PROCESS FOR THE LYSIS OF A CULTURE OF LACTIC ACID BACTERIA BY MEANS OF A LYSIN, AND USES OF THE RESULTING LYSED CULTURE

Abstract: The invention provides a process for the lysis of a culture of lactic acid bacteria, or a product containing such culture e.g. cheese, by means of a lysin through the in situ production of a homologous autolysin, or a heterologous autolysin obtainable from Gram-positive bacteria esp. from lactic acid bacteria. The gene encoding said autolysin is controlled by a promoter, preferably regulated by food-grade ingredients or parameters, to achieve an enhanced lysis after induction resulting in an enhanced production of total autolysin compared with the natural production level of the homologous autolysin during fermentation or shortly thereafter. Other uses of the invention include preparing a mixture of peptides which are modified by peptidases freed after the lysis, using the autolysin as a bactericidal agent against spoiling bacteria or pathogenic bacteria for improving the shelf life of a product containing the lysed culture.

Description: Process for the lysis of a culture of lactic acid bacteria by means of a lysin, and uses of the resulting lysed culture Background of the invention and prior art The invention relates to a process for the lysis of a culture of lactic acid bacteria, or a product containing such culture, by means of a lysin e.g. in producing a fermented food product, e.g. in cheese-making. Such a process is known from WO 90/00599 (AGRICULTURAL & FOOD RESEARCH COUNCIL (AFRC), M.J. Gasson, published 25 January 1990, ref. 1). According to that patent specification the lysin from a Lactococcus (preferably prolate-headed) bacteriophage was used to lyse bacterial starter cultures during cheese-making. Exemplified was the lysin of the bacteriophage φvML3 of Lactococcus lactis ML3. In particular, the lysin can be added to a cheese product or a cheese precursor mixture, e.g. after whey removal, milling and salting. However, this solution has the disadvantage that thorough mixing of the contents of the lysed cells with the cheese product is not easily obtained. Another disadvantage is that the lysin was produced by Escherichia coli cells, which are not food- grade. It is explicitly stated if the cell wall of the host cell is not itself degraded by the lysin then the lysin secreting transformed host may be useful in suppressing populations of bacteria which are susceptible to lysis by the lysin. Nothing is mentioned regarding addition of a transformed host cell in improving cheese flavor, certainly not a transformed lactic acid bacterium. As an alternative it is suggested in that patent specification "to encapsulate the lysin so that the timing of its addition is not important. The encapsulating agent dissolves after the cheese-making process is complete thus not affecting the starter bacteria before their role in acidification was complete." This suggested alternative has the disadvantages, that (a) an encapsulating material has to be used, and (b) said material must not dissolve before the end of the cheese making process. Moreover, if the encapsulated lysin is added at the beginning of the cheese-making process, e.g. while adding the cheese starter culture to the milk, about 90U of it is removed with the whey. Thus one has to add about tenfold the required effective amount, which is economically not attractive. In a later publication CA. Shearman, K. Jury & M.J. Gasson (Feb. 1992, ref. 2) described an autolvtic Lactococcus lactis expressing a cloned lactococcal bacteriophage φvML * . lysin gene. In particular they stated that "(e)xpression of the cloned lysin did not impair the ability of Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremovis strains to metabolize lactose, to clot milk and produce acid (data not shown)". It was suggested that during the exponential phase the lysin would not, or would insufficiently be expressed. It would only be expressed in sufficient amounts to lyse an appreciable proportion of the cells during the stationary phase, which occurs at the end of the normal fermentation process. The article illustrates that maintenance of transformed lactococcal strains could be a problem. Maintenance at a temperature below 30°C slightly delayed the onset of lysis but at 30°C regrowth of lysin resistant bacteria occurred. As alternative buffering in a sucrose medium with a sucrose percentage higher than 20% was given. This does not seem to be suitable in a process of fermentation like cheese making where the fermentation step occurs at 30°C or higher and the presence of more than 20% sucrose is not acceptable. Furthermore, at the end of that publication it was indicated that expression in the stationary phase is not completely controlled. In addition the use of osmotic buffer in a cheese maturing process is probably not very efficient timewise. this can be illustrated by the length of time required for a Gouda cheese immersed in a brine bath to achieve the desired degree of salt flavour. For this cheese type for example the osmotic effect of salt concentration is not going to be very quick. The cheddar cheese making process would probably be more suitable as the salt addition step is more efficient, however, this still has the disadvantage of requiring a mixing step. Both disclosures described the use of a lysin originating from a lactococcal bacteriophage lysin, that means an enzyme produced in nature by an undesired substance like a bacteriophage, because bacteriophage contaminations are a major problem in large scale industrial dairy fermentation processes. In a review article R. Young (1992, ref. •*.) gives a survey of the state of the art on bacteriophage lysis, both mechanism and regulation. Especially in the section "Lysis in Phage Infections of Gram-Positive Hosts" on pages i .68--472 it was indicated that the DNA sequence found by Shearman c.s. (1989. ref. 5). which DNA sequence seems to be the same as that given in ref. 1, is probably not correct and that the deduced amino acid sequence might be quite different due to a mutation causing a phase shift in the reading frame. In a publication of Ward c.s. (1993 * re - 6) it is also suggested that the sequence of Shearman et al. (1989; ref. 5) is probably not correct. Comparison with a very similar phage lysin gene confirmed that a frame shift in the Shearman et al. (ref. 5) sequence is needed for aligning the two DNA sequences. Moreover, this comparison teaches that the real phage lysin is encoded by an ORF that is probably -.5 bases longer than disclosed by Shearman et al. (ref. 5)- C. Platteeuw and W.M. de Vos (1992, ref. 3) described the location, characterization and expression in Escherichia coli of lytic enzyme-encoding gt , lytA , of Lactococcus lactis bacteriophage φUS3- It was described that che φvML3 lysin, which is active on a wide range of lactococcal strains, lacked homology with known lytic enzymes. The bacteriophage φUS3 was identified during studying bacteriophages specific for the cheese-making strain Lactococcus lactis SK11 (NIZO) . The results showed that the deduced amino acid sequence of LytA shares similarities with that of an autolysin of Streptococcus pneumonia , suggesting that the bacteriophage φUS3 encodes an amidase rather than a lvsozvme-tvpe muramidase. The above illustrates the difficulties facing a person skilled in the art wishing to isolate DNA-sequences from different organisms. The lack of information regarding sequences and the lack of homology between known sequences makes use of probes and primers derived from known sequences quite unlikely to lead to successful isolation of a correct DNA sequence from different organisms. In EP-A2-0510907 (AFRC, M.J. Gasson, published 28 October 1992, ref. 7) the use of bacteriophages of food-contaminating or pathogenic bacteria or the lysins thereof to kill such bacteria was described. Examples included lysins from bacteriophages of Listeria monocytogenes (phage φLM*.) and Clostridium tyrobutyricum (phage φP) . Also tests for bacterial contamination can be made specific for specific bacteria by using the appropriate bacteriophage or lysin thereof and determining whether cells are lysed thereby. That European patent application thus describes the use of lysins obtained from phages of food-contaminating or even pathogenic bacteria, which is not desirable for food-grade applications. Moreover, the purpose of use of such lysins in that patent application is further away from the subject of this invention, which will be discussed below as it does not lie in improving flavour of food products by autolysis of lactic acid bacteria. Summary of the invention In contrast to these prior art developments the present invention relates to the use of a "real autolysin", i.e. a lysin obtainable from grampositive foodgrade bacteria preferably lactic acid bacteria, (i.e. not an autolysin derived or derivable from a bacteriophage) for improving food products and the processes for preparing food products. The maturation of cheese is a process of particlar relevance. In particular, the present invention is based on the identification and isolation of the gene of Lactococcus lactis subsp. lactis encoding a lysin that is capable of lysing the cell in which it is produced. During the normal fermentation process the amount of lysin should not be so large that it causes lysis of the cells in which it is produced. However, a limited constitutive production of the autolysin is needed to guarantee separation of cells and to prevent the formation of too strong cell walls. When a natural lactic acid culture is grown without addition of (sufficient) nutritional ingredients, after the exponential phase growth will decrease due to lack of nutritional ingredients and the culture will enter the stationary phase. Then after several days starvation will occur, and this event probably causes an increase in natural autolysin. Consequently during starvation enhanced amounts of the natural autolysin will be formed and will lyse the cells of the lactic acid bacteria. In the subject case the inventors faced the problem of devising a manner of lysing the foodgrade bacterium as soon as possible after the foodgrade bacterium enters the stationary phase and with a minimum of steps, without interfering with the normal activity of the foodgrade bacterium in the fermentation process in which it is involved. This has been achieved through recombinant DΝA technology on a foodgrade bacterium which leaves the foodgrade status and functionality of the bacterium intact and achieves regulatable lysis of the foodgrade bacterium in situ at any desired time. In this specification the following abbreviations of names of micro-organisms are used: E. = Esc erichia, e.g. E. coli , I. = Lactococcus, e.g. I. lactis , M. = Micrococcus, e.g. ti. lysodeikticus , S. = Streptococcus , e.g. S. faecalis and S. pneumonia. Brief description of the drawings FIGURE LEGENDS belonging to draft publication 1 FIG. 1. Cell wall hydrolase activity of L. lactis MG1363 in renaturing (12.5 ) SDS-PAGE. (A) gel containing 0.2% (wt/vol) M. lysodeikticus autoclaved cells (B) gel cr aining 0.2% (wt/vol) cell walls from L. lactis MGI363. WP, fractions of cultures grown in whey-based medium and GM17, respectively; C, cell-free extract; S, supernatant fraction. The amount of sample loaded was equalized according to the optical density of the cultures. Molecular masses of standard proteins are shown on the right, and the sizes of two of the lytic bands discussed in the text are indicated on the left (all in kilodaltons (kDa)). FIG. 2. Cell wall hydrolase activity in E. coli NM522 transformants by renaturing (12.5%) SDS-PAGE. The gel contained 0.2% (wt/vol) Λ. lysodeikticus autoclaved cells. 1,2: cell-free extract and supernatant of L. lactis MG1363 grown in whey-based medium, respectively; 3. cell-free extract of E. coli NM522; 4 and 5. cell-free extracts of two independent E. coli NM522 transformants producing a halo on TY plates containing N. lysodeikticus autoclaved cells. Molecular masses of standard proteins (in kDa) are shown on the right. Two lytic bands corresponding to sizes of -41 and 46 kDa are indicated on the left. FIG. 3. Restriction enzyme map of the -.137-bp Sαu3A DNA fragment of the chromosome of L. lactis MGI363 present in pALOl, showing the location of acmA , 0RFA and 0RFB. T, terminator; P iac , lac promoter and its direction of transcription; del., £coRV deletion made to construct pAL02. The Hindlll-Sacl fragments used to construct pALO-4 and pAL03 are indicated with (a) and (b) , respectively. The genetic analysis of the 2207-bp Sαu3A-SspI fragment will be presented elsewhere (Buist et al. , to be published) FIG. . Nucleotide sequence and deduced amino acid sequences of acmA , 0RFA and part of 0RFB of L. lactis MGI363. Putative ribosome binding sites (rbs and lower case), -10 and -35 sequences (shaded), startcodons (bold face), and stop codons (underlined) are indicated. A possible transcriptional terminator is indicated by horizontal arrows above the sequence. The synthetic primers PALA-*. and PALA-14 used in the PCR experiment are overlined. The possible signal peptide cleavage site is indicated by T. A number of relevant restriction enzyme sites are also indicated. The intergenic region between acmA and ORFA is shown double stranded. FIG. . (A.) Alignment of the deduced amino acid sequences of AcmA of L. lactis , the autolysin of S. faecalis (ref. 2), and muramidase-2 of Enterococcus hiτae (ref. 6) both of which are not foodgrade. The internally repeated regions of the three proteins are underlined. Identical (*) amino acids in all three proteins, identical (+) amino acids between muramidase-2 and the autolysin of S. faecalis in the C-terminal of the two enzymes not present in AcmA, and similar (.) amino acids are indicated. (*), signal peptide cleavage site in muramidase-2; (shaded and lower case), putative membrane spanning domains. (B.) Schematic presentation of the alignment shown in (A). Thick line, (putative) signal sequence. The two slashes indicate the sequence in the S. faecalis autolysin not present in AcmA and muramidase-2. Repeated sequences according to the consensus proposed by Joris et al . (20) are boxed. FIG. 6. Amino acid sequence alignment of the carboxy terminal repeats of AcmA (in the box) plus the preceding and intervening sequences. The consensus sequence proposed by Joris et al . (20) is shaded. (*) .identical amino acids; (.),similar amino acids. FIG. 7« PCR products formed on chromosomal DNA's of the lactococcal strains MG1363(1); ILl403(2); l8-l6s(3); AMI(4) and HP(5). Primers used were PALA-4 and PALA1*. shown in Fig. 4. M, molecular weight marker: bacteriophage SPPI DNA cut with EcoRI . Sizes indicated in the margins are in basepairs. FIG. 8. Schematic representation of the construction of the chromosomal acmAhl mutant of L. lactis using pINTAA. Black bar, insert of pAL09 in pORI28θ; acmAAl , deletion derivative of acmA ; Em r , erythromycin resistance gene; lacZ, β-galactosidase gene of E. coli expressed under the control of the lactococcal promoter P 32 (p) ; open square, origin of replication of the lactococcal plasmid pWVOl; 1 and 2, possible sites for the first crossover; x and y, primers PALA-19 and BK05AL, respectively, used to distinguish between the two types of Campbell integrants; A and B, possible regions for a second crossover and their products A and B. A number of relevant restriction enzyme sites are also shown. FIG. 9- Southern blot analysis of the mutant strain L. lactis MG1363 acmAAl . Chromosomal DNA's of MG1363U), MGI363 αcιτ_4Δ_-a(2) , and MG1363 αcm____-b(3) digested with Hαelll were separated on a 0.8% agarose gel, transferred to gene screen-plus membrane, and hybridized to the -.137 bp Sα 3A insert of pALOl. M, molecular weight marker: bacteriophage SPPI DNA cut with Z-CoRI, of which the sites (in bp) are given on the left. The sizes (in bp) of the hybridising bands of the chromosomal DNA's are shown in the right margin. FIG. 10. AcmA activity in L. lactis MGI363 (lanes 1,4) and deletion mutants MGI363 acmAAl -a (lanes 2,5) and acmAAl -b (lanes 3.6) by renaturin 12.5%) SDS-PAGE. The gel contained 0.2% (wt/vol) N. lysodeikticus autoclaved cells. Cell-free extracts (lanes 1,2,3) and supernatant fractions (lanes -.,5,6) after growth in GM17 were applied. The amount of sample was eαualized according to the optical density of the cultures. Molecular _r s of standard proteins are shown on the right and of mature AcmA ^-.ό) or its secreted form (4l) are shown on the left (all in kilodaltons) . FIG. 11. Light microscopic view of L. lactis MGI363 and MGI363 acmAAl at 1000-fold magnification. Both strains were grown overnight in GM17. FIG. 12.A-1/A-2. Scheme showing the construction of deletion derivatives of pALOl. Ap r , ampicillin resistance gene; Km r , kanamycin resistance gene; lacZ, α-lacZ; del., ZJcoRV deletion resulting in pAL02.Some restriction enzyme sites are given. The various subfragments of the 2850-bp Hi'ndlll fragment in pALOl obtained in separate subclones are indicated by various modes of shading. FIG. 12.B. Scheme showing the construction of the integration plasmid pINTAA. Ap r , ampicillin resistance gene; Black bar, insert of pAL09 in p0RI280; acmAAl(vertical lined), deletion derivative of acmA(dotted) ; Em r , erythromycin resistance gene; lacZ, β-galactosidase gene of E. coli expressed under the control of the lactococcal promoter P 32 (p) ; ori+, origin of replication of the lactococcal plasmid pWVOl. A number of relevant restriction enzyme sites are shown. FIGURE LEGENDS OF DRAFT PUBLICATION 2 FIG. 13. Growth measured by reading optical density readings at 600 nm of L. lactis subsp. cremoris strains MGI363 (Q) and MG1363αcm 2 (*). FIG. 14. (A) Nucleotide sequence of the promoter region of acmA . Putative ribosome binding site (rbs and lower case), -10 and -35 sequences (shaded), startcodon (bold face), and the Sspl and Seal restriction enzyme sites are indicated. (B) Subcloning of acmA , with (pGKALl) and without (pGKAL2) its own promoter, in ρGK13. Ap r , ampicillin resistance gene; Em r , erythromycin resistance gene; Cm r , chloramphenicol resistance gene; 0RI(open square), origin of replication of the lactococcal plasmid pWVOl; repA, gene encoding the replication protein of plasmid pWVOl; acmA, N-acetylmuramidase gene of L. lactis subsp. lactis MGI363. The Scal/BamΑI fragment containing the acmA gene is dotted and the Sspl/Scal fragment containg the acmA promoter is shown in black. Only relevant restriction enzyme sites are shown. FIG. 15. Analysis of halo formation of L. lactis MGI363 containing pGK13 (1), L. lactis MG1363αcm__Δ_ containing pGK13 (2), pAL2 (3), or pGKALl (•*.) on a GM17 plate containing N. lysodeikticus autoclaved cells. FIG. 16. Analysis of reduction of the optical density measured at 600 nm of L. lactis MGI363 containing pGK13 (*) and L. lactis MG1363αem__ΔZ containing pGK13 (■) or pGKALl (I) . FIG. 17. Analysis of reduction of the optical density measured at 600 nm of L. lactis MG1363 (0), L. lactis MG1363pepT (Δ) , L. lactis KG13β3acmAAl (D) and an end-exponentional phase mixed culture of L. lactis MG1363pepϊ" and L. lactis MG1363αcπ_Δ_ (I) . The numbers on top of the graph indicate the time points at which samples were taken from the cultures. FIG. 18. A. Detection of AcmA activity in a renaturing SDS-12.5% PAGE containing 0.15% (wt/vol) M. lysodeikticus autoclaved cells. B. Analysis of released intracellular proteins on a 12.5% PAA gel stained with Coomassie. C. Detection of PepT among the released intracellular proteins by using specific antibodies. Only the results of the samples of the supernatant fractions are shown. Numbers 1 to 1 , shown on top of the gels, are the time points, indicated in Fig. 17, at which the samples were taken. Molecular masses (in kilodaltons) of standard proteins are shown on the left. FIG. 19. Cloning scheme for the construction of pAL12, in which AcmA production is inducible. * > , promoters from bacteriophage Rl-t; Em r , erythromycin resistance gene; Ap r , ampicillin resistance gene; lacZ, 6- galactosidase gene of E. coli ; T, transcription terminator of prtP; rro , Rl-t repressor gene; tec , topological equivalent of lambda cro; 0RF29', N-terminal part of 0RF29 of phage Rl-t; 0RF29/lacZ, transcriptional fusion of 0RF29 and lacZ ; Sacl fragment, a 27l6-bp Sαcl fragment of 0RF1 of the srfA locus of Baci llus subti lis ; acmA , N-acetylmuramidase gene of L. lactis;^ ' ,43.44,-45,46' , indicating the ORF's of phage Rl-t which are located on the EcoRI fragment ofpEF+. Only relevant resriction enzyme sites are shown. FIG. 20. Effect of mitomycin C on OD 6oo of L. lactis MGI363 containing pGK13 (D) and L. lactis LL302 strains containing pIR12 (+), pIRlPR (•»■) and pAL12 (*). The culture of L. lactis LL302 cotaining pAL12 (I) was not induced. The cultures were induced with 1 μg/ml mitomycin C at time zero (dotted line). The arrow ( I ) indicates the time at which 1 ml samples were taken and processed for the analysis of AcmA activity (Fig.21) and protein and peptidase analysis (Fig's. 22 and 23, respectively). FIG. 21. Renaturing SDS-PAGE analysis of the autolysin activity of L. lactis LL302 containing pIR12 (lanes 1,2), pIRlPR (lanes 5,6) or pAL12 (lanes 3»*4). Samples of 1 ml were taken four hours after the addition of mitomycin C to exponentially growing cells (see Fig. 15). Cell-free extracts (1,3,5) and supernatant fractions (2,4,6) were loaded on PAA gels containing 0.2% (wt/vol) M. lysodeikticus autoclaved cells. Molecular masses of standard protc s (M) are shown on the left and on the right the clearing bands due to AcmA (40.3) and LytR (30.2) activit are shown (all in kilodaltons (kDa). A clearing band caused by a degradation product of AcmA is indicated with an arrow (<-) . FIG. 22. Liberation of proteins in the culture supernatant of induced (lanes 2,4,6) and non-induced (lanes 1,3,5) L. lactis LL302 containing pIR12 (1,2), pAL12 (3,4), or pIRlPR (5,6). Lane 9* cell-free extract of non-induced L. lactis LL302(pIR12) . Molecular masses (in kDa) of standard proteins (M) are shown on the left. FIG. 23. Western blot analysis of the gel shown in Fig. 22 using polyclonal antibodies raised against the lactococcal endopeptidase PepO. The arrowhead indicates the position of the endopeptidase. The order of the samples is identical to the order in Fig. 22. Table 3 Cell fraction Supernatant fraction Strain AcmA PepT AcmA PepT Protrel. WT ++ ++ ++ ++ ++ pepT ++ - ++ ++ - acmA - ++ - - - pepT/acmA + + + + + Detailed description of the invention It is an object of the invention to obtain lysis some hours after the cells have reached the stationary phase, the latter often coinciding with the end of the industrial fermentation. The enhanced induction of the autolysin according to the invention is performed within some hours after the fermentation is finished, in contrast to enhanced lysis after several days as occurs naturally. Alternatively, the enhanced induction can be achieved at the end of the fermentation, with the result that the fermentation is stopped due to the lysis of the cells. The lysis is suitably achieved before 12 hours of the stationary phase have lapsed, preferably before 4 hours of the stationary phase have lapsed. This deliberately induced production of a higher amount of autolysin at a relatively early stage is the enhanced lysis mentioned in the claims. Therefore, the gene encoding this lysin needs to be under control of a regulatable promoter, i.e. a promoter other than the promoter it is normally associated with. As stated before the presence of a low amount of constitutively produced autolysin is needed for normal cell growth. Ther. 3 the inducible gene must be additional to the existing autolysii. le. Thus in one aspect of the invention there is provided a process for the lysis of a culture of lactic acid bacteria by means of a lysin, which comprises the in situ production of an autolysin obtainable from Gram- positive foodgrade bacteria esp. from lactic acid bacteria, i.e. not a bacteriophage or active fragments of such autolysin still being capable of lysing the cell walls, the gene encoding said autolysin or a fragment thereof being under control of a regulatable promoter not normally associated with the autolysin gene, to achieve an enhanced lysis during fermentation or shortly thereafter after induction of said regulatable promoter due to an enhanced production of total autolysin compared with the natural production level of the homologous autolysin. Preferably, said regulatable promoter is regulatable by the use of food- grade ingredients or parameters, e.g. - by aeration to induce an oxygen-inducible promoter, - by changing the ionic strength or the water activity, in particular the salt content of the culture to induce a salt-inducible promoter, - by changing the pH of the culture to induce a pH-inducible promoter, or - by applying a lower or higher temperature than used for the growth of the lactic acid bacteria to induce a temperature-inducible promoter. The autolysin gene can be heterologous or homologous to the bacterium strain in which it is incorporated. Preferably it will be homologous. In particular with regard to legislation regarding recombinant DNA work the use of DNA homologous to a bacterium is much sooner approved than when applying heterologous sequences. An advantage of a process according to the invention is that no extra amount of lysin needs to be added, because during the fermentation the cells multiply and lysin is only formed at the time when it is needed as a consequence of the regulatable promoter, for example when the whey has already been removed. This is more economical in price and manpower than the state of the art processes for introducing lysin as such. Furthermore the lysm need not first be isolated or encapsulated as is needed according to the methods disclosed in WO 90/00599• Another advantage of a process according to the invention is that the time of lysis of the culture can be controlled rather precisely by choosing an adequate promoter or regulon that can be activated by the parameters of the production process for obtaining the product containing the culture. It should be noted that a regulon comprises a promoter and operator region, an operator region can be activated or deactivated by the action of a repressor. In this specification a regulatable promoter also comprises a regulatable regulon whereby the expression is regulated by a repressor-operator interaction. Finally this is the first foodgrade system that has been developed and thus can be applied in practice for producing products suitable for human consumption. The culture of lactic acid bacteria can be used as such, but can also form part of a product containing such culture. The latter occurs when the lactic acid bacteria culture is used for producing a fermented food product obtainable by the fermentative action of the lactic acid bacteria and subsequently the lactic acid bacteria in the fermented food product are lysed. In particular, the fermented food product can be a cheese product. In a preferred embodiment additionally a cheese ripening step is carried out, whereby some of the constituents after leaving the lysed cells will change the composition of the cheese product. According to another aspect of the invention a process for producing a mixture of peptides is provided, in which a mixture of peptides obtained by proteolysis of proteins is combined with a culture of lactic acid bacteria containing a lactic acid bacterium autolysin gene under control of a regulatable promoter and subsequently the promoter is induced to achieve production of the autolysin in such amount that the lactic acid bacteria are lysed and the contents of the cells containing peptidases will modify the composition of the mixture of peptides. Instead of carrying out the lysis in the peptide mixture itself, it is also possible to first carry out a process for the lysis of a culture of lactic acid bacteria by means of a lysin according to the invention, followed by adding the resulting lysed culture to a mixture of peptides obtained by proteolysis of proteins, and allowing the contents of the cells containing peptidases to modify the composition of the mixture of peptides. The starting materials for the mixture of peptides can be any protein or mixture thereof, but preferably the proteins comprise milk proteins or vegetable proteins, or both. According to a further aspect of the invention a process according to the invention is provided, in which first the autolysin is produced and subsequently used as a bactericidal agent against spoiling bacteria, e.g. certain Lactobaci llus species that can spoil a product, or pathogenic bacteria, e.g. Gram-positive Listeria species or Gram-negative Salmone lla species. To obtain improved results it is desirable that the production of autolysin is enhanced compared with the natural production level. Thus according to this aspect of the invention a process for improving the shelf life of a consumer product is also provided, in which a free autolysin-containing product obtained by a process according to the invention is incorporated into said consumer product in such amount that the growth of spoiling bacteria or pathogenic bacteria is inhibited in the resulting consumer product or that their viability is strongly reduced. The advantage is that no purification is required of the autolysin prior to use. Such consumer products comprise edible products, cosmetic products, and products for cleaning fabrics, hard surfaces and human skin. Examples of such products may be bread and bread improvers; bu t ter, margarine and low calorie substitutes therefor; cheeses; dressings -nd mayonnaise-like products; meat products; food ingredients containing peptides; shampoos; creams or lotions for treatment of the human skin; soap and soap- replacement products; washing powders or liquids; and products for cleaning food production equipment and kitchen utensils. Another aspect of the invention covers the nucleotide sequence encoding the lysin of a lactic acid bacterium. In particular the nucleotide sequence according to sequence id. no. 1 is claimed as are functional equivalents thereof. Variants of this nucleotide sequence e.g. other nucleotide sequences encoding the amino acid sequence according to sequence id. no. 2 as encoded by the nucleotide sequence according to sequence id. no. 1 are also claimed. Fragments of such sequences which upon expression exhibit the activity of a lysin which can be determined with an activity test as illustrated in the description also fall within the scope of the invention. Nucleic acid sequences encoding an N-terminal amino acid sequence that is more than 70%, preferably more than 80%, more preferably 90% homologous with the N-terminal amino acid sequence according to sequence id no 2 and encoding a polypeptide exhibiting lytic activity of an N-acetylmuramidase as can be determined by a standard assay also fall within the scope of the invention. Nucleic acid sequences in addition encoding an overall amino acid sequence that is more than 40%, preferably more than 50%, more preferably between 60-100% homologous with the overall amino acid sequence according to sequence id no 2 and encoding a polypeptide exhibiting lytic activity of an Ν-acetylmuramidase as can be determined by a standard assay also fall within the scope of the invention. In particular a nucleic acid sequence according to any of the above described embodiments further comprising at least 3 direct repeats in the C-terminal part of the amino acid sequence, said direct repeats being characterised by a homology of more than 75% at amino acid level falls within the scope of the invention. Preferably an amino acid sequence encoded by a nucleic acid sequence according to the invention will comprise serine, threonine and asparagine rich intervening sequences between the direct repeats. More specifically a nucleic acid sequence according to the invention will comprise a part encoding a membrane spanning domain as illustrated in Figure 5- A nucleic acid sequence according to any of the abovementioned embodiments further characterised by its ability to hybridize to a nucleic acid sequence according to sequence id no 1 and Figure 4 under normal to stringent conditions common to a person skilled in the art. Recombinant host cells comprising a sequence according to the invention in any of the abovementioned embodiments under control of a promoter other than that naturally occuring with the lysin gene, said promoter preferably being regulatable and most preferably derived from a foodgrade organism are also covered. Examples of suitable promoters are provided in the European Patent Applications EP-942013541.1 and EP- 94201355-8. The details regarding promoters, expression vectors in the various embodiments disclosed in the two cited European Patent Applications are hereby enclosed by reference. In particular foodgrade grampositive host cells such as lactic acid bacteria are suitable embodiments of host cells. A recombinant host cell according to the invention will also possess a native autolysin gene under control of it's native promoter such that the required degree of autolysin production during the exponential growth phase is ensured. The invention is further illustrated by two draft publications, which are given below. Draft publication 1 MOLECULAR CLONING, NUCLEOTIDE SEQUENCE, AND FUNCTIONAL ANALYSIS OF THE GENE FOR THE MAJOR PEPTIDOGLYCAN HYDROLASE GENE OF LACTOCOCCUS LACTIS SUMMARY A gene of L. lactis subsp. cremoris MGI363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening of E. coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized M. lysodeikticus cells. In cell- free extracts of L. lactis MG1363 and several halo producing E. coli transformants, lytic bands of similar sizes were identified by denaturing SDS-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding with the presence of lytic enzymes of 46 kDa and 41 kDa, the 4l kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2428-bp __ * coRV-5αu3A fragment. Sequencing of part of this fragment revealed a gene (acmA) of 1311 bp that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46.564 Da) corresponded with that of one of the lytic activities detected. Presume" *! y, the enzyme is synthesized as a precursor protein which is p. jcessed by cleavage after Ala at position 57, thus producing a mature protein of 40.264 Da, which corresponded with the size of the lytic activity present in culture supernatants of L. lactis . The Ν-terminal region of the mature protein showed 60% identity with the Ν-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of S. faecalis . Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these 3 repeats are separated by non-homologous intervening sequences highly enriched in serine, threonine, asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris using the SDS-PAGE detection assay and PCR experiments. Using replacement recorrhination, an acmA deletion mutant was constructed which grew as long chai. indicating that AcmA is required for cell separation. INTRODUCTION Bacteria produce several types of cell wall hyά_?oiases, enzymes capable of hydrolysing the peptidoglycan of the cell envelope. Based on their cleavage specificity, the enzymes are classified as N- acetylmuramidases (lysozymes), N-acetylglucosaminidases, N-acetylmuramyl- L-alanine amidases, endopeptidases, and transglycosylases (44). Cell wall hydrolases are thought to be involved in cell wall turnover (11), cell separation, competence for genetic transformation, formation of flagella, sporulation, and the lytic action of some general antibiotics (for reviews see (38,51)). Strains of the starter bacteria L. lactis are of eminent economic importance because of their worldwide use in cheese making. It is generally believed that during cheese maturation, autolysis of the bacteria results in release of intracellular proteolytic enzymes such as a variety of peptidases which digest casein-borne peptides and, thus, may contribute to cheese flavour development (48) . Some data are available on the biochemistry of autolysis and autolytic activity in lactococci. Thus, Mou et al . (31) and Νiskasaari (33) have shown that L. lactis subsp. cremoris shows maximal autolytic activity during exponential growth in media with a neutral pH. These authors could only detect an Ν-acetylmuramidase, which was present in the cell wall and supernatant fraction; no endopeptidase or glucosaminidase activity was detectable. The lactococcal autolytic activity was inhibited by lipoteichoic acid and cardiolipin and activated by trypsin (33)- Mou et al . (31) also showed that the cell walls of exponential-phase L. lactis subsp. cremoris cells autolyse most readily at the equatorial ring. McDonald (30) noted that in both L. lactis and L. cremoris filament formation was associated with decreased autolysin activity, an observation later confirmed by Langsrud et al . (25). These data suggest that autolytic activity in L. lactis is involved in cell separation. Although data on the biochemistry of lactococcal autolysins is gradually emerging, nothing is known about the genetic components governing autolysis in L. lactis . In this paper we report the cloning, expression and sequence of the gene for the major peptidoglycan hydrolase from L. lactis . In addition, a deletion mutant was constructed in order to elucidate the function of the autolysin. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions The strains and plasmids used in this study are listed in Table 1.1. L. lactis was grown in M17 broth (Difco, West Molesey, UK) or whey- based medium (9) at 30°C as standing cultures, or on M17 agar, all supplemented with 0.5% glucose. Erythromycin (Boehringer Mannheim, GmbH, Germany) and 5-bromo-4-chloro-3~indolyl-β-D-galactopyranoside (X-gal) (Sigma Chemicals Co., St.Louis, M0.) were added to 5 μg/ * * * -! and 0.008% respectively. E. coli was grown in TY (Difco laboratories, Detroit, Mich.) medium at 37°C with vigorous agitation, or on TY medium ~ idified with 1.5% (wt/vol) agar, containing 100 μg/ml of ampicillin (S. a), 50 μg/ml of kanamycin (Boehringer) or 100 μ/ml erythromycin (Boehringer) , when required. Isopropyl-β-D-thiogalactopyranoside(IPTG) and X-gal (both from Sigma) were used at concentrations of 1 mM and 0.002% (wt/vol), respectively. General DNA techniques and transformation Molecular cloning techniques were performed essentially as described by Sambrook et al . (39) • Restriction enzymes, Klenow enzyme, T4 DNA polymerase, T4 DNA ligase and deoxynucleotides were obtained from Boehringer Mannheim and were used according to the instructions of the supplier. Genomic DNA of L. lactis was isolated according to the method described by Leenhouts et al . (28) with one modification: cell pellets resuspended in lysis solution with lysozyme were incubated at 55°C for 15 minutes (4l). E. coli and L. lactis were transformed by electroporation using a Gene Pulser (Bio-Rad) as described by Zabarovsky and Winberg (55) and Holo and Nes (15) with the modifications suggested by Leenhouts and Venema (29) , respectively. Sample preparation for SDS-PAGE After overnight growth, the optical density of the L. lactis cultures was measured in a Philips PU8720 UV/VIS scanning spectrophotometer (Pye Unicam Ltd, Cambridge, UK) at 600 nm. TABLE 1.1. Bacterial strains and plasmids Strains Relevant phenotype or genotype Source or reference L. lactis subsp. cremoris MG1363 Plasmid-free strain (13) MG1363::pINTAA-l Em r , derivative of MGI363 with integrated pINTAA via portion 1 This work MG1363::pINTAA-2 Em r , derivative of MGI363 with integrated pINTAA via portion 2 This work MG1363αcm/Δ2 Derivative of MGI363 containing a 701 bp Scal/Spel chromosomal deletion in the acmA gene This work AMI Wild-type strain (12) HP Wild-type strain New Zealand Dairy Research Institute (NZDRI) L. lactis subsp. lactis IL1403 Plasmid-free strain (5) L. lactis subsp. lactis biovar diacetylactis 18-16S Sm r (1*4) E. coli NM522 supE, thi ,A( lac-proAB) ,_Vιsd5(r-,m—)[F' , proAB,lαcI q ZM15] Stratagene (La Jolla, CA.) EC1000 Km r , glgB , derivative of MC1000 containing the repA gene of the lactococcal plasmid pWVOl Laboratory collection TABLE 1.1. Bacterial strains and plasmids (continued) Plasmids Relevant phenotype or genotype Source or reference pBluescript SK+ Ap r Stratagene PUC19 Ap r (53) pUK21 Km r (-47) 10 P0RI280 Em r , β-GAL", ori * of pWVOl; integration vector which replicates in strains containing RepA (27,29) pALOl Ap r , pUC19 carrying a 4137 bp lactococcal chromosomal DΝA insert with the cell-wall 15 hydrolase gene This work PAL02 Ap r , pALOl with a 507 bp EcoRV deletion This work PAL03 Km r , pUK21 with a 1383 bp Hindlll/Sacl insert of pALOl This work PAL04 Km r , pUK21 with a 1467 bp Hindlll/Sacl 20 insert of pALOl This work PAL05 Ap r , SK+ with a 682 bp Hindlll/Spel fragment of pAL03 This work pAL06 Ap r , SK+ with a 701 bp Sacl/Spel fragment of pAL03 This work 25 PAL07 Ap r , SK+ with a 477 bp Scal/Clal fragment of pAL04 This work pAL08 Ap r , pALOl with a 16 bp Smal/EcoRl deletion This work pAL09 Ap r , pAL08 with a 701 bp Sacl/Spel deletion This work pINTAA Em r , β-GAL * . p0RI280 with a 2247 bp 30 EcoRV/Hι"ndIII fragment of pAL09 This work 5 millilitres of the cultures were subjected to centrifugation and the supernatant fractions were dialysed against several changes of 35 demineralized water, lyophilized, and dissolved in 1 ml denaturation buffer (2) . The cell pellets were resuspended in 1 ml denaturation buffer and cell free extracts were prepared as described by van de Guchte et al . (45). Cell free extracts of E. coli strains were made accordingly after pelleting 2 ml of overnight cultures. The samples were boiled for 5 40 minutes and centrifuged before loading. SDS-polyacrylamide (SDS-PAA) gel electrophoresis was carried out as described by Laemmli (24) with the Protean II Minigel System (Bio-Rad Laboratories, Richmond, CA.). Prestained mole ilar weight markers were obtained from Bio-Rad and Pharmacia (Pharmacia AB, Uppsala, Sweden). • 45 Detection of lytic activity in SDS-PAA gels Lytic activity was detected in situ by using 12.5% (wt/vol) SDS-PAA gels containing 0.2% (wt/vol) autoclaved, lyophilized M. lysodeikticus ATCC 4698 cells (Sigma), or cell walls of L. lactis MGI363. Cell walls of L. lactis MGI363 were isolated using the method of Potvin et al . (37) with the following modification: after resuspension in 4% (wt/vol) SDS, the cells were disrupted as described by van de Guchte et al . (45). After electrophoresis, the gels were gently shaken at room temperature for 24 to 48 h in 3 to 5 changes of 100 ml 25 mM Tris-HCI (pH 7) containing 1% (vol/vol) Triton X-100 to allow for protein renaturation (37). Bands of lytic activity were visualised by staining with 1% (wt/vol) Methylene Blue (Sigma) in 0.01% (wt/vol) K0H and subsequent destaining with demineralized water (17) ■ SDS-PAA gels without cell walls were stained with Coomassie Brilliant Blue or with the Bio-Rad Silver Stain Kit (Bio- Rad) . Construction of a chromosomal gene library of L. lactis MGI363 in E. coli screening for lytic activity against FI. lysodeikticus A library of L. lactis MGI363 chromosomal DΝA was constructed according to Zabarovsky and Allikmets (54) with some modifications. Genomic DΝA of L. lactis MGI363 was digested for 15 minutes with the appropriate amount of Sα 3A to generate partial digests. After electrophoresis in ultrapure agarose (Pharmacia) of the chromosomal digest, fragments of 4 to 10 kb in size were isolated from the gel using electroelution. The isolated chromosomal DΝA fragments, partially filled- in with dATP and dGTP, and pUC19 DΝA linearized with Sail and partially filled-in with dTTP and dCTP, were ligated, and the ligation mixture was used to electrotransform E. coli ΝM522. After electroporation the cells were plated on TY plates containing 0.2% (wt/vol) autoclaved, lyophilized M. lysodeikticus cells and placed at 37°C. After 2 days the plates were placed at room temperature and examined daily for the appearance of halos around the colonies. Molecular cloning and DNA sequencing. The two Hindlll-Sacl fragments of 1467 and 1383 bp of pALOl, indicated with (a) and (b) in Fig. 3, were subcloned into the Hindlll and Sαcl sites of pUK21 and the resulting plasmids were designated pAL04 and pAL03, respectively (see Fig. 12.A-1). The insert of pALθ4 was subcloned in two fragments using the unique Spel site: pAL05 was constructed by digestion of pAL03 with Spel and Hindlll and subcloning of the 682-bp fragment into the Hindlll and Spel sites of pBluescript SK+: the other part of the insert of pAL04 was subcloned as a Spel-Sacl fragment of 701 bp into the same sites of pBluescript SK+. This subclone was named pAL06. The 352-bp Scal-Sacl fragment of pAL03 was subcloned as a Scαl-CZαl fragment into the Smal and CZαl sites of pBluescript SK+, resulting in pAL07 (see Fig. 12.A-2). Both strands of the inserts of the various subclones were sequenced by the dideoxy-chain-termination method (40) with the T7 sequencing kit (Pharmacia) and double-stranded plasmid templates according to the manufacturer's instructions using universal and reverse pUC primers. The sequence was completed using synthetic DNA primers. Primers were synthesized with an Applied Biosystems 38IA DNA Synthesizer (Applied Biosystems, Inc., Foster City, CA. ) . DNA nucleotide and amino acid sequences obtained were analyzed with the PC/GENE (version 6.7) sequence analysis program (IntelliGenetics, Inc., Geneva, Switzerland). Protein homology searches were carried out with the data bases SWISSPROT (release 27) and the ATLAS of protein and genomic sequences (March, 1994) by means of the FASTA program (35). Southern transfer, DNA hybridisation, and PCR After agarose gel electrophoresis DNA was transferred to Gene- Screen Plus membranes (NEN Research Products, Boston, Mass.) by the protocol of Southern, as modified by Chomczynski and Quasba (4). Probe labelling and hybridisation was done with the ECL labelling and detection system according to the instructions of the manufacturer (Amersham International, Amersham, UK). PCR reactions were carried out with SUPER TAQ DNA-polymerase according to the instructions of the manufacturer (HT BIOTECHNOLOGY LTD, Cambridge, England) on chromosomal DNA using the primers combinations PALA-4 (5'-CTTCAACAGACAAGTCC) and PALA-14 (5'-GATAAATGATTCCAAGC) , both located within the acmA gene (Fig. 4), or PALA-19 (5'-CAAGGTTAAGTCCACG) , which is located upstream of the insert of pINTAA, and BK05AL (5'- ATTATTTGATTGGAGTT) , which is located in the origin of replication of pORI28θ, designated with x and y in Fig. 8, respectively. Construction of an acmA deletion strain A replacement recombination system developed by Leenhouts and Venema (27,29) was used to replace acmA by an acmA gene with an internal deletion on the chromosome of L. lactis MGI363. See Fig. 12.B for a scheme showing the construction of pINTAA. A unique Sαcl site in acmA was obtained by cutting pALOl with EcoRI and Smαl. The plasmid DNA was treated with Klenow enzyme, ligated, and used to electrotransform E. coli NM522. The resulting plasmid pALOδ was digested with Sαcl and Spel , treated with T4 DNA polymerase, ligated, and used to electrotransform E. coli NM522, giving plasmid pAL09. pAL09 was digested with BαmHI and EcoRV, and the DNA fragment containing the deleted acmA gene was subcloned into the SαmHI and Smαl sites of p0RI280. The ligation mixture was used to electrotransform E. coli EC1000. The resulting integration plasmid, named pINTAA, was used to electrotransform L. lactis MGI363. Selection of the second cross-over event was done as described by Leenhouts and Venema (29) . RESULTS Analysis of peptidoglycan hydrolase activity of L. lactis MGI363 The peptidoglycan hydrolysing activity of GM17 or whey-grown L. lactis MGI363 was examined by SDS-PAGE in the presence of either autoclaved, lyophilized PI. lysodeikticus cells, or isolated cell walls of Lactococcus lactis MGI363 as a substrate (Fig. 1). Eleven different clearing bands, corresponding to proteins ranging in size from 29 to 111 kDa, were detected in the supernatant fraction of the whey culture, but only three of these were detectable in this fraction of the GM17 culture when autoclaved ti. lysodeikticus cells were used as a substrate. Three activities above the 46 kDa band detected in the supernatant fraction of whey-grown cells were not detectable in the supernatant fraction of cells grown in GM17. With cell walls of L. lactis MGI363, only 3 bands could be detected in the supernatant of the whey-grown cells and only one in the GM17 culture supernatant. In the cell-free extracts of the two cultures only two clear bands were observed, corresponding to 41 and to 46 kDa proteins, when using N. lysodeikticus cells as a substrate. Only the smaller band was found in gels containing isolated lactococcal cell walls. All the activities could be detected when buffered Triton X-100 ranging from pH 3 to 10 was used in renaturation, but the staining conditions were optimal after renaturation at pH 7• When samples were run on SDS-PAA gels without substrate, protein bands corresponding with the clearing bands could neither be detected in the cell-free extract, nor in the supernatant fraction after staining with Coomassie brilliant blue and subsequent silver staining (results not shown). Peptidoglycan hydrolytic activity was also detected on GM17 agar plates in which 0.2 % (wt/vol) autoclaved, lyophilized N. lysodeikticus cells had been incorporated. Hydrolysis of the cell walls could be seen as zones of clearing around colonies of L. lactis MGI363 after 36 h of incubation at 30°C. When a proteinase-positive strain of L. lactis MGI363 was examined for peptidoglyca r hydrolase activity by SDS-PAGE only a few of the small clearing bands w i present in the supernatant fraction. The bands in the cell-free extract were the same as those seen in a proteinase-deficient strain (results not shown). Cloning of the gene of the major peptidoglycan hydrolase of L. lactis MG1363 The average insert size of the genomic library of ' actis MGI363 in pUC19 was 6 kt f approximately 8000 colonies test* produced a halo on TY plates containing ti. lysodeikticus cells. Re :ng SDS-PAGE on cell-fre*-- extracts of these E. coli clones showed t. I produced clearing ' : is of approximately 46 and 4l kDa (Fig. 2) -kDa band present 1 : ' ■ . coli was present in the lactococcal cell . ;ion only, whereas a -41-kDa band produced by E. coli was observed ia both the cell- free extract and supernatant fraction of L. lactis. Furthermore, several faster migrating bands were visible when cell-free extracts of the positive E. coli clones were analyzed. The sizes of the inserts in pUC19 of the 13 E. coli clones ranged from 4.1 to 9 kb. Restriction enzyme analysis showed that the plasmids isolated from all lysin-producing colonies shared a 4.1-kb Sα 3A insert. A clone containing only this 4.1- kb fragment showed growth problems. An overnight culture of the strain foamed heavily and flocculation of cells was observed in the culture by visual inspection and by light microscopy. This clone was selected for further experiments and its plasmid was designated pALOl. A restriction map of the insert is presented in Fig. 3- Various restriction enzyme digests of chromosomal DNA of L. lactis MG1363 were analyzed by Southern hybridization using the 4.1-kb Sα 3A insert of pALOl as a probe. No rearrangements were found as judged from a comparison of the restriction map of this fragment and the corresponding chromosomal DNA (results not shown) . The 507-bp EeoRV fragment of pALOl could be deleted without loss of any of the clearing bands. As the activity band with the highest molecular weight present in the E. coZi(pAL02) cell-free extracts was 46 kDa, and because the coding- capacity of the smallest of the Sαu3A-2_coRV fragments (1201-bp) was too little to encode a protein of 46 kDa, the 2434-bp _.coRV-Sαu3A fragment was subcloned and sequenced. Nucleotide sequence of the lactococcal gene for peptidoglycan hydrolase The two Sαcl-Hindlll fragments (a) and (b) of pALOl (see Fig. 3.) were subcloned into pUK21 and several smaller subclones were made using pBluescript SK+ (see Fig. 12.A-1 and 12.A-2). The nucleotide sequence of the 1930-bp SspI-Sαu3A fragment pALOl is presented in Fig. 4 and Sequence id no 1 and shows that it contains two complete open reading frames (ORF's), one of 1311 bp (acmA ) and one of 282 bp (ORFA) . Upstream of ORFA a part of a third ORF was found suggesting that ORFA is the last ORF of an operon. Both ORFA and acmA are preceded by putative ribosome-binding sites complementary to the 3' end of the lactococcal l6S rRNA (3) and have ΔG°'s of -9-7 and -9.6 kcal/mol, respectively (43). Also, a possible -10 and -35 region with a spacing of 23 bp is present upstream of acmA . The putative -10 region is preceded by the sequence TGN, which is found in more than 40% of the lactococcal promoters analyzed so far (8) . Downstream of this ORF an inverted repeat with a ΔG°= -16 kcal/mol is located that may function as a rho-independent terminator (36). The sequenced fragment has a G+C content of 36.2%, which is in agreement with the G+C content determined for L. lactis (38.6%; (32)). The codon usage of acmA and ORFA are in agreement with that calculated by van de Guchte et αZ.(46) from several sequenced L. lactis genes. Deduced amino acid sequences and homology comparisons acmA could encode a protein of 437 amino acids with a deduced molecular weight of 46.564. The first 57 amino acids constitute a putative signal peptide (49) and a membrane spanning domain was identified within this stretch of amino acids (Fig. 5 and sequence id no 3). Cleavage of this putative signal peptide would result in a protein of 40.264 Da. The entire protein has an isoelectric point (PI) of 10.45. The protein without the putative signal sequence has a PI of 10.12. A high degree of similarity of the N-terminal part of AcmA was found with muramidase-2 of Enterococcus hirae (6) , and the autolysin of S. faecalis (2) , and a low degree of similarity was observed with the flagellar protein FlgJ of Salmone lla typhimurium (19). An identity of approximately % was found between AcmA and the autolysins of Enterococcus hirae and S. faecalis between amino acids 65 and 220 (Fig. 5A and sequence id 2) . From these data, combined with the biochemical data available from literature (31.33). and the results presented below, we conclude that we have cloned the gene encoding the major lysozyme (N- acetylmuramidase) of L. lactis. Therefore the gene was called acmA . In the C-terminal part of the protein three repeated regions were present. The regions are 44 amino acids long and are separated by intervening sequences highly enriched for serine, threonine, and asparagine residues. The overall similarity between the repeated regions is approximately 75 % (Fig- 6) . At the DNA level the homology is only 45 %. The repeated regions in the C-terminal part of AcmA have homology with the C-terminal repeated regions of the cell wall hydrolases of Enterococcus hirae and S. faecalis . The organisation of these repeats in the three enzymes is schematically presented in Fig. 5*3- This part of AcmA also shows homology with protein P6θ of Listeria monocytogenes , which recently has been shown to possess peptidoglycan hydrolase activity (52). ORFA could encode a protein of 9*4 amino acids with a molecular size of 11.287 Da. The ORFA product has a homology of 64 % (19 % identity) with an 11.2 kDa hypothetical protein in the rfaH-fre intergenic region of the E. coli chromosome (1,7)- Detection of acmA and its gene product in other lactococcal strains. L. lactis subsp. lactis IL1403, L. lactis subsp. lactis biovar diacetylactis l8-l6S, and the L. lactis subsp. cremoris strains AMI, HP, and MG1363 were grown in GM17 for 3 hours. Cell-free extracts were prepared and analyzed by renaturing SDS-PAGE using ti. lysodeikticus cells as a substrate. Major bands of lytic activity at posi ' -ns corresponding to sizes of 4l and 46 kDa were detected in all strains results not shown) . This result was complemented with PCR using the two sequencing primers PALA-4 and PALA-14 (see Fig. 4) and the data are shown in Fig. 7- From the chromosomal DNA's of all strains the same 1131-bp DNA fragment was amplified. These results indicate that all strains contain the same major peptidoglycan hydrolase activity which, in strain AMI, has been identified as an N-acetylmuramidase (31). Construction and analysis of a chromosomal acmA deletion mutant To investigate the function of AcmA a deletion -.s introduced in t chromosomal copy of acmA by replacement recombinε :>n (27,29). After transformation of L. lactis MGI363 with pINTAA, which carries an acmA gene with an internal deletion (Fig. 12.B), the integrants (Em r and β- galactosidase-producing) were checked by PCR using the primers PALA-19 and BK05AL (x and y in Fig. 8, respectively). Integration via portion 1 of the insert of pINTAA resulted in a PCR product of 2550 bp, integration via portion 2 in a PCR product of 347 bp. Three quarters of the integrants obtained had integrated via portion 1 (results not shown) . One of these integrants was used for further experiments. Excision of the integrated plasmid was established after non-selective growth for 30 to 35 generations in GM17. Cells were plated on GM17 plates containing X-gal and 0.2% (wt/vol) autoclaved, lyophilized ti. lysodeikticus cells and screened for loss of blue staining and halo formation. The chromosomal DΝA's of two such colonies were analyzed by Southern hybridisation to show the loss of the Hαelll site located between the Sαcl and Spel sites in acmA (Fig. 8). As expected, in the chromosomes of both strains a fragment of 4057 bp instead of two fragments of 29 bp and 1599 bp (Fig. 9) was present. This result indicates that acmA is not an essential gene in L. lactis . Cell-free extracts and supernatant fractions of the two deletion mutants and MGI363 grown in whey-based medium or GM17 were analyzed on a renaturing SDS-PAA gel containing autoclaved ti. lysodeikticus cells as a substrate. Fig. 10 shows that in both deletion mutants no cell wall hydrolase activity was present, neither in the cell-free extracts nor in the supernatant fractions in GM17~grown cells. Also after growth in whey- based medium, no clearing bands could be detected (results not shown). The fact that all the clearing bands normally present in cell free extracts and supernatant fractions of L. lactis (see Fig. 1) had disappeared after inactivation of acmA shows that all originated from AcmA. After overnight growth in both media precipitation of MG1363αcm_4ΔZ was observed. The cells of MGI363 and of MG1363αcm_Δl were examined by light microscopy. MG1363αcm_4___ formed very long chains as compared to MGI363 (see Fig. 11), indicating that the major lactococcal peptidoglycan hydrolase, AcmA, is needed for cell separation. The data presented above, conclusively show that L. lactis contains only one major peptidoglycan hydrolase, which is a Ν-acetylmuramidase (lysozyme) needed for cell separation. DISCUSSION In this report we present the cloning of the first peptidoglycan hydrolase of the genome of a lactic acid bacterium. The gene encodes the major peptidoglycan hydrolase of Lactococcus lactis. Using PCR and denaturing SDS-PAGE the gene was detected in all L. lactis strains used and, in fact, in all strains tested so far (unpublished observation). Because the gene was also detected in strain AMI, in which Mou et al. (31) observed only a muramidase activity in the cell wall fraction, we conclude that the cloned gene encodes the lactococcal N-acetylmuramidase, an enzyme hydrolysing the linkages between N-acetylmuramic acid and N- acetylglucosamine moieties and was. Accordingly, the gene was designated acmA . Using a standardized assay to detect autolytic activity in a denaturing polyacrylamide gel, several lytic bands were found in both the cell and supernatant fractions of an L. lactis culture using ti. lysodeikticus cell walls as a substrate, but only a few of these bands were found when cell walls of the host were used. This result indicates that the lytic activities of L. lactis are far better detectable with ti. lysodeikticus cell walls as a substrate. This has also been observed by Leclerc et al . (26) when they analyzed bacterial extracts of Clostridium perfringens , Baci llus megaterium or S. faecalis . Comparison of the cell and supernatant fractions of the wild-type lactococcal strain and an acmA deletion mutant revealed that all the lytic bands present in the former originated from AcmA, as all had disappeared in the deletion mutant. As the smallest of the active bands corresponded to a molecular size of approximately 29 kDa, a large part of AcmA can be removed without major loss of activity. As the active site resides, most probably, in the N- terminus (see below) the deletions are thought to occur in the C-terminal repeated region. Degradation of cell wall hydrolases, without loss of activity, has been observed before in Bacillus licheniformis (34) and Bacillus subti lis (22,23.37). In cell-free extracts of E. coli cells expressing acmA , two bands, one of 46 and one of 4l kDa, and several smaller bands were present upon renaturing SDS-PAGE using cell walls of M. lysodeikticus . Bands of 4l and 46 kDa were also detected in cell-free extract of L. lactis but only the smaller of the two was found in the supernatant fraction of L. lactis. acmA encodes a protein of 437 amino acids and the calculated molecular mass of AcmA (46.564 Da) corresponds to the size of the largest clearing band in the lactococcal cell-free extract (46 kDa) . Within the deduced amino acid sequence of AcmA a putative signal sequence of 57 amino acids was identified using the rules of von Heijne (49) • In contrast to most signal peptides from Gram- positive bacteria, the n-region of this putative signal sequence consists of 29 amino acids with 12 charged amino acid residues instead of eight to twelve (50) . This is similar to the signal sequence of muramidase-2 of Enterococcus hirae which has 11 charged amino acids within the first 29 amino acids (6). Based on a signal peptide of 57 amino acids, the molecular mass of mature AcmA would be 40.264 Da, which corresponds to the size (4l kDa) of the major clearing band in the culture supernatant of L. lactis . Most probably, AcmA is produced as a preprotein and the secreted form is the 4l-kDa protein found in the supernatant of an L. lactis culture. The 46-kDa preprotein is either produced in lower amounts or has a reduced activity in the assay used, as the corresponding band of activity is always less clear. The 4l-kDa protein that is present in the cell-free extract is, most likely, the enzyme that is still attached (with the C-terminal repeat region (see below)) to the whole cell. The deduced amino acid sequence of AcmA shows overall similarity with muramidase-2 of Enterococcus hirae (6) and the autolysin of S. faecalis (2) . The identity was very high in the N-terminal regions of the three proteins which, most probably, encompass the active site (20). Within this region homology was also found with the flagellar protein FlgJ of Salmonella typhimurium (19)- In the C-terminal part of AcmA, three repeated regions were present separated by nonhomologous sequences rich in serine, threonine, and asparagine residues. Muramidase-2 possesses six of such repeated regions separated by the same kind of nonhomologous sequences as in AcmA. Within the repeats a consensus sequence was postulated by Joris et al . (20). From the homology comparison it is clear that AcmA, muramidase-2, and the autolysin of S. faecalis contain 3, 6, and 5 of such consensus sequence, respectively. All three cell wall hydrolases have a repeat at their extreme C-terminus. Similar repeats have also been detected in the Baci llus subti lis φPZA lysozyme, the homologous Baci llus gene 15 lysozyme, Listeria monocytogenes pathogenicity-associated protein p60, and Staphylococcus aureus protein A by Joris et al . (20), and in the sporulation related T-D-glutamyl- (L)meso-diaminopimelic-acid-hydrolysing peptidase I of Baci llus sphaericus by Hourdou et αZ.(l6). We also found this consensus twice in the N-terminal part of DniR of E. coli , a protein affecting the anaerobic expression of the hexaheme nitrite reductase (21), of which the second repeat showed some similarity with the first repeat of AcmA. The repeated regions are thought to be involved in substrate recognition and, thus, cell-wall binding (20) . The acmA deletion mutant grows in long chains causing precipitation of the culture after overnight growth. This result proves that AcmA is involved in cell separation as has been found previously by McDonald et al. (30) and confirmed by Langsrud et al . (25). The p6θ protein of Listeria monocytogenes (52) and muramidase-2 of Enterococcus hirae (10) were also found to have an essential role in cell separation. Further analysis of the deletion mutant will reveal whether acmA is the only lactococcal gene encoding a peptidoglycan hydrolase or whether L. lactis contains a second muramidase as is the case in Enterococcus hirae (42). No halos were present when the deletion mutant was plated on GM17 containing M. lysodeikticus cell walls indicating that L. lactis does not express hydrolases that would have been missed in the denaturing SDS-PAGE procedure used here. The results obtained with the proteinase positive L. lactis strain indicate that the activity of the autolysin is partly regulated by proteolytic degradation, as has been observed in Baci llus subti lis (18). The fact that in the supernatant of an L. lactis culture no protein could be detected in an SDS-PAA gel corresponding to the lytic bands observed in an activi gel suggests that the expression of the cell wall hydrolase is very i ., which is the subject of current research. Draft publication 2 AUTOLYSIS OF L. LACTIS CAUSED BY INDUCED OVERPRODUCTION OF THE MAJOR AUTOLYSIN AcmA SUMMARY Growth analysis of a culture of Lactococcus lactis subsp. cremoris MGI363 and a mutant of this strain, containing a deletion in the major autolysin gene acmA , showed that autolysis of the mutant strain was almost absent during prolonged stationary growth. In contrast, the optical density of the wild-type strain L. lactis MGI363 was reduced to more than 50% in the same period. Complementation studies taught that the acmA promoter was located within a 138 bp Sspl-Scal fragment upstream of the structural gene. An acmA mutant with a plasmid-encoded acmA gene and, thus containing more copies of acmA than the wildtype, lysed to a higher extent than the wildtype strain .luring prolonged stationary growth. In trans action of AcmA was shown by mixing end-exponential-phase cultures of an acmA deletion mutant and a tripeptidase (pepT) deletion mutant. PepT, produced by the acmA deletion strain, was detected in the culture supernatant of the mixed culture indicating that lysis of the acmA mutant was due to the action of AcmA secreted by the pepT minus cells. A plasmid was constructed in which the lactococcal major autolysin gene, lacking its own promoter, was placed downstream of an inducible promoter/operator region of the temperate bacteriophage Rl-t. After induction with mitomycin C of an early exponentially growing culture of L. lactis LL302, containing this plasmid, the cells became subject to autolysis, as shown by means of a denaturing SDS-polyacrylamide gel containing tiicrococcus lysodeiktidus cell walls as a substrate and the release of intracellularly located proteins. INTRODUCTION Autolysins are potentially lethal enzymes which hydrolyse the peptidoglycan of the cell wall. Recently we succeeded in cloning the major autolysin acmA of Lactococcus lactis subsp. cremoris MGI363. AcmA is a lysozyme-like enzyme (muramidase) that hydrolyses the N- acetylmuramyl,l,4,-β-N-acetylglucosamine bonds in the peptidoglycan. AcmA was shown to be required for cell separation (2) . Vagerud et al. (24) and Langsrud et al. (9) have monitored autolysis of group N streptococci for prolonged period and observed different rates of autolysis among the strains tested. Vagerud et al. (24) showed that autolysis of lactococci was maximal in buffered M17- medium with glucose as carbon source. Langsrud et al. (9) showed that the activity of intracellularly located lactate dehydrogenase could be detected in the culture supernatant after autolysis. The proteolytic activities of lactococci are involved in ripening and in flavour development in fermented milk products, especially cheese (16,22). Lactococci contain several intracellularly located peptidases (18,20). Release of these peptidases after spontaneous lysis of the cells is believed to be involved in cheese maturation and flavour development (29). Maturation is a slow and therefore, a costly process, which might be accelerated by enhanced lysis of cells and thus, the quick release of intracellular peptidases. In this report we show that, in principal enhanced autolysis can be obtained by using the recently characterized promoter/operator region which is controlled by the temperate lactococcal bacteriophage Rl-t repressor (14,28). MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The strains and plasmids used in this study are listed in Table 2. Lactococcus lactis was grown in £*M17 broth (Difco, West Molesey, United Kingdom) at 30"C or on M17 broth solidified with 1.5% agar, all of which were supplumented with 0.5% glucose. During the growth analysis of the MG1363 and MG1363αc AΔ2, both strains were grown on 1*M17- ! - en needed, 5 μg/ml erythromycin was added (Boehringer GmbH Mannheim, Germany). Escherichia coli was grown in TY (Difco laboratories, Detroit, Mich.) medium at 37°C with vigorous agitation, or on TY medium solidified with 1.5% (wt/vol) agar containing 100 μg of ampicillin (Sigma Chemical Co., St.Louis, MO.) or 100 μg of erythromycin per ml, when required. General DΝA techniques and transformation. Molecular cloning techniques were performed essentially as described by Sambrook et al. (19)- Restriction enzymes, Klenow enzyme, T4 DΝA polymerase, T4 DΝA ligase and deoxynucleotides were obtained from Boehringer Mannheim and were used according to the instructions of the supplier. E. coli and L. lactis were transformed by electroporation, using a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.), as described by Zabarovsky and Winberg (10) and Holo and Νes (5) with the modifications suggested by Leenhouts and Venema (11), respectively. Plasmid DΝA isolation of L. lactis was performed as described by Leenhouts and Venema (11) with some modifications. L. lactis was grown on J*M17 without DL- threonine and the incubation in solution A (without mutanolysin) was done at 5 °C for 15 minutes. Plasmid constructions. The plasmids pGKALl and pGKAL2 were constructed by subcloning of the 1942-bρ Sspl/BamKI and the 1804-bp Scαl/BαmHI fragments of pALOl, respectively, into the EcoRV/BαmHI sites of the lactococcal plasmid pGK13 (Fig.l4B). The ligation mixtures and pGK13 were transformed to L. lactis MG1363αcm Z . The construction of pAL12, in which acmA expression is inducible, is schematically presented in Fig. 16. All steps were performed in E. coli MC1000 unle r -tated otherwise. pALOl is a pUC19 derivative containing a 4l37-bp chrc -αal DΝA fragment from L. lactis MG1363 carrying the autolysin e^ .*7_4). The Sαcl site prer t in the multiple cloning site of pALOl was ed by cutting with EcoRi and Smαl. The plasmid was treated with K_._:.._>w enzyme, ligated, and used to electrotransform E. coli ΝM522, resulting in plasmid pALOδ. Because E. coli grows very poorly when it carries an intact acmA gene (2) , acmA was disrupted by cloning into the unique Sαcl site of pAL08 a 27l6-bp Sαcl fragment originating from the first ORF of the srfA operon of Baci llus s bti lis (27). This resulted in pALlO. Plasmid pIR12 contains a segment of DNA of the temperate lactococcal bacteriophage Rl-t encompassing the repressor gene (rro) , the promoter/operator of rro , tec , the topological equivalent of the E. coli lambda cro gene, and 0RF29. Expression from the promoter/operator region is induced upon addition of mitomycin C to pIR12-containing cells (14). One unique Sαcl site present in pIR12 were deleted by replacing the 2750- bp Sall/XhoII fragment by the 17δ5-bρ SαZI/BαmHI fragment, originating from phage Rl-t, of pEF+ (28) . The second Sαcl site in the resulting plasmid, pIR12EF, was removed by digestion with Sαcl and treatment with T4 DNA polymerase. After self-ligation, pIRlEF was obtained. The 1764-bp EcoRV/XhoII fragment of pIRlEF was replaced by the the 4520-bp Scαl/BαmHI fragment of pALlO containing the interrupted acmA gene. The resulting plasmid, pALll, was digested with Sαcl to remove the fragment interrupting acmA , After self-ligation the mixture was used to transform L. lactis LL302 and plasmid pAL12 was obtained. Mitomycin C induction. An overnight culture of L. lactis was diluted 100-fold in GM17 and grown to an optical density at 600 nm of 0.2. The culture was divided into two portions, to one of which mitomycin C (Sigma) was added to a final concentration of 1 μg/ml, and incubation was continued at 30°C. Optical densities were measured in a Philips PUδ720 UV/VIS scanning spectrophotometer (Pye Unicam Ltd, Cambridge, UK) at 600 nm. SDS-polyacrylamide gel electrophoresis (SDS-PAGE). For the analysis of in trans action of AcmA two milliliters of culture were subjected to centrifugation. One milliliter of the supernatant fraction was dialysed against several changes of demineralized water, lyophilized, and dissolved in 0.5 ml denaturation buffer (1). The cell pellet was resuspended in 1 ml denaturation buffer and cell-free extracts were prepared as described by van de Guchte et. al. (26) . The samples were boiled for 2 min and centrifuged before loading. 30 μl of the mid- exponentional phase samples and 15 μl of the other samples were loaded. For the analysis of induced lysis one milliliter of sample was treated as described above and the supernatant- and cell fractions were dissolved in 0.2 μl denaturation buffer. The amount of sample loaded was equalized according to the measured optical density. SDS-PAGE was carried out according to Laemmli (δ) with the Protean II Minigel System (Bio- Rad) . The low range and prestained low and high range SDS-PAGE standards obtained from Bio-Rad were used. Detection of lytic activitiy in SDS-PAA gels. Lytic activity was detected in situ by using 12.5% (wt/vol) SDS-PAA gels containing 0.15% (wt/vol) autoclaved, lyophilized ti. lysodeikticus ATCC 469δ cells (Sigma). After electrophoresis, the gel was gently shaken at room temperature for 14 h in 200 ml 25 mM Tris-HCI (pH 7) containing 1% (vol/vol) Triton X-100 to allow protein renaturation (17). Bands of lytic activity were visualised by staining with 1% (wt/vol) Methylene Blue (Sigma) in 0.01% (wt/vol) K0H and destaining with demineralized water (6) . SDS-PAA gels without cell walls were stained with Coomassie Brilliant Blue (Bio-Rad) . Western blotting and immunodetection After SDS-PAGE the proteins were transferred to BAδ5 nitrocellulose membranes (Schleicher and Schull, Dassel, Germany) as described by Towbin et al. (23). Endopeptidase antigen and tripeptidase antigen were detected with polyclonal antiendopeptidase antibodies (12), diluted l:δ000, and polyclonal antitripeptidase antibodies (20), diluted 1:4000, respectively, and alkaline phosphatase-conjugated goat anti-rabbit antibodies (Promega Corporation, Madison, Wis.) using the Western-Light Chemiluminescent detection system of TR0PIX (TR0PIX, Inc., Bedford, Mass.) according to the manufacturer's instructions. RESULTS AcmA is involved in autolysis of L. lactis during stationary phase. Overnight cultures of L. lactis MG1363 and its acmA deletion mutant MG1363αcm/_ΔZ (2) were diluted 200-fold in fresh prewarmed M17. During the first 9 hours of growth and hourly sampling, the cultures were gently shaken at 30°C to prevent settling of MG1363αcm__ΔZ, which grows as long filaments due to improper cell separation (2). During further incubation, without shaking, samples were taken with larger intervals. Fig.13 shows the changes in OD 6oo of MGI363 and MG1363αcm/LΔ_ during 320 hours of incubation. Whereas the wild-type and mutant cells grew equally well, the decrease in OD 6oo of the mutant was much less pronounced than that of the wild-type strain. Apparently, the major autolysin is not only required for cell separation (2) , but is also responsible for cell lysis upon prolonged incubation. Complementation of the acmA deletion, and localisation of the acmA promoter. To examine wether the 13δ-bp Sspl/Scal fragment containing a putative -35 and -10 sequence upstream of the startcodon of the acmA gene, was the promoter of this gene, pGKALl and pGKAL2 were constructed (Fig.l4B). pGKALl contains this putative promoter fragment whereas pGKAL2 lacks this sequence. Strain L. lactis MGI363 containing pGK13 and strain L. lactis MG1363αcmlΔ_ containing pGK13, pGKALl or pGKAL2 were plated on GM17 plates containing 0.2 % tiicrococcus lysodeikticus autoclaved cells and incubated for 36 hours at 30°C. The results are presented in Fig.3 and shows that no halo was obseved around the cells containing pGKAL2, but that a large halo was present around the cells containing pGKALl. The halo was even larger than the one formed by L. lactis MGI363. This result indicates that the 13δ-bρ Sspl-Scal fragment is required for acmA expression, most likely because it contains the -35 and -10 promoter- specific sequences shaded in Fig.l4A (corresponding to Sequence id no 5) . Increasing the copy number of acmA results in more autolysis. Overnight cultures of strains MGI363 containing pGK13 and MG1363αcm_Δ_ containing the plasmids pGK13 or pGKALl were diluted 100- fold in fresh medium. During the exponentional growth phase the strains grew equally fast (see Fig. 16) . In the following 70 hours of incubation, the reduction in optical density of MG1363αcπ_ΔZ (pGKALl) was much higher than that of MGI363 (pGK13). As expected during the same period of growth nearly no reduction in 0D 600 was observed with the deletion mutant containing pGK13. Secreted AcmA can act in trans. Overnight cultures of MG1363αcm_ΔZ and MG1363pepT were diluted 100- fold in fresh medium. At the end of the exponentional phase of growth equal fractions of both strains were mixed and incubated at 30 β C. The optical density was followed for 70 hours and samples were taken at various time points to analyze AcmA activity, the presence of PepT, and the release into the culture medium of intracellular proteins. The reduction of 0D 600 during prolonged stationary phase of the mixed culture is nearly equal to that of the cultures of MGI363 and the pepT deletion mutant (see Fig. 17) , indicating that the degree of autolysis is nearly the same in these three cultures. Also the average chainlenght in the mixed culture was equal to that of the lenght of the chains of MGI363 and MG1363pep2\ Figures 18 (A.B.C) and Table 3 show the analysis of AcmA activity and the presence of PepT in the cell and supernatant fractions while the release of intracellular proteins into the culture medium is presented in Fig. 18B. From these results it can be concluded that in the control strain MGI363 AcmA is produced, secreted and causes autolysis of cells from the moment the culture enters the stationary growth phase. Autolysis results in the release of intracellular proteins into the culture medium, see Fig. lδB, of which one, PepT, was visualized using specific antibodies (see Fig. lδC) . Also in MG1363pepT autolysis and subsequent release of intracellular proteins occurs, as expected no PepT was detected in the supernatant fraction. MG1363αcmΛΔ2 produces PepT but no autolysis occurs and, therefore, no PepT or other intracellular proteins are released (Fig. lδB and lδC) . Induced expression of AcmA. To examine whether overexpression of AcmA would lead to cell lysis, the acmA gene lacking its native promoter but retaining its own RBS (2) was cloned in pIR12 (15), resulting in plasmid pAL12 (Fig.19). pAL12 contains rro, tec , and the promoter/operator of the repressor protein Rro " the L. lactis subsp. cremoris bacteriophage Rl-t. The expression of > nes cloned downstream of tec is repressed by the Rro repressor (15) but is induced upon addition of mitomycin C. Plasmid pAL12 was transformed to L. lactis subsp. cremoris LL302 which contains a copy of the pWVOl repA gene on the chromosome to ensure efficient replication. Mitomycin C (lμg/ml) was added to L. lactis MGI363 containing pGK13 and L. lactis LL302 containing pIR12, pAL12, or pIRlPR, in which β-galactosidase, AcmA, and LytP and LytR are expressed, respectively. With intervals of one hour the optical density at 600 nm was measured. The results are presented in Fig. 20 and show tha. starting at approximately 2 to 3 hours after the addition of mitomycin C, the optical density of strain LL302(pAL12) gradually and steadily decreased. The strain carrying the bacteriophage Rl-t lysis cassette LytP/LytR also showed a distinct reduction in OD 6oo upon addition of mitomycin C, as shown by Nauta et al. (14). In contrast, strain MG1363(pGK13) and LL302(pIR12) , which produces E. coli β- galactosidase from the same promoter/operator cassette, did not show detectable lysis in this assay. The data presented in Fig. 20 clearly shows that addition of 1 μg/ml of mitomycin C resulted in a severe inhibition of growth of the cells. Taken together, the results presented in Fig. 16 strongly suggest that overproduction of AcmA by the addition of mitomycin C to exponentially growing L. lactis LL302(pAL12) causes lysis of the cells. To examine whether under the conditions employed, production of AcmA was indeed induced, samples were taken four hours after mitomycin C addition and inspected by renaturing SDS-PAGE for lytic activity in the cell-free and supernatant fractions. The results are presented in Fig. 21 and show that increased activity of AcmA was present in an L. lactis LL302(pAL12) cell-free extract as compared to cell-free extracts of L. lactis LL302(pIR12) and L. lactis LL302(pIRlPR) . Qualitatively, the same applied to the supernatant fractions of the three strains: only L. lactis LL302(pAL12) produced an excess of a clearing band at a position corresponding to a protein of 30 kDa, which has been shown to be an active degradation product of AcmA (2). Both in the cell-free extract and the supernatant of strain LL302(pIRlPR) the phage lysin specified by pIRlPR was present, together with the lytically active breakdown products of AcmA produced from the chromosome of the strain. We conclude from these data that the synthesis of AcmA was increased upon induction with mitomycin C. Cytoplasmic proteins are released in the culture medium upon induction of AcmA overexpression. Although AcmA production was clearly induced, the 0D measurements did not conclusively show that increased production of the autolysin resulted in lysis of the host cells. To examine this in a more direct way, the supernatant fractions of induced cultures were assayed for the presence of intracellular proteins by SDS-PAGE. The results, presented in Fig. 22, show that in uninduced cultures only one protein is present. Most probably, this protein is the previously described Usp45 (25). Upon induction, proteins normally present in the cell-free extracts only, are extruded into the culture medium, especially in L. lactis(pIRlPR) , overexpressing the bacteriophage lysis cassette LytP/LytR (Fig. 19 lane 6). To a considerable extent this also applies to L. Zactis(pAL12) , overexpressing acmA (Fig. 22, lane 4). To a lesser extent, L. lactis(pIR12) extruded proteins into the supernatant, indicating that, as has been observed before (14), the presence of mitomycin C and/or the overproduction of high amounts of β-galactosidase, caused some lysis. To ascertain that cytoplasmic proteins were extruded into the culture medium after mitomycin C induction, immunoblots were performed on supernatants of cells carrying the various plasmids. The results in Fig. 20 show that antibodies raised against the cytoplasmic lactococcal peptidase PepO (13) gave a strong signal with the supernatant of L. Zαc£is(pAL12) and only a weak one with that fraction of L. Zαctis(pIR12) . DISCUSSION The observation that nearly no cell lysis was observed in a culture of the Lactococcus lactis MG1363αcm_4Δ_! during prolonged stationary growth, indicates that AcmA, is, in addition to cell separation (2), also responsible for autolysis. This result also shows that no other major autolysin activity, resulting in autolysis, is present in L. lactis MGI363. No halo could be detected, not even after one week of incubation at 30 °C (result not shown) , when L. lactis MG1363αcτπ_ΔZ was plated on a GM17 plate containing tiicrococcus lysodeikticus cell walls as a substrate, showing that no other major autolysin is present. In contrast, when this mutant strain contained plasmid pGKALl a large halo was produced, but no halo was formed when it contained pGKAL2. This result shows that the acmA promoter is located within the 138-bp Sspl-Scal fragment upstream of acmA . When the mutant strain contained pGKALl this led to a higher reduction of the optical density as compared to the wildtype strain, indicating that a higher expression of AcmA, because of the presence of more copies of the gene in this strain, results in more cell lysis. The acmA gene, lacking its own promoter, downstream of the promoter/operator region of the temperate lactococcal bacteriophage Rl-t was subcloned. In this plasmid the expression of acmA was inducible upon addition of mitomycin C, which was shown in a denaturing SDS-PAA gel containing ti. lysodeikticus cell-walls as a substrate. Four hours after induction increased expression of AcmA was observed in the lactococcal strain containing pAL12 while a natural level of expression was found in the strain containing either pGK13, pIR12, or pIRlPR. Growth analysis of these strain showed that the overproduction of AcmA leads to a cell lysis. Although limited lysis of cells was observed in the strain overexpressing β-galactosidase, which might be due to the high level of mytomicin C used, a higher level of reduction of the optical density was observed in cultures of the strain overexpressing AcmA. Cells containing pIRlPR lysed more readily upon induction than cells containing pAL12 (Fig. 20). Although other conclusions cannot be excluded, this difference may be caused by a higher lytic activity of LytR than that of AcmA. From the growth analyses it is clear that mitomycin C inhibits the growth of cells. To circumvent this undesirable side effect of this DNA-damaging substance, research was focussed on the isolation of a temperature sensitive repressor (Rro) mutation, so that the system can be made temperature-inducible. The promoter is present on plasmid pIRl4 deposited at the Centraal Bureau voor Schimmelcultures in Baarn, The Netherlands in accordance with the Budapest Treaty on May 11, 1995 with accession number CBS 327.95. Release of the intracellularly located lactococcal endopeptidase, which might well be involved in flavour development during cheese maturation, was demonstrated using antibodies against this protein. Strong signals were found in samples of the culture supernatant of the two strains that lysed upon induction, viz. LL302(pIRlPR) and LL302(pAL12). In trans action of AcmA was shown by analysing a mixed culture of acmA and pepT minus cells during prolonged incubation in stationary phase. Release of PepT, expressed by acmA minus cells, into the culture medium was shown to occur as a result of the expression and secretion of AcmA by pep T minus cells. From these data we conclude that we have succeeded in the development of a system in which the expression of the major autolysin AcmA of L. lactis was made inducible, and that the production of AcmA leads to lysis of L. lactis with concomitant release of intracellular proteins and enzymes in the culture medium. Table 2. Bacterial strains and plasmids Strain or plasmid Relevant phenotype or genotype Source or reference Strains L.lactis subsp. cremoris MG1363 Plasmid-free strain (4) 10 MG1363acmAΔl MGI363 derivative containing a 701 bp Sacl/Spl chromosomal deletion in the acmA gene (2) MG1363pepT MGI363 derivative containing a deletion in the pepT gene (12) LL302 MGI363 carrying the pWVOl repA gene on the chromosome Culture Collection PU Groningen 15 E. coli NM522 supE.thi.Δ(lac-proAB) ,Δhsd5(r-,m-)[F' ,proAB,lacI q ZM15] Stratagene (La Jolla, MC1000 araD139, lacx74, (ara,leu)7697. galU, galK, strA (3) 20 Plasmids pALOl Ap r , pUC19 carrying a 4137 bp lactococcal chromosomal DNA (2) insert with the peptidoglycan hydrolase gene acmA pALOδ Ap r , pALOl with a Smal/EcoRI deletion (2) 25 pALlO Ap r , pALOδ containing a 27l6-bp Sad fragment of ORFl of This work the srfA operon of B.subtilis. pALll Em r , SK+ with a 710 bp Sacl/Spel fragment of pAL03 This work pAL12 Em r , SK+ with a 77 bp Scal/Clal fragment of pAL04 This work (Continuation Table 2) Strain or plasmid Relevant phenotype or genotype Source or reference pIR12 Em r , carrying the regulatory region of phage Rl-t (15) 10 pEF+ Ap r , SK+ containing a 174θ-bp EcoRI fragment of phage Rl Plasmid Collectio RU Gronin PIR12EF Em r pIR12 containing a 17δ5~bp Sall/BamHI fragment of pEF+ This wor pIRlEF Em r pIR12EF with a deleted Sad site This work 15 pIRlPR Em r pIR12 derivative carrying the lytP and lytR gene of Rl-t (14) pGK13 Em r Cm 1" , pWVOl based lactococcal plasmid. (7) pGKALl Em r Cm r , pGK13 containing the 1942 bp SspI/BamHI fragment of pALOl This work pGKAL2 Em r Cm r , pGK13 containing the lδθ4 bp ScalBamHI fragment od pALOl This work 20 List of references • 1. WO 90/00599 (AGRICULTURAL & FOOD RESEARCH COUNCIL; M.J. Gasson) published 2 January 1990; Uses of viral enzymes 2. CA. Shearman, K. Jury & M.J. Gasson (AFRC) ; Biotechnology 10 (Feb. 1992) 196-199; Autolytic Lactococcus lactis expressing a lactococcal bacteriophage lysin gene 3. C. Platteeuw and W.M. de Vos (NIZO) ; Gene llδ (1992) 115-120; Location, characterization and expression of lytic enzyme-encoding gene, lytA , of Lactococcus lactis bacteriophage φUS3 4. R. Young; Microbiol. Reviews 56 (1992) 430-4δl; Bacteriophage Lysis: Mechanism and Regulation; esp. pages 46δ-472: Lysis in Phage Infections of Gram-Positive Hosts, and Perspectives 5. C. Shearman, H. Underwood, K. Jury, and M. Gasson (AFRC); Mol. Gen. Genet. 2lδ_ (19δ9) 214-221; Cloning and DNA sequence analysis of a Lactococcus bacteriophage lysin gene 6. L.J.H. Ward, T.P.J. Beresford, M.W. Lubbers, B.D.W. Jarvis and A.W. Jarvis; Can. J. Microbiol. 2 (1993) 767~774; Sequence analysis of the lysin gene region of the prolate lactococcal bacteriophage c2 7. EP-A2-0510907 (AGRICULTURAL & FOOD RESEARCH COUNCIL; M.J. Gasson) published 2δ October 1992; Bacteriophage lysins and their applications in destroying and testing for bacteria Literature references of draft publication 1 1. Bailey, M.J.A., V. Koronakis, T. Schmoll, and C. Hughes. 1992. Escherichia coli HlyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH(sfrB) locus required for expression of sex factor and lipopolysaccharide genes. Mol. Microbiol. 6:1003-1012. 2. Beliveau, C. , C. Potvin, J. Trudel, A. Asselin, and G. Bellemare. 1991. Cloning, sequencing and expression in Escherichia coli of a Streptococcus faecalis autolysin. J. Bacteriol. 173*5619-5623. 3- Chiaruttini, C. and M. Milet. 1993- Gene organization,primary structure and RNA processing analysis of a ribosomal RNA operon in Lactococcus lactis . J. Mol. Biol 230:57~76. 4. Chomczynskl, P. and P. K. Quasba. 19δ4. Alkaline transfer of DNA to plastic membrane. Biochem. Biophys. Res. Commun. 122:340-344. 5. Chopin, A., M. -C. Chopin, A. Moillo-Batt, and P. Langella. 19δ4. Two plasmid-determined restriction and modification systems in Streptococcus lactis . Plasmid 11:260-263. 6. Chu, C.-P., R. Kariyama, L. Daneo-Moore, and G. D. Shockman. 1992. Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae . J. Bacteriol. 174:l6l9 _ l625. 7. Daniels, D.L., G. Plunkett III, V. Burland, and F. R. Blattner. 1992. Analysis of the Escherichia coli genome: DΝA sequence of the region from δ4.5 to δ6.5 minutes. Science 257:771-77δ. δ. de VOS, W.M. and G. F. M. Simons. 1993. Gene cloning and expression systems in lactococci, p. 52-10 • In M. J. Gasson and W. M. de Vos (eds.), Genetics and Biotechnology of Lactic Acid Bacteria. 9. de VOS, W.M., P. Vos, H. de Haard, and I. Boerrigter. 19δ9. Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase. Gene 8 *169-176. 10. Del Mar Lleo, M. , P. Canepari, and G. Satta. 1993- Thermosensitive cell growth mutants of Enterococcus hirae that elongate at non-permissive temperature are stimulated to divide by parental autolytic enzymes. J. Gen. Microbiol. 139:3099-3107. 11. Doyle, R.J., J. Chaloupka, and V. Vinter. 19δ8. Turnover of cell walls in microorganisms. Microbiol. Rev. 52:554-567- 12. Exterkate, F.A. 1976. Comparison of strains of Streptococcus cremoris for proteolytic activities associated with the cell wall. Νeth. Milk Dairy J. 30:95-105. 13- Gasson, M.J. I983. Plasmid complements of Streptococcus lactis ΝCD0 712 and other lactic Streptococci after protoplast-induced curing. J. Bacteriol. 154:1-9. 14. Hill, C, C. Daly, and G. F. Fitzgerald. 19δ5. Conjugative transfer of the transposon Tn919 to lactic acid bacteria. FEMS Microbiol. Lett. 30:115-119- 15. Holo, H. and I. F. Νes. 19δ9- High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol. 55:3119-3123- 16. Hourdou, M.-L., M. Guinand, M. -J. Vacheron, G. Michel, L. Denoroy, C. Duez, S. Englebert, B. Joris, G. Weber, and J. -M. Ghuysen. 1993. Characterization of the sporulation-related τ-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus ΝCTC 9602 as a member of the metallo(zinc) carboxypeptidase A family (Modular design of the protein) . Biochem. J. 292:563-570. 17. Jayaswal, R.K. , Y. -I. Lee, and B. J. Wilkinson. 1990. Cloning and expression of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity. J. Bacteriol. 172:57δ3~57δδ. lδ. Jolliffe, L.K. , R. J. Doyle, and U. Ν. Streips. 19δ0. Extracellular proteases modify cell wall turnover in Baci llus subti lis . 3. Bacteriol. l4l:1199-1206. 19. Jones, C.J., M. Homma, and R. M. Macnab. 19δ9- L-.P-, and M-ring proteins of the flagellar basal body of Salmonella typhimurium: gene sequences and deduced protein sequences. J. Bacteriol. 171:3δ90~3900. 20. Joris, B. , S. Englebert, C. -P. Chu, R. Kariyama, L. Daneo-Moore, G. D. Shockman, and J. -M. Ghuysen. 1992. Modular design of the Enterococcus hirae muramidase-2 and Streptococcus faecalis autolysin. FEMS Microbiol. Lett. 91:257-264. 21. Kaije, S.-I., R. Ideta, I. Yamato, and Y. Anraku. 1991- Molecular cloning and DΝA sequence of dniB , a gene affecting anaerobic expression of the Escherichia coli hexaheme nitrite reductase. FEMS Microbiol. Lett. 83:205-212. 22. Kuroda, A. and J. Sekiguchi. 1990. Cloning, sequencing and genetic mapping of a Baci llus subti lis cell wall hydrolase gene. J. Gen. Microbiol. 136:2209-2216. 23. Kuroda, A. and J. Sekiguchi. 1991- Molecular cloning and sequencing of a major Baci llus subti lis autolysin gene. J. Bacteriol. 173:7304-7312. 24. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature(London) 227:680-665. 25. Langsrud, T. , A. Landaas, and H. B. Castberg. 1967- Autolytic properties of different strains of group N streptococci. Milchwissenschaft 42:556-560. 26. Leclerc, D. and A. Asselin. 19δ9. Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis. Can. J. Microbiol. 35:749-753- 27. Leenhouts, K. and G. Venema. 1992. Molecular cloning and expression in Lactococcus. Med. Fac. Landbouww. Univ. Gent. 57-2031-2043. 2δ. Leenhouts, K.J., J. Kok, and G. Venema. 1990. Stability of integrated plasmids in the chromosome of Lactococcus lactis . Appl. Environ. Microbiol. 56:2726-2735- 29. Leenhouts, K.J. and G. Venema. 1993- Lactococcal plasmid vectors, p. 6 -9^- In K. G. Hardy (ed.), Plasmids, a practical approach. Oxford University Press, Oxford. 30. McDonald, I.J. 1971- Filamentous forms of Streptococcus cremoris and Streptococcus lactis . Observations on structure and susceptibility to lysis. Can. J. Microbiol. 17:δ97~902. 31. Mou, L., J. J. Sullivan, and G. R. Jago. 1976. Autolysis of Streptococcus cremoris . J. Dairy. Res. 43:275 -282. 32. Mundt, J.O. 1986. Lactic acid streptococci, p. IO65-IO66. In P. H. A. Sneath, Ν. S. Mair, M. E. Sharpe, and J. G. Holt (eds.), Bergey's manual of systematic bacteriology,vol.2. Williams and Wilkins, Baltimore. 33- Νiskasaari, K. 1989. Characteristics of the autolysis of variants of Lactococcus lactis subsp. cremoris . J. Dairy. Res. :639- 49. 34. Oda, Y. , R. Νakayama, A. Kuroda, and J. Sekiguchi. 1993- Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwlL, of Baci llus licheniformis. Mol. Gen. Genet. 241:360-366. 35- Pearson, W.R. and D. J. Lipman. 19δδ. Improved tools for biological sequence comparison. Proc. Νatl. Sci. USA 85:2444-2448. 36. Platt, T. 1986. Transcription termination and the regulation of gene expression. Ann. Rev. Biochem. 55:339-372. 37- Potvin, C. , D. Leclerc, G. Tremblay, A. Asselin, and G. Bellemare. 198δ. Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli . Mol. Gen. Genet. 214:241-248. 38. Rogers, H.J. 1979. The function of bacterial autolysins, p. 237-268. In R. C. W. Berkeley, G. W. Gooday, and D. C. Ellwood (eds.), Microbial polysaccharides and polysaccharidases. Academic Press, Inc.(London) ,Ltd. , London. 39- Sambrook, J., E.F. Fritsch, and T. Maniatis. I989. Molecular cloning - a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,Ν.Y.. 40. Sanger, F. , S. Νicklen, and A. R. Coulson. 1977- DΝA sequencing with chain-terminating inhibitors. Proc. Νatl. Acad. Sci. USA 74:5463-5467. 41. Seegers, J.F.M.L., S. Bron, C. M. Franke, G. Venema, and R. Kiewit. 1994. The majority of lactococcal plasmids carry a highly related replicon. Microbiol. (in press,: 42. Shockman, G.D. 1992. The autolytic ('suicidase' ) system of Enterococcus hirae: From lysine depletion autolysis to biochemical and molecular studies of the two muramidases of Enterococcus hirae ATCC9790. FEMS Microbiol. Lett. 100:261-266. 43. Tinoco, jun.I., P. Ν. Borer, B. Dengler, M. D. Levine, 0. C. Uhlenbeck, D. M. Crothers, and J. Gralla. 1973- Improved estimation of secondary structure in ribonucleic acids. Nature New Biol. 246:40-41. 44. Tomasz, A. 1984. Building and breaking of bonds in the cell wall of bacteria-the role for autolysins, p. 3"12. In C. Nombela (ed.), Microbial cell wall synthesis and autolysis. Elsevier Science Publishers, Amsterdam. 45. van de Guchte, M. , J. Kodde, J. M. B. M. van der Vossen, J. Kok, and G. Venema. 1990. Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, .secretion, and processing of the Bacillus subtilis neutral protease. Appl. Environ. Microbiol. 56:2606-2611. 46. van de Guchte, M. , J. Kok, and G. Venema. 1992. Gene expression in Lactococcus lactis . FEMS Microbiol. Rev. 88:73-92. 47. Vieira, J. and J. Messing. 1991- New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189-194. 48. Visser, F.M. . 1977- Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 2.Development of bitterness and cheese flavour. Neth. Milk Dairy J. 31:l8δ-209- 49. Von Heijne, G. 1986. A new method for predicting signal peptide cleavage sites. Nucl. Acids Res. 14:4683-4690. 50. Von Heijne, G. and L. Abrahms' n. 1989. Species-specific variation in signal peptide design; Implications for protein secretion in foreign nosts. FEBS Letters 244:439-446. 51. Ward, J.B. and R. Williamson. 19δ4. Bacterial autolysins: specificity and function, p. 159-175. In C. Nombela (ed.), Microbial cell wall synthesis and autolysis. Elsevier Science Publishers, Amsterdam. 52. Wuenscher, M.D. , S. KOhler, A. Bubert, U. Gerike, and W. Goebel. 1993- The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity. J. Bacteriol. 175:3491-3501. 53- Yanish-Perron, C, J. Vieira, and J. Messing. 1985- Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103-119- 54. Zabarovsky, E.R. and R. L. Allikmets. 1986. An improved technique for efficient construction of gene libraries by partial filling-in of cohesive ends. Gene 42:119-123. 55- Zabarovsky, E.R. and G. Winberg. 1990. High efficiency electroporation of ligated DΝA into bacteria. Νucl. Acid Res. 18:5912. LITERATURE OF DRAFT PUBLICATION 2 1. Beliveau, C. , C. Potvin, J. Trudel, A. Asselin, and G. Bellemare. 1991. Cloning, sequencing and expression in Escherichia coli of a Streptococcus faecalis autolysin. J. Bacteriol. 17356l9- 623. 2. Buist, G. , J. Kok. K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrikman. 1995. Molecular cloning and nucleotide sequence of the gene encoding the major petidoglycan hydrolase of Lactococcus lactis, a muramidase neede for cell separation. J. Bacteriol 177:1554-1563- 3- Casadaban, M.J. and S. N. Cohen. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli . J. Mol. Biol. 138:179-207. 4. Gasson, M.J. 1983. Plasmid complements of Streptococcus lactis NCD0 712 and other lactic Streptococci after protoplast-induced curing. J. Bacteriol. 154:1-9. 5. Holo, H. and I. F. Nes. 1989. High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol. 3119-3123- 6. Jayaswal, R.K. , Y. -I. Lee, and B. J. Wilkinson. 1990. Cloning and expression of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity. J. Bacteriol. 172:5783-5786. 7. Kok, J. 1992. Special-purpose vectors for lactococci, p. 97-102. In G. Dunny, P. P. Cleary, and L. L. McKay (eds.), Genetics and molecular biology of Streptococci, Lactococci, and Enterococci. American Society for Microbiology, Washington,D.C.. 8. Laemnli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature(London) 227:680-665. 9. Langsrud, T. , A. Landaas, and H. B. Castberg. 1987. Autolytic properties of different strains of group N streptococci. Milchwissenschaft 42:556-560. 10. Zabarovsky, E.R. and G. Winberg. 1990. High efficiency electroporation of ligated DNA into bacteria. Nucl. Acid Res. 18:5912. 11. Leenhouts, K.J. and G. Venema. 1993- Lactococcal plasmid vectors, p. 65-94. In K. G. Hardy (ed.), Plasmids, a practical approach. Oxford University Press, Oxford. 12 Mierau, I., A. J. Haandrikman, 0. Velterop, P. S. T. Tan, K. J. Leenhouts, W. N. Konings, G. Venema, and J. Kok. 1994. Tripeptidase gene (pepT) of Lactococcus lactis : molecular cloning and nucleotide sequencing of pepT and construction of a chromosomal deletion mutant. J. Bacteriol 176:2854-2861. 13. Mierau, I., P. S. T. Tan, A. J. Haandrikman, J. Kok, K. J. Leenhouts, W. N. Konings, and G. Venema. 1993- Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase. J. Bacteriol 175-'2087-2096. 14. Nauta, A., D. Van Sinderen, H. A. Karsens, J. Kok, and G. Venema. 199*4. Inducible lysis of Lactococcus lactis mediated by the Lactococcus lactis subsp. cremoris bacteriophage Rl-t functions. (EP 94201353-3) 15. Nauta, A., D. Van Sinderen, J. Kok, and G. Venema. 1994. Inducible gene expression mediated by a repressor-operator system isolated from Lactococcus lactis subsp. cremoris bacteriophage Rl-t. (EP 94201353-3) 16. Olson, Ν.F. 1990. The impact of lactic acid bacteria on cheese flavour. FEMS Microbiol. Rev. 87:131-148. 17. Potvin, C. , D. Leclerc, G. Tremblay, A. Asselin, and G. Bellemare. 1988. Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli . Mol. Gen. Genet. 214:241-248. 18. Pritchard, G.G. and T. Coolbear. 1993. The physiology and biochemistry of the proteolytic system in lactic acid bacteria. FEMS Microbiol. Rev. 12:179-206. 19. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning - a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,Ν.Y.. 20. Tan, P. S. T., M.-P. Chapot-Chartier, K. M. Pos, M. Rousseau, C.-Y. Boquien, J.-C. Gripon, and W. N. Konings. 1992. Localization of peptidases in lactococci. Appl. Environ. Microbiol. 58:285-290. 21. Tan, P.S.T., B. Poolman. and W. N. Konings. 1993- Proteolytic enzymes of Lactococcus lactis. J. Dairy Res. 60:269-286. 22. Thomas, T.D. and G. G. Pritchard. 1987. Proteolytic enzymes of dairy starter cultures. FEMS Microbiol. Rev. 46:245-268. 23. Towbin, H., T. Staechelin, and J. Gordon. 1979- Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 24. Vagerud, G. , H. B. Castberg, and T. Langsrud. 1983. Autolysis of group N Streptococci. Effects of media composition and temperature. J. Dairy. Sci 66:2294-2302. 25. Van Asseldonk, M. , G. Rutten, M. Oteman, R. J. Siezen, W. M. de V0S, and G. Simons. 1990. Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MGI363. Gene 95-155-160. 26. van de Guchte, M. , J. Kodde, J. M. B. M. van der Vossen, J. Kok, and G. Venema. 1990. Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Baci llus subti lis neutral protease. Appl. Environ. Microbiol. 56:2606-2611. 27. Van Sinderen, D. , G. Galli, P. Cosmina, F. De Ferra, S. Withoff, G. Venema, and G. Grand!. 1993- Characterization of the srfA locus of Baci llus subti lis : only the valine-activating domain of srfA is involved in the establishment of genetic competence. Mol. Microbiol. 8:833"84l. 2δ. Van Sinderen, D. , H. A. Karsens, G. Venema, and A. Νauta. 1994. (unpublished results). 29. Visser, F.M.W. 1977- Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 2.Development of bitterness and cheese flavour. Νeth. Milk Dairy J. 31:166-209. SEQUENCE LISTING Sequence id no 1 is the nucleotide sequence of figure 4. Sequence id no 2 is the amino acid sequence of the lysin of figure 4 and also corresponds to the lysin of L. lactis amino acid sequence of figure 5A. Sequence id no 3 is the membrane spanning domain identified within the stretch of amino acids forming lysin of figure 5- Sequence id no 4 is an amino acid sequence comprising the 3 direct repeats of figure 6. Sequence id no 5 is the nucleotide sequence of the promoter region of acmA as in figure 14. Sequence id no 6-9 are primers or probes. Sequence id no 10 is the amino acid sequence of ORF A. Sequence id no 11 is the nucleic acid sequence of figure 4-0RF B nt 1823-1928. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Quest International (B) STREET: Huizerstraatweg 28 (C) CITY: Naarden (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP): l4ll GP (G) TELEPHONE: 02159 - 99111 (H) TELEFAX: 02159 - 46067 (ii) TITLE OF INVENTION: Process for the lysis of a culture of lactic acid bacteria by means of a lysin, and uses of the resulting lysed culture. (iii) NUMBER OF SEQUENCES: 14 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER: (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1930 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Lactococcus lactis (B) STRAIN: MGI363 (vii) IMMEDIATE SOURCE: (B) CLONE: Fig. (publ.l)-acmA and ORFA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 178..1489 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: complement (1542..1824) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATTTTTGTTT TAAATCAGAT TTTTTAGATT AAAGGCAAAA AGTTTTTACA AATATGAATC 60 CTTAACGGAA AAACGTTTAC AAACCGCCAC CAAATTGACA TCTTTTTTTA GCTTGAGGCG 120 TGGTAGAATA AAGATAGTAC TTATTATATT TTGTAATCTT TAGAAAGGTA ATTATTT 177 ATG CCA GTA TCA CGT GTT AAA GTT AAA AAT AGA CAT TTA AAA AAG AAA 225 Met Pro Val Ser Arg Val Lys Val Lys Asn Arg His Leu Lys Lys Lys 1 5 10 15 ACT AAA AAA CCA CTC GCT TTT TAT AAA CCA GCC ACA AAA TTT GCT GGC 273 Thr Lys Lys Pro Leu Ala Phe Tyr Lys Pro Ala Thr Lys Phe Ala Gly 20 25 30 GCT GTT CTT ATT GCC GGA ACA TTG ACA ACC ACA CAT GAA CTT CTT CTT 321 Ala Val Leu He Ala Gly Thr Leu Thr Thr Thr His Glu Leu Leu Leu 35 40 45 CAA CAG ACA AGT CCA ATG GTT CAA GCA GCG ACT AAC TCA TCA GAG GTT 369 Gin Gin Thr Ser Pro Met Val Gin Ala Ala Thr Asn Ser Ser Glu Val 50 55 60 TTT ATT GAA AGT ATT GCC GCA TCA GCA AAA CCT GTG GCA GAT GCT AAT 417 Phe He Glu Ser He Ala Ala Ser Ala Lys Pro Val Ala Asp Ala Asn 65 70 75 80 GGC TTA TAT CCT TCG GTC ATG ATT GCC CAA GCT ATT TTG GAA AGC AAC 465 Gly Leu Tyr Pro Ser Val Met He Ala Gin Ala He Leu Glu Ser Asn 85 90 95 TGG GGC TCA AGT CAG CTT TCA CGA GCT CCC TAT TAT AAT TTA TTT GGT 513 Trp Gly Ser Ser Gin Leu Ser Arg Ala Pro Tyr Tyr Asn Leu Phe Gly 100 105 HO ATT CAA GGT ACT TAT CAA GGA AAG AGC GTC GTA TTT AAA ACT CAA GAG 56l He Gin Gly Thr Tyr Gin Gly Lys Ser Val Val Phe Lys Thr Gin Glu 115 120 125 TAT CTC AAT GGT AAA TGG GTG ACT AAA GAT ATG CCC TTT AGG GTC TAT 609 Tyr Leu Asn Gly Lys Trp Val Thr Lys Asp Met Pro Phe Arg Val Tyr 130 135 140 CCT TCC TTT AAT CAA AGT TTT CAA GAC AAT GCT TAT GTT CTA AAA ACA 657 Pro Ser Phe Asn Gin Ser Phe Gin Asp Asn Ala Tyr Val Leu Lys Thr 145 150 155 160 ACA AAC TTT GGG AAT GGT CCC TAT TAC GCT AAG GCT TGG CGG GCC AAT 705 Thr Asn Phe Gly Asn Gly Pro Tyr Tyr Ala Lys Ala Trp Arg Ala Asn 165 170 175 GCT GCC ACC TAT CAA GAC GCT ACT GCT GCT TTG ACG GGC AGA TAT GCT 753 Ala Ala Thr Tyr Gin Asp Ala Thr Ala Ala Leu Thr Gly Arg Tyr Ala 180 185 190 ACC GAC CCA AGT TAT GGC GCT TCA CTG AAT CGC ATT ATT TCT CAA TAT 801 Thr Asp Pro Ser Tyr Gly Ala Ser Leu Asn Arg He He Ser Gin Tyr 195 200 205 AAT TTG ACT CGT TTT GAC GGA GCT TCT TCA GCT GGA AAT ACT AAT TCT 849 Asn Leu Thr Arg Phe Asp Gly Ala Ser Ser Ala Gly Asn Thr Asn Ser 210 215 220 GGT GGC TCG ACA ACC ACA ATT ACG AAT AAT AAT TCT GGA ACC AAT AGC 897 Gly Gly Ser Thr Thr Thr He Thr Asn Asn Asn Ser Gly Thr Asn Ser 225 230 235 240 AGT TCA ACT ACT TAT ACC GTC AAA TCT GGT GAT ACT CTT TGG GGA ATC 94 Ser Ser Thr Thr Tyr Thr Val Lys Ser Gly Asp Thr Leu Trp Gly He 245 250 255 TCA CAA AGA TAT GGA ATT AGT GTC GCT CAA ATT CAA AGT GCG AAT AAT 993 Ser Gin Arg Tyr Gly He Ser Val Ala Gin He Gin Ser Ala Asn Asn 260 265 270 CTT AAA AGT ACC ATT ATC TAC ATT GGT CAA AAA CTT GTA CTG ACA GGT 1041 Leu Lys Ser Thr He He Tyr He Gly Gin Lys Leu Val Leu Thr Gly 275 280 285 TCA GCT TCT TCT ACA AAT TCA GGT GGT TCA AAC AAT TCC GCA AGC ACT 1089 Ser Ala Ser Ser Thr Asn Ser Gly Gly Ser Asn Asn Ser Ala Ser Thr 290 295 300 ACT CCA ACC ACT TCT GTG ACA CCT GCA AAA CCA ACT TCA CAA ACA ACT 1137 Thr Pro Thr Thr Ser Val Thr Pro Ala Lys Pro Thr Ser Gin Thr Thr 305 310 315 320 GTT AAG GTT AAA TCC GGA GAT ACC CTT TGG GCG CTA TCA GTA AAA TAT H85 Val Lys Val Lys Ser Gly Asp Thr Leu Trp Ala Leu Ser Val Lys Tyr 325 330 335 AAA ACT AGT ATT GCT CAA TTG AAA AGT TGG AAT CAT TTA AGT TCA GAT 1233 Lys Thr Ser He Ala Gin Leu Lys Ser Trp Asn His Leu Ser Ser Asp 340 345 350 ACC ATT TAT ATT GGT CAA AAT CTT ATT GTT TCA CAA TCT GCT GCT GCT 1281 Thr He Tyr He Gly Gin Asn Leu He Val Ser Gin Ser Ala Ala Ala 355 360 35 TCA AAT CCT TCG ACA GGT TCA GGC TCA ACT GCT ACC AAT AAC TCA AAC 1329 Ser Asn Pro Ser Thr Gly Ser Gly Ser Thr Ala Thr Asn Asn Ser Asn 370 375 380 TCG ACT TCT TCT AAC TCA AAT GCC TCA ATT CAT AAG GTC GTT AAA GGA 1377 Ser Thr Ser Ser Asn Ser Asn Ala Ser He His Lys Val Val Lys Gly 385 390 395 -+00 GAT ACT CTC TGG GGA CTT TCG CAA AAA TCT GGC AGC CCA ATT GCT TCA 1 25 Asp Thr Leu Trp Gly Leu Ser Gin Lys Ser Gly Ser Pro He Ala Ser 405 410 415 ATC AAG GCT TGG AAT CAT TTA TCT AGC GAT ACT ATT TTA ATT GGT CAG 1473 He Lys Ala Trp Asn His Leu Ser Ser Asp Thr He Leu He Gly Gin 420 425 ^30 TAT CTA CGA ATA AAA T AAATTATTAT TAATGAACTT TTAGTTAATA AAAAAAGAAT 1529 Tyr Leu Arg He Lys 435 GAAATTCAAT TCATTCTTTT TTTAATTCTT GTAAAACCGC TTGAAGATGG ATAGAACGGG 1589 TCGGTTCTGA CATTCCAACA GTCATTTCCC AAGGAGTTGT TTCTTTTTCA AGAGCCAATT 1649 CAAGAAAAAC ACGTTGAAAT GAAGTTAATT CTTCACAGTT CGTTAAATAG TCAATCTCTT 1709 TTTTAGAGAG CAA TTTTG TCACGTTGAA TCACTTTGTT TAGGTAGCGA ACCCAAAGTT 1769 CTAAATTCCA TTTGGATTTT TGGGCAGCCT GCAAGAGATA TTCAATCCAC GTCATTGTAA 1829 TAACCTCAGT ATTTTTGGAT TTTTTCGTAA ATMTTTTTT AAAGCTTCTT CACCATAAGC 1869 CAAACTATAA GCTTCTAAAA TAACCGCTGG GTCTGCCGAT C 1930 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 437 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Pro Val Ser Arg Val Lys Val Lys Asn Arg His Leu Lys Lys Lys 1 5 10 15 Thr Lys Lys Pro Leu Ala Phe Tyr Lys Pro Ala Thr Lys Phe Ala Gly 20 25 30 Ala Val Leu He Ala Gly Thr Leu Thr Thr Thr His Glu Leu Leu Leu 35 40 45 Gin Gin Thr Ser Pro Met Val Gin Ala Ala Thr Asn Ser Ser Glu Val 50 55 60 Phe He Glu Ser He Ala Ala Ser Ala Lys Pro Val Ala Asp Ala Asn 65 70 75 80 Gly Leu Tyr Pro Ser Val Met He Ala Gin Ala He Leu Glu Ser Asn 85 90 95 Trp Gly Ser Ser Gin Leu Ser Arg Ala Pro Tyr Tyr Asn Leu Phe Gly 100 105 110 He Gin Gly Thr Tyr Gin Gly Lys Ser Val Val Phe Lys Thr Gin Glu 115 120 125 Tyr Leu Asn Gly Lys Trp Val Thr Lys Asp Met Pro Phe Arg Val Tyr 130 135 140 Pro Ser Phe Asn Gin Ser Phe Gin Asp Asn Ala Tyr Val Leu Lys Thr 145 150 155 160 Thr Asn Phe Gly Asn Gly Pro Tyr Tyr Ala Lys Ala Trp Arg Ala Asn 165 170 175 Ala Ala Thr Tyr Gin Asp Ala Thr Ala Ala Leu Thr Gly Arg Tyr Ala 180 185 190 Thr Asp Pro Ser Tyr Gly Ala Ser Leu Asn Arg He He Ser Gin Tyr 195 200 205 Asn Leu Thr Arg Phe Asp Gly Ala Ser Ser Ala Gly Asn Thr Asn Ser 210 215 220 Gly Gly Ser Thr Thr Thr He Thr Asn Asn Asn Ser Gly Thr Asn Ser 225 230 235 240 Ser Ser Thr Thr Tyr Thr Val Lys Ser Gly Asp Thr Leu Trp Gly He 245 250 255 Ser Gin Arg Tyr Gly He Ser Val Ala Gin He Gin Ser Ala Asn Asn 260 265 270 Leu Lys Ser Thr He He Tyr He Gly Gin Lys Leu Val Leu Thr Gly 275 280 285 Ser Ala Ser Ser Thr Asn Ser Gly Gly Ser Asn Asn Ser Ala Ser Thr 290 295 300 Thr Pro Thr Thr Ser Val Thr Pro Ala Lys Pro Thr Ser Gin Thr Thr 305 310 315 320 Val Lys Val Lys Ser Gly Asp Thr Leu Trp Ala Leu Ser Val Lys Tyr 325 330 335 Lys Thr Ser He Ala Gin Leu Lys Ser Trp Asn His Leu Ser Ser Asp 340 34 350 Thr He Tyr He Gly Gin Asn Leu He Val Ser Gin Ser Ala Ala Ala 355 360 365 Ser Asn Pro Ser Thr Gly Ser Gly Ser Thr Ala Thr Asn Asn Ser Asn 370 375 380 Ser Thr Ser Ser Asn Ser Asn Ala Ser He His Lys Val Val Lys Gly 385 390 395 400 Asp Thr Leu Trp Gly Leu Ser Gin Lys Ser Gly Ser Pro He Ala Ser 405 410 415 He Lys Ala Trp Asn His Leu Ser Ser Asp Thr He Leu He Gly Gin 420 425 430 Tyr Leu Arg He Lys 435 (2) INFORMATION FOR SEQ ID NO: 3 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Phe Ala Gly Ala Val Leu He Ala Gly Thr Leu Thr Thr Thr 1 10 15 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 216 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Thr Asn Ser Gly Gly Ser Thr Thr Thr He Thr Asn Asn Asn Ser Gly 1 5 10 15 Thr Asn Ser Ser Ser Thr Thr Tyr Thr Val Lys Ser Gly Asp Thr Leu 20 25 30 Trp Gly He Ser Gin Arg Tyr Gly He Ser Val Ala Gin He Gin Ser 35 40 45 Ala Asn Asn Leu Lys Ser Thr He He Tyr He Gly Gin Lys Leu Val 50 55 60 Leu Thr Gly Ser Ala Ser Ser Thr Asn Ser Gly Gly Ser Asn Asn Ser 65 70 75 80 Ala Ser Thr Thr Pro Thr Thr Ser Val Thr Pro Ala Lys Pro Thr Ser 85 90 95 Gin Thr Thr Val Lys Val Lys Ser Gly Asp Thr Leu Trp Ala Leu Ser 100 105 HO Val Lys Tyr Lys Thr Ser He Ala Gin Leu Lys Ser Trp Asn His Leu 115 120 125 Ser Ser Asp Thr He Tyr He Gly Gin Asn Leu He Val Ser Gin Ser 130 135 140 Ala Ala Ala Ser Asn Pro Ser Thr Gly Ser Gly Ser Thr Ala Thr Asn 145 150 155 160 Asn Ser Asn Ser Thr Ser Ser Asn Ser Asn Ala Ser He His Lys Val 165 170 175 Val Lys Gly Asp Thr Leu Trp Gly Leu Ser Gin Lys Ser Gly Ser Pro 180 185 190 He Ala Ser He Lys Ala Trp Asn His Leu Ser Ser Asp Thr He Leu 195 200 205 He Gly Gin Tyr Leu Arg He Lys 210 215 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 183 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Lactococcus lactis (B) STRAIN: MGI363 (vii) IMMEDIATE SOURCE: (B) CLONE: Fig.l4a acmA promoter region (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: MTATTTTTG TTTTAAATCA GATTTTTTAG ATTAAAGGCA AAAAGTTTTT ACAAATATGA 60 ATCCTTAACG GAAAAACGTT TACAAACCGC CACCAAATTG ACATCTTTTT TTAGCTTGAG 120 GCGTGGTAGA ATAAAGATAG TACTTATTAT ATTTTGTAAT CTTTAGAAAG GTAATTATTT l8θ ATG 183 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Lactococcus lactis (B) STRAIN: MGI363 (vii) IMMEDIATE SOURCE: (B) CLONE: primer PALA-4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTTCAACAGA CAAGTCC 17 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Lactococcus lactis (B) STRAIN: MGI363 (vii) IMMEDIATE SOURCE: (B) CLONE: primer PALA-14 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 GATAAATGAT TCCAAGC 17 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DΝA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Lactococcus lactis (B) STRAIN: MG1363 (vii) IMMEDIATE SOURCE: (B) CLONE: PALA-19 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CAAGGTTAAG TCCACG 16 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Lactococcus lactis (B) STRAIN: MG1363 (vii) IMMEDIATE SOURCE: (B) CLONE: primer BK05AL (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : ATTATTTGAT TGGAGTT 17 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 94 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Thr Trp He Glu Tyr Leu Leu Gin Ala Ala Gin Lys Ser Lys Trp 1 5 10 15 Asn Leu Glu Leu Trp Val Arg Tyr Leu Asn Lys Val He Gin Arg Asp 20 25 30 Lys He Leu Leu Ser Lys Lys Glu He Asp Tyr Leu Thr Asn Cys Glu 35 40 45 Glu Leu Thr Ser Phe Gin Arg Val Phe Leu Glu Leu Ala Leu Glu Lys 50 55 60 Glu Thr Thr Pro Trp Glu Met Thr Val Gly Met Ser Glu Pro Thr Arg 65 70 75 80 Ser He His Leu Gin Ala Val Leu Gin Glu Leu Lys Lys Glu 85 90 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1930 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Lactococcus lactis (B) STRAIN: MGI363 (vii) IMMEDIATE SOURCE: (B) CLONE: Fig.4 - ORFB (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: complement (I823..I928) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATTTTTGTTT TAAATCAGAT TTTTTAGATT AAAGGCAAAA AGTTTTTACA AATATGAATC 60 CTTAACGGAA AAACGTTTAC AAACCGCCAC CAAATTGACA TCTTTTTTTA GCTTGAGGCG 120 TGGTAGAATA AAGATAGTAC TTATTATATT TTGTAATCTT TAGAAAGGTA ATTATTTATG l80 CCAGTATCAC GTGTTAAAGT TAAAAATAGA CATTTAAAAA AGAAAACTAA AAAACCACTC 240 GCTTTTTATA AACCAGCCAC AAAATTTGCT GGCGCTGTTC TTATTGCCGG AACATTGACA 300 ACCACACATG AACTTCTTCT TCAACAGACA AGTCCAATGG TTCAAGCAGC GACTAACTCA 3 O TCAGAGGTTT TTATTGAAAG TATTGCCGCA TCAGCAAAAC CTGTGGCAGA TGCTAATGGC 420 TTATATCCTT CGGTCATGAT TGCCCAAGCT ATTTTGGAAA GCAACTGGGG CTCAAGTCAG 480 CTTTCACGAG CTCCCTATTA TAATTTATTT GGTATTCAAG GTACTTATCA AGGAAAGAGC 5*40 GTCGTATTTA AAACTCAAGA GTATCTCAAT GGTAAATGGG TGACTAAAGA TATGCCCTTT 600 AGGGTCTATC CTTCCTTTAA TCAAAGTTTT CAAGACAATG CTTATGTTCT AAAAACAACA 660 AACTTTGGGA ATGGTCCCTA TTACGCTAAG GCTTGGCGGG CCAATGCTGC CACCTATCAA 720 GACGCTACTG CTGCTTTGAC GGGCAGATAT GCTACCGACC CAAGTTATGG CGCTTCACTG 78O AATCGCATTA TTTCTCAATA TAATTTGACT CGTTTTGACG GAGCTTCTTC AGCTGGAAAT 840 ACTAATTCTG GTGGCTCGAC AACCACAATT ACGAATAATA ATTCTGGAAC CAATAGCAGT 900 TCAACTACTT ATACCGTCAA ATCTGGTGAT ACTCTTTGGG GAATCTCACA AAGATATGGA 960 ATTAGTGTCG CTCAAATTCA AAGTGCGAAT AATCTTAAAA GTACCATTAT CTACATTGGT 1020 CAAAAACTTG TACTGACAGG TTCAGCTTCT TCTACAAATT CAGGTGGTTC AAACAATTCC 1080 GCAAGCACTA CTCCAACCAC TTCTGTGACA CCTGCAAAAC CAACTTCACA AACAACTGTT ll4θ AAGGTTAAAT CCGGAGATAC CCTTTGGGCG CTATCAGTAA AATATAAAAC TAGTATTGCT 1200 CAATTGAAAA GTTGGAATCA TTTAAGTTCA GATACCATTT ATATTGGTCA AAATCTTATT 1260 GTTTCACAAT CTGCTGCTGC TTCAAATCCT TCGACAGGTT CAGGCTCAAC TGCTACCAAT 1320 AACTCAAACT CGACTTCTTC TAACTCAAAT GCCTCAATTC ATAAGGTCGT TAAAGGAGAT 1380 ACTCTCTGGG GACTTTCGCA AAAATCTGGC AGCCCAATTG CTTCAATCAA GGCTTGGAAT l44θ CATTTATCTA GCGATACTAT TTTAATTGGT CAGTATCTAC GAATAAAATA AATTATTATT 1500 AATGAACTTT TAGTTAATAA AAAAAGAATG AAATTCAATT CATTCTTTTT TTAATTCTTG I56O TAAAACCGCT TGAAGATGGA TAGAACGGGT CGGTTCTGAC ATTCCAACAG TCATTTCCCA 1620 AGGAGTTGTT TCTTTTTCAA GAGCCAATTC AAGAAAAACA CGTTGAAATG AAGTTAATTC 1680 TTCACAGTTC GTTAAATAGT CAATCTCTTT TTTAGAGAGC AAAATTTTGT CACGTTGAAT 1740 CACTTTGTTT AGGTAGCGAA CCCAAAGTTC TAAATTCCAT TTGGATTTTT GGGCAGCCTG l800 CAAGAGATAT TCAATCCACG TCATTGTAAT AACCTCAGTA TTTTTGGATT TTTTCGTAAA i860 T TTTTTTA AAGCTTCTTC ACCATAAGCC AAACTATAAG CTTCTAAAAT AACCGCTGGG 1920 TCTGCCGATC 1930 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 amino acids ) TYPE: amino acid ) TOPOLOGY: linear (ii JLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Ser Ala Asp Pro Ala Val He Leu Glu Ala Tyr Ser Leu Ala Tyr Gly 1 5 10 15 Glu Glu Ala Leu Lys Asn Tyr Leu Arg Lys Asn Pro Lys He Leu Arg 20 25 30 Leu Leu Gin 35 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 671 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus faecalis (vii) IMMEDIATE SOURCE: (B) CLONE: Fig.5a (S. faecalis) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Met Lys Lys Glu Ser Met Ser Arg He Glu Arg Arg Lys Ala Gin Gin 1 5 10 15 Arg Lys Lys Thr Pro Val Gin Trp Lys Lys Ser Thr Thr Leu Phe Ser 20 25 30 Ser Ala Leu He Val Ser Ser Val Gly Thr Pro Val Ala Leu Leu Pro 35 40 45 Val Thr Ala Glu Ala Thr Glu Glu Gin Pro Thr Asn Ala Glu Val Ala 50 55 60 Gin Ala Pro Thr Thr Glu Thr Gly Leu Val Glu Thr Pro Thr Thr Glu 65 70 75 80 Thr Thr Pro Gly He Thr Glu Gin Pro Thr Thr Asp Ser Ser Thr Thr 85 90 95 Thr Glu Ser Thr Thr Glu Ser Ser Lys Glu Thr Pro Thr Thr Pro Ser 100 105 110 Thr Glu Gin Pro Thr Val Asp Ser Thr Thr Pro Val Glu Ser Gly Thr 115 120 125 Thr Asp Ser Ser Val Ala Glu He Thr Pro Val Ala Pro Ser Thr Thr 130 135 140 Glu Ser Glu Ala Ala Pro Ala Val Thr Pro Asp Asp Glu Val Lys Val 145 150 155 160 Pro Glu Ala Arg Val Ala Ser Ala Gin Thr Phe Ser Ala Leu Ser Pro 165 170 175 Thr Gin Ser Pro Ser Glu Phe He Ala Glu Leu Ala Arg Cys Ala Gin 180 185 190 Pro He Ala Gin Ala Asn Asp Leu Tyr Ala Ser Val Met Met Ala Gin 195 200 205 Ala He Val Glu Ser Gly Trp Gly Ala Ser Thr Leu Ser Lys Ala Pro 210 215 220 Asn Tyr Asn Leu Phe Gly He Lys Gly Ser Tyr Asn Gly Gin Ser Val 225 230 235 240 Tyr Met Asp Thr Trp Glu Tyr Leu Asn Gly Lys Trp Leu Val Lys Lys 245 250 255 Glu Pro Phe Arg Lys Tyr Pro Ser Tyr Met Glu Ser Phe Gin Asp Asn 260 265 270 Ala His Val Leu Lys Thr Thr Ser Phe Gin Ala Gly Val Tyr Tyr Tyr 275 280 285 Ala Gly Ala Trp Lys Ser Asn Thr Ser Ser Tyr Arg Asp Ala Thr Ala 290 295 300 Trp Leu Thr Gly Arg Tyr Ala Thr Asp Pro Ser Tyr Asn Ala Lys Leu 305 310 315 320 Asn Asn Val He Thr Ala Tyr Asn Leu Thr Gin Tyr Asp Thr Pro Ser 325 330 335 Ser Gly Gly Asn Thr Gly Gly Gly Thr Val Asn Pro Gly Thr Gly Gly 340 34 350 Ser Asn Asn Gin Ser Gly Thr Asn Thr Tyr Tyr Thr Val Lys Ser Gly 355 360 365 Asp Thr Leu Asn Lys He Ala Ala Gin Tyr Gly Val Ser Val Ala Asn 370 375 380 Leu Arg Ser Trp Asn Gly He Ser Gly Asp Leu He Phe Val Gly Gin 385 390 395 400 Lys Leu He Val Lys Lys Gly Ala Ser Gly Asn Thr Gly Gly Ser Gly 405 410 415 Asn Gly Gly Ser Asn Asn Asn Gin Ser Gly Thr Asn Thr Tyr Tyr Thr 420 425 430 Val Lys Ser Gly Asp Thr Leu Asn Lys He Ala Ala Gin Tyr Gly Val 435 440 445 Thr Val Ala Asn Leu Arg Ser Trp Asn Gly He Ser Gly Asp Leu He 450 455 460 Phe Val Gly Gin Lys Leu He Val Lys Lys Gly Thr Ser Gly Asn Thr 465 470 475 480 Gly Gly Ser Ser Asn Gly Gly Ser Asn Asn Asn Gin Ser Gly Thr Asn 485 490 495 Thr Tyr Tyr Thr He Lys Ser Gly Asp Thr Leu Asn Lys He Ala Ala 500 505 510 Gin Tyr Gly Val Ser Val Ala Asn Leu Arg Ser Trp Asn Gly He Ser 515 520 525 Gly Asp Leu He Phe Ala Gly Gin Lys He He Val Lys Lys Gly Thr 530 535 540 Ser Gly Asn Thr Gly Gly Ser Ser Asn Gly Gly Ser Asn Asn Asn Gin 545 550 555 560 Ser Gly Thr Asn Thr Tyr Tyr Thr He Lys Ser Gly Asp Thr Leu Asn 565 570 575 Lys He Ser Ala Gin Phe Gly Val Ser Val Ala Asn Leu Arg Ser Trp 580 585 590 Asn Gly He Lys Gly Asp Leu He Phe Ala Gly Gin Thr He He Val 595 600 605 Lys Lys Gly Ala Ser Ala Gly Gly Asn Ala Ser Ser Thr Asn Ser Ala 610 615 620 Ser Gly Lys Arg His Thr Val Lys Ser Gly Asp Ser Leu Trp Gly Leu 625 630 635 640 Ser Met Gin Tyr Gly He Ser He Gin Lys He Lys Gin Leu Asn Gly 645 650 655 Leu Ser Gly Asp Thr He Tyr He Gly Gin Thr Leu Lys Val Gly 660 665 670 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 666 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Enterococcus hirae (vii) IMMEDIATE SOURCE: (B) CLONE: Fig.5a (E. hirae) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Met Glu Asn He Ala Arg Lys Glu Arg Arg Arg Leu Asn Glu Thr Lys 1 5 10 15 Arg Phe Arg Lys Val Lys Arg Ser Ala Ala Leu Val Gly Thr Ala Met 20 25 30 Val Gly Cys Ser Val Ala Ala Pro Leu He Gin Pro Val Gin Val Asp 35 40 45 Ala Asp Gin Thr Pro Thr Gin Phe Gly Ala Arg He Asn Thr Ala Ala 50 55 60 Phe He Ala Glu He Ala Thr Tyr Ala Gin Pro He Ala Gin Ala Asn 65 70 75 80 Asp Leu Tyr Ala Ser Val Met He Ala Gin Ala Val Val Glu Ser Gly 85 90 95 Trp Gly Ser Ser Ala Leu Ser Gin Ala Pro Tyr Tyr Asn Leu Phe Gly 100 105 110 He Lys Gly Ser Tyr Gin Gly Gin Thr Val Tyr Met Asp Thr Leu Glu 115 120 125 Tyr Leu Asn Asn Lys Trp Val Ser Lys Lys Glu Pro Phe Arg Gin Tyr 130 135 140 Pro Ser Phe Ala Glu Ser Phe Asn Asp Asn Ala Tyr Val Leu Arg Asn 145 150 155 160 Thr Ser Phe Gly Asn Gly Tyr Tyr Tyr Ala Gly Thr Trp Lys Ser Asn 165 170 175 Thr Lys Ser Tyr Thr Asp Ala Thr Ala Cys Leu Thr Gly Arg Tyr Ala 180 185 190 Thr Asp Pro Gly Tyr Ala Gly Lys Leu Asn Asn He He Thr Thr Tyr 195 200 205 Gly Leu Thr Lys Tyr Asp Thr Pro Ala Ser Gly Asn Ala Gly Gly Gly 210 215 220 Val Thr He Gly Asn Gly Gly Asn Thr Gly Asn Thr Ser Asn Ser Gly 225 230 235 240 Ser Thr Ser Gly Asn Ser Gly Gly Ser Ala Thr Thr Thr Gly Thr Thr 245 250 255 Tyr Thr Val Lys Ser Gly Asp Ser Val Trp Gly He Ser His Ser Phe 260 265 270 Gly He Thr Met Ala Gin Leu He Glu Trp Asn Asn He Lys Asn Asn 275 280 285 Phe He Tyr Pro Gly Gin Lys Leu Thr He Lys Gly Gly Gin Ser Ala 290 295 300 Gly Ser Ser Thr Thr Asn Thr Gly Asn Asn Ala Ser Ser Gly Asn Thr 305 310 315 320 Ser Gly Asn Thr Asn Thr Ser Gly Ser Thr Gly Gin Ala Thr Gly Ala 325 330 335 Lys Tyr Thr Val Lys Ser Gly Asp Ser Val Trp Lys He Ala Asn Asp 340 345 350 His Gly He Ser Met Asn Gin Leu He Glu Trp Asn Asn He Lys Asn 355 360 365 Asn Phe Val Tyr Pro Gly Gin Gin Leu Val Val Ser Lys Gly Ser Ser 370 375 380 Ser Ala Ser Gly Ser Thr Ser Asn Thr Ser Thr Gly Asn Thr Ser Ser 385 390 395 400 Asn Thr Ala Asn Thr Gly Ser Thr Thr Ser Gly Ser Thr Tyr Thr Val 405 410 415 Lys Ala Gly Glu Ser Val Trp Ser Val Ser Asn Lys Phe Gly He Ser 420 425 430 Met Asn Gin Leu He Gin Trp Asn Asn He Lys Asn Asn Phe He Tyr 435 440 445 Pro Gly Gin Lys Leu He Val Lys Gly Gly Ser Ser Ser Ser Asn Ala 450 455 460 Ser Thr Ser Thr Ala Asn Asn Lys Asn Thr Ala Ser Ser Asn Thr Ser 465 470 475 480 Ser Thr Ala Thr Gly Gin Ala Thr Tyr Thr Val Lys Ala Gly Glu Ser 485 490 495 Val Trp Gly Val Ala Asn Lys Asn Gly He Ser Met Asn Gin Leu He 500 505 510 Glu Trp Asn Asn He Lys Asn Asn Phe He Tyr Pro Gly Gin Lys Leu 515 520 525 He Val Lys Gly Gly Ser Ser Lys Ala Ser Ala Thr Ala Thr He Lys 530 535 540 Pro Thr Ala Ser Thr Pro Ala Ser Thr Thr Pro Thr Ala Ser Ser Thr 55 550 555 560 Gly Asp Thr Lys Tyr Thr Val Lys Ala Gly Glu Ser Val Trp Gly Val 565 570 575 Ala Asn Lys His His He Thr Met Asp Gin Leu He Glu Trp Asn Asn 580 585 590 He Lys Asn Asn Phe He Tyr Pro Gly Gin Glu Val He Val Lys Lys 595 600 605 Gly Thr Ala Gin Ser Thr Pro Ala Lys Ser Asp Glu Lys Thr Tyr Thr 610 615 620 Val Lys Ala Gly Glu Ser Val Trp Gly Val Ala Asp Ser His Gly He 625 630 635 640 Thr Met Asn Gin Leu He Glu Trp Asn Asn He Lys Asn Asn Phe He 645 650 655 Tyr Pro Gly Gin Gin Leu He Val Lys Lys 660 665 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 93 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Lactococcus lactis phage Rl-t (vii) IMMEDIATE SOURCE: (B) CLONE: Fig.lib (publ.2) fusionpoint orf29 and lacZ gene (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..93 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: ACA ATC CGA AGC ACG GAG TAC ATG ACG GAT GCG AAG CTT GCA TGC CTG 48 Thr He Arg Ser Thr Glu Tyr Met Thr Asp Ala Lys Leu Ala Cys Leu 1 5 10 15 CAG GTC GAC TCT AGA GTC GGG GCC GTC GTT TTA CAA CGT CGT GAC 93 Gin Val Asp Ser Arg Val Gly Ala Val Val Leu Gin Arg Arg Asp 20 25 30 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 16 : Thr He Arg Ser Thr Glu Tyr Met Thr Asp Ala Lys Leu Ala Cys Leu 1 5 10 15 Gin Val Asp Ser Arg Val Gly Ala Val Val Leu Gin Arg Arg Asp 20 25 30

Claims: C L A I M S 1. Process for the lysis of a culture of lactic acid bacteria by means of a lysin, which comprises the in situ production of a homologous autolysin, or a heterologous autolysin obtainable from foodgrade Gram- positive bacteria esp. from lactic acid bacteria, or active fragments of such autolysin, preferably a homologous autolysin, the gene encoding said autolysin or a fragment thereof being under control of a regulatable promoter, to achieve an enhanced lysis during fermentation or shortly thereafter after induction of said regulatable promoter due to an enhanced production of total autolysin compared with the natural production level of the homologous autolysin, said regulatable promoter not normally associated with said autolysin encoding gene and wherein shortly thereafter implies before 12 hours in the stationary phase have lapsed, preferably before 4 hours in the stationary phase have lapsed. 2. Process according to claim 1, in which said regulatable promoter is regulatable by the use of food-grade ingredients or parameters. 3. Process according to claim 1, in which the culture of lactic acid bacteria is part of a product containing such culture. 4. Process according to claim 3. in which the lactic acid bacteria culture is used for producing a fermented food product obtainable by the fermentative action of the lactic acid bacteria and subsequently the lactic acid bacteria in the fermented food product are lysed. 5. Process according to claim 4, in which the fermented food product is a cheese product. 6. Process according to claim 5, in which additionally a cheese ripening step is carried out, whereby some of the constituents after leaving the lysed cells will change the composition of the cheese product. 7. Process according to claim 1 for modifying a mixture of peptides, in which a mixture of peptides obtained by proteolysis of proteins is combined with a culture of lactic acid bacteria containing a lactic acid bacterium autolysin gene under control of ~ regulatable promoter and subsequently the promoter is induced to achieve production of the autolysin in such amount that the lactic acid bacteria are lysed and the contents of the cells containing peptidases will modify the composition of the mixture of peptides. δ. Process for modifying a mixture of peptides, which comprises treating a mixture of peptides obtained by proteolysis of proteins with a lysed culture obtained by a process according to claim 1. 9- Process according to claim 7 or 8, in which the proteins comprise milk proteins or vegetable proteins, or both. 10. Process according to claim 1, in which the autolysin is first produced and subsequently used as a bactericidal agent against spoiling bacteria or pathogenic bacteria. 11. Process for improving the shelf life of a consumer product, in which a product obtained by a process as claimed in claim 1 and containing free autolysin is incorporated into said consumer product in such amount that in the resulting consumer product the growth of spoiling bacteria or pathogenic bacteria is inhibited or that their viability is strongly reduced. 12. Process according to claim 11, in which the consumer product is selected from the group consisting of edible products, cosmetic products, and products for cleaning fabrics, hard surfaces and human skin. 13- A nucleotide sequence encoding the lysin of a lactic acid bacterium, in particular an Ν-acetylmuramidase. 14. A nucleotide sequence encoding a polypeptide or protein having lytic activity, in particular a nucleotide sequence according to claim 13, said nucleotide sequence being a sequence according to sequence id. no. 1 or a variant nucleotide sequence encoding a functional equivalent of the amino acid sequence encoded by sequence id no 1. 15. A nucleotide sequence according to claim 14, said nucleotide sequence encoding the amino acid sequence according to sequence id. no. 2. 16. A nucleotide sequence according to any of claims 13~15 encoding an Ν- terminal amino acid sequence that is more than 70%, preferably more than 80%, more preferably 90% homologous with the Ν-terminal amino acid sequence according to sequence id no 2 and encoding a polypeptide exhibiting lytic activity of an Ν-acetylmuramidase as can be determined by a standard assay. 17- A nucleotide sequence according to any of claims 13-16 encoding an overall amino acid sequence that is more than 40%, preferably more than 50%, more preferably between 60-100% homologous with the overall amino acid sequence according to sequence id no 2 and encoding a polypeptide exhibiting lytic activity of an Ν-acetylmuramidase as can be determined by a standard assay. lδ. A nucleotide sequence according to any of claims 13-17 comprising at least nucleic acid sequences encoding 3 direct repeats in the C-terminal part of the amino acid sequence encoded by the nucleotide sequence, said direct repeats being characterised by a homology of more than 75% at amino acid level, the amino acid sequence preferably comprising serine, threonine and asparagine rich intervening sequences between the direct repeats. 19. A nucleotide sequence according to any of claims 13-lδ comprising at least a nucleic acid sequence encoding a membrane spanning domain as illustrated in Figure 5 or Sequence id no 3- 20. A nucleotide sequence according to any of claims 13-19 characterised by its ability to hybridize to a nucleic acid sequence according to sequence id no 1 and Figure 4 under normal to stringent conditions common to a person skilled in the art. 21. A recombinant vector comprising a nucleotide sequence according to any of claims 13-20, said nucleotide sequence preferably being under control of a promoter other than the promoter normally associated with the lysin encoding nucleotide sequence said promoter preferably being regulatable and most preferably derived from a foodgrade organism. 22. A recombinant host cell comprising a nucleotide sequence according to any of claims 13-20 or a recombinant vector according to claim 21, said 7δ nucleotide sequence being under control of a promoter other than that naturally occuring with the lysin gene, said promoter preferably being regulatable and most preferably derived from a foodgrade organism. 23. A recombinant host cell according to claim 22, said host cell being foodgrade grampositive such as a lactic acid bacterium. 24. A recombinant host cell according to claim 22 or 23 also possessing a native autolysin gene under control of it's native promoter such that the required degree of autolysin production during the exponential growth phase is ensured. 25- Use of a recombinant host cell according to any of claims 22-24 or a recombinant vector according to claim 21 in a manner known per se for expressing a nucleotide sequence according to any of claims 13-20 for the production of an autolysin in a process according to any of claims 1-12.

Inventors: BUIST GIRBE, KOK JAN, LEDEBOER AAT M, VENEMA GERARD, BUIST, GIRBE, KOK, JAN, LEDEBOER, AAT, M., VENEMA, GERARD
Applicants: BUIST GIRBE, KOK JAN, LEDEBOER AAT M, QUEST INT, VENEMA GERARD, BUIST, GIRBE, KOK, JAN, LEDEBOER, AAT, M., QUEST INTERNATIONAL B.V., VENEMA, GERARD
Application year: 1995
Main classification: C12N15
IPC codes: A23C19, A61K8, A61Q19, C07H21, C12N1, C12N9, C12N15, C12P21, C12R1