A degU-containing SPß prophage complements superactivator mutations affecting the Bacillus subtilis degSU operon

Res. Microbiol. 1992, 143, 559-567

© INSTITUTPASTEUR/ELSEVIER Paris 1992

A degU-containing SP[~ prophage complements superactivator mutations affecting the Bacillus subtilis degSU operon L. Podvin and M. Steinmetz

Laboratoire de G~ngtique des Microorganismes, lnstitut National Agronomique Paris-Grignon, CNRS and INRA, 78850 fhivervaI-Grignon (France) SUMMARY The Bacillus subtilis degSU two-gene operon encodes a zwo-component regulatory system which positively controls expression of a variety of genes encoding degradative enzymes. Point superactivator mutations in either degS or degU increase the production of these enzymes. A specialized transducing SP~ phage which partially complements several of these mutations was isolated from a phage library of the B. subtilis chromosome. This phage was shown to contain the degU (wild-type) gene. This unexpected co-dominance is discussed.

Key-words: Operon, Bacillus subtilis, Transduction, Bacteriophage SP~; Cloning vectors, Signal transduction, Two-component signalling systems, degU gene, Co-dominance.

INTRODUCTION In Bacillus subtilis, the rate of synthesis of a class of degradative enzymes, both secreted and intracellular, is controlled by a signal transduction pathway involving at least 4 regulatory genes: degS, degU, degQ and degR (Steinmetz and Aymerich, 1990; Kunst et al., 1991). The expression of genes encoding the exoenzyme levansucrase and several proteases has been studied to analyse their control by the products of the deg genes. Increased expression is observed in strains carrying missense nmtations designated degS(Hy) and degU(Hy). The degS and degU genes constitute an operon (Henner et al., 1988 ; Msadek et al., 1990) and their products share sequence similarities with two families of pro-

Submitted April 3, 1992, accepted May 29, 1992.

teins that mediate responses to environmental stimuli. Members of these two families are often associated in pairs, for example NtrB-NtrC, CheA-CheY and EnvZ-OmpR (Stock et al., 1990). The mode of action of the DegS-DegU pair is similar to those of other such pairs : DegS is a kinase which phosphorylates the regulatory DegU protein (Mukai et ak, 1990; Dahl et aL, 1991) and can also catalyse dephosphorylation of the phosphorylated form of DegU (Tanaka et al.~ 1991). Phosphorylated DegU appears to be required to activate degradative enzyme genes (Kunst et al., 1991). Therefore, the degS(Hy) mutants may contain a constitutively active kinase or be deficient in dephosphorylation ; in degU(Hy) mutants, DegU could be active without ph~,sphorylation, phosphorylable by

560

L. P O D V I N A N D M. S T E I N M E T Z

kinases other than DegS, or resistant to dephosphorylation. At present, k'. is u n k n o w n whether DegU directly interacts with the cis-rcgulatory regions of the genes it controls and what signal(s) activate DegS.

type allele had been cloned. Experiments presented in this paper showed that d e g U + can partly complement degU(Hy) mutations.

This paper describes observations concerning the deg genes which have been serendipitously obtained by screening secretory mutants in B. subtilis. In Escherichia coli, hybrid proteins containing NH2-terminal sequences of secreted proteins fused to [3-galactosidase have proved useful in the genetic analysis of secretion. The toxicity generally associated with the expression of such hybrid gene has been used to select mutants (Bankaitis et al., 1985). A similar approach has been initiated for B. subtilis in our laboratory using the sucrose-inducible sacB gene, which encodes the exoenzyme levansucrase. A sacB'lacZ translational fusion, containing the 5' end of sacB fused to the lacZ gene, was constructed. High level expression of the hybrid gene resulted in an inhibition o f secretion and, subsequently, cell death (Zagorec and Steinmetz, 1990). This high (inducible) expression required both amplification of the fusion and the presence the degU32(Hy) mutation, which strongly enhances sacB transcription (Shimotsu and Henner, 1986). We have used this construct to identify B. subtilis genes involved in secretion.

MATERIALS AND METHODS

For this purpose a cloning system based on "prophage transformation" (Fujita et al., 1983 ; P o t h and Youngman, 1988) was constructed : a library of recombinant transducing phages was constructed, using a novel SP[3 phage cloning system and fragments o f B. subtilis chromosomal D N A cloned downstream from an I P T G dependent promoter. The library was then used to select suppressors of the sucrose-dependent lethality. Characterization of a suppressor recombinant phage showed that the d e g U wild-

bla Cam cat erm A Ery

= = = = = =

gene coding for ~,-lactamase(BLA). chloramphenicol. genecodingfor chloramphenicolacetyltransferase. gei~econferringresistanceto erythromycin. deletion. erythromycin.

Strains

B subtilis strains used in this study are listed in table I. E. coil strains TGI [A(lac-proAB) supE thi hsdDS, F'(traA36 proAB + laql q lacZAMlS)] (Gibson, 1984) and its derivative TG90 were used for plasmid construction and preparation. TG90 was obtained by introducing into TG1 the pcnB80 (low plasmid copy number) mutation present in P. Youngman's KE94 strain (Quinn et al., 1991) by cotransduction with a TnlO insertion (P. Stragier, personal communication). Genetic techniques

LB medium (Miller 1972) supplemented with appropriate antibiotics was used for selection of B. subtilis and E. coli transformants. Preparation of competent cells and transformation of B. subtilis were carried out as described by Anagnostopoulos and Spizizen (19~,1), 2~. subtilis transductants and transformants were selected on LB plates containing erythromycin (0.4 rag/i) or neomycin (5 mg/l). E. coli transformants were selected on LB plates containing ampicillin (50 mg/l). The alkaline lysis procedure of Birnboim and Doly (1979) was used for the extraction of plasmid DNA from E. co.q. Techniques for the use of B. subtilis phage SP[3 were those described by Poth and Youngman 0988), Rosenthal et al. (1979) and Zahler et al. (1982). The SP~ prophage carried the c2 mutation, which makes the phage repressor thermolabile. Lysates were therefore produced by a simple thermo-induction procedure (incubation at 50°C for 8 min). Strains containing an SP~ prophage with the c2 mutation can be easily cured of the prophage by the method described by Rosenthal et al. (1979). Lysogens were streaked for single colonies on LB plates at 52°C. Cured colonies were further checked for the loss of

IPTG LB Ned pcn Suc

= = = = =

isopropylthiogalactoside. Luria-Benani(medium). neomycin. plasmidcopy number. sucrose.

C O M P L E M E N T A T I O N O F degU(Hy) M U T A T I O N S I N B. SUBTILIS

561

Table !. B. subtilis strains used. Strain

Genotype

Source of reference

QBII2(II) n QBI27a QBI28a QBI53 PY480 G M 130 GM287 GM632 GM815

Zagorec and Steinmetz (1990) Aymerich et aL (1986) Aymerich et aL (1986) Laboratory collection Poth and Youngman (1988) Steinmetz et al. (1989) Crutz et al. (1990) Figure 1

LP0

trpC2 degU32(Hy) sacB::(pLZ2)n trpC2 leuA 8 degS2OO(Hy) sacB: :(pl ,G l 31) trpC2 leuA8 sacB::(pLGl31) trpC2 leuA8 degU31(Hy) SP~c2A2::Tn917 deg U32(Hy) sacB'-lacZ trpC2 sacB'-IacZ A(sacXY) A(ptsGHl)::erm PY480 with SP~c2A2::Tn917::'pTV21A2ABg' = SP~3rX0 PY480 with SP~c2A2::Tn917::'pTV21A2ABg'::erm = SP~3rX PY480 with sacB::(pLZ2)n and degU32(Hy)

LPI

LP0 without SP~ prophage

Figure l DNA from QB112(II) n was used to transform PY480 LP0 cured of the prophage

sacB::(pLGl31) signifiesthat one copy of the pLGI31 plasmid was inserted into the sacB gene; sacB::(pLZ2)~,signifiesthat several copiesof the pLZ2plasmidwereinsertedinto the sacBgene.The deg mutationshavebeensequenced(Henneret al., 198g; Msadeket al., 1990).

antibiotic resistance conferred by the prophage. The SP~ phage D N A was extracted as described by Poth and Youngman (1988). GM815 was poorly transformable (with a frequency 30 to 300 times lower than those of efficiently transformable B. subtilis strains). This property was also observed with other PY480 derivatives and other B. subtilis strains c o n t a i n i n g derivatives o f SP~c2A2::Tn917 (Steinmetz, Podvin and Bezzate, unpublished observations). Plasmids Plasmids pLGI 31 and pLZ2 containing translational s a c B ' l a c Z fusions have previously been described (Aymerich et al., 1986; Zagorec and Steinmetz, 1990). pLG 131 and pLZ2 contain, respectively, the first codon and the first 216 codons ofsacB upstream from (and fused in-frame to) lacZ. Plasmid pLP3 (fig. 2) is an integrative derivative of the replicative plasmid pDG148 (Stragier et aL, 1988). The 4.3-kb EcoR1 fragment containing the origin of replication of pUB1 l0 was deleted and replaced by the Neo R EcoRl cassette from pBEST502 (ltaya et al., 1989). Plasmid pTV21A2ABg is pTV21A2 (Yougman, 1987) deleted of a 1,878 bp Bg/II fragment. Plasmid pIC90 (fig. l), a derivative of pSL20 (Aymerich and Steinmetz, 1987), was constructed by substituting the ClaI-Sall fragment o f pSLI32 (Crutz et al., 1990) containing the pE194 erm gene for the SallEcoRI fragment containing the cat gene. The singlestrand termini of DNA cut with EcoRI or Clal were

end-filled using Klenow fragment before digestion with Sail.

Insertion of genomic fragments into pLP3 Total genomic DNA of B. subtilis GM287 was prepared and partially digested with Sau3A. Fragments between 3 and 10 kb were isolated by ultracentrifugation on a sucrose gradient and inserted into the Sail site located downstream from the spac promoter in pLP3. Sail- and Sau3A-generated cohesive ends were made compatible through partial fillingin with Klenow fragment (Zabrovsky and Allikments, 1986). The partial filling-in prevented self-ligation of either vector or inserts. The ligation was carried out at 200 mg/l DNA with similar amounts of insert and vector.

Media and phenotypic characterization on solid media C mineral medium has previously been described (Aymerich et aL, 1986). CG medium was C medium supplemented with 1 g/1 glucose and 10 mg/l tryptophan. Sucrose sensitivity was tested by replica plating clones on CG medium plates with or without sucrose (20 g/l) and with or without 1 mM IPTG. Proteolytic activity was assessed by the size of proteolysis halos around colonies grown or. LB plates supplemented with 5 g/l skimmed milk powder (Difco).

L. P O D V I N A N D M. S T E I N M E T Z

562

Determination of enzymatic activities in liquid medium Growth was monitored by optical density measurentents at 600 nm (OD6oo). 13-Galactosidase activity was determined in cells grown in C medium supplemented with 10 g/I sorbitol, 50 mg/l tryptophan and 2 g/l casein hydrolysate; expression of the sacB'-IacZ fusions was induced by addition of sucrose (20 g/l) at 0.20D6o o. At 10D60o, a 1-ml sample was removed; cells were then lysed as described by Aymerich et al. (1986); ~-galactosidase activity was determined by the method of Miller (1972), and expressed as Miller units/OD. Proteolytic activity: ceils were inoculated at 0.15 OD6oo and grown to an OD of 6 in double-strength sporulation medium (Schaeffer et al., 1965); extracellular proteolytic activity was then measured in supernatants by the method described by Kunst et al. (1974) except that azocoll (Sigma) was used as the substrate instead of azocasein; an increase in OD520 of 1 unit was arbitrarily defined as 1 unit of proteolytic activity. RESULrS Cloning by " p r o p h a g c t r a n s f o r m a t i o n " involves the ligation of genomic DNA fragments to fragments o f phage DNA, followed by transformation o f a host lysogenic for that phrage. The ligated phage ~equences allow genomic fragments to become integrated into the resident prophage by homologous recombination (Fujita et al., 1983; Poth and Youngman, 1988). In the variation o f this technique that we used (fig. 2), pBR322 sequences present both in the prophage, SP[3rX, and in the plasmid vector, pLP3, served as regions o f h o m o l o g y that allow "Campbell-type" insertion of recombinant plasmids into the prophage platform. SP[3rX is a derivative o f the t h e r m o - i n d u c i b l e S P ~ c 2 A 2 : : T n g l 7 prophage (Poth and Youngman, 1988), constructed as described in figure 1. The plasmid pLP3 contains the IPTG-inducible spac promoter, the lacI gene encoding the repressor o f s p a t (Yansura and Henner, 1984), pBR322 sequences and a gene conferring resistance to neomycin (fig. 2).

Construction of a B. subtilis genomic library in SP[~rX phage A genomic library was constructed in SP[3rX as shown in figure 2. DNA fragments were gene-

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Fig. l. Construction of the SPi3rX phage. GMSI5 carrying SP[3rXwas constructed in two transformation steps of strain PY480 using pTV21A2ABgand plC90. In the first step, GM632 was derived from PY480 (containing the thermo-inducible SP~c2A2::Tn917 prophage) by transformation with pTV21A2ABg to CamR and Erys, by a double recombinationevent. In the second step, GM815 was derived from GM632 by transformation with plC90 to EryR Cams, by a double recombination event. E and S = EcoRI and Sail sites, respectively;E/C = site of EcoRI and Clal extremitiesligated to generate plC90; ABg= position of the Bgnl deletion in pTV21A2ABgand plC90.

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GENOMIC LIBRARY= PHAGELYSATE

Fig. 2. Schemeof the construction of the genomiclibrary in SPi3rX. Chromosomal DNA fragmentswere generatedby partial digestion with Sau3A (1); they were inserted downstream from the spac promoter (2); the ligationmixturewas used to transform the lysogenicstrain GM815, the recombinant plasmids were integrated into SP~rX by "Campbell insertion" (3); and the resultingtransformantsselected for NeoR(4). Pools of Neort transformants were induced to produce phage lysates (5).

COMPLEMENTA TION OF degU(Hy) MUTATIONS IN B. SUBTILIS rated by partial Sau3A digestion of B. subtilis chromosomal DNA. Size selected fragments (3-10 kb) were inserted downstream from the spac promoter as described in "Materials and Methods". The ligation mixture was used to transform the lysogenic B. subtilis strain GM815. Transformants resulting from insertion of recombinant plasmid were selected for Neo R. Five independent pools of 103 to 3 × 103 transformants were obtained and were thermoinduced to produce lysates. The transducing phages contained in these lysates constituted the phage library (fig. 2). Bacteria transduced by this library were first selected on erythromycin, since selection by neomycin was unreproducible; second, the Ery R colonies were replicated on plates containing neomycin.

563

nal tests were performed: I, transductants were cured of their prophage and the loss of the Suc R phenotype was checked ; 2) a transducing lysate was prepared from individual clones and used tt~ transduce the LPI strain. Five transductant~ from 3 independent pools werc kept after these analyses. The suppression in the other 13 transductants could have resulted from a partial, previously undetected, deletion of pLZ2 sequences. We estimated that the transductants whose Sue R phenotype was due to a suppressor prophage represented about 0.05 % of Neo R LPI transductants selected in the absence of sucrose. One of the 5 suppressors, giving a complete Suc R phenotype, was the SP[~rX::pLP210 pl',age.

Characterization of the recombinant prophage SP~rX::pLP210 Screening of the library for suppressors of the

sacB'lacZ-dependent lethality The phage library was screened for clones suppressing the sucrose-dependent lethality of strain LP1 which carries the degU32 mutation and several copies of plasmid pLZ2 (bearing the sacB'lacZ fusion and a Cam R gene). Strain LP1 was infected with the lysate library and part of the transduction mixture plated on LB plates containing erythromycin. The remainder was used to select Suc a (sucrose-resistant) transducrants by plating on LB plates containing sucrose (20 q/l), erythromycin and 1 mM IPTG. The Suc R transductants were subsequently replicated on plates containing neomycin. The phenotypes of the NeoRSuc R transductants ranged from a complete resistance to sucrose to intermediary phenotypes (partial, but reproducibly observed, resistance). The Suc R phenotype could have been due to the loss of some or all of the copies of pLZ2 plasmid, inserted into the recipient chromosome (Zagorec and Steinmetz, 1990); most of these transductants were eliminated after identification by testing their growth on LB plates containing 50 mg/1 chloramphenicol. Of the remaining SucRNeo R transductants, 18 were chosen for further study. In order to identify transductants whose phenotype was due to the presence of a suppressor prophage, two additio-

SP~rX::pLP210 and the other 4 selected prophages conferred the Suc R phenotype to LPI in the absence or presence of IPTG. They also reduced the extraceUular proteolytic activity of strain LPI as assessed on solid medium. Because the cloned insert was flanked by two homologous pBR322 regions in recombinant phages (fig. 2), it was theoretically possible to recover the inserted plasmid by homologous recombination after transformation of a RecA + E. coli st~'ain with phage DNA. E. coli TG1 was used as the recipient but the plasmids obtained carried deletions, probably due to the toxicity of the insert. The deletions affected bo[h the lacI gene and most of the insert as demonstrated by the restriction map analysis. We therefore used TG90, a TGl derivative containing the pcnBSO mutation which decreases the copy number of pBR322 derivatives, an5 obtained the plasmid pLP210. The insert in pLP210 was 3.2 kb in length; its restriction map, established using 8 enzymes, showed that it contained part of the degS-degU locus; the insert (fig. 3) contained the degUand degS genes, including the minor promoter of degU present in the 3' end of degS (Msadek et al., 1990). Careful examination of the restriction map of pLP2!0 strongly suggested that it~ insert began at a Sau3A site between the (major) degS promoter of the operon and the degS

L. PODVIN AND M. STEINMETZ

564

ribosome-binding site; the absence of the promoter on the insert was attested by the absence of a Xbal site present just upstream from the relevant Sau3A site in the chromosome (Msadek et aL, 1990).

(fig. 3) did not play a role in the suppression and strongly suggested that the wild-type DegU protein, encoded by pLP210, acted as a suppressor.

A back-cross was performed to verify that pLP210 was responsible for the suppression of sucrose-induced lethality, pLP210 was inserted into SP[~rX by transformation of strain GM815, The resulting prophage suppressed the sucrosedependent lethality of LP 1. Two deletion derivatives of pLP210 were constructed with the aim of mapping the region responsible for the suppression. One derivative, pLP2100, was constructed by deletion of the 1-kb region downstream from the degU gene and a second, pLP2101, by deletion of the same region plus the spac promoter (fig. 3). These plasmids were integrated into SP~3rX by transformation of strain GM815. The resulting prophages retained the ability to suppress the sucrose-dependent lethality of LPI. This showed that the spac promoter and the region downstream from degU

Mechanism of suppression by SP~rX::pLP210

~11),~ Sphl

",, "', A2 ~1 '~

(Sail) / ~ J ~ .

NeoR ~

Fig. 3. Structureof plasmidpLP210rescued from phage SP~,rX::pLP210DNA after transformationof TG90. PlasmidspLP2100and pLP2101 wereconstructedby deletion of fragmentHindIII-HindIll(Al) and fragment HindllI-Xbal(A2), respectively.Pm indicatesthe minor promoter of degU. The shadowed regionscorrespondto the pLP3 vector.

Because DegU controls transcription ofsacB, we tested the hypothesis that the suppression by SP[3rX::pLP210 could be due to a reduction of the transcription of the sacB'-lacZ fusion in strain LPI. SP[~rX::pLP210 was used to transduce strain GMI30 containing a (non-lethal) sacB'-IacZ fusion and the degU32 mutation. The ~-galactosidase activity, after induction of the fusion by sucrose, and the proteolytic activity of transductants was measured. As shown in table II, the presence of SP[3rX::pLP210 in GMI30 resulted in a 5-fold reduction in the expression of the fusion and a 2-fold reduction in proteolytic activity. This result supported the idea that the suppression of LPI sucrose sensitivity by SP[3rX::pLP210 was due to reduced expression of the toxic fusion protein. It should be noted that, in a haploid background, the degU32 mutation results in a 100-fold increase in the expression of the sacB'-lacZ fusion (table II; Aymerich et al. (1986). Therefore, complementation of degU32 by SPI3rX::pLP210 is only partial. The suppressor activity of SPI3rX::pLP210 was tested in different deg(Hy) mutants. Both the degU31 and degS200 mutants were unambigously suppressed (table II). Suppression of degS200 suggested that the degS gene present in SPI3rX::pLP210 was not completely silent, despite being devoid of its promoter (Msadek et al., 1991). In a recent study, Tanaka et al. (1991) showed that the DegS200 protein was defective for the dephosphorylation of DegU and proposed that the phenotype of the relevant mutant was due to this defect. The partial complementation of degS200 by SPI3rX::pLP210 suggests that, even when weakly expressed (presumably from a promoter present in pLP3 or in the insertion platform), the wild-type degS gene can code for phosphatase activity sufficient to dephosphorylate DegU.

COMPLEMENTA TION OF degU(Hy) MUTATIONS IN B. SUBTILIS

565

Table 11. Expression of sacB'-lacZ and proteolytic activity in different genetic backgrounds.

Recipient

Relevant genotype

GMl28a GM130 GMI30 QBI53 QB153 QBI27a QBI27a

sacB'-IacZ degU32(Hy) sacB'lacZ de~'U32(Hy) sacB'-IacZ degU31(Hy) degU31(Hy) degS2OO(Hy) sacB'-IacZ degS2OO(Hy) sacB'-IacZ

Prophage

[3-galactosidase activity (Miller units/OD)

Proteolytic activity (unit/ml)

No prophage SP~rX::pLP3 $P[3rX::pLP210 SPi3rX::pLP3 SP~rX::pLP210 SP[3rX::pLP3 SPi3rX::pLP210

7 793 162 ND ND 1,012 188

ND 47 25 62 31 74 47

Strains weregrown, extracts were prepared and enzymaticactivitiesassayed as describedin "Materialsand Methods". ND= not determined.

Other suppressing prophages The plasmids contained in two other SP[3rX recombinant prophages suppressing sucrose sensitivity in LP1, and obtained from independent pool transformants, were cloned in E. coli TG90; both plasmids contained the degU gene; the inserts were different from each other and from that in pLP210 (data not shown). DISCUSSION The SP~c2A2::Tn917 phage and its derivatives are very useful tools: specialized transduction using these phages permits the introduction of genes into mutants deficient in transformation (such as degU mutants) or in recombination, or resistant to PBSl-mediated generalized transduction; strains containing those prophages can be easily cured. The engineered SP[3rXO and SP[3rX prophages present in strains GM632 and GMS15, respectively (fig. 1), contain a pBR322 region which can serve as an "insertion platform" for integrative plasmids derived from pBR322. Therefore, there are a variety of possible -'ases of these thermo-inducible prophages. Experiments were performed with the SPI3rX phage carried by the strain GM815. We constructed a phage library based on SPI3rX and an integrative plasmid containing fragments of the B. subtilis chromosome. One advantage of this

system is that plasmids present in the recombinant prophage can be rescued without restriction or ligation (see "Results"). E. coli TG90 could be used for the cloning and the maintenence of the degU gene on a multicopy pBR322 derivative. As confirmed here, this appears impossible in the more commonly used E. coil strains (Henner et al., 1988). In E. coli, the overexpression of heat-shock genes can facilitate the export of LacZ-hybrid proteins and can suppress the associated lethality (Philips and Silhavy, 1990). We initiated an approach in B. subtilis to clone genes possessing similar properties. We used a phage library constructed in SP~3rX to screen for suppressors of the toxicity of a sacB'-IacZ fusion. The observat_':on presented in this paper is an indirect spin-off of this approach which failed to identify genes involved in secretion. Recombinant suppressor prophages were selected and one was characterized in detail. This phage, SP~rX::pLP210, contained the degU ger.~.. Deletion experiments demonstrated that spac did not play a role in the suppression and strongly suggested that the degU + gene (under the control of its minor promoter) was sufficient to partially suppress the phenotype of the degU32 mutant. By studying the effet of SPI3rX: :pLP210 in different genetic backgrounds, we observed pardal suppression of two other deg(Hy) mutations, degU31 and degS200. 1) The suppression of degU32 (or

566

L. P O D V I N A N D M. S T E I N M E T Z

degU31) by d e g U + could be the result o f c o m petition between the m u t a n t a n d wild-type p r o teins for a D N A (or protein) target ; it is k n o w n that some bacterial transcriptional activators can bind to their D N A target in b o t h the active a n d inactive f o r m (Schleif, 1987; Storz et aL, 1990). 2) A n o t h e r possibility is that D e g U acts as an o l i g o m e r ; oligomers containing wild-type a n d m u t a n t subunits could be totally or partially inactive. A third possibility is that the two forms (phosphorylated or u n p h o s p h o r y l a t e d ) o f DegU could be involved (positively and negatively, respectively) in the regulation o f target genes including sacB; careful examination o f published data s h o w s that, even t h o u g h p h o s p h o r y l a t e d D e g U is very p r o b a b l y an activator o f d e g r a d a t i v e enzyme genes, an (additional) negative effect o f the u n p h o s p h o r y l a t e d f o r m has not been ruled out. These hypotheses are largely speculative and others could be imagined.

In conclusion, we would like to underline that genetic studies based on the characterization o f merodiploid strains can provide information that is praetica~ty impossible to obtain f r o m h a p l o i d strains an6 m a y provide guidance to in vitro approaches.

Acknowledgements We 'hank Mitsuhiro ltaya for the gift of plasmid pBEST502. Patrick Stragier for plasmid pDGI48 and strain TGg0, Alex Edelman for correcting the manuscript and Dominique Le Coq for his help in this work. This work was supported by funds from the Minist~re de la Recherche Scientifique et Technologique and the Centre National de la Recherche Scientifique. Laurence Podvin was supported by the Contrat Couples H6tes-Vecteurs Performams.

Un prophage contenant le g~ne degU compl6mente des mutations super-activatrices affectant I'op6ron degSU de Bacillus subtilis L'op6ron bi-cistronique degSU de Bacillus subtilis code pour un syst~me rdgulateur/t deux composants qui r6gule positivement l'expression de plusieurs g~nes d'enzymes d6gradatives. Des mutations superactivatrices ponctuelles dans degS ou degU augmentent le niveau de synth~se de ces enzymes. Un bact6-

riophage tranducteur sp6cialis6, qui compl6mente partiellement plusieurs de ces mutations, a 6t6 isol~ partir d'une biblioth6que g6nomique du chromosome de B. subtilis construite dans le bact6riophage SP~. Ce bact6riophage recombinant contient le g/me degU sauvage. Cette observation inattendue de codominance est discut6e. Mots-clds: Op~'~on, Bacillus subtilis, Tranduction, Bact6riophage SPI3; Vecteurs de clonage, Transduction signal, Syst6me/t 2 composants, G~ne degU, Codominance.

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