AU703298B2 - Plasmids encoding bacteriophage resistance - Google Patents

Description

AUSTRALIA

Patents Act 1990 THlE UNIVERSITY OF NEW SOUTH WALES BURNS 1IILI1" COMPANY LIMItTED BURNS PHLILP RESEARCHI DEVELOPMENT iPTY LTD COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RIESEARCH ~*ORlGANISATIjON

*~:ARNOTT

t S BISCUITS LIM1ITED GOODMAN FIELDER INGRZEDIENTS LIM ITED ORIG INAL COMPLETE SPECI FICATION STANDARD P~ATENT Invention Title: Plasm ids Encoding Bacteriophage Resistance The following statement Is a fuill description of this Invention including the best method of performing it known to us,-- Field of the Invention The present invention relates to plasmids that encode lbcteriophage resistance and in particular, such plasmids for use in lactic acid bacteria.

Background of the Invention Lactic acid bacteria are used inter clia 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; 3, Use of strains in admixture and in rotation to minimise problems of bacteriophage infection: 4. Identification of plasmids that encode bacteriophage resistance then introducing the plasmid into a bacteriophage sensitive strain to generate a resistant variant: and 5, 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. HIost range mutants of bacteriophage or new bacteriophage can overcome the resistance niechaiisnisms and in and constructs mayI exhibit a degree of genetic instabilitv whlich may facilitate bacteriophage infection.

The present ilnvenitors have been able lo reduce genetic instability and achieve the introduction of inultiple phage resistant determinants into host bacteria by the ldevelopinnt of new plasmids.

Summary of the Invention Accordingly, in a first aspect the present invention consists inll a plasrmid encoding resistance to bacteriolphage selected from the group consisting of pND801, pNND802. pND809. pND8 11. pND85 1. pND852.

pND853, pND859. pND860. pND8G2 and functional equivalents thereof.

In a second aspect, thlie present invention consists in an isolated nucleic acid molecule including a niicleotide sequence encoding resistance to bacteriophage, wherein the nucleotide sequence is derived fromn a plasmid selected from the group consisting of pND801, pND802, pND809, pND811.

pND851, pND852, pND853, pND859, pND860, pND862 and functional equivalents thereof.

In a preferred embodiment of the second aspect of the present inveintion, the isolat nucleic mniolecule includes the nucleotide sequence substantially corresponding to the inucleotide 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 bacteriumn comprising introducing into the bacterium at least one plasinid 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 plasmiid consisting of lactic acid bacterial DNA or chemically svnthesised DNA for use in increasing or conferring phage resistance to lactic acid bacteria, the plasmiid ncluding at least two Iiucleotide 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 yet 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 mechanismis 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, Preferably 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 bacteriu at least one plasmid of the fifth aspect of the present invention.

In a vet further preferred embodiment of the present invention, the plasmids of the first and/or fifth aspect of the present invention are introduced singularly or ini vnrioulS cominiations into I bac teriuiu sgo as to icreaise or coiifer lbactel'iopllage resistance to t he lbacteriiiii.

As usedl herein, thie phrise3 "functional eqluivahlnts therofl is intended to cover plasmids or nucleic acid seqluences derived therefroml that have slightly altered nucleic acid sequences fromn the plasnhiis set out above but which retain substantially the samne biological activity. Thisj miay be achieved by various changes. such as insertions, deletions andl substitutions, either conservative Or 11on-conlservative whlere siich changes do not s ub stantLially alter the biological activity of the protein or proteins encoded by the lplasinilcls or inucleic acid sequences derived therefroi 28Samples of each of the plasinids set out above were deposited at the *Australian Government Analytical Laboratories (AGAI4 on 2Septemnber 1995 and 5 October '1995. The respective accession numbers of each deposit are set out below: PLASMID ACCESSION DATFE

NUMBER

pN..0 N9/81 8/99 pND80'I N95/58019 28/09/95 pND809 1N,95/58020 28/09/95 ND809 N95/58021 28/09/95 pND8ii N95/58022 28/09/95 pND852 N95/59300 05/10/95 pND853 N95/58023 28/09/95 pND859 N95/58024 28/09/95 pND860 N95/59301 05/10/95 pMD862 N95/58025 28/09/95 I order that the nature of the present: invention may be miore clearly understood, preferred forms thereof will be described with reference to the following examiples and the accompanying drawings.

Brief Description of Drawings Figure 1 shows restricion maps of pND801 and 1 )ND802; Figure 2 shows restriction map of pND861; Figures 3, 4, 5, 6 and 7 are schematic representations of the cloning of phage resistant mechanisms fromin pND801, pND852, pND853, pND859 and pND82 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 minechanism isolated from pND850; of 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.

Description of the Invention IvATE1UALS Lactococcal Strains For convenience the following terminology is used: L, lactis refers to *i the species Lactococcus lactis subsp lactis; L. cremois refers to the species Lactococcus lactis subsp cremoris and L. lactis var diaceijlactis refers to the species Lactococcus lactis subsp lactis var diacetylactics.

The following strains were used as donors for the isolation of plasmids that encode bacteriophage resistance by either conjugation, mobilization or co-transformation: L. lactis strains M195, M175, M113, M138, M1181; L. cremois strains M502, M111 and L. lactis var diacetidactis UK12922, UK19161 and UK1392 were all originally isolated from mixed strain starter cultures.

The followiig strains wvore used as recipients ini coliuigat1101 mnobilisation or elecirotraiisforinatioti experimlnlts: L. luctis.NM 1363 is a plasmici-free derivative of L. kidis 71t2 obtainled from MI. Gasson, Food Research Institute, Norwich, UK.

L. lujtiS MGi1363Sni is a plasii-f rep derivative of L bucls 712 made resistant to streptomvcini L. loctis LM10230 is a plasmnid-free derivative of L. luclis C2 obtainied from L.L. McKa.,, University of MNinniesota, St Paul, Mimn., USA.

L. lactis LTNO230Sni is a 1 laslid-free derivative of L. lactis C2 obtained from L.L. McKay, University of Milnnesota, St Paul, Minn., USA, and made resistant to streptomycin in the present inventor's laboratory.

L. bactis LN1023OFus is a pinsinid-free derivative of L. luctis C2 obtained from L.L. MNcKav, University of MNinniesota, St Paul, Mimni, USA, and made resistant to fusidic acid in thie present iinventor's laboratory.

Othier 1lactococcal strains used: L. cemoils FG2 was used as a standard for estimiating the size of plasiid. DNA in other lactococcal strains. It hiarbours plasinids of thie following sizes -135, 67, 50, 27, 14, 12.9, 11.9. 10.1, 8.8, 5.2, 3.

These sizes were calculated vs the reference s train E.coli V5i17 (Macrina et 1978).

Other Strains Refer'en ce Ecoli V517 Macrinia et 1978 Ecoli 1-11 101 Pro'. Leut, Thi-, Boyer RoullandrecA' plasinid-free Duissoix. '1969 Ecoli NM1522 Thif, Lac", r' in' Gough and -Mnrray (1983) -(lac pro AB) 11 Bacteriopliages ciD 7 12 Small isonietric-headed phiage (nsson. 1983 propagated onl JA.10230 and MG 1363 ciC2V Prolate-headed phiage p~rop~agated{ Jai vis S& onl LTN0230 anid MG,136)3 Klamilianinier, 1986 III Plasinids conferring bacteriopliage resistance p.ND80i is a 12 kb jplasnnd originallv isolatedc from L. luctis NM195 by co- transformiation into L. lactis 1,,%1230 pND8O2 is a 19 kb plasinid originally isolated from L. cremoris M1502 ~.by co-transformation into L. loctis LN10230 pND8O3 is a 29.2 kb plasmid constructed by cloning S Al linecarised pND8O2 into the SpliT site of pSA3 pND8O4 is a 18.9 kb plasnid. constructedt by cloning the 8.7 kb Bgl1 11 fragment fccom pND802 into the IBamilI site of pSA3 pND805 is a 22,2 kb plasmic constructed by cloning B oRI linearised pND80I into the EcoRi site of p)SA3 pND8O9 is a 65 kb plasniid originally isolated from L. loctis M1175 (pND300) by the miobilization procedure. This pasmici canl be conjugatively transferred to ILactococcal recipients where it confers insensitivity to bacteriop)hage infection and resistance to nisin pND8ii is a 75 kb plasinid originally isolated from L. ci'emor!s N1lI (pND3 00) by the mobilization p~rocedurile. This plasnid canl be conjugatively ti, nsferred to Lactococcat recipients where it confers insensitivity to bacteriophage infection, resistance to nisin and pr*oteinas e activity pND851 is a 46 kb Iplasmidt originially isolated from L. locUS N113 by co- transformation into L. lactis LN10230 pND852 is a 56 kb tplasnnd originialY isolated from L. lactis N1138.

This plasinid can be conjugativelv transferred to Lactococcal recipients where it confers insensitivity to ldcteriop)hage infection and also resistance to nisin pND85 3 is a 5 7 kb plasii originiallIv isolated fromn L. Ictis M118 1.

This plasmnid can be conjugatively transferred to Lactococcal recipients where it confers insensitivity to bacteriophage infection and also resistance to nisin pND859 is a 16 kb plasmnid originally isolated fromn L. lactis var dfacetviactis UK -J-2922. This plasmnid can be. conjug'ativelv transferred to Lactococcal recipients where it confers insensitivity to bacteriophiage infection and also resistance to cadniftn (12) pND86O is a 56 kb plasinid originally isolated fromi L. luctis var diacetlactis UK 196.This plaslnidl can be conjugativelv transferred to Lactococcal recipients where it confers insensitivity to bacteriophage infection (13) pND861 is an'in vivo' deletion derivative of p)ND860. 35 kb, obtained during a plasmnid curing experiment. This plasinid confers insensitivity to bacteriophiage infection (14) pND862 is a 65 kb plasmid originally isolated fromi L. loctis var diacetylactis UK 1392. This plasmid can, be onjugativelv trans.ferred to Lactococcal recipients where it confers insensitivity to bacteriophage infection and also encodes lactose utilisation and protrcinase production pND8i7 is a 12.3 kb pie d constructed by cloning of the 2.6 kb L1-h II fragmient fromi pNDj852 into the H-pa II site of pG13301 (16) pND825 is a 34 kb plasinid constructed by cloning of the 2.7 and 1.6 kb flpa 11 fragments from pNDB53 into the H-pa II site of pGB3O1.

(17) pND863 is a 9.5 kb plasinid constructed by cloning of a 2.9 kb H-ind III fragment from pN1J859 into the Hind III site of pDL278 (18) pND826 is a 23.5 kb plasmnid coinstructed by cloning of a '16 kb BamJ-HI fragmnent froin pND862 into the Banilil1 site of pNMU1328.

(19) pND821 is a 5.6 kb plasmid conistruc ted byv sulb-cloing the 2.6i kb IMpal fragment from pND8 17 into the CPUl site of' pGENC-7zf(+) pND806) is a 46 kb) Lactococcal plasinid that confers nsensi tivity to bacteriophiage infection and foundl to posses,, thle Abi A genle reported by Hill et ('1990). This plasnid was used as a positive control for hvbridisa tion experinmentis.

Otlier plasmids: pND325 a.

*..aaa pSA3 4.8 kb, hlybrid plasmAd containing E 1 1 of S~uu'eus plasmid )El 9 4 and a replicon from a crvp tic plasmici of L. lactis M1127 -10,2 kb. hybrid vector encoding Cm Tc (Eco~i selection) 'and Em R ictcis selection) 9.8 kb streptococcal vector encoding Em Cn 1 1 3 kb Exco~i vector encoding AipR and LacZ selection.

60 kb. nis 5 ,Ira promoter probe plasinici Em' promnoterless cat 6.6 kb SpeclR Lac Z' Mlai:; Laboratories Dao Ferretti.

1985 Belmnke &k Gilmnore.

1t981 Proniega Harvey and Dunn. '1989 Achen et 1986 Dmun et 199 1 pGEMI"-7zf(+) pND300 p~vui3 28 pDL278 13. Media M117 nmedia (Terzaghi Sandine, '1975) wtas used primarily for the growth of Lactococal strains, containing either 0.5%y glucose. M117(G). or lactose. N1'17(L). To distinguish lactose-positive and lactose-negative isolates. the indicator bromocresol-purple was added to NM17(L-) media. The antibiotic ervythronmvcin was used at a coilneitration of 5 pg/inl, nisin 300 ITJU/il, streptomycin 500 uagJ/ml, fusidic acid 40jag/inl aInd spectinonlyci n 100 ptg/uil for Lactococcal strains. FSDA (IHugginiis Sandine, 1984) plus 0.5% glucose was used to distinguish between pi inase-positive and pro teinase-negative Lact ococcal isolates.

METHODS

A. Isolation of plasmids that encodle 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: lactose utilization selected on N117(L) plates containing bronmocresol-purple (0.004%) plus appropriate antibiotic proteinase utilization selected on FSDA plus appropriate antibiotic nisin resistance selected on M1:7(G) plates containing nisin (300 IU/nil) plus appropriate an tibiotic cadlniium resistance selection on M17(G) plates containing cadmiuni (1 miiM) plus appropriate antibiotic.

Filter-Mate Method Overnight cultures of donor and recipient strains were grown in either M17(G) or MN17(L) broth. The donor and recipient cultures were mixed in the ratio of 1:2. A volume of 0.2 nil of the niating mixture and individual recipient and donor cultures (controls) were placed on 0.45 tn filters within sterile glass petri dishes containing No.1 Whatnan filter paper. The filters were allowed to blot dry and were then transferred to M17(G) agar plates. After incubation overnight, the 0.45 Ln filters were removed and suspended in 3 nil saline, Cells were washed from the filter by vortexing. For the selection of nisin resistance, filters were suspended and vortexed in ml saline. This cell suspension was centrifuged (10 iiins. 6.000 rpm) and momm 111011 rostusll1id ill .3 *iil 11es tiosli ln(% 'IlIs additi)!1'l kva'd'lig step Trhe. 105 nItanl IsulspenOsionl Wa~s ser iaIltv dliu ted ad 1 150 ii ahI(jUols were plte tol se 01lcive 11101 fi. Thiese f)LaU I 0 aere incubattid at (3 for 1t'-2 days. Trauisconijiiganis were screenied for phiage r~esi s taice l)v crossstreaking.

11 Co-transformation L. lactis MG 1363 (or LMtJ230 were traiisformed by electroporationl according to die procedure given ini the molecular techiniques section. The transforming DNA was die total plasii DNA from the wvild type strains.

Seeding of this DNA (ratio 4:1) with a- stiall plasmidL pND325. which encodes Enii resistance was also used to aid selection of transformants.

Either lactose utilization or ervthroiinvc.in resistance were uised for **the selection of transfornmnts. Transformnts were. screenied for acquired phiage resistance byv cross-streaking.

III Mobilization b~y pNl)300 SThe nisin resistance plasnud, IpND300. has been dlenrons trate d to nmobilise non-conjugative traits (1-larvey Dunn, '1989). This plasinid, ie., pND300. is transferred into the wild type strain to be investigated via conjugation selecting for lac' nis Rtransconjugants only app~licable to ii xt strains). A nisin resistant tranisconjug-ant from this mate is then used as a *.donor to LM10230 SniR. selecting for the transfer of nisin resistance (also lac' and prt"). Tranisconljugants are then screened for acquired plhage resistance.

IV Plasmid Curing Curing of plasmids from lactococci was achieved by either of the following methods: Elevated temperature the strain was cultured uinder non-selective conditions at a temperature which p)ermitted slighit but not turbid growth in M117 broth (usually 40"'C L. loctis; 36/37"C L. cr~emoris). The strain investigated was submultured three. or mnore timies at this temperature m 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.

Electroporation- modified procedure of Heery D.M. et L. (1989) Curing of a plasmid from E.coli using high-voltage electroporation. Nucleic Acids Research 17 10131.

B. Bacteriophage Techniques Cross-strealking 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 (CaCl,) plate. Atter 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 30 0 C or 37°C overni id 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 resuspendea, by vortexing, in a 3 ml saline solution. Two millilitres of this phage suspension was inoculated into 20 ml of an early log phase culture hours) of the phage sensitive host. CaC, and NlgC were added to a final concentration of 10 mM. This phage/ host culture was incubated at 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 rpm, 10 mins) and stored at 4°C.

Single Plaque Pmuification of Bacteriophages A lawn of the sensitive host culture was prepared by the addition of 3 ml of molten 0.8% top layer agar to 0.2 nml of an overnight culture plus one a drop of 1 MI CaC12. This was poured over a M17 (CaCI,) 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 30'C 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 pl) to 0.2 ml of the sensitive host (overnight culture] plus one drop of 1 M CaC2. After five mnins at room temperature. 3 ml of .nolten 0.6% top layer agar was added to the tube, quickly mixed and poured onto an M17 (CaCl,) plate. After setting, plates were incubated overnight at 30°C or 37 0 C. The titre was calculated as pfu/ml (plaque forming units/ml) for the phage preparation.

~Efficiency of plaquing (EOP) was calculated by dividing the 0 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 c( 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 Determiiiing Bacteriophage Resistance Mechanisms Bacteriophage adsorption was investigated using the method of Sanders Klaenhanmmer (1980) (ii) Res triction/Modification Restriction and modification mechanisms were determined by phage reciprocal titering. LM0230 was infected with 0712 or IC2V to make high titre phage preparation (step The resulting phage preparation was then titred onto LM0230 (used as control) and the other strains for testing (step Single phage preparation from the plates from

II

step 2 was made and used for re-titering back onto all the strains (step Phage preparations from step 2 and step 3 were compared by titering, phage from R/M strains should show an increased titre, (iii) Burst size experiments were performed according to a modified procedure of Durmaz et al (1992) and calculated according to the method Casey et al (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 a] (1988) with some modifications. The cells were washed twice with ice-cold sucrose solution (0.5 M) and resuspended in ml of sucrose solution (0.5 The DNA solutions, usually 5 tl containing 1 tg of DNA, and 50 Ll of cell suspension were mixed; and electroporation was carried out at 12,500 v/cm in a Gene-Pulser cuvette (electrode separation, 2 nmm). An electric pulse (2.5 kV, [IF, 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 immediately. After standing for 2 hr at 30°C, the cells were spread-plated onto M17GS agar containing selective antibiotics.

For E.coli, the CaC12 transformation method of Dagert Ehrlich (1979) was used without the extended pre-incubation in CaCl., (ii) Plasmid DNA isolation, restriction endonuclease analysis molecular cloning and agarose gel electrophoresis Lactococcal plasmid DNA was isolated by the method of Anderson McKay (1983). Plasmids from E.coli were isolated as described by Birnboim Doly (1979). Plasmid DNA was purified by caesium chloride-othidium bromide deiAi ty gradient centrifugation and desalted by dialysis in TE buffer. Restriction digests and molecular cloning were performed by the methods described in Sarnbrook et al (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 E.coli strain V517 (Macrina et al, 1978) was initially used as a reference for estimating lactococcal plasmid size. From this information, L. cremnoris FG2 was subsequently used as a standard for estimating plasmid sizes.

(iii) Hybridisation A modification of the procedure outlined in Sambrook et al (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 G-3' (position 2677-2698) r: 5'-CTT CGT TTT GAA GTA AGC CAG C-3' (position 2896-2875) oligo probe from 456-bp XbaI fragment (position from 2450 to 2471: 22 bp) TCT TGA AAG CTG ACG ACC A-3' Abi C (Durmaz, E, et al, 1992) The oligonucleotide from Abi C structural gene (1,056 bp) was synthesized (position from 426 to 455: 30 bp).

I

GGC AAT TAA CGA CAA ACA TGG CAA CTC3' Abi 416 (Cluzel, P-J, et al. 1991) The oligonucleotide froi Abi 416 structural gene was synthesized (position from 973 to 1003: 31 bp).

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 c35-dATP (Sanger et al. 1977). Double-stranded DNA sequencing was carried out for the phage resistant determinants and the sequ- icing 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, Becknan) 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 pGEM vector (Promega Corporation) and transformed into E.coli NM522, 'o 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 Biosystenms Inc., Calif).

In brief, the DNA was used in dideoxy nucleotide chain termination sequencing reactions with Taq DNA polymerase and fluorescent dye-coupled terminators (Dye Deoxy''" Terminator Cycle Sequencing Kit, Applied Biosystems, 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"I DNA rsequence assembly software (Applied Biosvstems. Inc,, Calif.) and Angis Software System (as described previously),

RESULTS

Isolation of Bacteriophage Resistant Plasmiids from Wild Type Strains Plasmid transfer techniques were performed according to that described in the methods section. Transformants/transconjugants were screened for resistance to (j)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, cotransformation 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 recipient strain 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 (1)712 (isometric type phage) and/or (C2V (prolate type phage).

p 19 TIablIe Summary of'plasmids thiat encode 4) resistanlce Nfuchazhisum 1'a"sInlir I lost C )igimi SiA( Isolatio~n 1{osistifl I NI(A3t33 L- luG/is NJ 15 12kb (C -timauibhiilion RIMf IpND802 LM023() L. cramoris 5(02 1 )kh Co-I ra I SI onlah loll RIMf p)ND139 U0230Sm' L. Inc/is NJ 175 (15kt NfohiI isainil R/M 4- Abdi* )NID81 Ui L0230) I, crnnoris NI' 11I 75 kb MobdIisautiou R/M Abi W p)Ni 85 1 LM10230 L. Inc/is NJ 113 4 (i kb >t h-nstrnmi (u Ab) i p)NIM52 LNIM3Sui 5 L. INn/ 1I38 k1 Con jugatinu AMi pI )85:3 LN(O23(J91ui1 I L t I. Pin/is NI 181 57kb C onj ugation Ab i I)859 LN1023O0uus Pin/is Vil 1(ikb) (nIipugibmi AMi ('K12922 LNMO23O0uus 5 L. luccs V'11 5(1kb Conugal~ lol A) Ai( W I (Jlia (I GI5 I K 19161 PND/1(12 U%1023()Smil L Incis Via (13kb C ouijiugil[unmi Abi R/MN dIwm(I'0/VhiG) 1,111<i392 IR/Mv restriction mndification; Abi abortive infection inechauiisin data on phage resistance conferred by these plasmids indicates that mnore than one mechanism is operating, possibly a R/M systenm in combination with an Abi system rablo 2: EOP/plaque size/+ morphology (1)712 C( 2 V Plasmid 30"C 37"C 30"C 37"C Control 1 1 1 I 2 11111- 1-2111111 3111111 3-41m11 pND8O1 2 x 10 _106 1 0.9 2111111 2mnin 3111111 3111111 pND802 10 10" to' 2nim1 2111111 3Inm 3 inn I pND8O9 10" 10' 10 10-2 2nim 2m11 31nni 3111111 pND811 10- 10 -1 1"10' 2 10-2# 1mm 1111 1-2 1-2mm *ND852 103 10 10 NA 0.5111111 mix: <0.,/lmmin 1-1.5 4 I# pND853 10 10 10 NA 1mm <0.51m1 2 pND859 10" 10 1 1 <1111111 <1m1m11 3 nmi 31n ii.

2mni pND861 <10'0 100 <10-8 10-4 21nni1 pND862 10,1 10-8 10-1 10-1 3 111111 3mrin pND83/ 101 NA 103 NA p pND8o4 2mm 3mmn 10,6 NA 1 NA 2mm 3mmni )ND817 NA 102# NA Im pND823 10,1 <10W1 0.2 0.5 mm1111 2mm pND863 10'2" 10'z* 1 1 11mn <11111m 3nini 31mm pND826 109 101 3111111 3 num )[Ij)LfJ Il[ j)IMqll(IS hazy 1111(o'S. VoI' vy (JilIicIIH [C t o ll NA da a 1ot0 availa) to EUMil PA icioncy of' plafu ing 111111 d otolio 4) bm~altoriopliage! .15 TraI)I 3: Adsorptioni ol Ibacterioplhages 712 and C2V to LM'0230 host and LM0230/LM0230Sin' host liarbotiring plasmnids I)NI)852, pND853, pND859, pND860. p)ND86i and J)NI)862 IN/o adsorp~tion Straini/Plasinid cD 12 C2 LM10230 (control) 99.6 08.6 LM10230 SniR (pND852) 97 99.8 LM10230 SniR (pND853) 99 99 LNM0230 (pND859) 99.2 NA LM10230 (pND860) 99.5 99.6 LM0230 (pND86i) 99.6 99.8 LM0230 (pND862) 99.6 NA ''pp p NA not applicable.

From this and oilher data it has been established that none of the plasinids inves tigatedi encode for p~reven tion of phiage aclsorp tion, denionstrating 96% adsorption of phages; 712 and/or C2V (where applicable).

Restriction and Modification Activity Phage resistance miechanisms encoded byv pNVD8O1 and pNVD8O2 The efficiency of plaquing of 01712 onl strains harbotiring pND8O1 and pND802 are given in Table 4. Phage cD7I2 propagated onl strain LM0230 showed an EOP of 4 x 1 0 o-'vwheni plated on strain MIG1363 (pND8O1). Phages picked from the rare plaques formned on tha t s train were no longer res tric ted by the same strain, if repropagateci on sLrain LM10230. howevfer, the phage showed thie same low efficiency of plating onl strain MIG1363 (pND801) as the original stock. Derivatives of IG'1363 (.pND80'I), whichi were cured of pNI)801I did not exhiibit the reduiction in phiage p~~.Reciprocaul titerinig of c1712 onl LM10230 (p)ND802) showed siniflar results as that onl MIG1363 (pNDBO1). These results indicate that host-controlled restriction modification systemns are operating in MIG1363 (pND8OlJ and( LM10230 (pND8O2). Phiage 0712 propagated onl MG1363 (pND80i) showed a low efficiency of plating oin strain LM10230 (pND8O2) and c1712 propagated onl LM0230 (pND8O2) was restricted by straini MNG1363 (pND8Olj illustrating that the specificity of RINI activities encoded hy pND80i and p)ND8O2 are dlifferen t.

pND8O1 and pNDBO2 were also tested for encoded ph.,ge resist'ance ~*onl the prolate phage (DC2V in anl experiment similar to that described above *and it was found that the ruestriction system of pND8Oi was not active onl thE.

*prolate phiage (DC2V, Plasniid pNDBO2 encoded! restriction towards both the isometric 0712 and the prolate phiage cDC2V.

Trable 4: Example of reciprocal plating for plasmnids pND8Oi and pND8O2 *c C CEOP of isomietric 0712 and prolate cIC2V on strains harbotiring plasmids pND8Oi, pND8O2 and 1 )ND803 Bactoriai I-lost off, NIC- 303 LM0230) 1A10230) Phago IA10230 NR;1303 (jpNI)U0i) (p)NIDU2J (tpNl)03) q)7121LM0230 1.0 1.0 4x lo' Ox 'I o- 7 x 'LO-' *7'12 IAM023() (pNI)0i) 1.0 1 .0 1.0 4 x l0- 5 x P712 LNI0230 (p)N1802) 1.0 1.0 2 x 10" 1.0 *7'12 LM10230 (I)D00 A- *1.0 '1.0 1L0 1.0 j)ND803) DC2V U10230 '1.0 1.0 1.0 2 x '1(0 3 x q'C2V LNM0230 tp)NIM02I 1. 0 1.0 1.0 1.0 Buirst size is definied as the average number of bacteriophage particles released per infected cell following the lytic infection of a population of sensitive cells, (For plasinids that did not display reciprocal plating, ie, RM systems oly: burst size experim-entis or infective centre assays were perform1ed, These results are giveni in Tables 5 a and 0*

C.

23 Tfables 5a and 51) Example of dlemonstrating the presence of aborelitf inflection mechianistms by reduction in burst size or inifective centre formation Table 6a Buirst Size* 712(D C2V(j Strain/Plasiniid 30 0 C 39 0 C 300C 39 0

C

LM10230 166 200 100 100 10 LM0230 (pND851) ND ND 5 200 LM0O2 30 (pNID 85 2) 5 100 ND N-D LM0230 (pND853) 11,6 2 ND ND LM10230 (pND8G2) 7.5 11.2 NA NA NA not applicable; ND not determinied Results given in Table 5a show that plasnids pND85i, pND852, pND853 and pND8B62 reduce the buLrst size of (1712 or ()C2V (where applicable) when present inl a LM0230 host, Burst size results at 39 0 C for LM0230 (pND85i) and LM0230 (pNDB52) indicate that the resistance mechanisms operating inl these hosts may be temperature sensitive.

The infoctive centre formationl (e.oxc.) was determined for plasnids pND859, itsr subclone derivative pND863 and pND86i as given in Table 51).

Table 51) [lost Iiiuc live Cmd re f'urimiiia ut~c. for (1)712 LN10230 FLIS' R LM,0230 Fut (pND863) 0.05 LM0230 Fits R(pND861) 0.1.5 The number of infective centres formed onl LM0230 Ftis R(pND859) or LNO23O Fus R (pNDB63) relative to the control ie U1,0230 Fits R was '10 reduced 95% for phiage 7'12 while a 85% e~o.c. reduction was observed for 712,!) when LM10230 Fusl (pND86i) was used as host.

Table 6: Milk Activity Test (Modified, Heap Lawrence, 1976) Example: Comiparison of L. iccis LMPY0230 SilR and L, lactis LM10230 Silm (pND852) and L. loctis LM0230 SmR (pND853) to redtuce the pH- of milk in the presence of pliage 7'12. NB: Milk (RSM) was fortified with glucose and yeast exL-ract (1I) Straiin LM0230 Still LM40230 Smnl LM10230Sil 2) (pND853) Cycle -01712 +01712 -(1)712 +C1)712 -0)712 +01712 1 4.44 6.09 4.60 4.68 4.64 4.70 2 4.68 G.08 4.90 4.88 4.90 4.78 3 4.65 6.12 4.79 4.74 4.84 4.79 4 4.55 6.08 4.82 4.83 4.85 4.83 (pHINI 6. 2) 0,1 ml of 7120 (8 x pfu/ml) and 0.2 nil of an O/N milk culture of the appropriate strain was added to Cycle t. In subsequent cycles, 0.1 ml of the whey from the previous cycle was added plus 0.2 ml of milk culture of the appropriate strain.

No disturbance was observed for strains LM0230 SM" (pND852) and LM0230 SM'" (pND853) in the presence of (712 over four cycles. This coniitrasts with the phage sensitive control strain, LM0230 SM N which was disturbed from the first cycle onwards when ()712 was present. Disturbance is indicated when the ApI-I is the L\pH (pH of test (pH- of test the larger the ApH the greater the disturbance or reduction in acid production.

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

15 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 20 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 map of the plasmids were constructed. The restriction maps of pND801, pND802 and pND861 are shown in Figures 1 and 2.

Iable 7: Reshi' ic tioii ana" lysis Of Pip I'(mSis lll jasi lli(d pr\I)8() I j)N]J802

I

pND809 pND811 pND851 pND852 pND853 pND859 pND861 Clul I coR I EcoRV 1HvliuIl EcoRI EcoRV Psti z dIll EcoRI EcoRI Clal IlJuI EcoRI il 1 (1111 Clctl PsLI

HII

1Hp all1 EcoRI Mi cll r

I

12.0 8.9, 2.0, I.1I 12.0 12.() 8 L3, 2.7 11. 8, 9,.2 16.1, 3.9 18.2, 1.8 11.6. 4.3. i. 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 >23, 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.U >23, 15, 13, 3.6, 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. 1, 16, 23, 23 38 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, 14.4, 11.2, 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. 8.0, 3.7, 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 snearing sinall fragments 22.5, 21, 1, 5.2. 1.5. 1. 0.7 19, 13, 9. 8.4, 4. 3.3, 2.2, 2,1.5, 0.6 pND862 PstI HIl i dmll Clar SGc Acci BsaBl YbaI Scal

I,

27 Cumulative Effect froin Introducing Various Phiage Resistant Plasinids in the Samne I-lost Various plasmnids encoding phiage resistance were introduced in different combinations into MG1363Sm or 1,M\423OFus by conjugation and electroporation to studty the cumulative effects of phiage resistance. The various constructs were cross-streaked and titred against (1712 (phage titre,= x 1i" pfu mil-i) and cDC2V (phage titre 2.0 x 101 pfu mi-i) determine ,he cunmlative phage resistance accrued to the various plasinici comibinations in them, The results are shown in Table 8. The effect of 10 additive phage resistance due to the acquisition of multiple piasmids was evident in most plasmii comibinations. Additive phage resistance was manifested in a reduction in the phiagu titre, EOP and the plaque size. in particular, MIGi363Sni (pND851 pND803 pND85 MVGi363Sni pND803 pNDB53), MGi1363Si (IpND8O 3 pND85i) and LM0230Fus (pND85i pND8O5) were observed to show complete resistance to both isometric 0712 and prolate (DC2V, Table 8: Cross-streaking p~rofles, plaqune size and POP of the constructs Examples of'cumutlative effects of bacteriophage resistance of single, doubleI and triple plasmid enlcoded bacteriopliage resistance mechanisms st rmk ii ig (1712 cD)C2V PI)dji1o (haul (11ul11) (D 712 1)C2V 1)7 12 *G'2V Strini Donors MG1,303 ()Ni)00i) LN40230(pNfl803) LN40230 (IpNl)05) JA40230 (p)NIJOO) U,10230 (p)NJJO11) LM10230 (I)D8,1) btv10230 (p)N!)52) Lfvt0230 (I)D53) L'40O230 (I)D859 LfvN1023() ND8612 0.7 0. 1 0. t +h Nil

NJ)

S

:3 4 3 (0.3 1 0.5 5 Nil 2 5- 2\x i x I10* I x 4 x 10- 2 x to", 5 x I10' 1 x 10- 1(, I x 1 4 t o" 3 x (3 x 1o-' 59 x Io 1 x 2 x

(V]

<to I x Finial I tost MG-1303 $11 LN10230Fits I I Ii x lo, 'I Final Comstrticts MIG-1363 (pND801 +p)Ni)03) LN10230Ftis (p)ND803 +pNIJO09) MG1363 Sin, ()ND03 IpNiJ8l) MG1363 Sin (pNIO51 p)ND852) NIGI3G3Sin(pND85I+pND853) MIG1363 Sin (pNDO852 p)NiJO3) M'vG1303 Sni (p)N1J53 +IpND803) M(G1363 Sm (pNIJ5+pNI)803+ I)D52) MIG1363 Smu (pNI05 1+pNI)003 1 'N11853) MIG1303Sini(pNI003-1 NI]i5 1) 1,0230 Ftis (p)NJ851 I)D05) NIG1363 Sun (p)NIJO3 p)NiJO2) 7 x 10_ 7 X 10*8 5 X I 0 9 x -to-' I x 10' "10 6, x 3\x 'I x 8 x 0" x 0i x 1I x o- Nil NJ' <10" 0 Nil NI' NJ) Ni' NI) 2 1 ,t X 1 4 Key: phage resistant; phiage sensitive;-, partial (weak) phage resistant; NP no plaque observed. All incubationis were at Stability of plasmids harboured by constructs MG1363Sni(pND803 pND851) The construct of MG1363 Sm (pND803 pND851) was observed to exhibit complete resistance to both isometric $712 and prolate OC2V. 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 1 0 1 cells ml-1), diluted to 10" cells ml' and grown up again. This was 10 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 over 100 generations of growth in liquid broth without selection pressure.

15 Cloning of Phage Resistant Genes from pND801, pND802, pND852, pND853, pND859 and pND862 i) pND802 Both pND802 and the vector pSA3 contain a single Sphl site and were digested to completion with Sphl then ligated together with T4 DNA 20 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 0712. Of the transformants tested, 60% showed pND802 type resistance to D712. One of the colonies was purified and the plasmid 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 (p712 and the prolate iC2V to a similar extent as did strains harbouring the parent plasmid pND802.

Additional subcloning reduced the R/MI systemn in ipND802 to a 9kb fragment by the construction of pND804. pND804 is a reconilnant plasmid constructed by cloning a 9 kb BglII fragment from pND802 into the unique Biami-I site of pSA3 and confers phage resistance to phage 712 and phage C2V when introduced into L. lactis LM0230.

(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 EcoRI. The two digests were then ligated. The ligation mixture was transformed into LM0230 by electroporaion. Transfornants were selected and challenged with 0712. The resultant recombinant plasmid pND805 was checked by *,restriction analysis (refer Figure 3).

(iii) pND852 pND852 contains eight Hpall sites. Using a short gun cloning technique it was attempted to clone the fragments using the streptococcal vector, pGB301, which contains a unique Hpall site. Following restriction and ligation, the mix was electroporated into LM0230. Transfornants arose from selection medium M17G Suc Em and were tested for phage 20 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 I-Ipall fragment. A representative subclone pND817, conferred complete resistance (EOUP 10) to J712 and reduced EOP to dJC2V as the parent plasmicid, pND852.

(iv) pND0853 The plasmid pND853 was partially digested with Hpall and then ligated with -Ipall digested pGB301. Transformnants were selected on M17G Suc Em plates and cross-streaked agains t j1712. A variety of plasmid sizes were isolated from the phage resistant transformnants. These were analysed by restriction endonuclease digestion. It was found all plasinids contained a 2.7 kb HpaIl fragment in addition to other different sized fragments. This result indicates that the commonly isolated 2.7 kb Hpall 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 pND859 The plasmid pND859 was digested with HindIII and ligated with 10 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 HlindIII fragment. An example is pND863 as illustrated in Figure 6.

The plasmid pND862 was digested with BamHI and ligated with BamII digested pMU1328. Erythromycin resistant transformants were screened for resistance to (h712 by cross-streaking. Phage resistant transformants were found to harbour a 16 kb BamHII 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 3 32 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 frame of about 846 bp in length which is proceeded by a SD sequence and several potential promoter regions (as shown in

I

Figure 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 BglII fragment from pND802 into the unique BamnHI 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 BcurHI site. This 10 plasmid confers complete phage resistance to 712 when introduced into L.

lactis LM0230.

pND824 was digested withXbaI and Sinal and the 9 kb DNA band was recovered from the agarose gel. pND827 was linearised byXbal and PruUII and mixed with the 9 kb DNA. Ligation was performed with T4 DNA 15 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 minimum size. Initially plasmids may be as large as 60kb to 100kb although the sequence encoding bacteriophage resistance may be from 2kb to 10kb 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 inot interfere 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, (Klaenhanner, 1984). A plasmid according to the present invention can be introduced into one strain of each bacteriophage unrelated group. This therefore provides the natural 10 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, they 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 Bgl II fragment derived from pND802 and is suitable for incorporation into a plasmid according to the present invention.

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1-Harvey, ML1. and Dunn, NW (1989) Identification and characterisation of a Slactis mobilizing plasmid which encodes for nisin :15 resistance, Proc. Eighth Aust. Bioteclh. Couf,, UNSW, 6-9 Febi'uarv, 3 -356.

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

e-

Claims (12)

1. A plasmid encoding resistance to bacteriophage, the plasmid selected from the group ctsisting 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 functionally equivalent plasmids 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), pND859 (N95/58024), pND860 (N95/59301), pND862 (N95/58025), and functionally equivalent plasniids thereof. 15 3. The isolated nucleic acid molecule according to claim 2 such that the nucleotide sequence encoding resistance to bacteriophage substantially Scorresponds 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. A method of increasing or conferring bacteriophage resistance to a S" bacterium, the method 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. 25 6. The method of claim 5 wherein the bacterium is a lactic acid bacterium.

7. A bacterium having increased or conferred bacteriophage 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 bacteriophage resistance to lactic acid bacteria, the plasmid including at least two nucleotide sequences encoding bacteriophage defence mechanisms selected from the group consisting of host controlled restriction, host controlled modification, abortive infection, and prevention of bacteriophage absorption, wherein the at least two nucleotide sequences are derived from plasmids selected from the group consisting of pND801 (N95/58018), pND802 L (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 functionally equivalent plasmids thereof.

9. The plasmid according to claim 8 such that the plasmid includes minimal DNA and substantially excludes DNA not required for maintenance of the plasmid or for encoding the bacteriophage defence mechanisms. 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.

11. 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.

12. A plasmid according to any one of claims 8 to 11 including at least 5 three nucleotide sequences encoding bacteriophage defence mechanisms.

13. The plasmid according to any one of claims 8 to 12 further including a nucleotide sequence encoding at least one selectable marker. S* 14. A bacterium including at least one plasmid according to any one of claims 8 to 13.

15. The bacterium of claim 14 being a lactic acid bacterium.

16. A method of increasing or conferring bacteriophage resistance to a bacterium comprising introducing into the bacterium at least one plasmid •according to any one of claims 8 to 13.

17. The method according to claim 16 wherein the bacterium is a lactic 25 acid bacterium. DATED this 20th day January of 1999 UNIVERSITY OF NEW SOUTH WALES *BURNS PIILP COMPANY LIMITED BURNS PHILP RESEARCH DEVELOPMENT PTY LTD GIST-BROCADES AUSTRALIA PTY LTD COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION ARNOTTS BISCUITS LIMITED and GOODMAN FIELDER INGREDIENTS LIMITED Patent Attorneys for the Applicants: F.B. RICE CO. I~ I -e 1 1 1 -I Plasmidcs are provided HiM;1 encode bacteriophagelag rosistauce. The'll plasmidts are useful for icreasilg or cofe~ring bacteriopiage resistance to bacteria and in particular to lactic acid bacteria. L