Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C19/00—Cheese; Cheese preparations; Making thereof
  • A23C19/02—Making cheese curd
  • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
  • A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C2220/00—Biochemical treatment
  • A23C2220/20—Treatment with microorganisms
  • A23C2220/202—Genetic engineering of microorganisms used in dairy technology

Definitions

• 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.
• E. Escherichia, e.g. E. coli
• L . Lactococcus , e.g. L. lactis
• M. Micrococcus , e.g. M. lysodeikticus
• S. Streptococcus , e.g. S. faecalis and S. pneumonia.
• 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.
• 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.
• a lysin e.g. in producing a fermented food product, e.g. in cheese-making.
• 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 ⁇ vML3 of Lactococcus lactis ML3.
• the lysin can be added to a cheese product or a cheese precursor mixture, e.g. after whey removal, milling and salting.
• this solution has the disadvantage that thorough mixing of the contents of the lysed cells with the cheese product is not easily obtained.
• 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. None is mentioned regarding addition of a transformed host cell in improving chees flavor, certainly not a transformed lactic acid bacterium.
• Lactococcus lactis subsp. cremo is strains to metabolize lactose, to clot milk and produce acid
• 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.
• 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.
• holin also means a protein or peptide required for the access of a lysin to its substrate, the murein part of the cell wall.
• 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 ⁇ LM4) and Clostridium tyrobutyricum (phage ⁇ P) . 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.
• the invention in another aspect relates to a process for inhibiting the growth of a culture of lactic acid bacteria without lysing the cells.
• lactic acid bacteria can be inhibited in several ways.
• 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.
• 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 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.
• 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.
• the second regulatable promoter is the same as the first regulatable promoter (claim 3), a d 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 4). It is advantageous for food fermentations when said first or second promoter or both can be regulated by food-grade ingredients or parameters (claim 5)-
• 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 6) .
• 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 7) •
• a specific example of such process is one in which the fermented food product is a cheese product (claim 8) . 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).
• 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 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 (1) 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 (2) 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).
• the host cell 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 15).
• 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 2 -27 fall within the intended scope of the invention.
• an alternative suitable embodiment of a process according to the invention can be directed at the inducible expression of 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.
• recombinant vectors comprising any of the nucleic acid sequences in any of the claimed embodiments, said vector preferably further being foodgrade.
• 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 pIRl4.
• 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 acid bacterium, in particular a L.
• a recombinant host cell 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.
• Fig. 1 A) Schematic outline of the PCR reactions used for the amplification of lytP, lytR , and the combination of lytP and lytB. 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) .
• Fig. 3- Nucleotide sequence of a 1200 bp DNA fragment of Rl-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) or pAG5 ⁇ R (lanes 3 and 4) were obtained two hours after the addition of IPTG.
• the arrow indicates the position of a clearing zone as a result of lytic activity exhibited by the lytR gene product.
• Fig. 6. The effect of expression of lytR, lytR , or the combination of lytR and lytR on the optical density of E. coli MC1000 cells.
• Optical density measurements of E. coli cells carrying either pAG58 (a), pAG5 ⁇ R (b) , pAG58P (c) or pAG5 ⁇ PR (d) , 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).
• Fig. 7- The effect of the induced expression of lytR, lytR , or the combination of lytR and lytR on the optical density of L. lactis subsp. cremoris LL302 cells.
• Optical density measurements of induced L. lactis cells carrying either pIR12 (•), pIRIP ( ⁇ ) , pIRlR (*). or pIRlPR (°) respectively, are indicated as a function of growth.
• the optical density (0D) measurements of L. lactis carrying pIRlPR, not exposed to mitomycin C, are represented by (°) . Time scale is in hours after the time of induction with mitomycin C (1 ⁇ g/ml).
• Both lytR 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.
• the small single- stranded DNA phage ⁇ X174 encodes a protein which forms a channel to transport complete phage particles from the cytoplasm of the host to the environment (4, 7, 27).
• most of the known phages encode an enzyme with murein-degrading 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 (30).
• Em r , Ap r , and Cm 1* represent resistances to erythromycin, ampicillin, and chloramphenicol, respectively.
• LytR requires an additional gene product, specified by lytR upstream of lytR , for efficient in vivo lysis of E. coli and L. lactis.
• Bacterial strains Bacterial strains, phage, plasmids, and media
• E. coli was grown in TY broth (17) or on TY broth solidified with 1.5% agar.
• L. lactis was grown in glucose M17 broth (21), or on glucose M17 agar.
• Erythromycin was used at 100 ⁇ g/ml and 5 US/ml for E. coli and L. lactis , respectively.
• ampicillin and chloramphenicol were used at a concentration of 100 ⁇ g/ml and 5 Ug/ m l» respectively.
• Plasmid DNA was isolated essentially by the method of Birnboim and Doly (1). 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 3 ⁇ lA DNA synthesizer (Applied Biosystems Inc., Foster City, Calif.). Polymerase chain reactions were performed using Vent polymerase (New England Biolabs Inc., Beverly, MA.).
• E. coli was used as a host for obtaining recombinant plasmids. Transformation of E. coli was performed by the method of Mandel and Higa (12). Plasmids were introduced in L. lactis subsp. cremoris LL302, which contains a copy of the pWVOl repA gene on the chromosome to ensure efficient replication, by means of electroporation (23).
• 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 ⁇ -turn search program BETATURN. Table 2. Primers used for amplification of lytP and lytR
• the lytic activity assay was performed essentially as described by Potvin et al . (15) with some minor adjustments as reported by Buist et al. (2).
• the lytR and lytR containing fragments of the Rl-t genome were amplified using polymerase chain reactions (PCR's).
• PCR's polymerase chain reactions
• a 4.1-kb Xbal/Nhel fragment of the Rl-t genome containing both lytR and lytR was subcloned in the unique Xbal site of pUCl ⁇ resulting in the plasmid pXNB.
• pXNB as a template, amplification with three different primer combinations (lytl-lyt2, Iyt3-lyt4, and lytl-lyt4; see Fig. 1A) yielded three DNA fragments carrying either lytR, lytR , or the combined lytR and lytR , respectively.
• the DNA fragments, carrying lytR, LytR and the combination of lytR and LytR were first subcloned in Sphl/S ⁇ ZI-cut pUCl ⁇ .
• the Hindll /Hindlll fragments of these three constructs designated pUCl ⁇ P, pUCl ⁇ R, and pUCl ⁇ PR, were cloned in the iVruI and Hindlll sites of pIR12, resulting in the plasmids pIRIP, pIRlR, and pIRlPR, containing lytR, lytR , and both lytR and lytR, respectively, under the transcriptional control of the Rl-t promoter-operator region (Fig.
• Plasmid pIR12 L. lactis subsp. lactis strain LL302 which contains a copy of the pWVOl 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.
• ORF 23 specifies an amino acid sequence similar to the amino acid sequences of the N- terminal portions of the amidase Hbl of the Streptococcus pneumoniae bacteriophage HB-3 (16), and the S. pneumoniae LytA autolysin (5). 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.
• lytR was cloned into the IPTG inducible expression-vector pAG5 ⁇ , resulting in pAG5 ⁇ R (Fig. IB).
• Cell- free extracts of E. coli cells containing pAG5 ⁇ R were assayed for lytic activity on an SDS-polyacrylamide gel in which Micrococcus lysodeikticus 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 incorporated cell walls.
• Figure 4 in cell free extracts of pAG5 ⁇ -containing E.
• ORF 22 which is situated upstream of lytR , specifies a protein of 75 amino acids with a predicted molecular weight of 7.6 ⁇ Da (sequence id no 6) . 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 lytR, predicted structural similarities with these proteins.
• the protein specified by lytR, 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. 5)- 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 Rl-t encoded LytR.
• LytP and lytR are required for lysis in Escherichia coli
• lytR, lytR , and the combination of lytR and lytR were subcloned in the inducible expression- vector pAG5 ⁇ , resulting in pAG5 ⁇ P, pAG5 ⁇ R and pAG5 ⁇ PR, respectively (Fig. IB).
• Induction studies were performed with E. coli MClOOO 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.
• plasmids pIRIP, pIRlR, and pIRlPR were constructed (Fig. IB). Transcription of lytR, lytR , and both lytR 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. IB) . Expression was induced by the addition of the DNA damaging substance mitomycin C. Induction studies were performed with L.
• LytR 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 (16), the C- terminal domain specifying the binding site to the murein substrate and the N-terminal domain determining the specificity of the enzyme.
• LytA is an iV-acetylmuramoyl-L-alanine amidase (6)
• LytR is also an W-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 lytR , which is situated immediately upstream of lytR , can specify a protein of 75 amino acids with the characteristics of a so-called holin which, for other phages, was shown to render the murein substrate accessible to lysins which lack a signal peptide (30) .
• 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 lytR , lytR , and the combined lytR and lytR in L. lactis . Expression of the combined lytR and lytR in L. lactis resulted in lysis of the cells. In contrast to E. coli , lysis was also observed when only 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 .
• 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.
• bacteriophage Rl-t was taken.
• plasmid pBTSl was constructed ( Figure 8).
• pBTS2 will be constructed in which rro is replaced by rro ⁇ s and therefore can be used to make this system thermo-inducible.
• Bacterial strains Bacterial strains, phage, plasmids, and media
• Escherichia coli JM101 was grown in TY broth (Rottlander and Trautner, 1970) with vigorous agitation, or on TY agar, at 37 °C.
• IPTG isopropyl- ⁇ -D-thiogalactopyranoside
• X-gal 5-bromo-4-chloro-3 _ indolyl ⁇ -galactopyranoside
• cremoris was grown in M17 broth (Terzaghi and Sandine, 1975), or on M17 agar, supplemented with 0. % glucose or lactose at 30 °C.
• erythromycin Boehringer Mannheim, GmbH, Germany
• X-gal were used at concentrations of ⁇ g/ml and 0.004 * 5. (wt/vol), respectively.
• Plasmid DNA was isolated by the method of Birnboim and Doly (1979) and by using QIAGEN Midi-Plasmid isolation columns (Qiagen Inc..Chatsworth, Ca. ) . 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. coli was performed as described by Mandel and Higa (1970). L.
• lactis LLlO ⁇ was transformed by electroporation using a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.), as described by Holo and Nes (19 ⁇ 9) with the modifications suggested by Leenhouts and Venema (1993) ' Electroporation of L. lactis Rl, R131 and R1K10 was done as decribed by van der Lelie et al. (19 ⁇ ). Oligonucleotides were synthesized using an Applied Biosystems 3 ⁇ lA DNA synthesizer (Applied Biosystems, Inc., Foster City, Calif.). Polymerase chain reactions (PCR's) were performed using Vent polymerase (New England Biolabs, Inc., Beverly, MA.).
• target DNA was amplified in 30 subsequent cycles under the following conditions: 9 °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.). Isolation of Rl-t phage particles and DNA
• the bacteriophage Rl-t suspension was dialysed against several changes of 150 mM NaCl, 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 8,0.
• the attachment sites attL and attR of the bacteriophage Rl-t lysogen L. lactis Rl 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 Xib ⁇ l and PstI sites (Table 4) were used for cloning the attB site of L.
• lactis MG1363- The 272-b ⁇ PCR fragment obtained with attBL and attBR was cut with Xbal and PstI and cloned in the Xbal/PstI sites of pUCl ⁇ and sequenced using the dideoxy-chain-termination method (Sanger et al. , 1977) and the T7 sequencing kit (Pharmacia AB, Uppsala, Sweden).
• the indicator strain L. lactis R1K10 was grown in GM17 until the 0D600 was 0.7 ' 2 ml of culture were centrifuged and cells were resuspended in 2 ml 1 mM MgSO ⁇ . An aliquot of 100 ⁇ l diluted phage-particles were added to 200 ⁇ l cells. After incubation at room temperature for 20 minutes 3 m l Top agar (0.7# GM17 agar, 0.25% glycine, 10 mM CaCl 2 ) were added, mixed, and poored on a GM17 agar-plate ( 1.5% ) containing glycine (0.25%) and CaCl 2 (10 mM) . The plates were incubated overnight at 30 °C and the number of plaques were determined. Rely sogeni sat ion of L. lactis R1K10
• the plasmid pORIRlPR was constructed in L. lactis by subcloning the 2 ⁇ 64-bp £c_>RI/SphI-fragment of pIRlPR into pORI28 ⁇ restricted with J-coRI and Sphl ( Figure 8) .
• Homology analysis showed that ORF 25 of the Rl-t genome shared 9 % 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 S ⁇ cl and Xt> ⁇ l sites using PCR and primers intl and int2 (Table 4).
• Plasmid pUCl ⁇ Int was constructed by cloning the resulting 1326-bp PCR-fragment digested with S ⁇ cl and Xbal into the Sacl/Xbal sites of pUCl ⁇ .
• a 1047-bp Hindll fragment of pUCl ⁇ Int, which contains the 5'-truncated intR was subcloned into the alkaline phosphatase-treated Sm ⁇ l-site of pUCl ⁇ .
• Both the resulting plasmid pUCl ⁇ lntd and pUCl ⁇ Int were constructed in E. coli JM101 ( Figure 9).
• Plasmid pBTSl was introduced in L. lactis LLlO ⁇ . As can be seen in figure 10, pBTSl is still able to give inducible lysis after mitomycin C induction.
• a second recombination step in the region C or A will delete the whole prophage and plasmid from the chromosome of strain Rl, 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 .
• the second recombination step will not result in the substitution of intR and rro for the 5'-truncated intR and rro ⁇ s (future work), respectively.
• the integrase deletion is needed to prevent intR catalysed excision.
• L. lactis Rl Because of the extremely low transformation efficiency of L. lactis Rl (less than 1 transformant/ ⁇ g 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 Rl on glucose and incubation at 37 "C. The resulting strain L. lactis R131 was shown by UV- induction to still contain Rl-t prophage.
• pBTSl and pVE6007 will be introduced together in L. lactis R1K10.
• pVE6007 encodes a temperature sensitive RepA protein enabling pBTSl to replicate.
• pBTSl will integrate into the chromosome when raising the temperature to 37 °C.
• the second way to obtain a 'food-grade' inducible lysis system is to introduce pBTSl and pVE6007 together in L. lactis MGI363.
• FIG. 8 Cloning scheme for the construction of pBTSl.
• Electroporation cuvette (2-mm electrode gap); 25 ⁇ F 200 ⁇
• 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.
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• ORGANISM Lactococcus phage Rl-t
• MOLECULE TYPE DNA (genomic)
• ORGANISM Lactococcus phage Rl-t
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• ORGANISM Lactococcus phage Rl-t
• C INDIVIDUAL ISOLATE : Fig . 3 cds lytP and cds lytR
• MOLECULE TYPE protein
• ORGANISM Lactococcus lactis subsp. cremoris
• EP-A2-0 510 907 (AGRICULTURAL _. FOOD RESEARCH COUNCIL; M.J. Gasson) published 2 ⁇ October 1992; Bacteriophage lysins and their applications in destroying and testing for bacteria
• M13 phage cloning vectors and host strains nucleotide sequences of the M13mpl ⁇ and pUC19 vectors. Gene 33: 103-119.

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• 1995
  • 1995-05-12 EP EP95917522A patent/EP0759999A1/de not_active Withdrawn
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  • 1995-05-12 AU AU23540/95A patent/AU702604B2/en not_active Ceased
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