GB2294463A - Plasmids encoding bacteriophage resistance for use in lactic acid bacteria - Google Patents

Description

PLASMIDS ENCODING BACTERIOPHAGE RESISTANCE FOR USE IN LACTIC ACID BACTERIA

DECSCRIPTION 2294463 The present invention relates to plasmids that encode bacteriophage resistance and in particular. such plasmids for use in lactic acid bacteria.

Lactic acid bacteria are used intar alia extensively in the dairy food industry for the production of dairy products including cheeses and yoghurts. Lactic acid bacteria however can succumb to bacteriophage infection during the production of fermented products thus resulting in a lower effectiveness of the fermentation process. Improvement of resistance to bacteriophage poses an on-going problem and is an extensively researched area in the lactococcal dairy starter culture industry. Approaches to developing bacteriophage resistance in lactic acid bacteria include:

1. Isolation of bacteriophage resistant mutants: 2. Isolation of new strains from the environment which have enhanced natural resistance.:

Use of strains in admixture and in rotation to minimise problems of bacteriophage infection; Identification of plasmids that encode bacteriophage resistance then introducing the plasmid into a bacteriophage sensitive strain to generate a resistant variant. and Introduction of more than one plasmid into a bacteriophage sensitive strain where the cumulative resistances give enhanced bacteriophage resistance.

All of the above approaches offer short term protection of the cultures to bacteriophage infection but none have been able to withstand prolonged exposure to bacteriophage in the commercial environment. A principal reason for this is that each method introduces usually one. or possibly. two different mechanisms of bacteriophage resistance. Host range mutants of bacteriophage or new bacteriophage can overcome the resistance 3.

4.

5.

mechanisms and in (4) and (5) constructs may exhibit a degree of geneti instability which may facilitate bacteriophage infection.

The present inventors have been able to reduce genetic instability and achieve the introduction of multiple phage resistant determinants into host bacteria by the development of new plasmids.

Accordingly, in a first aspect the present invention consists in a plasmid encoding resistance to bacteriophage selected from the group consisting of pND801, pND802. pND809, pIND811. pND851, pND852, pNW53. pND859. pND860. pIND862 and functional equivalents thereof.

In a second aspect. the present invention consists in an isolated nucleic acid molecule including a nucleotide sequence encoding resistance to bacteriophage, wherein the nucleotide sequence is derived from a plasmid selected from the group consisting of pND801. piNI)802, pND809. pND811, pNE851. pND852, pND853. pIND859, pND860, pND862 and functional equivalents thereof.

In a preferred embodiment of the second aspect of the present invention, the isolated nucleic acid molecule includes the nucleotide sequence substantially corresponding to the nucleotide sequence of Figure 8 or Figure 9.

In a third aspect. the present invention consists in a method of increasing or conferring phage resistance to a bacterium comprising introducing into the bacterium at least one plasmid or at least one nucleotide sequence derived from at least one plasmid of the first aspect of the present invention. Preferably the bacterium is a lactic acid bacterium.

In a fourth aspect. the present invention consists in a bacterium having increased or conferred phage resistance produced by the method of the third aspect of the present invention.

In a fifth aspect. the present invention consists in a lactic acid 'bacterial plasinid consisting of lactic acid bacterial DNA or cheinicalIv 3 - svnthesised DNA for use in increasing or conferring phage resistance to lactic acid bacteria, the plasmid including at least two nucleotide sequences encoding phage defence mechanisms selected from the group consisting of host controlled restriction. host controlled modification. abortive infection and prevention of phage adsorption. Preferably the plasmid includes minimal DNA and substantially excludes DNA not required for maintenance of the plasmid or for encoding the phage defence mechanisms.

In a preferred embodiment of the fifth aspect of the present invention, the plasmid includes at least three nucleotide sequences encoding phage defence mechanisms.

In a further preferred embodiment of the fifth aspect of the present invention. the nucleotide sequences encoding resistance to bacteriophage are derived from the plasmids of the first aspect of the present invention.

In a vet further preferred embodiment of the fifth aspect of the present invention, the plasmid further includes a nucleotide sequence encoding at least one selectable marker.

It will be appreciated that there are a number of different phage defence mechanisms of lactic acid bacteria and that the plasmids of the present invention may include at least two different nucleotide sequences encoding the same type of mechanism.

In a sixth aspect. the present invention consists in a bacterium including at least one plasmid of the fifth aspect of the present invention.

Preferablv the bacterium is a lactic acid bacterium.

In a seventh aspect. the present invention consists in a method of increasing or conferring phage resistance to a lactic acid bacterium comprising introducing into the bacterium at least one plasmid of the fifth aspect of the present invention.

In a yet further preferred embodiment of the present invention, the plasinids of the first and/or fifth aspect of the present invention are introduced singularly or in various combinations into a bacterium so as to increase or confer bacteriophage resistance to the bacterium.

As used herein. the phrase "functional equivalentg thereof' is intended to cover plasmids or nucleic acid sequences derived therefrom that have slightly altered nucleic acid sequences from the plasmids set out above but which retain substantially the same biological activity. This may be achieved by various changes. such as insertions. deletions and substitutions, either conservative or non-conservative where such changes do not substantially alter the biological activity of the protein or proteins encoded by the plasmids or nucleic acid sequences derived therefrom.

Samples of each of the plasmids set out above were deposited under the provisions of the Budapest Treaty at the Australian Government Analytical Laboratories (AGAL) on 28th September 1995 and 5th October 1995. The respective accession numbers of each deposit are set out below:

PLASMID ACCESSION DATE NUMBER pND801 N95/58018 28/09/95 pND802 N95/58019 28/09/95 pND809 N95/58020 28/09/95 pND811 N95/58021 28/09/95 pIND85 1 N95/58022 28/09/95 pND852 N95/59300 05/10/95 pND853 N95/58023 28/09/95 pND859 N95158024 28/09/95 pND860 N95/59301 05/10/95 pNB862 N95/58025 28/09/95 In order that the nature of the present invention may be more clearly understood. preferred fornis thereof will be described with reierence to the following examples and the accompanying drawings.

Figure 1 shows restriction maps of (a) pND801 and (b) pND802; Figure 2 shows restriction map of pND861; Figures 3, 4, 5. 6 and 7 are schematic representations of the cloning of phage resistant mechanisms from pND801, pND852. pNB853, pND859 and pND862 respectively.

Figure 8 shows the nucleotide sequence and inferred amino acid sequence of the Abi determinant isolated from pND852; Figure 9 shows the nucleotide sequence and inferred amino acid sequence of the Abi mechanism isolated from pND859; Figures 10 and 11 are schematic representations for the construction of plasmids encoding bacteriophage resistance according to the present invention; Figure 12 is a schematic representation of the production of plasmids encoding bacteriophage resistance according to the present invention; and Figure 13 is an example of a DNA fragment encoding phage resistance derived from pND802.

MATERIALS Lactococcal Strains For convenience the following terminology is used: L. lactis refers to the species Lactococcus lactis subsp lactis; L. cremoris refers to the species Lactococcus lactis subsp cremoris and L. lactis var diacet.vlactis refers to the species Lactococcus lactis subsp lactis var diacet.i.,lactis.

The following strains were used as donors for the isolation of plasinids that encode bacteriophage resistance by either conjugation, mobilization or co-transformation:

L. lactis strains X1195. M175. N1113, M138, N1181, L. cremoris strains X1502. N1111 and L. lactis var diciceti,,lactis UK12922, L1K19161 and UK1392 ivere all originalIv isolated from mixed strain starter cultures.

The following strains were used as recipients in conjugation mobilisation or electrotransformation experiments:

L. lactis MG1363 is a plasmid-free derivative of L. lactis 712 obtained from M. Gasson. Food Research Institute, Norwich. UK. L. lactis MG1363Srn is a plasmid-free derivative of L. lactis 712 made resistant to streptomycin, L. lactis LM0230 is a plasmid-free derivative of L. lactis C2 obtained from L.L. McKay, University of Minnesota, St Paul, Minn.. USA. L. lactis LM0230Srn is a plasmid-free derivative of L. lactis C2 obtained from L.L. McKay, University of Minnesota. St Paul, Minn., USA. and made resistant to streptomycin in the present inventor's laboratorv. L. lactis LM0230Fus is a plasmid-free derivative of L. lactis C2 obtained from L.L. McKay, University of Minnesota, St Paul, Minn., USA, and made resistant to fusidic acid in the present inventor's laboratory.

Other lactococcal strains used: L. cremoris FG2 was used as a standard for estimating the size of plasmid DNA in other lactococcal strains. It harbours plasmids of th following sizes (kb): -135. 67,50. 27. 14, 12.9, 11.9. 10.1. 8.8, 5.2, 3.

These sizes were calculated vs the reference strain E.coli V517 (Macrina et a], 1978).

Other Strains Reference E.coli V517 Macrina et a]. 1978 E.coli HB 101 Pro. Leu-. Thi-. Bover & Roulland recA- plasmid-free Dussoix. 1969 E.coll NN1522 Tlii-. Lac'-. r- ni- Gough and Murray (1983) A(lac - pro AB) (2) (3) (4) 11 Bacteriophages 0712 Small isometric-headed phage propagated on LM0230 and MG1363 0C2V Prolate-headed phage propagated on LM0230 and MG1363 Gasson, 1983 jarvis & Klaenhammer, 1986 111 Plasmids conferring bacteriophage resistance (1) pND801 is a 12 kb plasmid originally isolated from L. lactis M195 by co-transformation into L. lactis LM0230 pND802 is a 19 kb plasmid originally isolated from L. cremoris M502 by co-transformation into L. lactis LM0230 pND803 is a 29.2 kb plasmid constructed by cloning iphI linearised pND802 into the iphI site of pSA3 pND804 is a 18.9 kb plasmid constructed by cloning the 8.7 kb 13&1111 fragment from pND802 into the BamBI site of pSA3 pND805 is a 22.2 kb plasmid constructed by cloning EcoRI linearised pND801 into the EcoRi site of pSA3 pND809 is a 65 kb plasmid originally isolated from L. lactis M175 (pND300) by the mobilization procedure. This plasmid can be conjugatively transferred to Lactococcal recipients where it confers insensitivity to bacteriophage infection and resistance to nisin (7) pNB811 is a 75 kb plasmid originally isolated from L. cremoris Mill (pND300) by the mobilization procedure. This plasmid can be conjugatively transferred to Lactococcal recipients where it confers insensitivity to bacteriophage infection, resistance to nisin and proteinase activity (8) pNID851 is a 46 kb plasmid originally isolated from L. lactis M113 by co-trans formation into L. lactis LM0230 (9) pND852 is a 56 kb plasmid originally isolated from L. lactis N1138.

This plasmid can be conjugatively transferred to Lactococcal (5) (6) (13) recipients where it confers insensitivity to bacteriophage infection and also resistance to nisin (10) pND853 is a 57 kb plasmid originally isolated from L. lactis M181.

This plasmid can be conjugatively transferred to Lactococcal recipients where it confers insensitivity to bacteriophage infection and also resistance to nisin (11) pND859 is a 16 kb plasmid originally isolated from L. lactis var diacetylactis UK 12922. This plasmid can be conjugatively transferred to Lactococcal recipients where it confers insensitivity to bacteriophage infection and also resistance to cadmium (12) pND860 is an - 56kb plasmid originally isolated from L. lactis var diacet.vlactis UK 19161. This plasmid can be conjugatively transferred to Lactococcal recipients where it confers insensitivity to bacteriophage infection pND861 is an'in vivo'deletion derivative of pND860, 35 kb, obtained during a plasmid curing experiment. This plasmid confers insensitivity to bacteriophage infection (14) pND862 is a 65 kb plasmid originally isolated from L. lactis var diacet.vlactis UK 1392. This plasmid can be conjugatively transferred to Lactococcal recipients where it confers insensitivity to bacteriophage infection and also encodes lactose utilisation and proteinase production (15) pND817 is a 12.3 kb plasmid constructed by cloning of the 2.6 kb Hpa 11 fragment from pND852 into the Hpa II site of pGB301 (16) pND825 is a 34 kb plasinid constructed by cloning of the 2.7 and 1.6 kb Upa II fragments from pND853 into the Hpa II site of pGB301 (17) pND863 is a 9.5 kb plasinid constructed by cloning of a 2.9 kb Hind III fragment from pND859 into the Hind III site of pDL278 (18) pND826 is a 23.5 kb plasinid constructed by cloning of a 16 kb BaniHI fra2inellt froin pND862 into the BamHI site of p-MU1328.

- 9 (19) pND821 is a 5.6 kb plasmid constructed by sub-cloning the 2.6 kb HpaII fragment from pND817 into the Clal site of pGEM-7zf(+) (20) pND806 is a 46 kb Lactococcal plasmid that confers insensitivity to bacteriophage infection and found to possess the Abi A gene reported by Hill et al, (1990). This plasmid was used as a positive control for hybridisation experiments.

Other plasmids:

References Mauri Laboratories pND325 pSA3 pGB301 pGEN1 -A - 7zf (+) pND300 pMul328 pDL278 B. Media 4.8 kb, hybrid plasmid containing Em R of S.aureus plasmid pE194 and a replicon from a cryptic plasmid of L. lactis M127 10.2 kb. hybrid vector R encoding Cm ' TcR (E.coli selection) and R Em (L. lactis selection) 9.8 kb streptococcal vector R R encoding Em ' Cm 3 kb E.coli vector encoding AMPR and LacZ selection.

kb, nis R, tra+ promoter probe plasmid R Em, promoterless cat 6.6 kb SpeJ Lac Z Dao & Ferretti, 1985 Behnke & Gilmore, 1981 Promega Harvey and Dunn, 1989 Achen et al.. 1986 Dunnv et aL 1991 N117 inedia (Terzaghi & Sandine, 1975) was used primarily for the growth of Lactococal strains. containing either 0.5% glucose. M17(G), or 0.5% lactose, N117(L). To distinguish lactose-positive and lactose- negative isolates. the indicator broinocresol-purple (0.004%) was added to N117(L) media. The antibiotic ervthromycin was used at a concentration of 5 ig/mi.

nisin 300 IU/ml. streptomwin 500 VLg/M1, fusidic acid 40PLg/nll and spectinoinvein 100 ptg/ml for Lactococcal strains. FSDA (Huggins & Sandine, 1984) plus 0.5% glucose was used to distinguish between proteinase- positive and prote inase- negative Lactococcal isolates.

METHODS A. Isolation of plasmids that encode phage resistance I Conjugation Filter-mate conjugations were carried out as described below.

Depending on the phenotype of the wild type donor strain. transfer of the following traits were selected for:

(a) lactose utilization - selected oil M17(L) plates containing broinocresol-purple (0.004%) plus appropriate antibiotic (b) proteinase utilization - selected on FSDA (G) plus appropriate antibiotic (c) nisin resistance - selected on M17(G) plates containing nisin (300 IU/ml) plus appropriate antibiotic (d) cadmium resistance - selection on M17(G) plates containing cadinium (1 rriNI) plus appropriate antibiotic.

Filter-Mate Method Overnight cultures of donor and recipient strains were grown in either M17(G) or M17(L) broth. The donor and recipient cultures were mixed in the ratio of L2. A volume of 0.2 ml of the mating mixture and individual recipient and donor cultures (controls) were placed on 0.45 LM filters within sterile glass petri dishes containing No. 1 Whatman filter paper. The 45 1m filters were allowed to blot dry and were then transferred to M17(G) agar plates. After incubation overnight, the 0.45 tm filters were removed and suspended ill 3 inl saline. Cells ivere washed from the filter by vortexing. For the selection of iiisin resistance. filters were suspended and vortexed ill 10 nil saline. This cell suspension was centrifuged (10 mills. 6.000 rpm) and then resuspended in 3 ml of fresh saline. This additional washing step reduced the number of nisin resistant mutants appearing.

The resultant suspension was serially diluted and 150 d aliquots were plated on selective media. These plates were incubated at 300C for 1-2 days. Transconjugants were screened for phage resistance by crossstreaking. II Co-transformation L. Jactis MG1363 or LM0230 were transformed by electroporation according to the procedure given in the molecular techniques section. The transforming DNA was the total plasmid DNA from the wild type strains.

Seeding of this DNA (ratio 4:1) with a small plasmid, pND325., which encodes Em resistance was also used to aid selection of transformants.

Either lactose utilization or erythromycin resistance were used for the selection of transformants. Transformants were screened for acquired phage resistance by cross-streaking. III Mobilization by pND300 The nisin resistance plasmid. pND300, has been demonstrated to mobilise non-conjugative traits (Harvey & Dunn. 11989). This plasmid, ie., pND300, is transferred into the wild type strain to be investigated via conjugation selecting for lac' nisF' transconjugants (.-. only applicable to nis s wt strains), A nisin resistant transconjugant from this mate is then used as a donor to LM0230 SmR, selecting for the transfer of nisin resistance (also lac' and prt+). Transconjugants are then screened for acquired phage resistance.

IV Plasmid Curing Curing of plasmids from lactococci was achieved by either of the following methods:

(a) Elevated temperature - the strain was cultured under non-selective conditions at a temperature which permitted slight but not turbid growth in Nil 7 broth (usuallv 400C L. lactis; 36/37'C L, cremoris). The strain investigated was subcultured three or more times at this temperature and then tested for the loss of the relevant phenotype and/or screening of isolates by plasmid extraction procedure. Subculturing would continue until curing was achieved.

(b) Elec tropo ration- modified procedure of Heery D.M. et al. (1989) Curing of a plasmid from E. coli. using high-voltage electroporation. Nucleic Acids Research 17: 10131.

B. Bacteriophage Techniques Cross-streaking This method was employed for the initial screening of phage resistant and phage sensitive isolates. A sterile cotton wool applicator was soaked with a high titre phage preparation, and streaked along a M17 (CaC12) plate. After the phage streak line had dried, colonies to be tested were streaked across the phage line. A phage sensitive control was always included on each plate. Plates were incubated at 3WC or 37"C overnight and results recorded the following morning. Sensitive isolates were identified by no growth of the culture after meeting with the bacteriophage line.

High Titre Bacteriophage Preparation A single purified plaque was cut from a plate. using a sterile scalpel and resuspended. by vortexing, in a 3 nil saline solution. Two millilitres of this phage suspension was inoculated into 20 nil of an early log phase culture (l:t2 hours) of the phage sensitive host. CaC12andM9C12were added to a filial concentration of 10 mM. This phage/ host culture was incubated at WC overnight and then titred as described below.

Titres of > 108 pfu/ml were usually obtained by this method.

Alternatively appropriate levels of phage and log culture were combined to give a MOI of 0.1 to 0.01. The phage preparation was then filter sterilised, after centrifugation (6.000 rpin. 10 mills) and stored at 40C.

Single Plaque Purification of Bacteriophages A lawn of the sensitive liost culture was prepared by the addition of nil of inolten 0.8% top laver agar to 0.2 mI of an overnight culture plus one 3 - 13 drop of 1 M CaC12. This was poured over a M17 (CaC12) plate and allowed to set. Using a platinum wire loop a single plaque was picked and streaked gently over this top layer. Plates were incubated at WC overnight. Bacteriophages were purified by streaking for single plaques at least two to three times. Titering Bacteriophage Preparations Appropriate serial dilutions of the phage preparation were added (100 tl) to 0.2 mI of the sensitive host (overnight culture) plus one drop of 1 M CaCh. After five mins at room temperature, 3 mI of molten 0.6% top layer agar was added to the tube. quickly mixed and poured onto an M17 (CaC12) plate. After setting, plates were incubated overnight at WC or 370C. The titre was calculated as pfui'mi (plaque forming units/ml) for the phage preparation.

Efficiency of plaquing (EOP) was calculated by dividing the (D titre obtained on the host being tested, by the (D titre obtained on the sensitive or control host.

Milk activity tests were carried out according to the method of Heap & Lawrence (1976) to assess the effectiveness of CDR plasmids in simulated laboratory cheese-making trials. Growth curves in the presence and absence of bacteriophage were performed according to the method of Sanders and Klaenhammer (1984).

C.Methods for Determining Bacteriophage Resistance Mechanisms (i) Bacteriophage adsorption was investigated using the method of Sanders & Klaenharnmer (1980) Restriction/Modification Restriction and modification mechanisms were determined by phage reciprocal titering. LNI0230 was infected with $712 or (DC2V to make high titre phage preparation (step 1). The resulting phage preparation was then titred onto LX110230 (used as control) and the other strains for testing (step 2). Single pliage preparation from the plates from step 2 was made and used for re-titering back onto all the strains (step 3). Phage preparations from step 2 and step 3 were compared by titering. phage from RIM strains should show an increased titre.

(iii) Burst size experiments were performed according to a modified procedure of Durmaz et a] (1992) and calculated according to the method Casev et a] (1992). A reduction in burst size indicated the presence of an abortive infection mechanism.

(iv) Infective-centre assays and cell lysis/survival measurements after phage infection were performed according to Sing and Klaenhammer (1990).

D.Molecular Techniques Transformation L. lactis ssp lactis was transformed by electroporation as described by Powell et al (1988) with some modifications. The cells were washed twice with ice-cold sucrose solution (0.5 M) and resuspended in 0. 5 ml of sucrose solution (0.5 M). The DNA solutions, usually 5 A containing I pg of DNA, and 50 pl of cell suspension were mixed; and electroporation was carried out at 12,500 v/cm in a Gene-Pulser cuvette (electrode separation. 2 mm). An electric pulse (2.5 M 25 pF. 200 ohms) was then applied to generate a peak field strength of 12.5 kV/cm with the Pulse Controller (Bio-Rad). After the electric pulse. the cuvette was then removed from the chamber and 1 ml of M17G-sucrose (M17GS) medium was added immediatelv. After standing for 2 hr at 30'C. the cells were spread- plated onto M17GS agar containing selective antibiotics. For E. coh, the CaC12 transformation method of Dagert & Ehrlich (1979) was used without the extended pre-incubation in CaCl2' (ii) Plasinid DNA isolation. restriction endonuclease analysis molecular cloning and agarose gel electrophoresis - 15 Lactococcal plasmid DNA was isolated by the method of Anderson & McKay (1983). Plasmids from Exoli were isolated as described by Birnboirn & Doly (1979). Plasmid DNA was purified by caesium chloride-ethidium bromide density gradient centrifugation and desalted by dialysis in TE buffer. Restriction digests and molecular cloning were performed by the methods described in Sambrook et a] (1989). DNA was fractionated using 0.6% agarose in horizontal gels in Tris-acetate buffer (pH 8.0) at 4 V/cm, followed by staining in ethidium bromide (0.5 mg/ml). The Exoli strain V517 (Macrina et al, 1978) was initially used as a reference for estimating lactococcal plasmid size. From this information, L. cremoris FG2 was subsequently used as a standard for estimating plasmid sizes.

Hybridisation A modification of the procedure outlined in Sambrook et a] (1989) was used. Oligonucleotide probes were constructed for probing the presence of Abi A, C and 416 by selecting a specific sequence suitable for use as a probe. These sequences are as follows:

Abi A (Hill. C, et al, 1990) reported that a probe consisting of an internal 456-bp XbaI fragment (positions 2067 to 2523) was specific for the hsp sequence. The primers for PCR. however. were selected from the position 2677 to 2698 (22 bp) and 2875 to 2896 (22 bp) creating total 222 bp PCR product for the appropriate primer selection.

f: 5'-TTT GAT AGG TTT GTT GCT CAC G4(position 2677-2698) r: T-CTT CGT TTT GAA GTA AGC CAG C4(position 2896-2875) oligo probe from 456-bp XbaI fragment (position froin 2450 to 2471:

22 bp) T-CTA TCT TGA AAG CTG AW ACC A-3' Abi C (Durinaz. E. et a], 1992) The oligonucleotide from Abi C structural gene (1.056 bp) was synthesized (position from 426 to 455: 30 bp).

0 - 5'-GAG GGC AAT TAA CGA CAA ACA TGG CAA CTCT Abi 416 (Cluzel. P+ et al. 1991) The oligonucleotide from Abi 416 structural gene was synthesized (position from 973 to 1003: 31 bp).

5'-GTT GGC TTT CTT TTG ATA ACA TGC CAA TTC C-3' (iv) DNA nucleotide sequencing Nucleotide sequencing were determined by the dideoxy chain termination method with a35-dATP (Sanger et al. 1977). Double-stranded DNA sequencing was carried out for the phage resistant determinants and the sequencing reactions were performed with the T7 sequencing" kit (Pharmacia Biotech). Sequencing of the phage resistant determinants was initiated using the T7 and Sp6 primers of pGEM-7Zf(+), then continued using oligonucleotides which were based on sequences obtained. The oligonucleotides for DNA sequencing were synthesized on a DNA synthesiser (Oligo 1000, Beckman) according to the manufacturer's instruction. The DNA sequence data were analysed using the ANGIS software system operated by the Australian Genomic Information Centre of the University of Sydney.

Alternatively another procedure was used for DNA sequencing as follows:The smallest fragment encoding phage resistance was subcloned into pGENI vector (Promega Corporation) and transformed into Exo1i NM522, with blue/white screening for recombinants. Double-stranded DNA was sequenced on both strands essentially according to the protocol accompanying the 377 DNA sequencer (Applied Biosystems. Inc.. Calif).

In brief, 'the DNA Nvas used in dideoxy nucleotide chain termination sequencing reactions with Taq DNA polymerase and fluorescent dye-coupled terminators (Dve Deoxv ' Terminator Cycle Sequencing Kit. Applied Biosystenis. Inc., Calif.). 20 - nier oligonucleotide primers were synthesised and used to "walk" along the template DNA. The recording and analysis of the nucleotide sequence was carried out using the Auto Assembler TNI DNA sequence assembly software (Applied Biosystems, Inc., Calif.) and Angis Software System (as described previously).

RESULTS Isolation of Bacteriophage Resistant Plasmids &om Wild Type Strains Plasmid transfer techniques were performed according to that described in the methods section. Transformants/transconjugants were screened for resistance to 712 and C2V by the cross streaking method.

Phage resistant isolates were then subjected to plasmid analysis and acquired plasmids identified.

A summary of the 10 plasmids isolated by either conjugation, co transformation or mobilisation from the wild type strains is given in Table 1.

Confirmation that the plasmids identified in Table 1 encoded phage resistance was obtained by subsequent transfer into another recipientstrain by either transformation or conjugation techniques. The second-round transformants or transconjugants were screened for phage resistance by cross-streaking and plasmid content.

Physical Characterisation of Plasmids that Encode Phage Resistance A summary of the phage resistance of the plasmids of the present invention is given in Tables 2 to 6. In addition, where applicable. the phage resistance of sub-clones from these plasmids is also given in Tables 2 to 6.

The data given in Table 2 demonstrates that the plasmids of the present invention, and their sub-cloned derivatives, either reduce the efficiency of plaquing (EOP) and/or interfere with plaque morphology and/or plaque size when compared to the plasmid-free controls. Table 2 illustrates that these plasmids encode for some type of bacteriophage resistance mechanism(s) that is effective against either 712 (isometric type phage) and/or C2' (prolate type phage).

Table 1: Summary of plasmids that encode resistance

Mechanism Nlethod of of Plasinid [lost Origin Size Isolation Resistance pND801 NIG1363 L. lactis NI 195 12kb Co-transformation RIM pND802 LM0230 L cremoris 502 19kb Co-transformation RINI pND809 LNI023OSMR L. lactis M175 65kb Mobilisation RIM (+Abi) pND811 LM0230 L. cl-em oris NI 111 75kb I'Vlobilisation R/M (+Abi) pND851 LM0230 L. lactis N1113 46kb Co-transforination Abi pND852 LM0230SniR L. lactis N1138 56kb Conjugation Abi pND853 LM0230SMR L. lactis N1181 5 7kb Conjugation Abi pND859 LM0230Fus-R L. Jactis var 16kb Conjugation Abi diacel " V1actis UK12922 pN13860 LM0230Fus R L. lactis var 56kb Conjugation Abi(+RIM) diacet ' V1actis UK19161 pND862 LNI023OSml L. lactis var 63kb Conjugation Abi + R/M diacet - Yjactis UK1392 R/M restriction modification: Abi - abortive infection mechanism data on phage resistance conferred by these plasmids indicates that more than one mechanism is operating, possibly a R/M system in combination with an Abi system Table 2: EOP/plaque size/+ morphology 712 C2V Plasmid W1C 3 70C 30"C 3 70C Control 1 1 1 1 2mm 1-2mm 3mm 3Amm pND801 2 x 10-6 10-6 1 0.9 2mm 2mm 3mm 3mm pND802 10-3 10-3 10-3 10-3 2mm 2mm 3mm 3mm pND809 10-6 10-0 10-2 10-2 2mm 2mm 3mm 3mm pND811 10- 7 10,4 10-2 10"2 lmm lmm 3mm 3mm pND851 10-2 0.16 10,4# 10-4# lmm lmm 1-2mm 1-2mm pND852 10-3# 10-3' 10^2# 1T 1 NA 0.5mm mix:<0A/lmm 1-1.5 pND85 3 10-4# 10-4# 10^ 1# 10- 1 NA lmm <0.5mm 2 pND859 10-4# 10-4# 1 1 < lmm < lmm 3mm 3mm pND860 < 10-, < 10-, < 10-, 10-3 2mm pND861 < 10-, < 10-, < 10-, 10-3# 2mm pND862 < 10-, < 10-, 10-1 10,1 3mm 3mm pND803/ 10,3 NA 10-3 NA pND804 2mm 3mm pND805 10,6 NA 1 NA 2mni 3mm pND817 < 10-, NA 10-2# NA < lmm pND823 < 10-, < 10-, 0.2 # 0,5 <0.5mrn 2mm pND863 10-2' 10-2 1 1 < lmm < lmm 3mm 3mm pND 826 < 10-, < 10-, 10- 1 10- 1 3mm 3mm NA EOP Iiiiii JI pinpoint plaques Ilazy placlues. very difficult to count data nut available Efficiency ni'l)laqiiiiig,Iiaiiic,t(ti 0 pla(ples (in 1) ac t eri o' 1) lia Q-C Table 3: Adsorption of bacteriophages 712 and C2V to LM0230 host and LM0230/LM0230SIUR host harbouring plasmids pND852, pND853. pND859. pND860, pND861 and pND862 % adsorption (30T) Strain/Plasmid $712 (DC2V LM0230 (control) 99.6 98.6 LM0230 SmR (pND852) 97 99.8 LM0230 SmR (pND853) 99 99 LM0230 (pND859) 99,2 NA LM0230 (pND860) 99.5 99.6 LM0230 (pND861) 99.6 99.8 LM0230 (pND862) 99.6 NA NA not applicable.

From this and other data it has been established that none of the 10 plasmids investigated encode for prevention of phage adsorption? demonstrating 2: 96% adsorption of phages 712 and/or C2V (where applicable).

Restriction and Modification Activity Ph ageresistan ce m ech anisms en coded by pND801 an d pND802 The efficiency of plaquing of (D712 on strains harbouring pND801 and pND802 are given in Table 4. Phage (D712 propagated on strain LM0230 showed an EOP of 4 x lo-c'when plated on strain NIG1363 (pND801). Phages picked from the rare plaques formed on that strain were no longer restricted by the same strain. If repropagated on strain LM0230. however. the phage showed the sanie low efficiency of plating on strain NIG1363 (pND801) as the orieinal stock. Derivatives of MG1363 (pND801). which were cured of pND801 did not exhibit the reduction in phage plating. Reciprocal titering of (D712 on LM0230 (pND802) showed similar results as that on MG1363 (pND801). These results indicate that host-controlled restriction modification systems are operating in MG1363 (pND801) and LM0230 (pND802). Phage (D712 propagated on MG1363 (pND801) showed a low efficiency of plating on strain LM0230 (pND802) and (D712 propagated on LM0230 (pND802) was restricted by strain MG1363 (pND801) illustrating that the specificity of R/M activities encoded by pND801 and pND802 are different.

pND801 and pND802 were also tested for encoded phage resistance on the prolate phage 0C2V in an experiment similar to that described above and it was found that the restriction system of pND801 was not active on the prolate phage 0C2V. Plasmid pND802 encoded restriction towards both the isometric (D712 and the prolate phage (DC2V. Table 4: Example of reciprocal plating for plasmids pND801 and pND802 EOP of isometric 0712 and prolate 0C2V on strains harbouring plasmids pND801, pND802 and pND803 Phage 0712LM0230 1.0 tv712 LNI0230 (pND801) 1.0 0712 LNI0230 (pND802) 1.0 t712 LNI0230 (pND801 + 1.0 pND803) Bacterial Host MG1363 LIA0230 UY10230 LNI0230 NIG1363 (pND801) (pND802) (pND803) -6 9 10-4 4X 10 1.0 4 X 10-3 2 X 10-6 1.0 1.0 1.0 7 X 1T4 5 X 10-, 1.0 1.0 1.0 1.0 1.0 1.0 (DC2V LIM0230 1.0 1.0 1.0 2 x 10' 3 X 10,3 (DC2V LM0230 (pND802) 1.0 1.0 1.0 1.0 1.0 Burst size is defined as the average number of bacteriophage particles released per infected cell following the lytic infection of a population of sensitive cells. (For plasmids that did not display reciprocal plating. ie. R1\4 systems only; burst size experiments or infective centre assays ivere performed. These results are given in Tables 5a and 5b.

Tables 5a and 5b Example of demonstrating the presence of abortive infection mechanisms by reduction in burst size or infective centre formation Table 6a Burst Size 7120 C2V0 Strain/Plasmid 300C 390C 300C 390C LM0230 166 200 100 100 LM0230 (pND851) ND ND 5 200 LM0230 (pND852) 5 100 ND ND LM0230 (pND853) 11.6 2 IND IND LM0230 (pND862) 7.5 11.2 NA NA NA not applicable: ND not determined Results given in Table 5a show that plasmids pND851, pND852, pND853 and pND862 reduce the burst size of 712 or C2V (where applicable) when present in a LM0230 host. Burst size results at 390C for LX10230 (pND851) and LN10230 (pND852) indicate that the resistance mechanisms operating in these hosts may be temperature sensitive.

The infective centre formation (e.o.c.) was determined for plasmids pND859, its subclone derivative pND863 and pND861 as given in Table 5b.

Table 5b

Host LM0230 Fus R LM0230 Fus R (pND859) LM0230 Fus R (pND863) LM0230 Fus R (pND861) Infective Centre formation (e.o.c.) for 712 1 0.05 0.05 0.15 The number of infective centres formed on LM0230 FUSR (pND859) R or LM0230 FuS (pND863) relative to the control ie LM0230 FusR was reduced 95% for phage 712 while a 85% e.o.c. reduction was observed for 712 when LM0230 Fus R (pND861) was used as host.

Table 6:

Milk Activity Test (Modified, Heap & Lawrence, 1976) Example: Comparison of L. lactis LM0230 SrnR and L. lactis LM0230 Srn R (pND852) and L. lactis LM0230 SmR (pND853) to reduce the pH of milk in the presence of phage 712. NB: Milk (RSM) was fortified with glucose (0. 50/6) and yeast extract (1 %).

Strain LM0236 Sm LM0230 SrnK (pND852) -0712 +0712 -0712 +0712 4.44 6.09 4.60 4.68 4.68 6.08 4.90 4.88 4.65 6.12 4.79 4.74 4.55 6.08 4.82 4.83 LM0230 Sm (pN1D853) -0712 +0712 Cvcle 1 2 3 4 4.64 4,70 4.90 4.78 4.84 4.85 4.79 4.83 (pH RSM = 6.2) 0.1 mI of 7120 (8 x 109 pfu/ml) and 0.2 mI of an OIN milk culture of the appropriate strain was added to Cycle 1. In subsequent cycles, (2-4), 0.1 mI of the whey from the previous cycle was added plus 0.2 mI of milk culture of the appropriate strain.

No disturbance was observed for strains LM0230 SM R (piND852) and LM0230 SM R (pND853) in the presence of 712 over four cycles. This R contrasts with the phage sensitive control strain, LM0230 SM which was disturbed from the first cycle onwards when 712 was present. Disturbance is indicated when the ApH is >0.2, the ApH = (pH of test +712) - (pH of test - 712), the larger the ApH the greater the disturbance or reduction in acid production.

Hybridisation Data Plasmids were probed with oligonucleotides constructed as specified in materials and methods.

The plasmid pND806 served as a positive control for the detection of AbiA.

None of the plasmids of the present invention hybridised with the AbiA probe concluding that they do not possess the AbiA gene. Similarly no hybridisation was observed for AbiC or Abi416 probes, however positive controls were not available. These three Abi genes were the only Abi gene DNA sequences available at the time of this work.

Restriction Analysis and Physical Mapping of the Phage Resistance Plasmids Restriction analysis of the plasmids was carried out using a series of restriction enzymes and the restriction data is listed in Table 7. Plasmids were extracted from strains harbouring phage resistant plasmids and subjected to single and double digestion with a variety of restriction enzymes. DNA fragments generated were visualised on agarose gels and restriction inap of the plasinids were constructed. The restriction rnaps of pND801. pND802 and pND861 are shown in Figures 1 and -1.

Table 7: Restriction analysis of phage resistant plasmids Plasmid Enzyme Molecular weights of fragments (kb) pND801 pND802 pND809 pND811 pND851 pND852 pND853 pND859 pND861 pND862 BstI EcoRI EcoRV PvUII XbaI Bg1II ClaI EcoRI EcoRV HindIII SphI EcoRI EcoRI Psti PvUil EcoRI Psti HpaII EcoRI ClaI HpaII EcoRI HindIII ClaI Psti Bg1II BcunHI BamHI Bg1II EcoRI HpaII Psti Hin dIII ClaI SCOI AccI 8Sa81 Xb u I SCUI 12.0 8.9. 2.0. 1.1 12.0 12.0 8.3, 2.7 11.8,9.2 16.1.3.9 18.2,1.8 11.6, 4. 3, 4.1 20.0 18.9, 1.1 39.0, 11.2, 7.6, 5.8, 1.4 >23, 15, 9, 6, 2.2 >23, 18, 9 doublet >23, 15 doublet, 2.5, 2.2 16.7, 9.5, 5.3, 4.8, 4.15, 3.8, 2. 2, 0.7 20.0,9.0, 7.5, 6.2, 4.1 13.0, 9.6. 8.9, 7.4, 6.0, 5.4, 3.7, 2.8 > 2 3, 11, 2. 0, 1.8 >23, 17, 5.5, 2.7, 2.1 22.0, 15.0, 8.9, 5.6, 2.7, 2.1, 1.6 > 2 3, 15, 13, 3.6, 2. 0 6.5, 4.5, 2.9, 2 - 18, 9.4, 9.2, 6 27,4.2 > 23, 6.3, 3.5, 3.2 14. 9.4, 8.4. 6.5 1. 16. 23, 23 25, 38 20, 10, 7.8, 7.2, 4.8, 4, 3.1, 2.3, 2, 1.6 16, 12.5, 8.5, 5.5, 3.65, 3.5. 3.4. 2.4, 1.8, 1. 65. 1.5, 1.5, 0.8 40, 13, 10 14.4, 11.21. 7.2, 5.8, 4.9, 4.8, 3.8, 3.2, 3, 1.7, 1.1, 0.9, 0.8 16, 15, 13. 11. 5, 3 20, 13, 9.8, 8.6, 3.71.3.1. 2.2. 2. 0.4 13, 11. 11. 4.5, 2.3. 2, 1.5, 1.2, 1 and multiple small fragments 20. 13, 9.3. 6. 5.2. 3.5. 1.8. 1.7. 0.9, 0.7 and smearing small fragments 22.5. 21. 11. 5.2. 1.5. 1. 0.7 19. 13. 9. 8.4. 4, 3.3. 2.2. 2. 1.5. 0.6 Cumulative Ellect from Introducing Various Phage Resistant Plasmids in the Same Host Various plasmids encoding phage resistance were introduced in different combinations into MG1363Srn or LM0230Fus by conjugation and electroporation to study the cumulative effects of phage resistance. The various constructs were cross-streaked and titred against 0712 (phage titre 2.0 x 109 pfu ml-l) and 0C2V (phage titre = 2.0 x 109 pfu ml-l) to determine the cumulative phage resistance accrued to the various plasmid combinations in them. The results are shown in Table 8. The effect of additive phage resistance due to the acquisition of multiple plasmids was evident in most plasmid combinations. Additive phage resistance was manifested in a reduction in the phage titre, EOP and the plaque size. In particular. NIG1363Srn (pN13851 + pND803 + pND852), MG1363Srn (pND851 + pND803 + pND853), MG1363Srn (pND803 + pND851) and LM0230Fus (pND851 + pND805) were observed to show complete resistance to both isometric 0712 and prolate 0C2V.

Table 8: Cross-streaking profiles. plaque size and EOP of the constructs Examples of cumulative effects of bacteriophage resistance of single, double and triple plasmid encoded bacteriophage resistance mechanisms Cross- Plaque diarn streaking (Inin) EOP Strain 0712 qC2V o712 0C2V 07 12 0C2V Donors NIG1363 (pND801) 1 5 2x 10-6 1 UY10230(pND803) 1 3 1 X 10-, 3 x 10-3 LM0230 (pND805) 1 5 1 X 10-, 1 LM0230 (pND809) 1 4 4 X 10-6 6 X 10^2 LM0230 (pND811) + 1 3 2 X 10-7 5 X 10,2 LM0230 (pND851) +I- + 0.7 0.3 5 X 10-, 1 X 101 LM0230 (pND852) + - 0.1 1 1 X 10-, 2 X W' LM0230 (pND853) + - 0.1 < 0.5 10 10-1 LM0230 (pND859) + - 1 5 1 X 10-, 1 LM0230 (pND861 + + NP NP < 10-, < 10-, L1Y10230 (pND862) + - NP 2 < 10-, 1 X 10-, Final Host NIG1363 Sin - - 1 5 1 1 LM0230Fus - - 1 5 8 x 10-1 1 Final Constructs NIC1363 (pND801 +pND803) + +/- 1 5 7 X 10-9 6 X 10-4 M0230Fus (pND803 +pND809) + 1 2.5 7 X 1T8 3 x 10-3 MG1363 Sin (pND803 + pND811) + 1 2 5 X 10'1? 1 X 10-1 NIG1363 Sin (851 + pND852) + +1- 0.1 0.5 9 X 10-, 8 X 10-, DvIG1363Sni(pND851+pND853) + +/- 0.1 1 1 X 10-1 6 x 10,3 NIG1363 Sin (852 + pND803) + + NP 0.1 < 10-, 6 x 10-8 MG1363 Sin (pND853 +pND803) + + NP 0.2 < 10-, 1 X 10 NIG1363 Sin (pND85l+pND803+ + + NP NP < 10-, < 10-, pND852) NIG1363 Sin (pND85l+pND803+ + + NP NP < 10-, < 10-, pND853) NIG1363Siii(I)ND803+pND851) + + NP NP < 10-, < 10-, L1Y10230 Fus (pND851 + pND805) + + NP NP < 10-, < 10-, NIG1363 Sin (803 + pND862) 1_ +/- NP 2 < 10-, 1 X 10-, Key: + phage resistant: - phage sensitive. +./- partial (weak) phage resistant: "JP no plaque observed, All incubations were at 30c'C.

Stability of plasmids harboured by constructs MG1363SND803 + pND851) The construct of MG1363 Sm (pND803 + pND851) was observed to exhibit complete resistance to both isometric (D712 and prolate (DC2V. Having achieved that combination of plasmids which gave maximal protection to both isometric and prolate phage the next step would be to test the stability of these plasmids after growth for many generations without selection. An overnight culture of the strain was grown in M17G broth to stationary phase (10" cells ml-l), diluted to 10' cells mFl and grown up again. This was repeated for 100 generations. At the end of that time, isolates were cross streaked and phage assayed to see if the phage resistance phenotype had been retained. It was observed that this combination of plasmids was relatively stable (98%) over 100 generations of growth in liquid broth without selection pressure.

Cloning of Phage Resistant Genes from pND801, pND802, pND852, pND853, pND859 and pND862 (i) pND802 Both pND802 and the vector pSA3 contain a single SphI site and were digested to completion with SphI then ligated together with T4 DNA ligase. The ligation mixture was precipitated with ethanol and electroporated into LM0230. Transformants were obtained on the M17G + 0.5 Suc + Em selection plates. Colonies that arose on the selection media were screened by cross streaking across (D712. Of the transformants tested, 60% showed pND802 type resistance to (D712. One of the colonies was purified and the plasinid harboured in the strain was designated pND803.

The expression of phage resistance encoded by pND803 in L. lactis was investigated by challenging transformants harbouring pND803 with different phage and the results are shown in Table 6. pND803 restricted the isometric 712 and the prolate C2V to a similar extent as did strains harbouring the parent plasmid pND802.

29 - Additional subcloning reduced the R/M system in pND802 to a 9kb fragment by the construction of pND804. pND804 is a recombinant plasmid constructed by cloning a 9 kb BgIII fragment from pND802 into the unique BamM site of pSA3 and confers phage resistance to phage 712 and phage C2V when introduced into L. lactis LN10230.

(ii) pND801 The whole of pND801 had been cloned into the unique EcoRI site of vector pSA3 to generate pND805. pND801 was digested partially with EcoRI to form a linear band. Vector pSA3 was digested completely with EcoRL The two digests were then ligated. The ligation mixture was transformed into LM0230 by electroporation. Transformants were selected and challenged with 0712. The resultant recombinant plasmid pND805 was checked by restriction analysis (refer Figure 3).

(iii) pND852 pND852 contains eight HpaII sites. Using a short gun cloning technique it was attempted to clone the fragments using the streptococcal vector, pGB301. which contains a unique HpaII site. Following restriction and ligation. the mix was electroporated into LM0230. Transformants arose from selection medium M17G + Suc + Em and were tested for phage resistance and plasmid size.

A schematic diagram showing the steps involved in cloning the Abi system from pND852 is given in Figure 4. Phage resistant transformants all harboured a 2.6 kb HpaII fragment. A representative subclone. pND817, conferred complete resistance (EOP <10-8) to 712 and reduced EOP to C2V as the parent plasmid, pND852.

(iv) pND0853 The plasmid pND853 was partially digested with HpaII and then ligated with LipaII digested pGB301. Transformants were selected on N117G - Suc + Em plates and cross-streaked against 712. A variety of plasmid sizes were isolated from the phage resistant trans formants, These were analysed by restriction endonuclease digestion. It was found all plasmids contained a 2.7 kb LipaII fragment in addition to other different sized fragments. This result indicates that the commonly isolated 2.7 kb HpaII fragment from pND853 encodes for abi. Representative clones e.g. pND825, were found to confer a higher level of resistance to 712 than the parent plasmid. A schematic diagram illustrating the steps for cloning the abi system from pND853 is given in Figure 5.

(v) pNW859 The plasmid pND859 was digested with HindIII and ligated with HindIII digested pDL278, spectinomycin resistant transformants were screened for resistance to 712 by cross streaking. Phage resistant transformants were found to harbour a 2.9 kb HindIII fragment. An example is pND863 as illustrated in Figure 6.

The plasmid pND862 was digested with Ban-M and ligated with Barn.HI digested pMU1328. Erythromycin resistant transformants were screened for resistance to 712 by cross-streaking. Phage resistant transformants were found to harbour a 16 kb Barnffi fragment as illustrated in Figure 7.

DNA Sequencing of abi Systems from pND852 and pND859 The DNA sequence data for the two abortive infection mechanisms isolated from pND852 and pND859 are given in Figures 8 and 9 respectively.

The nucleotide sequence of the 2.6 kb DNA insert in pND852 was determined. An analysis of the sequence, shown in Figure 8, revealed a large open reading frame (ORF) of 996 bp which corresponds to a 332 amino acid sequence. Upstream of the putative start codon a putative ribosome binding site and potential promoter regions were found. Downstream of the putative stop codon a terminator also exists.

DNA sequence analysis of the 2.9 kb HindIII fragment from pND859 revealed an open reading fraine of about 846 bp in length which is proceeded by a SD sequence and several potential promoter regions (as shown in 31 Figure 9). No homology with any published phage resistance genes was detected in the gene database for both pND852 and pND859 abi mechanisms. Construction of Plasmids Encoding Bacteriophage Resistance pND824 is a recombinant plasmid constructed by cloning a 9 kb BgIII fragment from pNTD802 into the unique BamHI site of pMU1328 and confers phage resistance to phage 712 and phage C2V when introduced into L. lactis LM0230.

pND813 is a recombinant plasmid which contains a 16 kb BamHI fragment from pND862 inserted into pND600 at its unique BamHI site. This plasmid confers complete phage resistance to 712 when introduced into L.

lactis LM0230.

pND824 was digested with XbaI and SmaI and the 9 kb DNA band was recovered from the agarose gel. pND827 was linearised by XbaI and PruUII and inixed with the 9 kb DNA. Ligation was performed with T4 DNA ligase. The ligation mixture was transformed into competent E.coli HB101.

Transformants arose on LB Amp 100 selection plates and were examined for the size of the insert obtained. The recombinant plasmid with a 25 kb insert was designated pND828 (refer to Figure 10 for diagram of this construction).

This 25 kb insert can be released from pND828 and cloned into a lactococcal food grade vector. Similarly, Figure 11 illustrates the construction of another multiple phage resistant plasmid using phage resistance mechanisms from pND852 and pND801.

Figure 12 illustrates the general concept of constructing plasmids encoding bacteriophage resistance in lactic acid bacteria. A specific DNA fragment that encodes bacteriophage resistance is identified and subcloned to a inininium size. Initially plasmids may be as large as 60kb to 1OOkb although the sequence encoding bacteriophage resistance may be from 2kb to 1Okb in size. The subcloned DNA fragments that encode bacteriophage resistance are then joined together and linked into an acceptable cloning vector that can be used in food-approved lactic acid bacteria. The plasmid is selected or manipulated so that, when in a host cell. it would not interfe with the replication of any natural resident plasmids in the host. Once constructed the plasmid can be transferred into different strains of lactic acid bacteria.

Lactic acid bacteria can be grouped according to their sensitivity to different bacteriophage. Approximately 25 groups of Lactococci are known that are bacteriophage unrelated. (Maenhammer, 1984). A plasmid according to the present invention can be introduced into one strain of each bacteriophage unrelated group. This therefore provides the natural resistance of each phage unrelated group together with the bacteriophage resistance mechanisms encoded by the plasmid.

As the plasmids of the present invention are composed solely of DNA derived from lactic acid bacteria, thev are suitable for use in lactic acid bacteria used for the production of human foodstuffs.

Figure 13 shows an example of a DNA fragment that encodes bacteriophage resistance to small isometric phage and prolate phage on Lactococcus lactis. The fragment may be a 9kb BgI II fragment derived from pNB802 and is suitable for incorporation into a plasmid according to the present invention.

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It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadlv described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (22)

Claims:

1. A plasmid encoding resistance to bacteriophage selected from the group consisting of pND801 (N95/58018), pND802 (N95/58019). pND809 (N95/58020), pND811 (N95/58021), pND851 (N95/58022), pND852 (N95/59300), pND853 (N95/58023), pND859 (N95/58024), pND860 (N95/59301), pND862 (N95/58025) and functional equivalents thereof.

2. An isolated nucleic acid molecule including a nucleotide sequence encoding resistance to bacteriophage, wherein the nucleotide sequence is derived from a plasmid selected from the group consisting of pND801 (N95/58018), pND802 (N95/58019), pND809 (N95/58020), pND811 (N95/58021), pND851 (N95/58022), pND852 (N95/59300), pND853 (N95/58023), pNB859 (N95/58024), pND860 (N95/59301), pND862 (N95/58025) and functional equivalents thereof.

3. The isolated nucleic acid molecule according to claim 2 such that the nucleotide sequence encoding resistance to bacteriophage substanatially corresponds to the nucleotide sequence of Figure 8.

4. The isolated nucleic acid molecule according to claim 2 such that the nucleotide sequence encoding resistance to bacteriophage substantially corresponds to the nucleotide sequence of Figure 9.

5. A method of increasing or conferring phage resistance to a bacterium comprising introducing into the bacterium at least one plasmid or at least one nucleotide sequence derived from at least one plasmid according to claim 1.

6. The method of claim 5 wherein the bacterium is a lactic acid bacterium.

7. A bacterium having increased or conferred phage resistance produced according to the method of claim 6. -

8. A lactic acid bacterial plasmid consisting of lactic acid bacterial DNA or chemically synthesised DNA for use in increasing or conferring phage resistance to lactic acid bacteria. the plasmid including at least two nucleotide sequences encoding phage defence mechanisms selected from the group consisting of host controlled restriction. host controlled modification, abortive infection and prevention of phage absorption.

9. The plasinid according to claim 8 such that the plasinid includes inininial DNA and substailtialIv excludes DNA not required for inaffitenance of the plasmid or for encoding the phage defence mechanisms.

37 -

10. The plasmid according to claim 8 or 9 wherein the at least two nucleotide sequences are derived from plasmids selected from the group consisting of pND801 (N95/58018), pND802 (N95/58019), pND809 (N95/58020), pND811 (N95/58021), pND851 (N95/58022), pND852 (N95/59300), pND853 (N95/58023), pND859 (N95/58024), pND860 (N95/59301), pND862 (N95/58025) and functional equivalents thereof.

11. The plasmid according to claim 8 or 9 wherein at least one of the two nucleotide sequences substantially corresponds to the nucleotide sequence of Figure 8 or Figure 9.

12. The plasmid according to claim 8 or 9 wherein the two nucleotide sequences substantially correspond to the nucleotide sequences of Figure 8 and Figure 9.

13. A plasmid according to any one of claims 8 to 12 including at least three nucleotide sequences encoding phage defence mechanisms.

14. The plasmid according to any one of claims 8 to 13 further including a nucleotide sequence encoding at least one selectable marker.

15. A bacterium including at least one plasrriid according to any one of claims 8 to 14.

16. The bacterium of claim 15 being a lactic acid bacterium.

17. A method of increasing or conferring phage resistance to a bacterium comprising introducing into the bacterium at least one plasmid according to any one of claims 8 to 14.

18. The method according to claim 17 wherein the bacterium is a lactic acid bacterium.

19. A plasmid encoding a bacteriophage, defence mechanism substantially as hereinbefore described.

20. An isolated nucleotide molecule including a nucleotide sequence encoding a bacteriophage defence mechanism substantially as hereinbefore described.

21. A method of increasing or conferring phage resistance to a bacterium comprising introducing at least one plasmid as claimed in claim 19 or at least one nucleotide sequence as claimed in claim 20 into the bacterium.

22. A bacterium including at least one plasmid as claimed in claim 19.