growth was observed throughout each experiment as long as nutrients were available. At temperatures from 38.9 °C up to 42.8 °C some minor transient mass growth was detected which had no counterpart in cell population growth (unbalanced growth). The combined ARRHENIUS plots of the specific rates of balanced exponential growth and of the specific thermal death rates, determined following pre-incubation at 25 °C, are depicted in Fig. 1. The maximum temperature for growth is 38.6 °C and the optimum temperature for growth lies in the neighbourhood of 37 °C; neither thermal death nor cell population growth were observed in the temperature interval 38.6 °C to 40.9 °C, a characteristic behaviour of a dissociatively-profiled yeast. The growth yield coefficients showed no significant variation from at least 28 °C up to about 39 °C, where they sharply declined down to zero (Fig. 2). Thus the temperature profile of the yield on carbon was similar with that found earlier in Lipomyces kononenkoae (SPENCER-MARTINS and VAN U D E N 1 9 8 2 ) and Hansenula polymorpha (CABEQA-SILVA and MADEIRA-LOPES 1 9 8 4 ) and thus seems to be characteristic for yeasts with a dissociative temperature profile. While in associative yeasts the yield starts declining above Tov, in dissociative yeasts the yield declines only at or near 27op.

Zeitschrift für allgemeine Mikrobiologie 2 4 (1984) 2, 1 3 3 - 1 3 5

Kurze

Originalmitteilung

{Laboratorio de Microbiologia, Institute Gulbenkian de Ciencia, Apartado 14, 2781 Oeiras Codex, Portugal)

The dependence on temperature of thermal death, growth and yield of Candida tropicalis A . MADEIRA-LOPES a n d C. CABEQA-SILVA

(Eingegangen

am, 23.

8.1983)

The temperature profile of thermal death and growth of a strain of the yeast Candida tropicalis was found to be dissociative. The yield coefficients on glucose did not significantly v a r y from 28 °C up to 39 °C, the maximum temperature for growth.

The combined graphical representation of the ARRHENIUS plot of the specific thermal death rates of a yeast species, together with the ARRHENIUS plot of its specific growth rates, either exhibits a conjunction of the two plots (associative profile), which was shown to indicate a concurrence of death with growth in the supraoptimal temperature range (VAN U D E N and MADEIRA-LOPES 1 9 7 0 ) , or displays separation of the plots, excluding such a concurrence: dissociative profile (MADEIRA-LOPES and VAN U D E N 1 9 7 9 , VIDAL-LEIRIA and VAN U D E N 1 9 8 0 ) . These findings of two types of response of yeasts to temperature have recently been reinforced by the additional study of temperature dependence of growth yield coefficients (SPENCER-MARTINS and VAN U D E N 1 9 8 2 , VAN U D E N 1 9 8 4 ) . Here we report on the temperature profile of Candida tropicalis, a yeast both of medical and industrial importance (ZIOBRO 1 9 7 9 , YAMADA etal. 1 9 8 0 ) . W e utilized the type-strain of Candida tropicalis (CBS 94), kept in our laboratory under IGC 3097, formerly isolated by CASTELLANI from human material (VAN UDEN and BUCKLEY 1970) and the methods described elsewhere (VAN UDEN et al. 1968, VAN UDEN and MADEIRA-LOPES 1970, SPENCEK-MARTINS and VAN UDEN 1982).

At temperatures from 27.9 °C up to 38.G °C, sustained balanced exponential growth was observed throughout each experiment as long as nutrients were available. At temperatures from 38.9 °C up to 42.8 °C some minor transient mass growth was detected which had no counterpart in cell population growth (unbalanced growth). The combined ARRHENIUS plots of the specific rates of balanced exponential growth and of the specific thermal death rates, determined following pre-incubation at 25 °C, are depicted in Fig. 1. The maximum temperature for growth is 38.6 °C and the optimum temperature for growth lies in the neighbourhood of 37 °C; neither thermal death nor cell population growth were observed in the temperature interval 38.6 °C to 40.9 °C, a characteristic behaviour of a dissociatively-profiled yeast. The growth yield coefficients showed no significant variation from at least 28 °C up to about 39 °C, where they sharply declined down to zero (Fig. 2). Thus the temperature profile of the yield on carbon was similar with that found earlier in Lipomyces kononenkoae (SPENCER-MARTINS and VAN U D E N 1 9 8 2 ) and Hansenula polymorpha (CABEQA-SILVA and MADEIRA-LOPES 1 9 8 4 ) and thus seems to be characteristic for yeasts with a dissociative temperature profile. While in associative yeasts the yield starts declining above Tov, in dissociative yeasts the yield declines only at or near 27op.

134

A . M A D E I R A - L O P E S a n d C . CABEÇA-SILVA >Î

ri x 105/k-1/ Fig. 1. Temperature profile of Candida tropicalis

Temperature

I'C)

Fig. 2. Temperature dependence of the growth yield of Candida tropicalis in mineral medium with 0.3% glucose (w/v) Y e a s t s with such a behaviour have been called "thermotolerant" yeasts ( P O Z M O G O V A 1978, K V A S N I K O V and I S A K O V A 1978) and are of practical interest for the production of single cell protein at supraoptimal temperatures. Acknowledgement The authors are indebted to Prof.

N . VAN U D E N

for advice and encouragement.

References and M A D E I R A - L O P E S , A . , 1984. Temperature relations of yield, growth and thermal death in the yeast Hansenula polymorpha. Z. allg. Mikrobiol., 24, 129—132. K V A S N I K O V , E. I. and I S A K O V A , D. M., 1978. The Physiology of Thermotolerant Microorganisms (in Russian). Verlag " N A U K A " , Moscow. M A D E I B A - L O P E S , A . and VAN U D E N , N . , 1 9 7 9 . Thermal association and dissociation in thermosensitive mutants of Saccharomyces cerevisiae. Z. allg. Mikrobiol., 19, 303—305. POZMOGOVA, I. N., 1978. The effect of above-optimal temperatures on bacteria and yeasts (in Russian). Izvestia Akademii Nauk SSR, Seria Biologitscheskaia, no. 4, 588—597. CABEÇA-SILVA, C.

T e m p e r a t u r e profil of C. tropicalis

135

and VAN U D E N , N . , 1 9 8 2 . The temperature profile of growth, death a n d yield of the starch-converting yeast Lipomyces kononenkoae. Z. allg. Mikrobiol., 22, 503—505. VAN UDEN, N., 1984. Temperature profiles of yeast. Adv. Microbiol. Physiology, 25, in press. SPENCER-MABTINS, I .

VAN UDEN, N . ,

ABEANCHES, P . a n d CABE^A-SILVA, C., 1968. T e m p e r a t u r e f u n c t i o n s of t h e r m a l

death in yeast and their relation to t h e maximum temperature for growth. Arch. Mikrobiol., 61, 3 8 1 - 3 9 3 . VAN U D E N , N . and B U C K L E Y , H . , 1 9 7 0 . Genus Candida B E R K H O U T . I n : L O D D E R (ed.), The Yeasts, a Taxonomic Study. North Holland Publishing Company, pp. 8 9 3 — 1 0 8 7 . VAN UDEN, N. and MADEIRA-LOPES, A., 1970. Concurrent exponential growth and death of cell populations of Saccharomyces cerevisiae a t superoptimal growth temperatures. Z. allg. Mikrobiol., 10, 515—526. V I D A L - L E I R I A , M. and VAN U D E N , N . , 1 9 8 0 . Thermal dissociation in t h e psyehrophilic yeast Candida curiosa. Z. allg. Mikrobiol., 20, 471—474. Y A M A D A , T . , N A W A , H . , K A W A M O T O , S., T A N A K A , A. and F U K U I , S., 1 9 8 0 . Subcellular localization of long-chain alcohol dehydrogenase and aldehyde dehydrogenase in ra-alkanol-grown Candida tropicalis. Arch. Microbiol., 128, 145 — 151. ZIOBBO, J . , 1979. Pilzflora bei Kranken mit Infektionen des Atmungssystems. Mykosen, 22, 454-458. Mailing address: Dr. A.MADEIRA-LOPES, Instituto Gulbenkian de Ciencia, Apartado 14, 2781 Oeiras-Codex, Portugal Zeitschrift f ü r allgemeine Mikrobiologie 24 (1984) 2, 136

Buchbesprechung P. W E S T B R O E K and E. W . D E J O N G (Editors), Biomineralization and Biological Metal Accumulation. Biological and Geological Perspectives (Papers presented a t the F o u r t h International Symposium on Biomineralization, Renesse, The Netherlands, J u n e 2—5, 1982). 533 S., 219 Abb., 37 Tab. Dordrecht-Boston-London 1983. D. Reidel Publishing Company. Dfl 160,-. Unter dem Begriff Biomineralisation wird die Akkumulation von Metallverbindungen durch lebende Systeme verstanden, wobei der Bildung von CaC0 3 die wichtigste Bedeutung zukommt. Zu diesem Thema fand vom 2.—4. J u n i 1982 in Renesse (Holland) ein Symposium s t a t t , das mehr als 70 Wissenschaftler verschiedenster Fachgebiete vereinte und das die Förderung der interdisziplinären Zusammenarbeit auf diesem Gebiet zum Hauptziel hatte. Das vorliegende Buch enthält neben Einleitung und Schlußbemerkung 48 Beiträge, deren Spektrum von medizinischen, palaeonthologischen, geologischen, physikalisch-chemischen, biochemischen, biotechnologischen bis zu mikrobiologischen Fragestellungen der Biomineralisation reicht. Als Objekte wurden dabei vor allem Muscheln und Schnecken mit ihren Kalkschalen und Wirbeltiere (Knochen- und Zahnbildung) untersucht, daneben aber auch verschiedene Mikroorganismen. Vom Umfang her im Mittelpunkt steht der 2. Teil der Publikation, wo in 27 Beiträgen verschiedenste Aspekte der Kalkbildung durch Organismen behandelt werden. Auf die Kalkfällung durch Mikroorganismen wird in 5 Artikeln eingegangen (Coccolithophoriden, Cyanobakterien/Stromatolithen, Foraminiferen). F ü r den Mikrobiologen empfehlenswert sind auch einige der 14 Beiträge des 3. Teils (Biological accumulation of metals other than calcium), die Angaben über die Beteiligung von Mikroorganismen bei der Ablagerung von Eisen, Mangan, Silicium und Gold enthalten. Darunter kommt dem Bericht von B R I E R L E Y , J . A. und C. L . B R I E R L E Y über die Anwendung der biologischen Akkumulation von Schwermetallen zur Gewinnung dieser Elemente aus verdünnten Lösungen und zur Reinigung von Abwässern eine zukunftsweisende Bedeutung zu. Durch das Bestreben, möglichst viele Aspekte der Biomineralisationsforschung sichtbar werden zu lassen, um eine Integration zu fördern, sind die mikrobiologischen Beiträge etwas zu kurz gekommen. Damit wird der großen ökologischen Bedeutung der Mikroorganismen f ü r die globalen Kreisläufe auf der Erde und den geomikrobiologischen Erkenntnissen in nicht genügendem Maße Rechnung getragen. Dennoch wird auch der Mikrobiologe, Geomikrobiologe oder Mikrobenökologe eine Reihe von interessanten Anregungen finden. J . HEYER ( J e n a )

HERBERT KLUG

Bau und Funktion von Zellen Eine Einführung in die medizinische Zellbiologie 1980. IX, 314 Seiten - 138 Abbildungen Bestell-Nr. 7627580 (6558)

14,7 X 21,5 cm — 2 2 , - M

Auf Grund der Anwendung hochentwickelter Methoden und Techniken ist es möglich geworden, in den molekularen und makromolekularen Bereich der Zellen vorzudringen. Dort haben sowohl die Histologie als auch die Biochemie ihre gemeinsame Wurzel. So konnten nicht nur ihre Struktur und Funktion, sondern auch die pathomorphologischen Veränderungen bei verschiedenen Erkrankungen weitgehend erfaßt werden, was nicht nur für den wissenschaftlich tätigen, sondern auch für den mehr praktisch arbeitenden Mediziner bedeutsam ist. Nach Darstellung der für die allgemeine Zellbiologie wichtigsten molekularen Bausteine zeigt der Autor die wesentlichsten Stoffwechselreaktionen und deren hormonale Regulation. Ebenso erörtert er die Struktur und Funktion der verschiedenen Zelltypen unter normalen und pathologischen Bedingungen und fügt ergänzend die Zelle der Prokaryonten und die Viren an. Dazu werden neben zahlreichen instruktiven elektronenmikroskopischen Abbildungen auch schematische Zeichnungen verwendet.

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AKADEMIE-VERLAG • B E R L I N DDR-1086 Berlin, Leipziger Str. 3 - 4

Buchhandlung

Z E I T S C H R I F T F Ü R ALLGEMEINE MIKROBIOLOGIE VOLUME 24

1984

NUMBER 2

CONTENTS Characterization ot aromatic aminotransferase activities from Candida maltosa Dissimilation of methanol in Acetobacter sp. MB 58 A new method for electronmicroscopical preparation of capsules from Bordetella bronchlseptica Alternative life cycles in Thermoac tinomyces vulgaris Characterization of aerial mycelium of Thermoactinomyces vulgaris Mixed culture kinetics of stringent and relaxed Escherichia coli cell in glucose-limited chemostat Plasmid pBR322: Formation of oligomers in dependence on the concentration of tetracyclin S h o r t Notes Use of nystatin for random spore selection in the yeast Saccharomycopsis lipolytica Temperature relations of yield, growth and thermal death in the yeast Hansenula polymorpha The dependence on temperature of thermal death, growth and yield of Candida tropicalis Book Reviews

R . B O D E AND D . B I R N B A U M

67

M . W . G B Ü N D I G AND N . V . DOKONINA

77

U . K L U D A S JUN.

85

AND W . R U D O L P H

.SIGRID R R E T S C H M E B

93

SIGRID K B E T S C H M E R

101

D . R I E S E N B E K G AND F . B E R G T E R

113

W , WIEHLE, M . HECKES, B . REICHS T E I N AND F . M A C H 119

G . B A R T H AND H . W E B E R

125

C . QABECA-SILVA AND A . M A D E I R A LOPES '

129

A . M A D E I R A - L O P E S AND C . £ABE{)ASILVA

133

7 6 , 1 1 2 , 1 1 8 , 128, 136

Zeitschrift für allgemeine Mikrobiologie is indexed in Current Contents/Life Sciences