Gene. 94 (1990) 61-67 Elsevier

GENE 03683

61

Cloning, expression and sequence analysis of an endolysin-encoding gene of Lactobacillus bulgaricus bacteriophage my1

(Recombinant DNA; lyric enzyme; muramidase; coliphage 4)

Brigitte Boizet, Yvette Lahbib-Mansais, Laurence Dupont, Paul Ritzenthaler and Mireille Mata

Centre de Recherche de Biochimie et de Gdndtique Cellulaires du CNRS, 31062-Toulouse (France)

Received by J.-P. Lecocq: 27 December 1989 Revised: 2 February 1990 Accepted: 20 May 1990

SUMMARY

The lysA gene specifying an endolysin of Lactobacillus delbrueckii subsp, bulgaricus bacteriophage mvl, was cloned and expressed in Escherichia coli. The 4.05-kb restriction fragment containing this gene was analysed by restriction and deletion mapping, and by subcloning. The nucleotide sequence of a 1150-bp fragment coding for an active lysin was determined. The lysA gene consists of 585 bp and codes for a protein of a deduced Mr of 21 120, which agrees with the size based on in vivo transcription/translation studies. The deduced amino acid sequence of the mvl lysin (LysA) was compared to that of other known lyric enzymes. Significant homology was observed with the N-terminal portion of the muramidase of the fungus Chalaropsis and that of the muramidase of the Streptococcus pneumoniae phage Cp-l, suggesting that LysA might be a muramidase. In E. coil, the cloned lysA gene was able to complement the muramidase-defective bacteriophage ~.Ram5, proving that the products of these two genes are interchangeable. The iysA gene is preceded by an open reading frame with unknown function and no characteristic prokaryotic promoter sequences could be detected upstream from lysA, suggesting that this gene is part of an operon.

INTRODUCTION

The last step of phage infection is the release of mature phage particles out of the cell by lysis of the cell wail. Most often, this process is carried out by bacteriophage lyric enzymes (endolysins) which depolymerize the peptidogly- can. Phage mvl is a temperate phage of L. delbrueckii

Correspondence to: Dr. M. Mata, Centre de Transfert-lNSA, avenue de Rangueil, 31077-Toulouse (France) Tel. 33 6155 96 57; Fax 33 6155 95 00.

Abbreviations: aa, amino acid(s); pGai, p-galactosidase; bp, base pair(s); d, deletion; kb, kilobase(s) or 1000 bp; IPTG, isopropyi./?-D-thiogaiacto- pyranoside; L., Lactobacillus; LysA, bacteriophage mv I endolysin; lysA, gene encoding mvl endolysin; Mur, muramidase(s); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; RBS, ribo- some-binding site(s); SDS, sodium dodecyl sulfate; UWGCG, University of Wisconsin Genetic Computer Group; wt, wild type; [ ], denotes plasmid-carrier state.

subsp, bulgaricus and was shown to be closely related t~ the temperate phage mv4 and to the virulent phage LL-H (Mata et al., 1986; Lahbib-Mansais et al., 1988). All these phages belong to a widespread species designated group a. The gene coding for an endolysin was mapped on a 4.35-kb fragment of the LL-H chromosome (Trautwetter et ai., 1986). A DNA homology was demonstrated between this LL-H lysin encoding gene and one restriction fragment from each phage of group a, strongly suggesting that all these phages possess closely related genes encoding lysins (Mata et al., 1986).

A cloned lysin encoding gene from a Lactobacillus phage can be used for the construction of a positive selection vector in Lactobacilli. Due to the potential toxicity of the lysin protein, only insertionai inactivation of this gene will result in a recombinant plasmid able to propagate into these bacteria. In biotechnology, the availability of a lysin protein provides the opportunity to eliminate specific contaminant

0378-1119/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

62

species and to facilitate the release ofintracellular enzymes in dairy fermentations.

The aim of present study was to clone a DNA fragment from phage mvl which encodes and expresses a phage lyric enzyme in E. coil, and to determine the nt sequence oflysA, the lysin encoding gene.

RESULTS AND DISCUSSION

(a) Cloning of the mvl lysin in Escherichia coil BamHl fragments from phage mvl DNA were cloned

into the plasmid vector pBR322. Among the E. coil HB 101 transformants, one clone had a Lys + phenotype, i.e., was able to produce a halo of lysis when spotted on a lawn of phage indicator strain L. delbrueckii subsp, lactis LKT. The recombinant plasmid present in this clone had a 6.15-kb BamHI insert (Fig. 1) and was retained for further study. This fragment was subcloned into different vectors. The 4.05-kb HindIII-BamHI fragment cloned in the two oppo- site orientations into pTZISR and pTZI9R gave rise to respectively pTl and pT2. The cloned lysA gene was expressed whatever its orientation with respect to the T7 promoter of the vector, suggesting that this gene was trans- cribed from its own promoter. The 4.05-kb HindIII-BamHI fragment was subjected to restriction endonuclease map- ping and various deletions inside this fragment were con- structed (Fig. 1). The resulting plasmids and their pheno- type are indicated in Table I and Fig. 1. Plasmid pT2-2

TABLE I

Plasmids u

B H C I I I

B H I I

H C

1 kb

H G I I

lysA , - r

A A B Lys kb • ' I + p A l 6.16

pN1 2.1 A A B • " " + p T 1 / p T 2 4 . 0 5

o

, pT2-1 2.2 A B ! I . pTI-1 3.45

H A A B i , ~ / " ' " + pT2-2 1.2 z~

Fig. 1. Physical map of the phage mvl lysin region. The size of the mvl DNA inserts in the recombinant plasmids and the Lys phenotype are indicated. The arrow indicates the transcription direction for lysA gene. A, AccI; B, BamHI; C, Clal; H, HindIII. The construction of the recombinant plasmids is indicated in Table I. Plasmid pTl-I is derived from pTl by deletion of the Accl fragment. Piasmid DNA isolation, restriction endonuclease digests, DNA ligation and gel electrophoresis were performed as described by Maniatis et al. (1982). The Lys phenotype was detected by a bioassay as followed: 5 #1 of a 50-fold concentrated culture (Aeoo n m = 0.6) of the E. coil-producing strain were spotted on a plate, which was previously seeded by a culture of L. delbrueckii subsp. lactis strain LKT and incubated overnight at 40°C. ARer 6 h incubation at 37 ° C, halos oflysis appeared around the lysin-producing clones. LKT is the propagating host for phage mvl (Mata et al., 1986).

(1.2kb) was the smallest one which conferred a Lys ÷ phenotype.

(b) The nt sequence of the mvl lysA gene The nt sequence of the 1152-bp fragment of pT2-2 and

the computer predicted aa sequence are shown in Fig. 2. Only one ORF 0f significant length was found, starting by

Plasmid Size mvl DNA restriction designation (kb) fragment b

Parental vector (source, reference)

pAl 6.15 BamHl pN ! 2. I BamHI.Hindlll pTl 4,05 BamHI-HindIll pT2 4.05 BamHI.Hindlil pTl-I 3.45 BamHI-Hindlll

A0.6-kb Accl pT2-1 2.2 BamHI.Clal pT2-2 !.2 BamHI.HindIll, ABAL 31

from Clal site pT2-21 0.704 pT2-2 ABAL 31

from BamHI site pT2-24 0.868 idem pT2-21 PT2-223 0.93 ! idem pT2-21 pT2-214 1.005 idem pT2-21 pGPl-2

pBR322 (Bolivar et ai., 1977) pNM480-482 (Minton, 1984) pTZI8R (Mead et al., 1986) pTZI9R (Mead et al., 1986) derived from pTI

derived from pT2 derived from pT2

derived from pT2-2

derived from pT2.2 derived from pT2-2 derived from pT2-2 pl5A (Tabor and Richardson, 1985)

" The E. coli strains were HB 101 (Boyer and Roulland-Dussoix, 1969) for the transformation experiments and K38 (Russel and Model, 1984) for the in vivo expression experiments. Culture conditions for E. coil were as described by Maniatis et al. (1982). Lactic acid bacteria were grown in MRS (DeMan et ai., 1960) for Lactobacilii or MI7 broth (Terzaghi and Sandine, 1975) for Lactococcus and Streptococcus. b The restriction map ofplasmids pTl, pT2 and derivatives is given in Figs. I and 3. The BAL 31-deleted plasmids differed by the extent of the deletion. Plasmid pGPI-2 codes for T7 RNA polymerase under PL control.

63

A C I a l - - - ~ O R F I

I GACTCACTATAGGGAAAGCTTCTGACGACGGCGGTAAACGAAGCCGAACACAGCGACTTGCGGGCG&TGCAA A V N E A E H S D L R A N Q

~3 AGCGGGAATATGCCGCAGAAATCA'I"rGCTGAACAACTCCAACGCAAAGGAATTACCGGCATTACCAAGTCAA S G N H P Q g S L L N N S N A K g L P A ~ P S Q

145 CGGTTTACGGGGCAATTCAAGCCGCTTGGAAGGCCGC&GACTTCAACCATGGGGAAAAGCCCGCAGAGAGCA R F T G Q F g P L G a P Q T S T ~ G g S P Q • A

21y CGAAC&CGGTTTCT&GCG&TTC&GCAAACCGCTTG&CT&TGG&TG&CGC&CC&GTGG&GGTTAA?AAGAAC& e T a F L A Z Q Q T A - L w U T H 0 w e L e a T

~ L y e A 289 kTG&CTAAAACTT&TGG&GT&GkCGT&GC&GTTT&TC&GCCT&T&G&CTT&GCGGCTT&CC&CAAGGCGGGC

H T g T Y G V D V A V Y Q P Z D L A A Y H g A G

as1 GCAAGTTTTGCGATCGT,I~IJ~CTGACGGAAGGGG'IVI'GACTATGTCAACCGAAGGGGGCCAAGC&GGTGG&CA A S F A l V g L T E G V D Y V N R R G P S R W T

433 GCTCCAGGGCTAACCACCTCTACACTCATGCCTACCATTTCGCGCAGTTTTGGCTCATCTGTTAGCCGCGCC A P G L T T S T L N P T Z S R S F G S S V S R k

sos AAAAAAGAAGCGGC~rL'AC'I"I'C CTTAAGGAAGCCAAOAAGCAAGACA TTAGCAAGAAACGGA'JL'GCTTTGGC'JL'A g g K A A Y F r. g E k K g Q D Z S g K R N_ L W L

5)'l GACTGGGAAGCCGGTAGCGGCAATGTGGT,~CTGGGTCAAAGTC&TCCAACACGGCGGC,~XTCCTGG&CTTT D W E k G S G N V V T G S g S S N T k A l L D F

04o ATGGACGCGATTAAAGCCGCAGGCTGGCGGCCGGGTCTCTATAGCGGTGCATCCCTGATGCGGACGGCGATT 14 D k Z g A A G W R P G h Y S G A S L N_ 1~ T k Z

Tal GACACC~GCAGGTGGTAAA~GTATGGCACCTGTCTCTGGGTGGC,IJ~GCTACCCGACCATGGCGGCAGTC D T g Q V V g g Y G T C L W V A S Y P T, H A A V

?ca TCCACGGCTGACTTTGGATACTTCCGTCAATGGACGGGGTCGCCATCTGGCAGTTTACCAGTAACTGGCATG s T A D F G Y F 1~ q W T m S P s • s r~ p v T G U

~ A 4 I R I R aos GCCTGGACGTAGACGGGAACG'L~GCTCTGG'L~GACCTCAAC&GCGAGAACAAGCCTAAAGCCG&GGTCAAGC

k W T - o A 2 8 1 ,n,

ealr CAAAGACAAAGCAAAAGGCCACOGCGACTGAC~I'CCATGGTG'LULtGTC/LAAGTAAAGAGCCTGGGGCTJ~ GCAA

1000 GGCCAGCTGGAAGG'L~CGGCTGCTCTCAAAGGATGGCCACTACACCGAAAGCTACGTACCACAAGGT&GTGC

1001 TGGKAG&CC&GCGAAGTG&CG&CC&TTIIJ, AGGTAAGAAGTGTT&TCTG&TCGGCAAGG&TC~G&TCCC C

Fig. 2. Nucleotide sequence of the 1.2-kb DNA fragment containing the lys,4 gene of phage mvl. The deduced aa sequence of LysA and that of ORFI are shown. The start and stop codons are indicated by asterisks. The possible RBS and inverted repeated sequences (IR) capable of forming hairpin loops are underlined./t Clal indicates the location of the deletion borne by plasmid pT2-2 and obtained after Clal linearization ofpT2 and BAL 31 digestion. Symbols (d !,/14, d23,/114) indicate the deletion end points ofplasmids pT2-21, pT2-24, pT2-223 and pT2-214, respectively (Table I and FiB. 3)~ The nt sequence of both strands was determined by the dideogy chain.te.rmination method (Sanger et al., 1977) using the Sequenase kit (United States Biochemical Corp., Cleveland, OH). MI3 primers were initially used. To continue the sequencing, synthetic primers (17-nt oligos) were designed using nt sequences as they became available. Sequence data have been deposited with GenBank under accession number M3523$.

either of the two ATG codons at nt 260 and 290. Using the program of Mulligan etal. (1984), a putative RBS (homology score of 0.76) (AAGAAGAA, nt 279 through 286) was found upstream from the ATG codon (nt 290). It showed complementarity with the 3' end of the 16S rRNA of Bacillus subtilis (3'-UCUUCCCUCC) (Graves and Rabinowitz, 1986), L. lactis (3 ' -UUUUCCUCC) (DeVos,

1987) and E. col/ (3 ' -AUUCCUCC)(Hawley and McClure, 1983). We therefore tentatively located the start codon for LysA at nt 289-291. To determine the C-terminal end of iysA, the size of plasmid pT2-2 was reduced by digesting the BamHl-linearized pT2-2 plasmid with BAL 3 I. The exact extent of the resulting deletions was deter- mined by sequencing the fragment (Fig. 3). Deletions

64

ORF1 ,,.._ lysA m.~ m . - -

Hindlll Accl Accl BamHI Lys I I I I , pT2-2

704 . pT2-21

868 +/- pT2-24

931 + pT2-g23

1005 + pT2-214

Fig. 3. Restriction maps ofthe my] insert in pT2-2 and its BamHI /BAL 31 suoclones. The construction of the BAL 3J-deleted plasmids is described in section b. Number (nt) on the right of each deleted plasmid indicates the deletion end point as determined by sequencing experiments (Fig. 2). The Lys phenotype is indicated for each subclone by + , + / - , or - signs. + / - corresponds to a very poor but detectable expression of lysA.

occurring downstream from nt 931 did not affect the lysin activity, whereas deletions from nt 868 resulted in a very poor lyric activity, suggesting that the lysA end might be located between nt 868 and 931.

The first stop codon (TAG) was found at nt 874-876 followed by three more in the same reading frame at nt 989 (TAA), nt 1004 (TAG) and nt 1076 (TAG). Downstream from this first stop codon, a region of dyad symmetry (nt 894 through 934) was identified. The two perfect comple- mentary inverted repeats of 8 bp separated by 26 bp could form a stem-loop secondary structure with a free energy of formation AG of -9.2 kcal/mol as calculated following the rules ofTinoco et al. (1973). This putative hairpin loop may play a role as a Rho-dependent terminator (Friedman et al., 1987).

Upstream from the putative start codon (nt 289-291), no promoter-like sequence was found but an ORF (ORFI)of 220 bp was identified (Fig. 2). ORFI was terminated by a stop codon at nt 251 and the beginning of this ORF was suppressed by the ¢/al deletion generated for the construc- tion of plasmid pT2-2. ORF 1 may represent the C-terminal part of a gene transcribed with/ysA on a same messenger RNA. The/ysA gene should be the last operon gene since a putative Rho-dependent terminator was detected down- stream from the lysin-coding sequence.

The (3 + C content (53.5~o) of lysA was slightly higher than the value of 50~o given for genomic DNA of L. de/brueckii subsp, bulgarlcus (London, 1976; Kandler and Weiss, 1986). A comparison of the codon usage showed a predominance ofguanine and cytosine in the third position as observed for L. de/brueckii subsp, bulgar/cus ~Gal (Schmidt et al., 1989), for L. easel phospho-~Gal (Porter and Chassy, 1988) and D-2-hydroxyisocaproate dehydrogenase (Lerch et al., 1989). This preference was not observed for the dihydrofolate reductase gene of L. easel (Andrews et al., 1985). Inspection of the Ile codons in/ysA

shows one case of AUA usage (nt 332). Only one example of lle codon AUA usage in Lactobacilli was previously described for phospho-/~Gal-encoding genes (Porter and Chassy, 1988).

(c) Express ion of the cloned lysA gene in Escherichta coil E. coil cells containing plasmids pTl, pT2 or pT2-2 had

very poor viability and were unusually susceptible to lysis. E. coli clones carrying these plasmids were tested for their ability to produce lysin, using a bioassay with L. delbrueckii subsp, lactis LKT as indicator strain. LKT is the pro- pagating host for the phage mvl. The bioassay was also used with other indicator strains and the lysin produced by E. cell was active on some strains of L. delbrueckii subsp. bulgaricus, L. helveticus and Streptococcus salivarius subsp. thermophilus. No effect ofthe lysin was detected on cultures of Lactococcus lactis.

An in vitro transcription/translation study was performed to identify the iysin product of gene lysA. The two plasmids pT1 and pT2 were separately introduced into E. coil K38[pGP1-2], which expresses the T7 RNA polymerase under temperature control (Tabor and Richardson, 1985). In clones K38[pGPI-2][pT2], the expression of lysA was significantly increased by heat (42 ° C) and/or IPTG induc- tion as judged by the increased size of the lysis halos (data not shown). In contrast, in clones containing pTl, the lysin synthesis remained low and was not affected by any of the two types of induction (IPTG and heat). Therefore, in both plasmids (pTl and pT2), lysA was probably expressed from a promoter present on the mvl cloned fragment. The enhancement of the lysin synthesis by transcriptional readthrough from the lactose and T7 promoters in front of the my I cloned fragment observed only with plasmid pT2, suggests that the direction of transcription oflysA was from HindIll to Bam HI sites.

TABLE I!

Complementation of phage ~. lytic functions by LysA

Plasmid" Phage (eop) b

~ a m $ ~Sam7

None < 10- e < 10- e pTZI9R < 10 -e < 10 -6 pT2 2 x 10 -3 < 10 -e pTl-I < 10 -e < 10 -e pT2-2 2 x 10- 3 < 10-6

a The plasmids used are described in Table I. b Strain MCI061 (sup°) (Minton, 1984), bearing plasmids specified in the first column, and strain ERI458 (supF) were grown to 2 x l0 s celis/ml and plated on LB plates. Dilutions of ,1Ram$ and ~c/857 Sam7 were spotted on these two strains. The efficiency of plating (eop) is the ratio of the plaque-forming units/ml on strain MCl061 bearing the indicated plasmids to that obtained on strain ER1458.

1 ] I0 20 30 ~ 40 50 Clml&ropsis TVQGF .... DISSYQPSVNFAGA.YSAGARFVIIKATEGTSYTNPSFSSQYNGATTATGN

: *: *** :::* *** . .:**** . . . .

m y 1 l y s i n MTKTYG..VDVAVYQP.IDI.AAY.HKAGASFAIVKLTE~Y~qRRGPSRT~TAPGLTTST . . *** *: . .:*: . .:. .

C P L NVKKNDLFVDVSSHNCCYDITGILEQMGTTNTIIKISESTTYLN...PCLSAQV~SNPI

65

60 70 8G 90 I00 II0 YFIRGGYHFAHPCETTGAAQADYFIAHGCG~SCDGITLPGIfLDLESEGSNPA~GLSAAS

. :* **: .: . ** .: . . .

LMPTISRSFG.SSVSRAKKEAAYFLKEA...KKQDISKKRMLWLD~--AGSGNW4TGSKSS . ** . . :** :** * : * ** * .

GFYHFAR. FG. GDVAEAEREAQFFLDNV... PMQV .... KYLVLDYEDDPS .... GDAqA

120 130 140 RV AWI KAFS DRYHAVTGRYPRLYTN

** ** * *: ***

NTAAILDFMDAIKA. AG~RPGLYSG. * * * * * * : * * * : * * *

NTNACLRFMQMIAD. AGYKPIYYSY

Fig. 4. Comparison of the lq termini ofthe Mur of Chal.ropsb (upper line), ofthe Mur ofpneumococcal phage Cp-I (CPL; lower line) and of LysA (middle line). Comparisons of the aa sequence data were performed either with the alignment method oflqeedleman and Wunsch 0970) (program GAP, UWGCG)

or with the multi sequence alignment program of Corpet (1988). Asterisks and colons indicate identical matches and conservative substitutions, respectively (Lipman and Pearson, 1985). Arrows indicate the two active-site aa ofthe Mur ofChalaropsis (Fouche and Hash, 1978). Last digits ofnumeral

are aligned with corresponding aa (Fig. 2).

Radioactively labelled proteins were synthesized from plasmid pT2-223 (Lysin + ) by in vivo transcription/transla- tion reactions (Tabor and Richardson, 1985). Under appropriate expression conditions, clone K38[pGPI-2] [pT2-223] synthesized a protein of apparent Mr of 24000 (data not shown). This protein was assumed to be the LysA protein.

(d) Eomplementation of phage A lysis functions The cell lysis induced by phage A involves protein S which

is thought to act by causing the formation of pores in the inner membrane (Wilson, 1982; Raab et al., 1988) and the product of the gene R, a transglycosylase which cleaves glycosidic bonds in the peptidoglycan (Bienkowska- Szewczyk and Taylor, 1980). E. coli MC1061 (sup °) strain carrying the plasmid pT2, pTl-I or pT2-2 (Fig. 1) was tested for its ability to complement the growth defect of phage AR and AS mutants (Table II). None of the recom- binant plasmids could complement S function; in contrast, surprisingly, AR mutants were able to propagate in strain MCI061 carrying plasmid pT2 or pT2-2. Deletion of the AccI fragment in pTl-I resulted in the inability to comple- ment the AR function. These results suggest that the 1.2-kb-mvl insert Of pT2-2 produces a lytic protein which can supply a function equivalent to that of gene R. Protein R of A and protein 19 of phage P22 were shown to be interchangeable (Rennell and Poteete, 1985); these results

were consistent with the fact that E. coli phage A and Salmonella phage P22 have a similar gene arrangement and are closely related. More interesting was the complementa- tion we found between AR protein and my I LysA (Table 11), since no sequence homology between the two proteins could be detected.

(e) Amino acid sequence analysis and comparison with other iytic enzymes

The lysin ORF encodes a protein of 195 aa residues. The M, of 21 120 calculated from the predicted aa sequence is in agreement with the size of 24 kDa deduced from transcription/translation studies. LysA aa sequence was compared to that of other known lyric enzymes. No homology was detected between LysA and the A protein R (Sanger et al., 1982), E. coli phage"l"4 protein e (Owen et al., 1983), Salmonella phage P22 protein 19 (Renneli and Poteete, 1985), B. subtilb phage ~29 protein 15 (Garvey etal., 1986) and Lactococcus lactb phage ML3 lysin (Shearman et al., 1989), even though some of these phage lysins (protein e of T4 and protein 19 of P22) have a Mur activity. A significant aa similarity was observed between the N ends of LysA, the Mur of fungus Chalaropsb (Flech et al., 1975) and the Mur CPL of Streptococcus pneumoniae phage Cp-I (Garcia et al., 1988) (Fig. 4). The 2 aa of Chalaropsis Mur that have been reported to be involved in the active center of the enzyme (Asp e and Giu 33) (Fouche

o~6

and Hash, 1978) have been found in the CPL protein (Aspro and GILl yt) (Garcia et al., 1987). It is noteworthy that in LysA, the two aa Asp s and Glu 3s were also separated by 25 aa. The homology of the N-end region of LysA with these two Mur suggests that LysA might act as a Mur and that the active center of the protein should be located in the N-terminal domain.

Very few data concerning other Mur of Lactobacilli phages are available. An N-acetyl Mur has been isolated from PL-I phage lysates of L. casei (Hayashida et al., 1987). Its size (37 kDa) is larger than that of LysA (21 kDa). The N-terminal sequence determined up to 37 aa residues showed no homology with that of LysA.

The C-terminal domain of LysA showed partial homology with the 98 aa of the S gene product of phage (Sanger et al., 1982; Altman et al., 1983) (data not shown). Of the last 90 residues of LysA 22 aa were identical (24 ~o) to protein S and 18 aa were conservative substitutions (20~) . By analogy with the localization in the inner membrane of protein S, the C-terminal end of LysA might be involved in the interaction of the lysin with the cytoplas- mic membrane of the ceil.

ACKNOWLEDGEMENTS

This work was supported by grants from the Centre National de la Recherche Scientifique (L.P. 8201 and action 'Bacttries Lactiques' du programme Biotech- nologies) and from the Rtgion Midi-Pyr(mtes (RECH/8800768). We thank M. Coddeville for technical assistance, A.R. Poteete tbr sending us A mutants and P. Soucaille for helpful discussions.

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