AU702604B2 - Process for inhibiting the growth of a culture of lactic acid bacteria, and optionally lysing the bacterial cells, and uses of the resulting lysed culture - Google Patents

Description

~ls~_ WO 95/31562 PCT/NL95/00171 Procecs for inhibiting the growth of a culture of lactic acid bacteria, and optionally lysing the bacterial cells, and uses of the resulting lysed culture Background of the invention and prior art The invention relates to a process for inhibiting the growth of a culture of lactic acid bacteria, and optionally lysing the cells of said bacteria.

In this specification the following abbreviations of names of micro-organisms are used: E. Escherichia, e.g. E. coli, L. Lactococcus, e.g. L. lactis, N. Micrococcus, e.g. M. lysodeikticus, S. Streptococcus, e.g. S. faecalis and S. pneumonia.

In one aspect 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 January 1990, ref. According to that patent specification the lysin from a Lactococcus (preferably prolate-headed) bacteriophage was used to lyse oacteri-l starter cultures during cheese-making. Exemplified was the lysin of the bacteriophage (vML3 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 hose 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 chees 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

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WO 95/31562 PCT/NL95/00171 2 thus not affecting the starter bacteria before their role in acidification was complete." This suggested alternative has the disadvantages, that an encapsulating material has to be used, and 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 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 C.A. Shearman, K. Jury M.J. Gasson (Feb. 1992, ref. 2) described an autolytic Lactococcus lactis expressing a cloned lactococcal bacteriophage (vML3 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. cremoris 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 fermen-" ion 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 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 when taking into consideration the length of time required for a Gouda cheese immersed in a brine bath to achieve the desired degree of salt flavour 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, still requires a mixing step.

WO 95/31562 PCT/NL95/00171 3 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. 4) 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 468-472 it was indicated that the DNA sequence found by Shearman c.s. (1989, ref. 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. More particularly it is speculated that the DNA sequence of the lysin gene like the pneumococcal phage associated lysin genes had no signal sequence which could account for secretion across the cytoplasmic membrane, however, this was puzzling in view of the absence of a typical N-terminal signal sequence which raises the question of how the lytic enzymes escape the cytoplasm and gain access to the cell wall.

In the above mentioned review of R. Young (1992, ref. 4, especially on page 469 in the paragraph bridging both columns and pages 472-473 in the section HOLIN FAMILY) it is argued that an additional protein is required for the action of bacteriophage lysins on the cell wall of infected cells. This additional protein is required for the access of the murein hydrolase, which is the more scientific name for the bacteriophage lysin, to its murein substrate. In that review the term "holin" was used for this additional protein. It was described in that review that the holin makes perforations in the cell wall enabling the lysins to pass the membrane so that subsequently the lysins can hydrolase the murein part of the cell wall. In this specification "holin" also means a protein or peptide required for the access of a lysin to its substrate, the murein part of the cell wall.

In the Young et al review it is stated that the realignment of the Shearman sequence and assumptions of a sequencing error obscuring a start codon does present a possible basis for establishing the requirement for a holin to effect the release of this phage encoded murein hydrolase. Young et al. further state "If our analysis so far has taught us anything it is that any phage with a lysozyme gene should have a holin gene". This statement is however contradicted somewhat further on

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WO 95/31562 PCT/NL95/00171 in the same article where a lys A clone of mvl which does not appear to possess a holin encoding sequence is illustrated as exhibiting lytic activity.

Young et al further examined the putative holin family and disclose 8 different proteins unrelated in primary sequence for which genetic or physiological evidence of holin function exists. Some postulations concerning structure and function are made, however nothing definite appears to be settled regarding this issue. They indicate that as these proteins are small, hydrophobic, without enzyme function and lethal this is array of characteristics not likely to attract legions of biochemists. This field is thus illustrated as being quite complex with little factual knowledge and a deal of speculation.

In a publication of Ward c.s. (1993; ref. 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 at. (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 45 bases longer than disclosed by Shearman et al. (ref. C. Platteeuw and W.M. de Vos (1992, ref. 3) described the location, characterization and expression in Eschevichia coli of lytic enzyme-encoding gene, lytA, of Lactococcus lactis bacteriophage (US3. It was described that the (vML3 lysin, which is active on a wide range of lactococcal strains, lacked homology with known lytic enzymes. The bacteriophage 4US3 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 US3 encodes an amidase rather than a lysozvme-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 encoding a holin from different organisms.

In EP-A2-0 510 907 (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

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I_ 1 WO 95/31562 PCT/NL95/00171 monocytogenes (phage 4LM4) and Clostridium tyrobutyricum (phage JP). 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 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 use of such lysins 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.

In another aspect the invention relates to a process for inhibiting the growth of a culture of lactic acid bacteria without lysing the cells.

The growth of lactic acid bacteria can be inhibited in several ways.

For example, in normal fermentations with lactic acid bacteria, e.g. for the production of yoghurt, when a certain low pH is obtained the high amount of lactic acid stops further fermentation. The growth changes from the log phase to the stationary phase which in effect is some sort of inhibition of the growth.

Another possibility is that the nutrients become scarce and the so-called starvation occurs, because the necessary ingredients are no longer available for growth of the bacteria. This means that no further growth occurs.

Still another possibility is the effect of pasteurization or sterilization causing cell death.

Summary of the invention It has now been found that holin on its own already has a bacteriostatic effect on Gramnegative bacteria like E. colt and Grampositive bacteria like lactic acid bacteria. Thus according to a first embodiment the invention provides a process as described in claim 1, i.e. a process for inhibiting the growth of a culture of lactic acid bacteria, which process comprises the in situ production in the cells of the lactic acid bacteria of a holin obtainable from bacteriophages of Gram-positive bacteria, esp. from bacteriophages of lactic acid bacteria, the gene encoding said holin being under control of a first regulatable promoter, said first regulatable promoter not normally being associated with said holin gene, said holin being capable of exerting a I I I ~pl WO 95/31562 PCT/NL95/00171 6 bacteriostatic effect on the cells in which it is produced by means of a system, whereby the cell membrane is perforated, while preferably the natural production of autolysin is not impaired.

According to a second embodiment the invention provides a process as described in claim 2, i.e. a process according to the first embodiment, which additionally comprises the in situ production in the cells of the lactic acid bacteria of a lysin obtainable from lactic acid bacteria other foodgrade grampositive microorganisms or their bacteriophages, the gene encoding said lysin being under control of a second regulatable promoter, whereby the produced lysin effects lysis of the cells of the grampositive or gramnegative bacteria, preferably the lactic acid bacteria.

Preferably the second regulatable promoter is the same as the first regulatable promoter (claim and more preferably the gene encoding the holin and the gene encoding the lysin are placed under control of the same regulatable promoter in one operon (claim It is advantageous for food fermentations when said first or second promoter or both can be regulated by food-grade ingredients or parameters (claim The processes according to the invention can be used in the culture of lactic acid bacteria as such, but they can also be used in a product containing such culture (claim A specific embodiment of this latter possibility is a process 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 (claim A specific example of such process is one in which the fermented food product is a cheese product (claim Then an additional cheese ripening step can be carried out, whereby some of the constituents after leaving the lysed cells will change the composition of the cheese product (claim 9).

A third embodiment of the invention relates to a process for combatting spoiling bacteria or pathogenic bacteria, in which a lysed culture obtained by a process according to the second embodiment of the invention is used as a bactericidal agent (claim 10). One way of use as a bactericidal agent is a process for improving the shelf life of a consumer product, in which a product obtained by a process according to either the first or the second embodiment of the invention and containing free holin or free lysin or both is incorporated into said consumer product in such amount that in the resulting consumer product the growth

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WO 95/31562 PCT/NL95/00171 7 of spoiling bacteria or pathogenic bacteria is inhibited or that their viability is strongly reduced (claim 11). Such consumer products comprise edible products, cosmetic products, and products for cleaning fabrics, hard surfaces and human skin (claim 12).

Examples of such products may be bread and bread improvers; butter, margarine and low calorie substitutes therefor; cheeses; dressings and 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.

A fourth embodiment of the invention is a process for modifying a mixture of peptides, which comprises combining a culture of lactic acid bacteria with a mixture of peptides obtained by proteolysis of proteins, the cells of said culture containing both a gene encoding a holin under control of a first regulatable promoter and a gene encoding a lysin under control of a second regulatable promoter, which second and first promoter can be the same and which first and second promoter are not normally associated with the respective genes, and effecting induction of the promoter or promoters for producing both the holin and the lysin in such amounts that the cells of the lactic acid bacteria are lysed and the contents of the cells containing peptidases will modify the composition of the mixture of peptides (claim 13). In order to achieve sufficient bacterium growth the host cell must not lyse too quickly, preferably lysis will occur at the end of the log phase or commencement of the stationary phase.

An alternative is a 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 the second embodiment of the invention (claim 14).

The proteins to be proteolysed can be, for example, milk proteins or vegetable proteins, or both (claim Any of the above-mentioned processes as claimed in claims 1-15, wherein the holin is encoded by a nucleic acid sequence according to any of claims 18-20 and/or is expressed from a recombinant vector according to any of claims 21-24 and/or is expressed by a recombinant cell according to any of claims 25-27 fall within the intended scope of the invention. In addition an alternative suitable embodiment of a process according to the invention can be directed at the inducible expression of

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~Bla~S1S~aa~wsaarrePI-~ WO 95/31562 PCT/NL95/00171 8 a lysin having the amino acid sequence of sequence id no 7 or being a functional equivalent thereof.

A nucleic acid sequence encoding a holin derivable from a grampositive bacterium such as a lactic acid bacterium, in particular a L. lactis or a bacteriophage derivable from such a grampositive bacterium also falls within the scope of the invention. Such a nucleic acid sequence can for example encode the amino acid sequence of sequence id no 6 or a functional equivalent thereof such as the nucleic acid sequence of nucleotides 103-328 of sequence id no 5. Any nucleic acid sequence according to the invention can further be operatively linked to a first regulatable promoter, said first regulatable promoter not normally being associated with the holin encoding sequence.

Also comprised by the invention are recombinant vectors comprising any of the nucleic acid sequences in any of the claimed embodiments, said vector preferably further being foodgrade. In addition such a recombinant vector according to the invention may suitably further comprise a nucleic acid sequence encoding a lysin, both the holin and the lysin being derivable from a grampositive bacterium such as a lactic acid bacterium, in particular a L. lactis or a bacteriophage derivable from such a grampositive bacterium, a preferred embodiment of a recombinant vector according to the invention further comprises the natural attachment/integration system of a bacteriophage. The natural attachment/integration system of a bacteriophage can comprise the bacteriophage attachment site and an integrase gene located such that integration of the holin and optionally lysin gene will occur, said system preferably being derived from a bacteriophage that is derivable from a food grade host cell, preferably a lactic acid bacterium. A suitable recombinant vector according to the invention comprises the nucleic acid sequence encoding the holin and the nucleic acid sequence encoding the lysin operatively linked to a foodgrade inducible promoter that can be induced via a food grade mechanism. Such a promoter system can for example be a thermosensitive complex inducible promoter as is disclosed in EP94201355 and is present on plasmid pIR 1 4. A recombinant host cell comprising a nucleic acid sequence according to any of claims 18-20 in a setting other than in its native bacteriophage and/or a recombinant vector according to any of claims 21-24 is claimed. Any of the abovementioned embodiments of recombinant host cell further comprising a nucleic acid sequence encoding a lysin, said lysin preferably being derivable from a grampositive bacterium such as a lactic

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WO 95/31562 PCT/NL95/00171 9 acid bacterium, in particular a L. lactis or a bacteriophage derivable from such a grampositive bacterium, said nucleic acid sequence encoding a lysin preferably being in a setting other than in its native bacteriophage or bacterium is also suitable. Preferably a recombinant host cell according to the invention will be a food grade host cell, preferably a lactic acid bacterium. Most preferably the host cell is of the same type from which the holin and/or lysin encoding nucleic acid sequences are derived.

Brief description of the drawings FIGURE LEGENDS belonging to the draft publication Fig. 1. A) Schematic outline of the PCR reactions used for the amplification of lytP, lytR, and the combination of lytP and lytR. The ORF's are indicated by hatched arrows. Sequences of the amplification primers 1-4 (lytl-lyt4) are given in Table 2 and as sequence id no 1-4 in the Sequence Listing. The scale is in kilobases (kb).

B) Schematic representation of the plasmid constructions. See for details Materials and Methods. Abbreviations: EmR, erythromycin resistance marker; Amp

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ampicillin resistance marker; Cm", chloramphenicol resistance marker; Pspac is a hybrid regulatory region, constructed by Yansura and Henner which contains the RNA polymerase recognition sequences of an early SP01 promoter and the lac operator; lacl, lac repressor under the control of the Bacillus licheniformis penicillinase transcriptional and translational signals, indicated as Ppen P, and promoters P, and P 2 of the bacteriophage R1-t; T, transcription terminator; ori, origin of replication; rro, R1-t repressor gene.

Fig. 2. Alignment of ORF 23 and the L. lactis subsp. cremoris c2 lysin Identical amino acid residues are indicated with asterisks, conserved changes by dots.

Fig. 3. Nucleotide sequence of a 1200 bp DNA fragment of R1-t carrying lytP and lytR as represented in sequence id no 5. The deduced amino acid sequences of lytP and lytR are indicated in sequence id no 6 and 7 respectively. The putative ribosomal binding sites (RBS) are underlined.

Asterisks represent stop codons. The stem-loop structure downstream of lytR is indicated by solid arrows.

Fig. 4. Analysis of the lytic activity of the lytR gene product. Cell free extracts of E. coli cells carrying the plasmid pAG58 (lanes 1 and 2)

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sDI~BAH~IBPB~BWPs~r~i~aaRllr~ WO 95/31562 PCT/NL95/00171 or pAG58R (lanes 3 and 4) were obtained two hours after the addition of IPTG. Abbreviations: ni, non-induced; i, induced. The arrow indicates the position of a clearing zone as a result of.lytic activity exhibited by the lytR gene product.

Fig. 5. A) Deduced amino acid sequence of the lytP gene product (sequence id no Predicted transmembrane segments are indicated by bars, the predicted 8-turn region by t's. Charged amino acid residues are indicated or depending on the sign of the charge.

B) Topological model of LytP based on the computer predictions.

The membrane-spanning amino acids are indicated.

Fig. 6. A) The effect of expression of lytP, lytR, or the combination of lytP and lytR on the optical density of E. codi MC1000 cells. Optical density measurements of E. coli cells carrying either pAG58 pAG58R pAG58P or pAG58PR with or without the addition of the inducer (IPTG) are indicated as a function of time. The time scale is in hours before and after the time of induction (indicated by arrow).

B) The number of colony Forming units per ml of E. coli cells carrying either pAG58 (empty bars), pAG58R (checkered bars), pAG58P (hatched bars), or pAG58PR (filled bars) before (left diagram), and after two hours after induction (right diagram).

Fig. 7. The effect of the induced expression of lytP, LytR, or the combination of lytP and lytR on the optical density of L. lactis subsp.

cremoris LL302 cells. Ortical density measurements of induced L. lactis cells carrying either pIR12 pIRIP pIR1R or pIRlPR (n) respectively, are indicated as a function of growth. The optical density (OD) measurements of L. lactis carrying pIR1PR, not exposed to mitomycin C, are represented by Time scale is in hours after the time of induction with mitomycin C (1 ug/ml).

The invention is illustrated by a draft publication, which is given below.

Inducible lysis of Lactococcus lactis mediated by the Lactococcus lactis subsp. cremoris bacteriophage Rl-t lysis functions.

SUMMARY

This work describes the involvement of two genes of the temperate Lactococcus lactis subsp. cremoris bacteriophage Rl-t, lytR and a WO 95/31562 PCT/NL95/00171 11 lyt in the lysis of its host. The gene product of lytR exhibits lytic activity as it hydrolysed Micrococcus lysodeiktic'is autoclaved cell walls. The gene product of lytP is required,in conjunction with lytR to obtain efficient lysis in vivo in Evcherichia coti as was shown by induction studies monitoring the optical density as a measure of cell lysis: expression of lytR alone did not cause significant lysis of E.

coli cells whereas simultaneous expression of lytP and lytR caused lysis of this bacterium. LytP therefore seems to have a similar function as the S protein of the E. coli phage lambda, i.e. rendering the murein substrate accessible to the lysin.

Both 19;? and lytR were subcloned in an inducible expression-vector for L. lactis. Induction of both genes in L. lactis was shown to result in cell lysis as monitored by a decrease in optical density.

INTRODUCTION

Host cell lysis by temperate bacteriophages is accomplished by at least two fundamentally different mechanisms The small singlestranded DNA phage X174 encodes a protein which forms a channel to transport complete phage particles from the cytoplasm of the host to the environment 7, 27). However, most of the known phages encode an enzyme with murein-deg! ding activity. These so-called lysins cause breakdown of the peptidoglycan layer which is followed by lysis of the host and the release of the phage particles.

Lysins of bacteriophages of Gram-negative bacteria so-far characterized lack a signal sequence needed for sec-dependent transport across the inner membrane. A second lysis function, encoded by a gene located immediately upstream of the lysin gene, is required for efficient lysis. This gene encodes a so-called holin which is believed to form holes in the cell membrane, thereby rendering the murein substrate accessible to the lysin Until recently it was believed that in Gram-positive bacteria, phage-mediated lysis was solely accomplished through the action of a phage-encodcd lysin. Transit of the phage-encoded lysin across the membrane was thought to occur via the general secretory route. However, the observation that a signal sequence required for this type of transport is absent in many of the identified lysins, raised the question how the murein-degrading activity gains access to the cell wall. There is now growing support for the idea that many of these phages require a second function for lysis of their Gram-positive host. Recently it was WO 95/31562 PCT/NL95/00171 12 shown that the Bacillus subtilis phage j29, gene 14, situated immediately upstream of the lysin gene, specifies a protein required for efficient release of the phage lysin to the substrate-containing environment Sequence analysis of the complete L. lactis subsp. cremoris bacteriophage R1-t genome has revealed the presence of an open reading frame (ORF) similar to several lysins of other bacteriophages. In this work we show that the corresponding gene, designated lytR, indeed encodes a protein with cell wall degrading activity in vitro, Table 1. Bacterial strains, plasmids and bacteriophage relevant features reference Bacterial strains L. lactis LL302 E. coli MC1000 subsp. cremoris MG1363 carrying the pWV01 repA gene on the chromosome to ensure efficient replication araD139, Alacx74, A(ara, leu)7697, gatU, galK, strA This work Plasmids pUC18 Apr pXNB Apr; pUC18 derivative, containing a 4.1-kb XI phage R1-t pAG58 Apr; Cmr pAG58P Apr; Cmr; pAG58 derivative carrying lytP pAG58R Apr; Cm'; pAG58 derivative carrying lytR pAG58PR Emr; Cmr; pAG58 derivative carrying ZytP and pUC18P Ap t pUC18 derivative carrying lytP pUC18R Apr; pUC18 derivative carrying ZytR pUC18PR Apr; pUC18 derivative carrying lytP and lytR pIR12 Emr; carrying the regulatory region of R1-t pIR1P Emr; pIR12 derivative carrying lytP pIRlR Emr; pIR12 derivative carrying lytR pIR1PR Emr; pIR12 derivative carrying lytP and lytR 28 baI/NheI-fragment of This work 8 ZytR This Thi Thi The,, This This This This This This This work work work work work work work work work work Bacteriophage R1-t type P335, small isometric lactococcal phage, isolated from L. lactis subsp. cremoris R1 9, 11 Em', Apr, and Cm' represent resistances to erythromycin, ampicillin, and chloramphenicol, respectively.

olif~~ PBII~ dllll~ I PI r~lll WO 95/31562 PCT/NL95/00171 13 With the use of two species-specific inducible expression systems we show that LytR require an addition ;ene product, specified by lytP upstream of lytR, for ef-.cient in.vivo lysis of E. coli and L.

lactis.

MATERIALS AND METHODS Bacterial strains, phage, plasmids, and media The bacterial strains, phage and plasmids used in this study are listed in Table 1.

E. coli was grown in TY broth (17) or on TY broth solidified with agar. L. Lactis was grown in glucose M17 broth or on glucose M17 agar. Erythromycin was used at 100 pg/ml and 5 pg/ml for E. coli and L.

Lactis, respectively. For E. coti, ampicillin and chloramphenicol were used at a concentration of 100 pg/ml and 5 pg/ml, respectively.

DNA techniques Plasmid DNA was isolated essentially by the method of Birnboim and Doly Restriction enzymes, Klenow enzyme, T4 DNA ligase, and T4 DNA polymerase were obtained from Boehringer GmbH (Mannheim, Germany) and used according to the instructions of the supplier. Synthetic oligonucleotides were synthesized using an Applied Biosystems 381A DNA synthesizer (Applied Biosystems Inc., Foster City, Calif.). Polymerase chain reactions were performed using Vent polymerase (New England Biolabs Inc., Beverly, Samples were heated to 94 "C for 2 min, after which target DNA was amplified in 25 subsequent cycles under the following conditions: 94 "C for 1 min; 50 *C for 2 min; 73 *C for 1 min. The primers used for amplification are listed in Table 2 and sequence id no 1-4 of the Sequence Listing. E. coli was used as a host for obtaining recombinant plasmids. Transformation of E. coli was performed by the method of Mandel and Higa Plasmids were introduced in L. lactis subsp. cremoris LL302, which contains a copy of the pWVO1 repA gene on the chromosome to enrure efficient replication, by means of electroporation DNA and protein sequences were analyzed using the programs developed by Staden Analysis of the LytP protein was computed with the PC/Gene program (version 6,7; IntelliGenetics, Inc., Geneva, Switzerland) using the membrane spanning domain search program SOAP, or the 8-turn search program BETATURN.

111 WO 95/31562 PCTINL95/00171 Table 2. Primers used for amplification of lytP and ZytR primer DNA sequence 5 Lyt AAAACCCGGGAAGCTTGTCGACAGCAGTGATTGGTTCAACG Lyt2 TTCTAGAAGCTTGCATGCCCCTTCTTTTTATTATTGAC Lyt3 AAAACCCGGGAAGCTTGTCGACGATAATACAGCAAGCCTAGTC Lyt4 TTTTTCTAGAAGCTTGCATGCGAAGCGGGGTTAATTTATCC HindIII (AAGCTT), SphI (GCATGC), e.id SaZI (GTCGAC) restriction enzyme sites are depicted boldfaced.

IPTG and mitomycin C induction Overnight cultures were diluted hundred-fold in fresh glucose M17 medium lactis) or TY medium supplemented with 0.5% glucose (E.

coti) and grown until the culture reached an OD600 of 0.3 at which point isopropyl-B-D-thiolgalactopyranoside (IPTG) or mitomycin C (Sigma Chemical Co., St.Louis, Mo.) was added to a final concentration of 5mM or 1 pg/ml, respectively. Before the addition of IPTG, E. coti cells were collected by centrifugation and resuspended in an equal volume of TY medium without additional glucose.

Lytic activity assay The lytic activity assay was performed essentially as described by Potvin et at. (15) with some minor adjustments as reported by Buist et at. Plasmid constructions The lytP and lytR containing fragments of the R1-t genome were amplified using polymerase chain reactions (PCR's). A 4.1-kb Xbal/Nhel fragment of the R1-t genome containing both lytP and LytR was subcloned in the unique XbaI site of pUC18 resulting in the plasmid pXNB. Using pXNB as a template, amplification with three different primer combinations (lytl-lyt2, lyt3-lyt4, and lytl-lyt4; see Fig. 1A) yielded three DNA fragments carrying either lytP, lytR, or the combined lytP and LytR, respectively. Following digestion with SphI and HindIII these three PCR products were subcloned in HindIII and SphI restricted pAG58, which r WO 95/31562 PCT/NL95/00171 resulted in plasmids pAG58P, pAG58R, and pAG58PR, carrying lytP, lytR, and the combined lytP and lytR, respectively, under the control of the IPTG inducible P.pac promoter (Fig. 1B). For-lysis studies in L. lactis plasmids pAG58P, pAG58R and pAG58PR were first restricted with SphI and Sail. Subsequently, the DNA fragments, carrying lytP, LytR and the combination of lytP and LytR, were first subcloned in SphI/SaiI-cut pUC18. The HindII/HindIII fragments of these three constructs, designated pUC18P, pUC18R, and pUC18PR, were cloned in the NruI and HtndIII sites of pIR12, resulting in the plasmids pIR1P, pIR1R, and pIR1PR, containing lytP, lytR, and both lytP and lytR, respectively, under the transcriptional control of the Rl-t promoter-operator region (Fig. 4B).

These plasmids were transformed to L. lactis subsp. lactis strain LL302 which contains a copy of the pWV01 repA gene on the chromosome to ensure efficient replication of pWVOl-derived plasmids.

The construction of plasmid pIR12 was described in a co-pending application EP-94201353.3, filed on the same date entitled: Process for the lysis of a culture of lactic acid bacteria by means of a lysin, and uses of the resulting lysed culture, the specification of which is incorporated herein by reference.

RESULTS

Cloning and sequence analysis of the R1-t lysis functions.

The switch from lysogenic to lytic life cycle of the temperate L. lactis subsp. cremoris phage Rl-t will ultimately result in host cell lysis caused by phage-encoded lysis function(s), followed by the release of phage particles. Inspection of the DNA sequence of Rl-t revealed that ORF 23, which specifies a protein of 270 amino acids with a calculated molecular weight of 30,214 Da, shows significant similarity with the lysin genes of the L. Lactis bacteriophages c2 (sequence id no 8) and 4ML3 (26, 18). The similarity between the deduced amino acid sequence of ORF 23 with the c2 lysin is shown in Figure 2. Moreover, ORF 23 specifies an amino acid sequence similar to the amino acid sequences of the Nterminal portions of the amidase Hbl of the Streptococcus pneumoniae bacteriophage HB-3 and the S. pneumoniae LytA autolysin Therefore, ORF 23, hereafter designated lytR (Fig. 3) (sequence id no 7), is a likely candidate for the phage-encoded lysin gene. This was not to be predicted as is apparent from the previously cited Young reference.

To test this supposition, lytR was cloned into the IPTG inducible expression-vector pAG58, resulting in pAG58R (Fig. 1B). Cell-

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%iPPllllilBBkllPlrsl~WI~ r~ I-' WO 95/31562 PCT/NL95/00171 16 free extracts of E. coli cells containing pAG58R were assayed for lytic activity on an SDS-polyacrylamide gel in which Micrococcus lysodeikttcus autoclaved cell walls were co-polymerized. After staining of the cell wall-containing gel with methylene blue, a clearing zone is expected at positions corresponding to lytic proteins due to the breakdown of incorporateu cell walls. As shown in Figure 4, in cell free extracts of pAG58-containing E. coli cells a clearing zone was absent at the position corresponding to a protein with the expected molecular weight of the lytR-encoded protein. Cell free extracts of pAG58R-containing cells, however, gave rise to a clearing zone at the expected position. A weak clearing zone was obtained with cell free extracts of uninduced cells due to limited expression of the lytR gene. Cell free extracts of induced pAG58R-containing cells showed an extended clearing zone, which became very large in extracts obtained two hours after induction.

According to the rules of Von Heijne LytR does not seem to contain a signal sequence specific for secreted proteins using the sec-dependent transport system. This apparent lack of a signal sequence has also been observed in lysins of other bacteriophages of both Gramnegative and Gram-positive bacteria. For host cell lysis to occur these phages require a protein that forms holes in the cytoplasmic membrane to render the host cell peptidoglycan layer accessible to the lysin ORF 22, which is situated upstream of lytR, specifies a protein of amino acids with a predicted molecular weight of 7,688 Da (sequence id no Although the predicted amino acid sequence shows no similarity with the putative hole-forming proteins of other phages, computer analysis of the protein product of ORF 22, designated hereafter as lytP, predicted structural similarities with these proteins. Computer analysis revealed that the protein, specified by lytP, has a high probability of containing a pair of transmembrane domains, separated by a sequence with a high probability of adopting a beta turn conformation (Fig. In addition it contains a charged C terminus and is highly hydrophobic. Therefore, this protein might function as a pore-forming protein required for the release across the cytoplasmic membrane of the R1-t encoded LytR.

LytP and lytR are required for lysis in Escherichia coli To determine whether the lytP and lytR gene products are involved in host cell lysis, lytP, lytR, and the combination of lytP and LytR were subcloned in the inducible expression- vector pAG58, resulting in pAG58P, pAG58R and pAG58PR, respectively (Fig. 1B). Induction studies

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WO 95/31562 PCT/NL')5/0171 17 were performed with E. coli MC1000 carrying these plasmids to examine the effects of the expression of the cloned genes on the optical density of the cells (Fig. 6A). Induction of lytR expression did not cause any lysis of pAG58R-containing E. coli cells as was determined by optical density measurements. The induction of lytP expression, however, almost immediately halted the increase in the optical density of pAG58Pcontaining cells. The expression of both lytP and lytR in E. coti caused lysis. Lysis occurred almost immediately after the addition of IPTG to pAG58PR-containing cells, as was demonstrated by the decrease in optical density which was associated with a dramatic decrease in colony forming units (CFU's) as compared to the uninduced control (Fig. 6B). No significant difference in CFU's between cells carrying pAG58 and pAG58R was observed. However, the induction of lytP had a significant effect on '.he viability of pAG58P-containing cells. The number of CFU's dropped more than 200-fold within two hours.

Expression of lytP and lytR in Lactococcus lactis In order to examine the effects of the expression of either lytP, lytR or the combined LytP and lytR on the optical density of L.

lactis cells, plasmids pIR1P, pIRlR, and pIR1PR were constructed (Fig.

1B). Transcription of lytP, lytR, and both lytP and lytR in these plasmids is controlled by the regulatory region of phage Rl-t, which incorporates the gene specifying the repressor (rro) of Rl-t in addition to its cognate operator region (see Fig. 1B). Expression was induced by the addition of the DNA damaging substance mitomycin C. Induction studies were performed with L. lactis subsp. cremoris LL302 cells carrying the plasmids described above. Figure 7 shows that the addition of mitomycin C to L. lactis cells carrying pIR12 slows down the increase in optical density similar to pIRiP-containing L. lactis cells. The addition of mitomycin C to genetically non-modified lactococci caused lysis of a small portion of the cells (results not shown). The expression of lytR as well as the simultaneous expression of lytP and lytR led to a decrease in optical density, as compared to pIR12-containing cells to which mitomycin C had been added, indicating cell lysis.

DISCUSSION

We recently determined the nucleotide sequence of the temperate L. Lactis subsp. cremoris bacteriophage Rl-t. On the basis of the similarity of the deduce amino acid sequence with various (auto)lysins ~II I rl Dlry(lpl~ ~''llll~ RC I~ WO 95/31562 PCT/NL95/00171 18 we postulated that ORF 23, designated LytR, could specify the phageencoded lysin. The ZytR gene product would consist of 270 amino acids with an estimated molecular weight of 30,214 Da. By assaying cell-free extracts of E. coli cells expressing lytR, it was shown that lytR indeed specified a protein with lytic activity.

The similarity of LytR is mainly limited to the C-terminal parts of the lysins of the lactococcal bacteriophages c2 and (vML3, whereas the N-terminal part of LytR is similar to the amino acid sequence of the N-terminal portion of the S. pneumoniae LytA autolysin. It has been proposed that LytA consists of two functional modules the Cterminal domain specifying the binding site to the murein substrate and the N-terminal domain determining the specificity of the enzyme. Since LytA is an N-acetylmuramoyl-L-alanine amidase it is tempting to speculate that LytR is also an N-acetylmuramoyl-L-alanine amidase.

Because of the lack of an apparent signal peptide, we hypothesized that, like many other phage-encoded lysins, LytR needs an additional factor in order to gain access to the cell wall. ORF 22, designated LytP, which is situated immediately upstream of lytR, can specify a protein of 75 amino acids with the characteristics of a socalled holin which, for other phages, was shown to render the murein substrate accessible to lysins which lack a signal peptide This hypothesis was corroborated by the observation that the expression of LytP is indeed needed for efficient lysis of E. coLi in vivo. In fact, induction of lytR expression did not result in lysis of E.

coli. However, E. coli did lyse when, in addition to lytR, lytP was also expressed. From these results it was concluded that the transit of LytR across the inner membrane is dependent on the lytP gene product. The induction of solely lytP almost immediately halted the increase in optical density and had a dramatic effect on the viability of the induced cells. This is probably caused by the spontaneous insertion of the protein into the lipid bilayer, inducing nonspecific lesions in the inner membrane and thereby dissipating the membrane potential Presumably LytP forms pores in the cytoplasmic membrane, thus allowing LytR to gain access to the cell wall.

An inducible expression system for Lactococci recently developed in our laboratory made it possible to examine the effects of expression of lytP, LytR, and the combined lytP and LytR in L. Lactis.

Expression of the combined lytP and lytR in L. Lactis resulted in lysis of the cells. In contrast to E. coli, lysis was also observed when only Ir PRCll~llal~ IIIIII Ima~ s~ -sllrP WO 95/31562 PCT/NL95/00171 19 lytR was expressed. Lysis of cells solely expressing lytR is probably caused by the combined effect of mitomycin C and LytR: Since mitomycin C lyses a small proportion of the cells (results not shown), LytR is extruded in the medium, thus acting upon the cell wall from without, and masking the additional requirement for LytP to effect lysis as was the case in E. coli.

For bacteriophages of both Gram-negative and Gram-positive bacteria, a system based on a murein hydrolase and a second protein required for the access of the hydrolase to its murein substrate, seems to be a general phenomenon in lysis strategies. Recently it was shown thpt, in addition to the B. subtilis phage )29-encoded lysin, efficient lysis of E. coti also required the gene 14 product. Also several lactococcal bacteriophages seem to encode an additional factor needed for host cell lysis. On the basis of structural similarity, it has been postulated that the bacteriophages c2 and (vML3 encode a holin (26, The deduced amino acid sequence of ORF2 of the virulent bacteriophage )U53, isolated from L. lactis SK11 also shares the characteristic structural traits of a holin, making it likely that it is involved in the translation of the phage-encoded lysin, LytA. This report, however, proves for the first time that a Lactococcus-specified holin is required for phage-induced lysis.

DRAFT PUBLICATION 2 Development of a food-grade, thermo-inducible lysis system using the regulatory region and lysis functions of the temperate Lactococcus lactis subsp. cremoris bacteriophage R1-t.

Introduction The system is based on the food-grade removal of most of the genomic DNA of a temperate lactococcal bacteriophage in such a way that an inducible regulatory region of the temperate bacteriophage is directly placed upstream of the lysis functions encoded by the prophage. As an examplP of the general applicability of this strategy to any prophage with a similar genetic structure, bacteriophage Al-t was taken. To obtain the desired delp 4jn, plasmid pBTS1 was constructed (Figure In the future pBTS2 will be constructed in which rro is replaced by rroTs and therefore can be used to make this system thermo-inducible.

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BIIPSlb~gl~an~asr~wa~a~a~~ WO 95/31562 PCT/NL95/00171 Experimental procedures Bacterial strains, phage, plasmids, and media The bacterial strains, phage, and plasmids used in this study are listed in Table 3. Escherichia coli JM101 was grown in TY broth (Rottlander and Trautner, 1970) with vigorous agitation, or on TY agar, at 37 When needed, ampicillin, isopropyl-B-D-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl 8-galactopyranoside (X-gal) (all from Sigma Chemical Co., St. Louis, MO.) were used at concentrations of 100 ug/ml, ImM and 0.002% (wt/vol), respectively. L. lactis subsp.

cremoris was grown in M17 broth (Terzaghi and Sandine, 1975), or on M17 agar, supplemented with 0.5% glucose or lactose at 30 When appropriate, erythromycin (Boehringer Mannheim, GmbH, Germany) and X-gal were used at concentrations of 5 pg/ml and 0.004% (wt/vol), respectively.

General DNA techniques and transformation General DNA techniques were performed as described by Sambrook et at. (1989). Plasmid DNA was isolated by the method of Birnboim and Doly (1979) and by using QIAGEN Midi-Plasmid isolation columns (Qiagen Inc.,Chatsworth, Restriction enzymes, alkaline phosphatase and T4 DNA ligase were obtained from Boehringer Mannheim and were used according to the instructions of the supplier. Transformation of E. colt was performed as described by Mandel and Higa (1970). L. lactis LL108 was transformed by electroporation using a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.), as described by Holo and Nes (1989) with the modifications suggested by Leenhouts and Venema (1993). Electroporation of L. lactis Rl, R131 and RIK1O was done as decribed by van der Lelie et al. (1988). Oligonucleotides were synthesized using an Applied Biosystems 381A DNA synthesizer (Applied Biosystems, Inc., Foster City, Calif.).

Polymerase chain reactions (PCR's) were performed using Vent polymerase (New England Biolabs, Inc., Beverly, After heating of the samples to 94 "C for two minutes, target DNA was amplified in 30 subsequent cycles under the following conditions: 94 "C for 1 min; 50 *C for 2 min; 73 °C for 3 min. PCR fragments were purified using the QIAEX DNA Gel Extraction Kit (Qiagen Inc.).

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1113s~ C~ ~llI~-- WO 95/31562 PCTINL95/00171 21 Isolation of R1-t phage particles and DNA An overnight culture of L. Lactis R1 was diluted hundred-fold in 500 ml fresh lactose M17 medium and grown until the culture reached an OD600 of 0.8 at which point mitomycin C (Sigma) was added to a final concentration of 2.5 pg/ml. Incubation at 30 *C was continued in the dark until lysis occurred. Cells debris was removed by centrifugation for min at 6000 rpm. Phage particles were precipitated by incubation with NaCI (0.5 M) and polyethylene glycol 6000 (10 [wt/vol]) for three hours on ice and purified by a CsCl step gradient as described by Sambrook et al. (1989). The bacteriophage Rl-t suspension was dialysed against several changes of 150 mM NaCI, 15 mM trisodiumcitrate. Phage DNA was obtained by extracting the suspension twice with phenol. The DNA solution was subsequently dialysed against 10 mM Tris-HCl/1 mM EDTA, pH Sequencing attB-sites The attachment sites attL and attR of the bacteriophage Rl-t lysogen L. lactis R1 were determined by means of cycle sequencing using the CircumVent Thermal Cycle Dideoxy DNA Sequencing Kit with Vent (exo') DNA Polymerase (Biolabs, New England). Primers attBL and attBR with flanking XbaI and PstI sites (Table 4) were used for cloning the attB site of L. lactis MG1363. The 272-bp PCR fragment obtained with attBL and attBR was cut with XbaI and PstI and cloned in the XbaI/PstI sites of pUCl8 and sequenced using the dideoxy-chain-termination method (Sanger et al., 1977) and the T7 sequencing kit (Pharmacia AB, Uppsala, Sweden).

Phage titre determination Supernatant taken from L. lactis R131 was diluted in 1 mM MgSO4.

The indicator strain L. lactis R1K10 was grown in GM17 until the OD600 was 0.7. 2 ml of culture were centrifuged and cells were resuspended in 2 ml 1 mM MgS04. An aliquot of 100 pl diluted phage-particles were added to 200 pl cells. After incubation at room temperature for 20 minutes 3 ml Top agar GM17 agar, 0.25% giycine, 10 mM CaCl 2 were added, mixed, and poored on a GM17 agar-plate containing glycine and CaCl 2 (10 mM). The plates were incubated overnight at 30 *C and the number of plaques were determined.

I I I BP~L1~PB~r~Y~RLlsrrrrrrur~~~s~ a- WO 95/31562 PCT/NL95/00171 22 Relysogenisation of L. lactis R1K10 After infection of L. lactis R1K10 with Rl-t phage particles, turbid plaques will be picked and tested for their ability to give UVinduction of prophage. Centrifuged cells of exponentially growing cultures will be resuspended in 1 ml 1 mM MgSOh and irradiated with a Mineralight u.v. lamp (model UVG-54, 2 5 4 nm, 3.2 Jm-2s-l: Ultra-violet Products Inc.) for 10 seconds, then 1 ml 2 times GM17 10 mM CaCl 2 will be added. The culture will be incubated at 30 "C until lysis occurs.

Mitomycin C induction Overnight cultures of L. lactis were diluted hundred-fold in fresh glucose M17 medium and grown until an 0D600 of 0.3 at which point mitomycin C was added to a final concentration of 1 pg/ml.

Plasmid constructions The plasmid pORIR1PR was constructed in L. Lactis by subcloning the 2864-bp EcoRI/SphI-fragment of pIRlPR into p0RI280 restricted with EcoRI and SphI (Figure Homology analysis showed that ORF 25 of the Rl-t genome shared 98% identity with the integrase-gene of bacteriophage phi LC3 (Lillehaug and Birkeland, 1993) and was therefore called intR.

The intR region was amplified with flanking SacI and XbaI sites using PCR and primers intl and int2 (Table Plasmid pUCl8Int was constructed by cloning the resulting 1326-bp PCR-fragment digested with SacI and Xbal into the SacI/XbaI sites of pUC18. A 1047-bp HindII fragment of pUCl8Int, which contains the 5'-truncated intR, was subcloned into the alkaline phosphatase-treated SmaI-site of pUC18. Both the resulting plasmid pUC18lntd and pUCl8Int were constructed in E.coti JM101 (Figure The intR was cut out of pUCl8Intd with EcoRI and BamHI and subcloned in the EcoRI and BamHI sites of p0RRlPR, resulting in pBTS1 (Figure The latter construction was done in L. lactis LL108.

In the future rro will be replaced by rroTs when this temperature inducible repressor is available. This will be done by replacing the 946-bp NcoI-EcoRI fragment of pBTS1 with the comparable fragment containing rrors. This will result in pBTS2. Plasmid pIR14 deposited at the Centraal Bureau voor Schimmelcultures in Baarn, The I I I I I I

UU~I~_

WO 9531562 PCT/NL95/00171 23 Netherlands comprises such a temperature sensitive rro. A detailed description is given in European Patent Application 94201355.8.

Results Plasmid pBTS1 was introduced in L. lactis LL108. As can be seen in figure 10, pBTS1 is still able to give inducible lysis after mitomycin C induction.

The initial idea was to introduce pBTS1 in L. lactis Rl. As pBTS1 cannot replicate in L. lactis (it lacks the gene for the plasmid replication protein RepA) it will integrate into the chromosome of R1 under selective conditions. The integration will take place at either of three homologous regions: the intR-region the regulatory-region (B) or the region of the lytic functions (Figure 11). With appropriate primer-sets the place of integration can be distinguished (Table 5 and Figure 11). After the first recombination step in the regions A or C, a second recombination step in the region C or A, respectively, will delete the whole prophage and plasmid from the chromosome of strain R1, except for the desired functions. These two recombination steps will place the lytic functions directly under control of the regulatory region of Rl-t, in a one copy situation at a well defined and stable place in the chromosome of L. lactis. If the integration takes place in region B (regulatory region), the second recombination step will not result in the substitution of intR and rro for the 5'-truncated intR and rrors (future work), respectively. The integrase deletion is needed to prevent intR catalysed excision.

Because of the extremely low transformation efficiency of L.

lactis R1 (less than 1 transformant/pg pVE6007) we tried to cure the strain of its natural plasmids. We succeeded in curing two plasmids of approximately 50 kb and 2 kb, by growing L. lactis R1 on glucose and incubation at 37 The resulting strain L. lactis R131 was shown by UVinduction to still contain Rl-t prophage.

Sofar we have not succeeded in introducing pBTS1 into L. lactis R131. It appears that the prophage is induced after the electroporation.

This, together with the presence of MgCl 2 and CaC12 in the recovery medium, makes the cells lyse (Table 6).

Therefore, we are currently trying to obtain the pBTSl integrant by two additional strategies. Firstly, pBTS1 and pVE6007 will be introduced together in L. lactis R1K10. pVE6007 encodes a temperature I I I ~XPW8PII~RPIQll~n~a~aPrsr~ WO 95/31562 PCT/NL95/001 7 1 24 sensitive RepA protein enabling pBTS1 to replicate. After relysogenisation of the resulting double transformant with phage Rl-t, pBTS1 will integrate into the chromosome when raising the temperature to 37 The second way to obtain a 'food-grade' inducible lysis system is to introduce pBTS1 and pVE6007 together in L. lactis MG1363. The attBregion of this strain has been sequenced and appeared to have an homology of 99% with the attB-region of L. lactis R1K10 (Figure 12). L. Lactis MG1363 will be transformed with Rl-t DNA ligated at its cos-sites. After intR-catalysed integration of the R1-t chromosome into the attachment site of MG1363 (Figure 12), pBTS1 can integrate when raising the temperature to 37 'C.

7 L~SPaarru ur~r~^lurs~--- WO 95/31562 PC'r/N5/00171 Figure legends Figure 8 Cloning scheme for the construction of pBTS1. EmR, erythromycin resistance gene; rro, Rl-t r *:essor gene; po, promoter(pl/p2)/operator region from bacteriophage Rl-t; tec, topological equivalent of lambda cro; lytP, Rl-t holin gene; lytR, Rl-t lysin gene; T, transcription terminator of prtP; ORI+, origin of replication of the lactococcal plasmid pWVO1; p32, promoter sequence of 0RF32 of L. lactis; lacZ, 8galactosidase gene of E. coli; 'intR, 5'-truncated Rl-t integrase gene; amp, ampicillin resistance gene. Only relevant restriction enzyme sites are shown.

Figure 9 Cloning scheme for the construction of pUCl8Intd in which the integrase gene of bacteriophage Rl-t is 5'-truncated. intR, Rl-t integrase gene; lacZ, 8-galactosidase gene of E. coli; amp, ampicillin resistance gene; 'intR, 5'-truncated R1-t integrase gene. Only relevant restriction enzyme sites are shown.

Figure Effect of mitomycin C on ODbOO of L. lactis LL108 containing pORI13 (Leenhouts and Venema, 1993) (open square) and pBTS1 (filled iangle). The cultures were induced with 1 pg/ml mitomycin C at time zero (dotted line).

Figure 11 Schematic representation of the food-grade removal of most of the genomic DNA of Rl-t prophage in such a way that the inducible regulatory region of the temperate bacteriophage is directly placed upstream of the lysis functions encoded by the prophage. As an example, the integration of pBTS1 in the integrase gene is depicted and the second recombination occurs in the region of the 1-.ic functions.

integrase-region; regulatory-region; region of the lytic functions; attR, 'right' phage-host junction; intR, Rl-t integrase gene; rro, Rl-t repressor gene; tec, topological equivalent of lambda cro; PROPHAGE, genomic DNA of R1-t prophage; lytP, R1-t holin gene; lytR, R1-t lysin gene; attL, 'left' phage-host junction; EmR, erythromycin resis- I- I WO 95/31562 PC77NI,95/00171 26 tance gene; IacZ, 1-galactosidase gene of E. coli; 'intR?, R1-t integrase gene. The genes derived from the plasmid pBTSl are shaded.

The primers: attBfl, R100, attBL and Rt40 are listed in table 4.

Figure 12 Comparison of the attB regions of L. lactis 11110 and MG1363.

The attfl site is shaded. Asterisks indicate identical nucleotides.

TABLE 3. Bacterial strains, phages, and plasmid LO Bacterial strain, phagc Relevant Source or or plasmid characteristic(s) reference Bacterial strains L. lactis subsp. cremoris LL108

RI

R13 1 RIK1O Escherichwa coli JM1OI Bacteriophage RI -t MG 1363 (plasmid-frec strain) carrying the pWVOI repA gene, and the Cm' gene on the chromosome (high copy) Original RI-t lysogenic L lactis subsp. cremoris strain L. lactis R1 cured of two natural plasmids RI-t indicator strain supE thi n~Qac-proAB-) [F :raD36 proAl)' IacP type P335, small isometric temperate lactococcal phage, isolated from L. lactis subsp. cremoris RI Leenhouts, unpublished results Lowrie, 1974 This study lab collection Messing, 1979 Lowrie, 1974 Plasmids plIPR p0 RI28 0 pORIR1PR PUC18 pUC18int pUC18lntd pBTS1 pVE6007 pOR113 Nauta et al., 1994 l Leenhouts and Venema, 1993 This study Yanisch-Perron et al., 1985 This study This study This study Maguin et al., 1992 Leenhouts and Venema, 1993 WO 95/31562 WO 95/ 1562PCTINL95/00171 27 TABLE 4. Nucleotide sequences of PCR primers Primer Sequence intl(Sacl) 5'-GCGCQA=fCCGCTCAAGTfI'GACGACAAGGG-3' (SEQ id no 9) int2(KbaI) 5'-GCGCT=TAGGATAGATGTGC=lAGATAATGGC-3' (SEQ id no attBL(Xba I) 5'-GCGCTIatAGACAGC1W1TCTATCT'CGTAAGGG-31 (SEQ id no 11) atnBR(Pstl) 5'-GCGCU:CLAGTACCTAAGCACACGAAGGCCI'AGG-3' (SEQ id no 12) 5'-GAAATTGGATAGTTAAGG-3* (SEQ id no 13) RIOO 5'-CTCGTGATrACTATTGG-3' (SEQ id no 14) *The restriction enzyme sites are underlined.

TABLE 5. Primer-sets to check integration Strain Primer-sets', 2 atiBR/IlOO L. lactis RI 2271 bp pBTS 1 integrated in: intR-region 1687 bp regulatory -region 2271 bp lytic-region 2271 bp 2249 bp After second recomb. 1687 bp 2249 bp Primers are listed in Table 4.

2The sizes of the expected PCR-fragments are given, WO 95/31562 WO 95/ 1562PCT/NL95/00 171 TABLE 6. RI-t induction by electrical pulse Electrical Medium' 0D600 of R13 1 3 PFU/mI pulse 1.5 hrs 4.5 hrs 21.5 hrs 4.5 hrs 0 GSM17 0.323 1.097 2.248 1.4 GSM17 0.228 0.783 2.000 4.4 0 GSM17MC 0.242 0.530 1.120 1.1 106 GSM17MC 0.190 0.189 0.107 1.9 01 Electroporat ion cuvene (2-mm electrode gap); 25 liF/200O2 2 obo and Ness, 1989.

W~ competent cells (van der Lelie et al., 1988), cells harsvested at 0D600 0,714 BIR1~ Wpai~erm~arrslonam~~ WO 95/31562 PCT/NL95/00171 29 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Quest International STREET: Huizerstraatweg 28 CITY: Naarden COUNTRY: The Netherlands POSTAL CODE (ZIP): 1411 GP TELEPHONE: 02159 99111 TELEFAX: 02159 46067 (ii) TITLE OF INVENTION: Process for inhibiting the growth of a culture of lactic acid bacteria, and optionally lysing the bacterial cells, and uses of the resulting lysed culture.

(iii) NUMBER OF SEQUENCES: 8 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) CURRENT APPLICATION DATA: APPLICATION NUMBER: INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

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WO 95/31562 PCT/NL95/00171 (vi) ORIGINAL SOURCE: ORGANISM: Lactococcus phage Rl-t INDIVIDUAL ISOLATE: primer LYT1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AAAACCCGGG AAGCTTGTCG ACAGCAGTGA TTGGTTCAAC G 41 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTFRISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Lactococcus phage Rl-t INDIVIDUAL ISOLATE: primer LYT2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TTCTAGAAGC TTGCATGCCC CTTCTTTTTT ATTATTGAC 39 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: 11__1_111_ WO 95/31562 PCT/NL95/00171 31 ORGANISM: Lactococcus phage R1-t INDIVIDUAL ISOLATE: primer LYT3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAAACCCGGG AAGCTTGTCG ACGATAATAC AGCAAGCCTA GTC 43 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Lactococcus phage Rl-t INDIVIDUAL ISOLATE: primer LYT4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TTTTTCTAGA AGCTTGCATG CGAAGCGGGG TTAAITTATC C 41 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1200 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Lactococcus phage R1-t I I I WO 95/31562 WO 95/1562 CT/N L95100 171 32 INDIVIDUAL ISOLATE: Fig.3 cds lytP and cds lytR (ix) FEATURE: NAME/KEY: CDS tB) LOCATION: 103. .328 (ix) FEATURE: NAME/KEY: CDS LOCATION: 331. .11141 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TCTACAGGTA CATGCCAAAA TATCGITCA GCAOTGATTO GTTCAACCAC AATATATTAT TGCAAACCAA CTCCATAAAA AATAAAAAAT AGGACAAAGA AC ATG AAA Met Lys ACA Mrr Thr Phe T'TT AAA OAT ATO OCA CAA COT CCC A'PT AAA Phe Lys Asp Met Ala Clu Arg Ala Ile Lys 10 ATTr CCC OCT TT17 GOT Ile Gly Ala Leu Cly CTT CAA GCC 'TCG ACT Leu Gin Ala Leu Ser OCT COT CCC ACA GOC Ala Cly Ala Thr Cly 30 ATC OCA CCC TI' OCA Ile Ala Cly Phe Ala ACA MI GCA CAA OCA ATO Thr Phe Ala Gin Ala Met 15 TTA ATT CCC GIT CAT TOG Leu Ile Gly Val Asp Trp ACA OTO OTA TCA AIT CTT Thr Val Val Se' Ile Leu ACT TCA 'PTA Thr Ser Leu OCA ACT COG A'PP CCC CCC OAT AAT ACA Ala Ser Cly Ile Pro Cly Asp Asn Thr 60 OCA AOC CTA CTC Ala Set' Leu Val AAT AAT Asn Asn AAA AAA GAA CCC CAA T AA ATC ACA AIT TAC CAC AAA ACO TITC Lys Lys Clu Cly Ciu Met Thr Ile Tyr Asp Lys Thr Phe 75 1 WO 95/31562 PCT/NL95/00171 CTA CTC GGC Leu Leu Gly ATC GTG ATT Ile Val Ile ACA GOT CAA GOT TCG TCA CAA AAG Thr Gly Gin Gly Ser Ser Gin Lys 15 CAC GAT ACC GCC AAT GAT AAT AAC His Asp Thr Ala Asn Asp Asn Asn 30 GCG AGT AAT CGA TAT Ala Ser Asn Arg Tyr CAA GGT GAT AAT ACT Gln Gly Asp Asn Ser GCC ACA AAT GAA GCG Ala Thr Asn Glu Ala AGT TAT ATG CAC AAT Ser Tyr Met His Asn 50 AAC TGG CAA AAT GCC TAT Asn Trp Gin Asn Ala Tyr ACT CAT GCC Thr His Ala GGA TAT GTT Gly Tyr Val ATT GCT GGC TGG GAT AAA le Ala Gly Trp Asp Lys 65 GCT TAT GGT OCA GGG AGT Ala Tyr Gly Ala Gly Ser 80 GTG TAT TTG GTA GGA GAA CCT Val Tyr Leu Val Gly Glu Pro CCA GCT AAT GAA CGC TCA CCG Pro Ala Asn Glu Arg Ser Pro TTC CAA Phe Gin ATC GAA CTC TCT CAC Ile Glu Leu Ser His 95 TAT TCA GAC CCA GCT AAA CAA CGT TCT Tyr Ser Asp Pro Ala Lys Gin Arg Ser 100

TCA

Ser 105 TAT ATC AAC TAT ATC Tyr Ile Asn Tyr Ile 110 AAT GCT GTG CGT GAA Asn Ala Val Arg Glu 115 CAA GCA AAA GTA TTC Gin Ala Lys Val Phe 120 GGT ATC CCT CTT ACT Gly lie Pro Leu Thr 125 CTT GAT GGA GCA GGT Leu Asp Gly Ala Gly 130 AAT GGT ATC AAA ACT CAT Asn Gly Ile Lys Thr His 135 AAA TOG GTT Lys Trp Val TAT TTA ACA Tyr Leu Thr 155 TCG GAT AAC CTT TGG GGA Ser Asp Asn Leu Trp Gly 140 145 CGC ATT GGT ATT AGC AAA Arg Ile Gly Ile Ser Lys 160 GAC CAT CAA GAC CCT TAC TCT Asp His Gin Asp Pro Tyr Ser 150 GAC CAA CTC GCC AAA GAC TTA Asp Gin Leu Ala Lys Asp Leu 165 Wo 95/31562 WO 95/31562 CTNL95O()171 GCA AAC Ala Asn 170 341 GOT AlT GGT 000 OCA TCG AAA TCT AAT CAA TCT AAT AAC GAT Gly Ile Gly Gly Ala Ser Lys Ser Asn Gin Ser Asn Asn Asp 175 .180 GAT TCA ACA CAC Asp Set' Thr His 185 ATG ACT TAT CT!' Met Thr Tyr Leu GCA ATC AAC TAC ACA CCT Ala Ile Asn Tyr Thr Pro 190 AT!' TIT OCA AAA GAC ACT Ile Phe Ala Lys Asp Thr 205 210 AAC ATO GAG GAA AAA GAA Asn Met Glu Glu Lys Glu 195 200 AAA CGC TOG TAO ATC ACA Lys Arg Trp Tyr Ile Thr 215 978 AAC GOT AT!' Asn Gly Ile TAT CAA AAT Tyr Gin Asn 235 GAA ATC COT TAT ATC AAA Oiu Ile Arg Tyr Ie Lys 220 225 CAA TOG TF!G AAA T!'C AAA Gin Trp Leu Lys Phe Lys 2t4o ACT GOT AGA 07!' CT!' OGA A.AT Thr Gly Arg Vai Leu Gly Asn 230 CT!' CCT OTO OAT ACT ATO 'TOC Leu Pro Val Asp Thr Met Phe 245 1026 1074~ CAA OCA Gin Ala 250 GAA OTC OAT AAA GAG Giu Vai Asp Lys Giu 255 TT!' GOA ACT OA OCA ACA AAT CCA AAT Phe Gly Thr Gly Ala Thr Asn Pro Asn 260 1122

COT

A rg 265 GAC AT!' TCA AAA OA Asp Ile Ser Lys Gly 270 T AAATTAACCC CGCT!'CGGCG GOTOTTFI 1171 TAAATATAAT 'ITAT!'CAAAT AACATTITTT 1200 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 75 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 95/31562 PCT/NL95/00171 Met 1 Ala (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Lys Thr Phe Phe Lys Asp Met Ala Glu Arg. Ala Ile Lys 5 10 Gin Ala Met Ile Gly Ala Leu Gly Ala Gly Ala Thr Gly Thr Phe Leu Ile Ala Thr Val Gly Val Asp Val Ser Ile Leu Gin Ala lie Ala Gly Leu Thr Ser Ser Gly lie Asp Asn Thr Ala Ser Leu Val Asn Lys Glu Gly INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 270 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Met Thr lie Tyr Asp Lys Thr Phe Leu Leu Gly Thr Gly Gin Gly Ser 1 5 10 Ser Gin Lys Ala Ser Asn Arg Tyr Ile Val Ile His Asp Thr Ala Asn 25 Asp Asn Asn Gin Gly Asp Asn Ser Ala Thr Asn Glu Ala Ser Tyr Met 40

'-I

WO 95/31562 9531562 CTINL95I00 171 His Asn Asn Trp Gin Asn Ala Tyr Thr His Ala Ala Giy Trp Asp Lys Val Tyr Leu Val Pro Ala Asn Giu Giu Pro Gly Tyr Ala Tyr Gly Ala Se r Arg Ser Pro Phe Ile Giu Leu Ser His Tyr Ser Asp Pro Val Arg Giu 115 Lys Gin Arg Ser Tyr Ile Asn Tyr Ile Asn Ala 110 Leu Asp Giy Gin Ala Lys Vai Giy Ile Pro Leu Ala Gly 130 Asn Giy Ile Lys Thr 135 His Lys Trp Vai Asp Asn Leu Trp Gly 1~45 Asp His Gin Asp Tyr Ser Tyr Leu Arg Ile Giy Ile Lys Asp Gin Leu Lys Asp Leu Aia Gly Ile Gly Gly Ala Ser 175 Lys Ser Asn Ser Asn Asn Asp Ser Thr His Ala Ile Asn Tyr Phe Ala Lys Thr Pro Asn Met 195 Giu Glu Lys Met Thr Tyr Leu Asp Thr 210 Lys Arg Trp Tyr Thr Asn Gly Ile Ile Arg Tyr Ile Lys 225 Thr Gly Arg Vai Gly Asn Tyr Gin Asn 235 Gin Trp Leu Lys Lys Leu Pro Vai Asp 245 Thr Met Phe Gin Giu Val Asp Lys Giu Phe 255 _____1111 WO 95/31562 PCT/NL95/00171 37 Gly Thr Gly Ala Thr Asn Pro Asn Arg Asp Ile Ser Lys Gly 260 265 270 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 241 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Lactococcus lactis subsp. cremoris INDIVIDUAL ISOLATE: Fig.2 c2 lysin (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Leu Phe Pro Tyr 1 Lys Thr lie lie Ile Gly Gly Gly Asn Ile Lys Val Ser Gin Asn Gly Leu Asn Ile Lys Glu Phe Glu Gly Cys Arg Leu Thr Ala Tyr Lys Pro Val 40 Pro Trp Glu Gin Met Tyr Thr Ile Gly Trp Gly His Tyr Gly Thr Ala Gly Thr Trp Thr Gin Ala Gin Ala Asp Ser Gin Met Val Asp Ala Tyr Glu lie Asp lie Asn 75 Asn Lys Tyr Ala Pro Val Lys Gly Lys Asn Gin Asn Glu Phe Asp

I

NVO 95/31562 WO 9531562PCT[/NL9S/OO 171 Ala Leu Val Ser Leu Ala Tyr Asn 100 Gly Asn Val Phe Val Ala Asp 110 Gly Trp Ala 115 Pro Phe Ser His Tyr Cys Ala Ser Ile Pro Lys Tyr Arg 130 Asn Ali' dy Gly Val Leu Gln Gly Val Arg Arg Arg Gln Ala Glu Leu Asn 145 Gin Asn Asn Gin Thr 165 Phe Asn Lys Pro Ser Ser Asn Ser Gly Gly Met Ile Met Tyr Leu Ile Ile Gly 175 Leu Asp Asn Ser Val Arg 195 Gly Lys Ala Lys His 185 Trp Tyr Val Ser Asp Gly Val 190 Tyr Gin Asn His Val Arg Thr Arg Met Leu Glu Lys Trp 210 Ala Lys Leu Asn Pro Val Asp Thr Met 220 Phe Ile Ala Giu Glu Ala Glu Phe Giu Aa Gl Phe Arg Lys Ile Asp AlSeGyGu Ala Ser Gly Glu WO 95/31562 13/TJN.L5/00() 171 39 List of references 1. WO 90/00599 (AGRICULTURAL FOOD RESEARCH COUNCIL; M.J. Gasson) published 25 January 1990; Uses of viral enzymes 2. C.A. Shearman, K. Jury M.J. Gasson (AFRC); Biotechnology 1Q (Feb. 1992) 196-199; Autolytic Lactococcus Zactis expressing a lactococcal bacteriophage lysin gene 3. C. Platteeuw and W.M. de Vos (NIZO); Gene 118 (1992) 115-120; Location, characterization and expression of lytic enzymeencoding gene, lytA, of Lactococcus lactis bacteriophage US3 4. R. Young; Microbiol. Reviews 56 (1992) 430-481; Bacteriophage Lysis: Mechanism and Regulation; esp. pages 468-4 7 2: Lysis in Phage Infections of Gram-Positive Hosts, and Perspectives 5. C. Shearman, H. Underwood, K. Jury, and M. Gasson (AFRC); Mol.

Gen. Genet. 218 (1989) 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. 3q (1993) 767-774; Sequence analysis of the lysin gene region of the prolate lactococcal bacteriophage c2 7. EP-A2-0 510 907 (AGRICULTURAL FOOD RESEARCH COUNCIL; M.J.

Gasson) published 28 October 1992; Bacteriophage lysins and their applications in destroying and testing for bacteria Literature references of the draft publication 1 1 Birnboim, and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA.

Nucleic Acids Res. 7: 1513-1523.

2 Buist, Haandrikman, Leenhouts, Venema, and Kok, J. (1994). Molecular cloning and nucleotide sequence of a major Lactococcus lactis peptidoglycan hydrolase gene (acmA).

(to be published).

3 Casadaban, and Cohen, (1980). Analysis of gene control signals by DNA fusion and cloning in Escherichia coli.

J. Mol. Biol. 138: 179-207.

4 Denhardt, Sinsheimer, R.L. (1965). The process of infection with bacteriophage X174, III. Phage maturation and lysis after synchronized

I

WO 95/31562 PCT/NL95/00171 infection. J. Mol. Biol. 12: 641-646.

Diaz, Lopez, and Garcia, J.L. (1992). Role of the major pneumococcal autolysin in the atypical response of a clinical isolate of Streptococcus pneumoniae. J. Bacteriol. 174: 5508- 5515.

6 Howard, and Gooder, H. (1974). Specificity of the aut-.'.in of Streptococcus (Diplococcus) pneumontae. J.

Bt er i 117: 794-804.

7 Hutchison, III, and Sinsheimer, R.L. (1966). The process of infection with bacteriophage )174. X. Mutations in a (X lysis gene. J. Mol. Biol. 18: 42,.447.

8 Jaacks, Healy, Losick, and Grossman, A.D. (1989).

Identification and characterization of genes controlled by the sporulation-regulatory hene spoH in Bacillus subtills. J.

Bacteriol. 171: 4121-4129.

9 Jarvis, Fitzgerald, Mata, et al. (1991).

Species and type phages of lactococcal bacteriophages.

Intervirology, 32: 2-9.

11 Lowrie, R.J. (1974). Lysogenic Strains of Group N Lactic Streptococci. Applied Microbiology, 27: 210-217.

12 Mandel, and Higa, A. (1970). Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53: 159-162.

14 Platteeuw, and De Vos, W.M. (1992). Location, characterization and expression of lytic enzyme-encoding gene, lytA, of Lactococcus lactis bacteriophage )US3. Gene 118: 115- 120.

Potvin, Leclerc, Tremblay, Asselis, and Bellemare, G. (1988). Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli. Mol. Gen.

Genet. 214: 241-248.

16 Romero, Lopez, and Garcia, P. (1990). Sequence of the Streptococcus pneumoniae bacteriophage HB-3 amidase reveals high homology with the major host autolysin. J. Bacteriol. 172: 5064-5070.

17 Rottlander, and Trautner, T.A. (1970). Genetic and transfection studies with Bacillus subtilis phage SP50. J.

Mol.Biol. 108:47-60.

IClsB- s" WO 95/31562 PCT/NL95/00171 41 18 Shearman, Underwood, Jury, and Gasson, M. (1989).

Cloning and DNA sequence analysis of a Lactococcus bacteriophage lysin gene. Mol.Gen.Genet. 218: 214-221.

19 Staden, R. (1982). Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing.

Nucleic Acids Res. 10: 4731-4751.

Steiner, Lubitz, and Blasi, U. (1993). The missing link in phage lysis of Gram-positive bacteria: gene 14 of Baciltus subtilis phage )29 encodes the functional homolog of the lambda S protein. J. Bacteriol. 175: 1038-1042.

21 Terzaghi, and Sandine, W.E. (1975). Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol.

29: 807-813.

22 Tinoco, Jr, Bore, Dengler, Levine., M.D., Uhlenbeck, Crothers, and Gralla, J. (1973).

Improved estimation of secundary structure in ribonucleic acids. Nature 246: 40-41.

23 Van der Lelie, Van der Vossen, J.M.B.M, and Venema, G.

(1988). Effect of plasmid incompatibility on DNA transfer to Streptococcus cremoris. Appl. Environ. Microbiol. 54: 865-871.

Von Heijne, G. (1986). A new method for predicting signal peptide cleavage sites. Nucleic Acids Res. 14: 4683-4690.

26 Ward, Beresford, Lubbers, Jarvis, B.D.W., and Jarvis, A.W. (1993). Sequence analysis of the lysin gene region of the prolate lactococcal bacteriophage c2. Can. J.

Microbiol. 39: 767-774.

27 Witte, Wanner, Blasi, Halfmann, Szostak, and Lubitz, W. (1990). Endogenous transmembrane tunnel formation mediated by 4X174 lysis protein E. J. Bacteriol. 172: 4109- 4114.

28 Yanisch-Perron, Vieira, and Messing, J. (1985).

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33: 103-119.

29 Yansura, and Henner, D.J. (1984). Use of the Escherichia coli Lac rrpressor and operator to control gene expression in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 81: 439-443.

Young, R. (1992). Bacteriophage lysis: Mechanism and regulation. Microbiol. Res. 56: 430-481.

UI Pls~Y(CIR(-s1-- _I I_ WO 95/31562 PCT/NL95/00171 42 References of draft publication 2 Birnboim, and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523.

Holo, H. and I.F. Nes. 1989. High-frequency transformation, by electroporation, of Lactococcus Zactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol.. 55:3119-3123.

Leenhouts, K.J. and G. Venema. 1993. Lactococcal plasmid vectors, p. 94. In K. G. Hardy Plasmids, a practical approach. Oxford University Press, Oxford.

Leenhouts, K. J. (unpublished results) Lillehaug, and N. K. Birkeland. 1993. Characterization of genetic elements required for site-specific integration of the temperate lactococcal bacteriophage (LC3 and construction of integration-negative 4LC3 mutants. J. of Bact.. 175:1745-1755.

Lowrie, R. J. 1974. Lysogenic strains of group N Lactic Streptococci.

Appl. Microbiol. 27:210-217.

Maguin, P. Duwat, T. Hege, D. Ehrlich, and A. Gruss. 1992. New thermosensitive plasmid for gram-positive bacteria. Journal of Bact.

174:5633-5638.

Mandel, and Higa, A. 1970. Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53:159-162.

Messing, J. 1979. A multipurpose cloning system based on the singlestranded DNA bacteriophage M13. Recombinant DNA technical Bulletin, NIH Publication No. 79-99, 2, No. 2, p. 43-48.

Nauta, A.M. Ledeboer, G. Venema, and J. Kok. 1994a. Inhibiting growth of lactic acid bacteria by a holin and optionally lysing the cells and uses of the resulting lysed culture. European Patent aanvraag nr. EP-PA 94201354.1 (T7038) WO 95/31562 PCT/N 195/00171 43 Nauta, A.M. Ledeboer, G. Venema, and J. Kok. 1994 b Complex inducible promoter system derivable from a phage of a lactic acid bacterium and its use in a LAB for production of a desired protein. European Patent aanvraag nr. EP-PA 94201355.8 (T7039) Rottlander, and T.A. Trautner. 1970. Genetic and transfection studies with BaciLLus subtilis phage SP50. J. Mol. Biol. 108:47-60.

Sanger, S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.

Sambrook, E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y..

Terzaghi, and W.E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29:807-813.

Van der Lelie, J. M. B. M. van der Vossen, and G. Venema. 1988.

Effect of plasmid incompatibility on DNA transfer to Streptococcus cremoris. Appl. Environ. Microbiol. 53:2583-2587.

Yanisch-Perron, 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.

IM

Claims (15)

1. Process for inhibiting the growth of a culture of lactic acid bacteria, which process comprises the in situ production in the cells of the lactic acid bacteria of a holin obtained from bacteriophages of Gram-positive lactic acid bacteria, the gene encoding said holin being under control of a first regulatable promoter, said first rec'l.atable promoter not normally being associated with the holin gene. said holin being capable of exerting a bacteriostatic effect an the cells in which it is produced perforating the cell membrane, while preferably the natural production of autolysin is not impaired.

2. Process according to claim 1. which additionally comprises the in situ production in the cells of the lactic acid bacteria of a lysin obtained from grampositive bacteria or their bacteriophages, the gene encoding said lysin being under control of a second regulatable promoter,said second regulatable promoter not normally being associated with the 0 lysin gene whereby the produced lysin effects lysis of the cells of the lactic acid bacteria.

3. Process according to claim 2, wherein the Gram-positive bacterium from which the lysin is derived is a lactic acid bacterium. C :*15 4. Process according to claim 2 or 3, in which the second regulatable promoter is the same as the first regulatable promoter.

5. Process according to claim 4, in which the gene encoding the holin and the gene encoding the lysin are placed under control of the same regulatable promoter in one operon.

6. Process according to any of claims 1-3, in which said first or second promoter or both are regulated by food-grade ingredients or parameters.

7. Process according to any of claims 1-3. in which the culture of lactic acid bacteria is part of a product containing such culture.

8. Process according to claim 7, 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.

9. Process according to claim 8, in which the fermented food product is a cheese product. Process according to claim 9, 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.

11. Process for combatting spoiling bacteria or pathogenic bacteria, in which a lysed culture obtained following the process steps as defined in claim 2 or 3 is used as a bactericidal agent.

12. Process for improving the shelf life of a consumer product, in which a product obtained by following the process steps as defined in any of claims 1-3. said product containing free holin or free lysin or both 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.

13. Process according to claim 12, in which the consumer 0 product is selected from the group consisting of edible products, cosmetic products, and products for cleaning ofabrics, hard surfaces and human skin.

14. Process for modifying a mixture of peptides, which comprises :25 combining a culture of lactic acid bacteria with a mixture of peptides obtained by proteolysis of proteins, tne cells of said culture containing both a gene encoding a holin obtained from a lactic acid bacterium under control of a first regulatable promoter and a gene encoding a lysin under control of a second regulatable promoter, which second and first promoter can be the same, and which second and first promoters are not normally associated with the respective genes and effecting induction of the promoter or promoters for producing both the holin and the lysin in such amounts that the cells of the lactic acid bacteria are lysed and the contents of the cells containing peptidases will modify the composition of the mixture of peptides. Process for modifying a mixture of peptides, which comprises treating a mixture of peptides obtained by .oo 5 0 ,AO N proteolysis of proteins with a lysed culture obtained by a process according to claim 2 or 3.

16. Process according to claim 14 or 15, in which the proteins comprise milk proteins or vegetable proteins, or both.

17. Process according to any of the preceding claims, wherein the holin is encoded by a nucleic acid sequence according to any of claims 19-22 and/or is expressed from a recombinant vector according to any of claims 23-31 and/or is expressed by a recombinant cell according to any of claims

32-40. 18. Process according to any of claims 2-17. wherein the lysin has the amino acid sequence of sequence id no 7 or is a functional equivalent thereof. 19. A nucleic acid sequence encoding a holin derived from a grampositive lactic acid bacterium or a bacteriophage derived from such a grampositive bacterium. A nucleic acid sequence according to claim 19. wherein the lactic acid bacterium is a L. lactis. 21. A nucleic acid sequence according to claim 19 or encoding the amino acid sequence of sequence id no 6 or a functional equivalent thereof such as the nucleic acid sequence of nucleotides 103-328 of sequence id no 22. A nucleic acid sequence according to any of claims 19-21,further being operatively linked to a first regulatable promoter, said first regulatable promoter not normally being associated with the holin encoding sequence. 23. A recombinant vector comprising a nucleic acid sequence according to any of claims 19-22, said vector preferably further being foodgrade. 24. A recombinant vector according to claim 23 further comprising a nucleic acid sequence encoding a lysin, with the holin being derived from a Gram-positive lactic acid bacterium and the lysin being derived from a Gram-positive bacterium or with the holin being derived from a bacteriophage of a Gram-positive lactic acid bacterium or the lysin being derived from a bacteriophage of a Gram-positive bacterium. A recombinant vector according to claim 24, wherein the Gram-positive bacterium from which the lysin is derived is a lactic acid bacterium. eooe ee e 46a 26. A recombinant vector according to claim 25 wherein the lactic acid bacterium is a L. lactis. 27. A recombinant vector according to any of claims 23-26 further comprising a atural attachment/integration system of a bacteriophage for example said system comprising the bacteriophage attachment site and an integrase gene located such that integration of the holin and optionally lysin gene will occur. 28. A recombinant vector according to claim 27. said system being derived from a bacteriophage that is derivable from a food grade host cell 29. A recombinant vector according to claim 28, said food-grade bacterium being a lactic acid bacterium. A recombinant vector according to any of the claims 23-29, ee o• e V K 4.y -I I I