Zeitschrift für Allgemeine Mikrobiologie: Band 23, Heft 9 [Reprint 2021 ed.] 9783112565544, 9783112565537

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ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE

AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS BAND 23 • 1983 • HEFT 9

AKADEMIE-VERLAG ISSN 0044-2208

Z. allg. Mikrobiol., Berlin 23 (1983) 9, 537 - 604

BERLIN EVP 2 0 , - M

Z E I T S C H R I F T FÜR A L L G E M E I N E M I K R O B I O L O G I E VOLUME 23

1983

NUMBER 9

CONTENTS 539

Double or triple sets oi replication functions as inverted and direct repeats on in vivo reconstructed streptococcal MLS resistance plasmids

D . BEHNKE a n d S. KLAUS

Influence of inorganic phosphate on the formation of tentoxin by Alternaria altemata ( F R . ) K E I S S L E R

B . BRÜCKNER, I . H Ä N E L , . F . H Ä N E L AND R . TRÖGER 549

Uncoupling of respiration in turimycin fermentations Bioconversions of a macrolide glycoside by growing L-form cells of Streptomyces hygroscopicus Action of supraoptimal temperatures on growth and development of a distillery yeast. I. Growth and development of the yeast Saccharomyces cerevisiae strain 193 in discontinuous culture Action of supraoptimal temperatures on growth and development of a distillery yeast. II. Growth and development of the yeast Saccharomyces cerevisiae strain 193 in continuous culture at pH-stat Alkane-mediated enzyme induction in the yeast Lodderomyces Microbiological implications of electric field effects. VI. Stimulation of plasmid transformation of Bacillus cereus protoplasts by electric field pulses

W . E F F E N B E R G E R , P . J . MÜLLER AND H . BOCKER

557

U . GRÄFE AND J . GUMPERT

565

L . MAVRINA, I . POZMOGOWA AND W.SCHADE

571

L . MAVRINA, I . POZMOGOWA, I . RABOTNOWA a n d W . SCHADE

581

H . - G . MÜLLER, S . MAUERSBERGER, W . - H . SCHUNCK a n d B . W I E D M A N N 5 8 9 N . SHIVAROVA, W . FÖRSTER, H . - E . JACOB a n d R . GRIGOROVA

595

Short N o t e Use of B-glucosamine and 2-deoxyglucose in the selective isolation of mutants of the yeast Lipomyces starkeyi derepressed for the production of extracellular endodextranase Book Reviews

A . L A I B E S , I . SPENCER-MARTINS a n d N.VAN UDEN 601

548, 570, 588, 594, 600, 604

ZEITSCHRIFT FÜR ALLGEMEINE MIKRO- BIOLOGIE AN INTERNATIONAL JOURNAL ON

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

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

unter der Chefredaktion von W. Schwartz, Braunschweig

MORPHOLOGY, PHYSIOLOGY, GENETICS,

und

AND ECOLOGY OF MICROORGANISMS

U. Taubeneck, Jena

U N T E R MITARBEIT VON

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

REDAKTION

BAND 23

1983

HEFT 9

AKADEMIE-VERLAG BERLIN

U. May, Jena

538 Die Zeitschrift für Allgemeine Mikrobiologie soll dazu beitragen, Forschung und internationale Zusammenarbeit auf dem Gebiet der Mikrobiologie zu fördern. Es werden Manuskripte aus allen Gebieten der allgemeinen Mikrobiologie veröffentlicht. Arbeiten über Themen aus der medizinischen, landwirtschaftlichen, technischen Mikrobiologie und aus der Taxonomie der Mikroorganismen werden ebenfalls aufgenommen, wenn sie Prägen von allgemeinem Interesse behandeln. Zur Veröffentlichung werden angenommen: Originalmanuskripte, die in anderen Zeitschriften noch nicht veröffentlicht worden sind und in gleicher Form auch nicht in anderen Zeitschriften erscheinen werden. Der Umfang soll höchstens l 1 ^ Druckbogen (24 Druckseiten) betragen. Bei umfangreicheren Manuskripten müssen besondere Vereinbarungen mit der Schriftleitung und dem Verlag getroffen werden. Kurze Originalmitteilungen über wesentliche, neue Forschungsergebnisse. Umfang im allgemeinen höchstens 3 Druckseiten. Kurze Originalmitteilungen werden beschleunigt veröffentlicht. Kritische Sammelberichte und Buchbesprechungen nach Vereinbarung mit der Schriftleitung. Bezugsmöglichkeiten der Zeitschrift für Allgemeine Mikrobiologie: Bestellungen sind zu richten — in der DDR an den Postzeitungsvertrieb, an eine Buchhandlung oder an den Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3 - 4 ; — im sozialistischen Ausland an eine Buchhandlung für fremdsprachige Literatur oder an den zuständigen Postzeitungsvertrieb; — in der BRD und Westberlin an eine Buchhandlung oder an die Auslieferungsstelle KUNST UND WISSEN, Erich Bieber OHG, Wilhelmstraße 4 - 6 , D-7000 Stuttgart 1; — in den übrigen westeuropäischen Ländern an eine Buchhandlung oder an die Auslieferungsstelle KUNST UND WISSEN, Erich Bieber GmbH, Dufourstraße 51, CH-8008 Zürich/Schweiz; — im übrigen Ausland an den Internationalen Buch- und Zeitschriftenhandel; den Buchexport, Volkseigener Außenhandelsbetrieb der Deutschen Demokratischen Republik, DDR-7010 Leipzig, Postfach 160, oder an den Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3—4. Zeitschrift für Allgemeine Mikrobiologie Herausgeber: Im Auftrag des Verlages von einem internationalen Wissenschaftlerkollektiv herausgegeben. Verlag: Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3 - 4 ; Fernruf 2236229 oder2236221 Telex-Nr. 114420; Bank: Staatsbank der DDR, Berlin, Kto.-Nr.: 6836-26-20712. Chefredaktion: Prof. Dr. U D O T A T J B E N E C K Prof. Dr. W I L H E L M SCHWARZ Anschrift der Redaktion: Zentralinstitut für Mikrobiologie und experimentelle Therapie der Akademie der Wissenschaften, DDR-6900 Jena, Beutenbergstr. 11; Fernruf: Jena 8 5 2 2 0 0 ; Telex-Nr. 058621. Veröffentlicht unter der Lizenznummer 1306 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: V E B Druckerei „Thomas Müntzer", DDR-5820 Bad Langensalza. Erscheinungsweise: Die Zeitschrift für Allgemeine Mikrobiologie erscheint jährlich in einem Band mit 10 Heften. Bezugspreise je Band 220, — M zuzüglich Versandspesen (Preis für die DDR 2 0 0 , - M). Preis je Heft 2 2 , - M (Preis für die DDR 2 0 , - M). Urheberrecht: Alle Rechte vorbehalten, insbesondere die der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translations into foreign languages). No part of this issue may be reproduced in any form, by photoprint, microfilms or any other means, without written permission from the publishers.

Erscheinungstermin: November 1983 Bestellnummer dieses Heftes 1070/23/9 © 1983 by Akademie-Verlag Berlin. Printed in the German Democratic Republic AN (EDV) 75218

Zeitschrift für Allgemeine Mikrobiologie

23

1983

9

539-547

(Akademie der Wissenschaften der D D R , Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und experimentelle Therapie, J e n a , Direktor: Prof. Dr. U . TAUBENECK)

Double or triple sets of replication functions as inverted and direct repeats on in vitro reconstructed streptococcal MLS resistance plasmids D . BEHNKE a n d S. KLAUS

(Eingegangen

am 2. 5.

1983)

In vitro rearrangement of plasmid pDBIOZ together with comparative studies of other streptococcal plasmids allowed the localization of replication and copy control functions on sequences and its naturally occurring ancestor p8M19035 as duplicates in which were present on pDB102 inverted orientation. E v i d e n c e is presented t h a t neither the presence of duplicate replication regions nor their arrangement in inverted orientation was essential for plasmid survival. A m o n g t h e in vitro reconstructed plasmids were several t h a t stably carried t w o or three sets of replication and copy control functions either as inverted or direct repeats or both. A copy control m u t a t i o n is described which led to a tenfold increase of copy number over t h a t of the naturally occurring plaspSM19035. mid

Among antibiotic resistance plasmids isolated from streptococcal strains the socalled MLS-resistance plasmids prevail (COTJRVALIN et al. 1972, C L E W E L L and F B A N K E 1974, HORODNICEANU et al. 1976, B E H N K E et al. 1979b). These plasmids mediate inducible or constitutive resistance to macrolide antibiotics, lincosamides, and streptogramin B. The mechanism of MLS-resistance has been shown to be a methylation of the 23 S rRNA — the target of the antibiotic action ( W E I S B L U M 1975, SHIVAKUMAE and D U B N A U 1981). DNA sequencing of an MLS-resistance gene from a staphylococcal plasmid has recently led to a model of translational attenuation being the mechanism of inducibility ( G R Y C Z A N et al. 1980, HORINUCHI and WEISLBTTM 1980). Excellent mutational analysis together with DNA sequencing has demonstrated the accuracy of this model ( H A H N et al. 1982). Several streptococcal MLS-resistance plasmids were found to have an interesting structural feature: the presence of long inverted repeat sequences ( B E H N K E et al. 1979b, B O I T S O V et al. 1979, GOLUBKOV et al. 1982). These inverted repeats covered as much as 80—90% of the plasmid molecules. In vitro and in vivo rearrangement of one such plasmid, pSM19035, has resulted in a set of deletion mutants which almost entirely consisted of sequences which were originally present as inverted repeats on the parental pSM19035 ( B E H N K E et al. 1980, 1979a). Only the MLS-resistance determinant which has been identified by cloning experiments ( B E H N K E and F E R R E T T I 1980) remained as a unique sequence on all plasmids. These data raised questions on the location and activity of functions involved in plasmid replication and maintenance. In this communication we describe the construction of small derivative plasmids of p8M19035 which allowed the localization of replication and copy control functions to a DNA sequence present on the parental plasmid in duplicate and in an inverted orientation. In addition small plasmids were obtained which carried double or even 36»

Zeitschrift für Allgemeine Mikrobiologie

23

1983

9

539-547

(Akademie der Wissenschaften der D D R , Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und experimentelle Therapie, J e n a , Direktor: Prof. Dr. U . TAUBENECK)

Double or triple sets of replication functions as inverted and direct repeats on in vitro reconstructed streptococcal MLS resistance plasmids D . BEHNKE a n d S. KLAUS

(Eingegangen

am 2. 5.

1983)

In vitro rearrangement of plasmid pDBIOZ together with comparative studies of other streptococcal plasmids allowed the localization of replication and copy control functions on sequences and its naturally occurring ancestor p8M19035 as duplicates in which were present on pDB102 inverted orientation. E v i d e n c e is presented t h a t neither the presence of duplicate replication regions nor their arrangement in inverted orientation was essential for plasmid survival. A m o n g t h e in vitro reconstructed plasmids were several t h a t stably carried t w o or three sets of replication and copy control functions either as inverted or direct repeats or both. A copy control m u t a t i o n is described which led to a tenfold increase of copy number over t h a t of the naturally occurring plaspSM19035. mid

Among antibiotic resistance plasmids isolated from streptococcal strains the socalled MLS-resistance plasmids prevail (COTJRVALIN et al. 1972, C L E W E L L and F B A N K E 1974, HORODNICEANU et al. 1976, B E H N K E et al. 1979b). These plasmids mediate inducible or constitutive resistance to macrolide antibiotics, lincosamides, and streptogramin B. The mechanism of MLS-resistance has been shown to be a methylation of the 23 S rRNA — the target of the antibiotic action ( W E I S B L U M 1975, SHIVAKUMAE and D U B N A U 1981). DNA sequencing of an MLS-resistance gene from a staphylococcal plasmid has recently led to a model of translational attenuation being the mechanism of inducibility ( G R Y C Z A N et al. 1980, HORINUCHI and WEISLBTTM 1980). Excellent mutational analysis together with DNA sequencing has demonstrated the accuracy of this model ( H A H N et al. 1982). Several streptococcal MLS-resistance plasmids were found to have an interesting structural feature: the presence of long inverted repeat sequences ( B E H N K E et al. 1979b, B O I T S O V et al. 1979, GOLUBKOV et al. 1982). These inverted repeats covered as much as 80—90% of the plasmid molecules. In vitro and in vivo rearrangement of one such plasmid, pSM19035, has resulted in a set of deletion mutants which almost entirely consisted of sequences which were originally present as inverted repeats on the parental pSM19035 ( B E H N K E et al. 1980, 1979a). Only the MLS-resistance determinant which has been identified by cloning experiments ( B E H N K E and F E R R E T T I 1980) remained as a unique sequence on all plasmids. These data raised questions on the location and activity of functions involved in plasmid replication and maintenance. In this communication we describe the construction of small derivative plasmids of p8M19035 which allowed the localization of replication and copy control functions to a DNA sequence present on the parental plasmid in duplicate and in an inverted orientation. In addition small plasmids were obtained which carried double or even 36»

540

D . BEHNKE a n d S. KLAUS

triple sets of these sequences either in direct or inverted orientation or both. From the data presented it was concluded that a single set of replication and copy control functions was sufficient for plasmid survival. Materials

and methods

Bacterial strains and media: Bacterial strains used in this study as a recipient in transformations or sources of plasmid DNAs are listed in Table 1. Brain heart infusion broth (DIFCO) supplemented with 2% of horse serum was used to grow all strains. Isolation of plasmid DNA: Plasmid DNA was isolated from C H A L L I S strains by the same procedures t h a t have been described previously ( B E H N K E and GILMORE 1 9 8 1 ) . Purified plasmid DNAs were stored at 4 °C in 10 MM Tris-HCL, p H 7.4. In vitro manipulation of plasmid DNA: Rearrangement of plasmid DNA sequences was accomplished by partial or complete cleavage with restriction enzymes specified in the Results section and subsequent treatment with T4 DNA ligase. Buffers and conditions used for restriction enzyme treatment and ligation were exactly as outlined in the Cold Spring Harbor "Manual of Advanced Genetics (R. W . D A V I S , D. B O T S T E I N , J . R. R O T H , Eds.) published by Cold Spring Harbor Laboratories, 1980. Transformation: Transformation procedures have been described in detail before (BEHNKE and GILMORE 1981).

Electron microscopy: The formation of fold-back molecules was accomplished by reannealing of denatured plasmid DNA for 5 — 10 min at 0 °C. Plasmid DNA was mounted for electron microscopy by the formamide method described elsewhere ( W E S T M O R E L A N D etal. 1 9 6 9 , D A V I S et al. 1 9 7 1 ) . The formamide concentration used for reannealing and spreading was 60% while the hypophase contained only 3 0 % formamide. Plasmid pBB 322 ( 4 3 6 2 b p ; STTTCLIFFE 1 9 7 9 ) served as an internal length standard. Table 1 Streptococcal strains used in this investigation Strain 8. 8. 8. S. S. 8.

sanguis sanguis sanguis sanguis sanguis sanguis

Designation

(CHALLIS) (CHALLIS) (CHALLIS) (CHALLIS) (CHALLIS) (CHALLIS)

Plasmid none pDB102 pDB1021 pDB1022 pDB10241 pDB1026

SM102 SM1021 SM1022 SM10241 SM1026

Reference

B E H N K E et al.

this this this this

(1979 a)

paper paper paper paper

Results O r i g i n a n d c h a r a c t e r i s t i c s of

pDB1021

The origin of p8M19035 and its deletion derivatives pDBlOl, pDB102, and pDB103 has been described previously (BEHNKE et al. 1979a, b). To further reduce the plasmid size and to obtain derivatives consisting of essential sequences only pDB102 (17. 4 kb) was digested with H i n d l l l to completion and subsequently treated with T4 DNA ligase (Fig. 1). Among the transformants one clone was identified which carried a considerably smaller plasmid designated as pDB1021. The size of pDB1021 was determined by contour length measurement to be 7.65 + 0.21 kb (n = 48). This value was in good agreement with estimations based on restriction enzyme analysis. Cleavage oipDB1021 with H i n d l l l revealed four fragments three of which were identical in size with the original H i n d l l l fragments B, D, and F of pSM19035 or pDB102, respectively. The fourth fragment turned out to be a second F complement which, however, had suffered a small deletion of about 80 — 100 bp (Fig. 1). Further restriction enzyme analysis of

Streptococcal MLS resistance Plasmids

Hindlll

541

cleavage

ligation

Fig. 1. Origin of plasmid pDB1021. Digestion of pDB102 by H i n d l l l to completion followed by ligation and transformation gave rise to a clone carrying pDB1021. Arrows indicate H i n d l l l cleavage sites. H i n d l l l fragments were designated by the same letters used for the parental plasmid pSM19035 (BEHNKE et al'. 1979a). Black regions indicate inverted repeats. Fragment X of pDB102 represents a fusion fragment most or all of which also contributed to the inverted repeats. The hatched area within the F fragment of pDB1021 corresponds to the region of nonhomology stemming from the 80 — 100 bp deletion in ZlF

pDB1021 with the enzymes Avail, B e l l , Haell, Hindll, Hpal, Kpnl, and PvuII proved the identity of the pDB1021 Hindlll fragments with the original fragments B, D, and F of pSM19035. A detailed map of restriction enzyme cleavage sites of plasmid pDB1021 is shown in Fig. 2. The two F complements on pDB1021 were present as inverted repeats. Besides that the Hindlll fragment D was known to carry short flanking inverted repeats (BEHNKE and FERRETTI 1980) which added to the total length of inverted sequences on pDB1021. The inverted orientation of these fragments was concluded from restriction enzyme analysis and directly shown by electron microscopy of selfannealed pDB1021 molecules (Fig. 3). The size estimations for the different domains of the selfannealed molecules corresponded well to the length which was predictable from restriction enzyme analysis (see legend to Fig. 3). Among the four Hindlll fragments comprising pDB1021 only B and F were linked as originally on the parentalpDBl02 (a,ndpSM19035, respectively). All other Hindlll sites represented artificial connections that destroyed sequence continuity initially present on pSM19035. This fact seems worth to be pointed out with respect to considerations on the location of biological functions on the pDB1021 molecule. L o c a t i o n of b i o l o g i c a l f u n c t i o n s on

pDB1021

The localization of replication and copy control functions on the pDB1021 map as outlined in Fig. 2 was derived from homology studies between pDB1021 and the wellcharacterized streptococcal cloning vector pGB301 (BEHNKE and GILMORE 1981). Comparison of the physical maps of pDB1021 and pGB301 revealed a striking similarity of arrangement and spacing of a number of restriction enzyme cleavage sites on Hindlll fragments F/AF of pDB1021 and that region of pGB301 where replication and copy control functions had been mapped by deletion analysis (Fig. 4 ; BEHNKE and GILMORE 1 9 8 1 ) . Hybridization of purified Hindlll F/AF fragments of pDB1021 to Hindlll cleaved pGB301 plasmid DNA by using the Southern technique revealed a strong homology of the two pDB1021 fragments with the pGB301 replication and copy control region (GILMORE etctl. 1 9 8 2 ) . The replication and copy control functions

542

D . BEHNKE a n d S. KLAUS

Hall

All

Avail

B1

Bel 1

HI

Hpa 1

H II

Hindll

Hill

Hindlll

Hall

Haell

K1

Kpn 1

Pll

Pvull

HI-HII Fig. 2. Restriction enzyme cleavage site map of plasmid pDB1021. The locations of biological functions on the pDB1021 molecule are indicated by the inner circle segments: Rep — replication functions, cop — copy control region, MLS R — MLS-resistance determinant. Since the exact boundaries of these functional regions were not known this is indicated by broken lines. Inverted repeats are shown as black areas (F and . IF referring to the H i n d l l l fragments). The hatched area within the F-fragment designates the region within which nonhomology was caused by the deletion on the second F complement

Fig. 3. Electron micrograph of a selfannealed pDB1021 molecule. Contour length measurements of the different domains of this structure gave rise to the following sizes US,: 2.49 ± 0.19 kb, US2 1.23 ~ 0.14 kb and ds-stem 1.75 ^ 0.17 kb (n = 30). Arrows indicate junction points between double-stranded regions and single-stranded loops

543

Streptococcal MLS resistance plasmids

_ xm }

x _ xxxix

x E x • •—•

|

i

• ^ /

i

i—•

t

pDB1021

• • • ^ i^j
silica gel sheets (25 x 25 cm, MERCK, F R G , precoated) a n d r u n in benzene/acetone (5:3, v/v). The products of b y c o n v e r s i o n were identified f r o m their pertinent Ry values (GRAFE et al. 1980 b) u p o n a u t o r a d i o g r a p h y . The radioactive zones were scraped off f r o m t h e c h r o m a t o g r a m a n d eluted w i t h 15 ml of methanol. T h e residue of extract was t r a n s f e r r e d t o 10 ml of scintillation cocktail (11 toluene, 5 g P P O , 0.3 g P O P O P ) a n d measured using a L K B 4100 LSC counter. Staining of D D A H - P l - l I on t h e t.l.c. sheets was carried out with a solution of 1 % (w/v) vanillin in concentrated H 2 S() 4 (GRAFE et al. 1980b).

Results and discussion I n the biogenesis of the leueomyeins containing 16-membered macrocyclic aglyeones, acetylation or propionylation of the 3—C—OH group has been established as one of the final steps of the biosynthetic pathway (KITAO et al. 1979a, b, c). Moderate specificity of the 3—O-aeylating enzyme has been suggested in our recent paper (GBAFK et al. 1980b) reporting on 3 —O-acylation and 14—C-hydroxylation of 5 - 0 (4', 6'-dideoxy-3'-C-acetyl-|5-i)-xylohexopyranosyl)-platenolide-II (DDAH-Pl-II, l . Fig. 2) by growing mycelium of S. hygrosr.opicus I M E T 10-2 2.5 • 10- 2

S t i m u l a t i o n of t r a n s f o r m a t i o n b y e l e c t r i c f i e l d p u l s e s The high resistance of protoplasts and the stimulation of protoplast regeneration by the electic field pulses suggested the application of comparatively high field strengths in the transformation experiments (10 and 14 kV/cm). In these experiments plasmid DNA from B. thuringiensis transformants was transferred into protoplasts of B. cereus by application of electric field pulses under optimum conditions for transformation, namely high protoplast titer (4 • 108/ml) and high concentration of plasmid DNA (50 (j.g/ml). The results of these electrically stimulated transformation experiments are given in Table 2.

598

N. SHIVABOVA et al.

Table 2 Dependence of transformation frequency of B. cereus protoplasts with plasmid DNA from B. thuringiensis on initial electric field strength Initial electric field strength

E 0 /kV • cm-

1

Transformation frequency

1.3 • 10-« 1.1 • io- 43 1.1 • io-

0 (control)

10 14

The incubation of protoplasts with the transforming DNA prior to the pulse application seems to be necessary for adsorbing the DNA on the protoplast surface. The enhancement of transformation frequency by about one order of magnitude after three electric field pulses of 14kV/cm probably is due to an increased penetration of extracellular DNA into the cell interior. However, the detailed mechanism of DNA transport through the cell membrane has not yet been established. NEUMANN et al. (1982) suggest the electroporation model of interaction of the external electric field with the lipid dipoles of a membrane pore configuration which induces and stabilizes penetration sites and thus enhances transport across the membrane. The kanamycine-resistant transformants still were stable after 10 passages on selective as well on non-selective media which proves that the pUB 110 is maintained steadily as an extrachromosomal element. Up to now the microscopic study of transformants obtained without and with electric field pulses did not show the presence of toxic protein crystals which are typical for B. thuringiensis. The results of an electrophoretic analysis of the obtained transformants are shown in Fig. 3. In (I) one can see the band of pUB 110 (A) and bands belongig to some smaller W

T

2T M

E

I

•0

'C

Fig. 3. Electrophoresis on 0.7% agarose gel in Tris-borate buffer, 35 V, 16 — 18 hours. I = The total DNA of the transformants of Bacillus thuringiensis (A — pUB 110, B, C, D, E — plasmids of the donor strain), I I = recipient strain Bacillus cereus, I I I — V I I = transformants of B. cereus

Electric field stimulated plasmid transformation

599

1er plasmids of t h e donor strain (B, C, D , E). N o plasmid D N A is present in t h e recipient strain B. cereus (II). All the transformants of B. ce.re.us (III t o V I I ) contain t h e p U B 110 D N A , but some of the smaller plasmids of the total plasmid D N A used for the transformation sometimes are lacking. W e are n o t able a t present t o decide whether the lack of some plasmids in the transformants is the reason for t h e lack of crystal protein formation. Nevertheless, our investigation clearly demonstrates t h a t t h e application of electric field pulses enhances the transformation frequency and t h a t this technique represents a v e r y promising tool in genetic transfer work.

A

cknowledgement

We thank Prof. H. BERG for valuable advice and fruitful discussions.

References CHANG, S. and COHEN, S. N., 1979. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen. Genetics, 168, 111 — 115. DEBABOV, V . G . , LINDEMAN, L . F . , HLEBALINA, R . R . a n d KOTOVA, T . C., 1979. U s i n g of t h e s y s t e m

transcription-translation for explanation of the role of some cryptic plasmids of Bacillus thuringiensis. Mol. Biol., IB, 1230—1236. FODOR, K. and ALEÖLDI, L., 1976. Fusion of protoplasts of Bacillus megatherium. Proc. Nat. Acad. Sei. USA, 73, 2 1 4 7 - 2 1 5 0 .

GÖNS ALES, J . M . J r . , DULMAGE, H . T . a n d CARLTON, B . C., 1981. C o r r e l a t i o n b e t w e e n s p e c i f i c

plasmids and endotoxin production in Bacillus thuringiensis. Plasmid, 5, 351—365.

GRYCZAN, T . J . , CONTENTE, S. a n d DTJBNAN,D., 197 8 . C h a r a c t e r i z a t i o n of Staphylococcus

plasmids introduced by transformation into B. subtilis. J . Bacteriol., 134, 318—329.

MARTIN, P . A. W., LOHR, J . R . a n d DEAN, D. H . , 1981. T r a n s f o r m a t i o n of Bacillus

protoplasts by plasmid DNA. J . Bacteriol., 145, 980—983.

aureus

thuringiensis

MITEVA, V . I . , SHIVAROVA, N . I . a n d GRIGOROVA, R . T . , 1981. T r a n s f o r m a t i o n of Bacillus

ringiensis protoplasts by plasmid DNA. FEMS Letters, 12, 253—256.

thu-

NEUMANN, E . M . , SCHAEEER-RIDDER, M . , WANG, Y . a n d HOESCHNEIDER, P . H . , 1 9 8 2 . G e n e t r a n s -

fer into mouse lyoma cells by electroporation in high electric fields. EMBO-J., 1, 841—845. SCHNEPF, H. E. and WHITELEY, H. R., 1981. Cloning and expression of the Bacillus thuringiensis crystal protein gene in E. coli. Proc. Nat. Acad. Sei. USA, 78, 2893—2897.

SHIVAROVA, N. I., GRIGOROVA, R . T. a n d MITEVA, V. I., 1983. T r a n s f o r m a t i o n of Bacillus

protoplasts with plasmid DNA of transformants of Bacillus thuringiensis. bulgarica, 13 (in press).

cereus

Acta microbiol.

WEBER, H., FÖRSTER, W., JACOB, H . - E . a n d BERG, H., 1981. Stimulation of y e a s t p r o t o p l a s t fusion

by electric field pulses. Z. Allg. Mikrobiol., 21, 553—562.

ZAKHARYAN, K . A., ARABALYAN, A. S. a n d CHIL-ACOPYAN, L. A., 1976. Possible role of extra-

chromosomal DNA in the formation of the entomocidal endotoxin of Bacillus Dokl. Acad. Nauk Arm. SSR, 61, 42—47.

thuringiensis.

ZIMMERMANN, U . , PTLWAT, G . , BECKERS, F . a n d RIEMANN, F . , 1976. E f f e c t s o f e x t e r n a l e l e c t r i c

fields on cell membranes. Bioelectrochem. Bioenergetics, 3, 58—83.

Mailing address: Dr. N. SHIVAROVA Institute of Microbiology of the Bulgarian Academy of Sciences 1113 Sofia, Bulgaria

Zeitschrift f ü r Allgemeine Mikrobiologie

23

9

1983

600

Buchbesprechungen

R . K . SCOPES, Protein Purification, SPrincipIes and Practice. X I I I + 282 S., 148 Abb., 23 Tab. Berlin-Heidelberg-New York 1982. pringer-Verlag. DM 79,00. Die Auftrennung komplexer Proteingemisehe und die Gewinnung hochreiner Einzelkomponenten gehören bereits zu den Routineaufgaben vieler, in der mikrobiologischen und molekularbiologischen Forschung angesiedelter biochemischer Laboratorien. E s ist insbesondere die stürmische Entwicklung der Gentechnologie, die die Ausweitung der Kenntnis und Anwendung präparativbiochemischer Methoden fördert. Das vorliegende, anschaulich illustrierte Lehrbuch vermittelt in systematisch gegliederter Form einen sachlich ausgezeichnet fundierten Überblick über analytische und präparative Methoden der Proteinbiochemie bis hin zu speziellen Kunstgriffen des experimentellen Arbeitens. E s empfiehlt sich daher vor allem f ü r Studenten biochemisch orientierter Forschungsrichtungen als eine Einführung in Laboratoriumstechniken, aber auch als Vorlage f ü r den Lehrenden zur Gestaltung von Vorlesungen und Praktika. Ausgehend von allgemeinen Informationen zur Ausstattung eines Labors f ü r Proteinbiochemie und den physikalisch-chemischen Prinzipien der Stofftrennung (Kap. 1) werden im K a p . 2 Methoden des Zellaufschlusses vorgestellt. K a p . 3 behandelt Proteinfraktionierungen durch Präzipitationsmethoden, während K a p . 4 der Theorie und Praxis chromatografischer Verfahren (Ionenaustausch- und Affinitätschromatografie, einschließlich Farbstoffliganden- und Immunoadsorbens-Techniken) gewidmet ist. K a p . 5 wurde der Gelfiltration sowie elektrophoretischen Trennntechniken vorbehalten (Gelelektrophorese, isoelektrische Fokussierung, Isotachophorese). K a p . 6 betrifft die Stabilisation von Proteinen, K a p . 7 dagegen die Maßstabsvergrößerung von Proteinreinigungsverfahren. K a p . 8 und 9 gehen schließlich auf die Enzymkinetik, Aktivitätsbestimmungen von Enzymen und analytische Methoden der Charakterisierung hochreiner Proteine ein. Appendices in Tabellenform geben Auskunft über die Herstellung von Ammoniumsulfatlösungen und Proteinbestimmungsmethoden. 204 spezielle Literaturstellen am Schluß des Buches sowie Literaturhinweise zu jedem Kapitel unterstützen die Aneignung vertiefender Informationen. U . GRÄFE ( J e n a )

E.SERFLING, S t r u k t u r und Expression der Gene höherer Organismen. 308 S., 58 Abb., 28 Tab. J e n a 1982. V E B Gustav Fischer Verlag. M 49,00. Der vorliegende B a n d gibt eine sehr ausführliche Übersicht des neuesten Wissensstandes zur S t r u k t u r und Expression eukaryotischer Gene. Notwendigerweise m u ß t e der Autor sich dabei auf eine Vorstellung weniger Gene beschränken. Die Auswahl der beschriebenen Gensysteme erfolgte so, daß ein repräsentativer Überblick über die Funktion u n d Organisation eukaryotischer Gene gegeben wird. Allerdings wäre eine Darstellung von gut untersuchten Genen niederer Eukaryoten (z. B. Alkoholdehydrogenase- oder Cytochrom c-Gene in Hefe) in einem gesonderten Kapitel eine wichtige Erweiterung. Auf die Beschreibung der Struktur und Expression extranucleärer Gene (z. B. mitochondrialen Genoms) wird verzichtet, vermutlich weil dies einer gesonderten Darstellung bedarf. Hervorzuheben ist das Bemühen des Autors, in einem vorangestellten allgemeinen Teil das zum Verständnis notwendige Umfeld unter Einbeziehung neuester Ergebnisse dem Leser nahezutringen. I m Teil C erfolgt vorteilhafterweise eine Vorstellung und Erläuterung molekulargenerischer Arbeitstechniken, und es werden die Möglichkeiten ihrer Anwendung beschrieben. Dies bemöglicht dem Leser, der nicht mit diesen Methoden vertraut ist, ein besseres Verständnis der dargestellten experimentellen Ergebnisse. G. BAETH (Jena)

Zeitschrift für Allgemeine Mikrobiologie

Kurze

23

9

1983

601—603

Originalmitteilung

(Laboratory of Microbiology, Gulbenkain Institute of Science, Oeiras, Portugal)

Use of D-glucosamine and 2-deoxyglucose in the selective isolation of mutants of the yeast Lipomyces

starkeyi

derepressed for the production of extracellular endodextranase

A . L A I R E S , I . S P E N C E R - M A R T I N S a n d N . VAN U D E N

(Eingegangen am 24.

3.1983)

The glucose analogues 2-deoxyglucose and D-glucosamine repressed the production of extracellular endodextranase in the yeast Lipomyces starkeyi. Though both analogues were successfully used for the selective isolation of derepressed and hyperproductive mutants, 2-deoxyglucose proved to be superior to D-glucosamine with respect to effective concentration and the avoidance of background growth. The glucose analogue 2-deoxyglucose has been used successfully for the selective isolation of mutants of yeasts and other fungi derepressed for the production of enzymes acting on carbohydrates: invertase (ZIMMERMANN and S C H E E L 1977), /5-glucosidase ( M O N T E N E C O U R T and E V E L E I G H 1979, B E J A DA COSTA and VAN U D E N 1980, L O U R E I R O - D I A S 1982), amylases (VAN U D E N et al. 1980, C A B E Q A - S I L V A 1982), maltase, invertase and galactokinase (BAILEY et al. 1982). M I C H E L S and R O M A N O W S K I ( 1 9 8 0 ) and H O C K N E Y and F R E E M A N ( 1 9 8 0 ) used the glucose analogue D-glucosamine in a similar way for the selective isolation of mutants of Saccharomyces cerevisiae resistant to carbon catabolite repression of a number of enzymes. Here we report on the use of D-glucosamine as compared with 2-deoxyglucose, for the selective isolation of mutants of the yeast Lipomyces starkeyi derepressed and hyperproductive with respect to the production of an extracellular endodextranase. This enzyme acts in a random way on dextrans producing glucose and isomaltooligosaccharides up t o isomaltohexaose ( W E B B and S P E N C E R - M A R T I N S 1 9 8 3 ) . Parent strain IGC 4047 of Lipomyces starkeyi was originally received from the Yeast Division of the Centraalbureau voor Schimmelcultures, Delft, The Netherlands, under number CBS 2512. The parent strain was grown with shaking at 25 °Cina liquid mineral medium with vitamins and 2 % (w/v) glucose (MV medium, VAN UDEN 1967). In the mid-exponential phase the cells were harvested by centrifugation, washed with sterile water and resuspended in distilled water. Of this suspension 0.1 ml samples containing approximately 3 x 106 viable cells were spread on the surface of plates of the following medium: 1 % (w/v) dextran T 70 (PHARMACIA), 0.3% (w/v) D-glucosamine (SIGMA) or 0.01% (w/v) 2-deoxyglucose (SIGMA), minerals and vitamins in water as in MV medium, 2 % (w/v) agar (DIFCO). The plates were irradiated with ultraviolet light during a period long enough (45 s under our conditions) to reduce the viability of the inoculum to about 10%. Incubation was at 25 °C in the dark. Colonies of mutants resistant to the repressor were picked, purified and maintained on the same medium containing the repressor. To test for derepression the mutant strains were plated on the following medium: 0.8% (w/v) B l u e d e x t r a n T 2 0 0 0 (PHARMACIA), 0 . 2 % (w/v) d e x t r a n T 2 0 0 0 (PHARMACIA), 1 % (w/v) g l u c o s e ,

MV medium, 2 % (w/v) agar. Derepressed mutant colonies produced colourless halos. To test for hyperproductivity the mutant strains were grown at 25 °C with shaking in liquid MV medium containing 0.5% (w/v) dextran T 70 (PHARMACIA). In the early stationary phase samples were centrifuged and endodextranase activity assayed in the supernatant.

Zeitschrift für Allgemeine Mikrobiologie

Kurze

23

9

1983

601—603

Originalmitteilung

(Laboratory of Microbiology, Gulbenkain Institute of Science, Oeiras, Portugal)

Use of D-glucosamine and 2-deoxyglucose in the selective isolation of mutants of the yeast Lipomyces

starkeyi

derepressed for the production of extracellular endodextranase

A . L A I R E S , I . S P E N C E R - M A R T I N S a n d N . VAN U D E N

(Eingegangen am 24.

3.1983)

The glucose analogues 2-deoxyglucose and D-glucosamine repressed the production of extracellular endodextranase in the yeast Lipomyces starkeyi. Though both analogues were successfully used for the selective isolation of derepressed and hyperproductive mutants, 2-deoxyglucose proved to be superior to D-glucosamine with respect to effective concentration and the avoidance of background growth. The glucose analogue 2-deoxyglucose has been used successfully for the selective isolation of mutants of yeasts and other fungi derepressed for the production of enzymes acting on carbohydrates: invertase (ZIMMERMANN and S C H E E L 1977), /5-glucosidase ( M O N T E N E C O U R T and E V E L E I G H 1979, B E J A DA COSTA and VAN U D E N 1980, L O U R E I R O - D I A S 1982), amylases (VAN U D E N et al. 1980, C A B E Q A - S I L V A 1982), maltase, invertase and galactokinase (BAILEY et al. 1982). M I C H E L S and R O M A N O W S K I ( 1 9 8 0 ) and H O C K N E Y and F R E E M A N ( 1 9 8 0 ) used the glucose analogue D-glucosamine in a similar way for the selective isolation of mutants of Saccharomyces cerevisiae resistant to carbon catabolite repression of a number of enzymes. Here we report on the use of D-glucosamine as compared with 2-deoxyglucose, for the selective isolation of mutants of the yeast Lipomyces starkeyi derepressed and hyperproductive with respect to the production of an extracellular endodextranase. This enzyme acts in a random way on dextrans producing glucose and isomaltooligosaccharides up t o isomaltohexaose ( W E B B and S P E N C E R - M A R T I N S 1 9 8 3 ) . Parent strain IGC 4047 of Lipomyces starkeyi was originally received from the Yeast Division of the Centraalbureau voor Schimmelcultures, Delft, The Netherlands, under number CBS 2512. The parent strain was grown with shaking at 25 °Cina liquid mineral medium with vitamins and 2 % (w/v) glucose (MV medium, VAN UDEN 1967). In the mid-exponential phase the cells were harvested by centrifugation, washed with sterile water and resuspended in distilled water. Of this suspension 0.1 ml samples containing approximately 3 x 106 viable cells were spread on the surface of plates of the following medium: 1 % (w/v) dextran T 70 (PHARMACIA), 0.3% (w/v) D-glucosamine (SIGMA) or 0.01% (w/v) 2-deoxyglucose (SIGMA), minerals and vitamins in water as in MV medium, 2 % (w/v) agar (DIFCO). The plates were irradiated with ultraviolet light during a period long enough (45 s under our conditions) to reduce the viability of the inoculum to about 10%. Incubation was at 25 °C in the dark. Colonies of mutants resistant to the repressor were picked, purified and maintained on the same medium containing the repressor. To test for derepression the mutant strains were plated on the following medium: 0.8% (w/v) B l u e d e x t r a n T 2 0 0 0 (PHARMACIA), 0 . 2 % (w/v) d e x t r a n T 2 0 0 0 (PHARMACIA), 1 % (w/v) g l u c o s e ,

MV medium, 2 % (w/v) agar. Derepressed mutant colonies produced colourless halos. To test for hyperproductivity the mutant strains were grown at 25 °C with shaking in liquid MV medium containing 0.5% (w/v) dextran T 70 (PHARMACIA). In the early stationary phase samples were centrifuged and endodextranase activity assayed in the supernatant.

602

A. LAIBES, I . SPENCER-MARTINS and N. VAN UDEN

Endodextranase activity was estimated by incubating 2 ml of a 2 % (w/v) solution of dextran T 70 in 0.05 M citrate-phosphate buffer (pH 5.5) with 1 ml of the culture supernatant for 10 min at 50 °C. Production of reducing sugar was determined using the 3,5-dinitrosalicylic acid method (BERNFELD 1955). One unit of endodextranase activity is the amount of protein that produces 1 [¿mole of glucose equivalent under these conditions.

Preliminary experiments revealed that the parent strain was unable to grow on dextran medium containing 0.01% (w/v) 2-deoxyglucose while growth in glucose medium was only slightly inhibited. To obtain similar repressing effects with Dglucosamine, concentrations at least ten times higher had to be employed. On a total of-.40 plates containing deoxyglucose an average of 8 well-isolated mutant colonies appeared. This corresponds to a mutation frequency of about 0.0027% of the cell population that was still viable after irradiation. About 5 0 % of the mutant strains resistant to 2-deoxyglucose displayed derepressed behaviour on glucose-blue dextran plates. On the plates containing D-glucosamine the mutant colonies were not always identifyable by visual inspection due to the occurrence of diffuse background growth. Such growth was absent on the deoxyglucose plates. Thus it was not possible to calculate the mutation frequency on the glucosamine plates. Again, about 5 0 % of the mutant strains that were isolated displayed derepressed behaviour. One of the derepressed mutant strains was compared with the parent strain in a growth experiment at 25 °C with aeration and stirring in 500 ml ERLENMEYER flasks containing 200 ml MV medium with 0.2% (w/v) glucose and 0.4% (w/v) dextran T 70.

B

2jO-

100

60

1.0 -

UO

?

% F h 50 &

-

§0.2

•I I

I I I

20 I

3j0 100

n.—MI

I

L

I

I I

A

2.0 to

to 50

0.5 20

0.2 - 0.1

I

I JWRA-C

10 15 20 Time(h)

26

30 35

Fig. 1. Growth of Lipomyces starkeyi in minimal medium with glucose (0.2%) and dextran (0.4%). (O) population density; (A)glucose concentration;(•) extracellulardextranase activity. A: parent strain, B : derepressed mutant

Selective isolation of Lipomyces starkeyi mutants

603

As is shown in Fig. 1 the parent strain displayed diauxic growth while the mutant strain grew in a continuous way, the dextran being degraded in the presence of glucose. Seven derepressed mutants isolated from glucosamine plates were compared with the parent strain with respect to hyperproductivity of extracellular endodextranase. The best produced about 1.5 times the amount of endodextranase produced by the parent strain. I t is concluded that D-glucosamine is a useful agent for the selective isolation of yeast mutants resistant to carbon catabolite repression but is inferior, at least in L. starkeyi, to 2-deoxyglucose. While use of the latter at low concentrations produced clean plates with well-isolated mutant colonies, with the former at much higher concentrations the results were less clear-cut due to background growth. Some yeasts are able to metabolize 2-deoxyglucose ( S P E N C E R - M A R T I N S , unpublished). In such cases glucosamine, provided this analogue is not metabolized, constitutes a useful substitute of 2-deoxyglucose for the selective isolation of derepressed mutants.

References R. B . , B E N I T E Z , T. and W O O D W A R D , A., 1982. Saccharomyces cerevisiae mutants resistant to catabolite repression: use in cheese whey hydrolysate fermentation. Appl. Env. Microbiol., 44, 631-639. B E J A DA COSTA, M . and VAN U D E N , N . , 1 9 8 0 . Use of 2-deoxyglucose in the selective isolation of mutants of Trichoderma reesei with enhanced /S-glucosidase production. Biotechnol. Bioeng., 22, 2429-2432. B E R N F E L D , P . , 1 9 5 5 . Amylases,