AU748950B2 - Zoocin A immunity factor - Google Patents

Description

rw H n nr t LT D/f9/'70fQIn n71 WU YYI7OYOY 1 ZOOCIN A IMMUNITY FACTOR TECHNICAL FIELD The invention relates to a factor which has activity in protecting a cell producing zoocin A, to the gene encoding that factor, to vectors and organisms containing the gene and the use of such organisms as anti-bacterial agents.

BACKGROUND ART Since the dawn of microbiology it has been observed that the growth of some strains of bacteria can interfere with the growth of other potentially harmful bacteria growing in the same medium. We now know that these inhibitory reactions are mediated by a range of metabolic and protein products produced by many different strains of bacteria. The "classical" antibiotics such as streptomycin and penicillin are metabolic (enzyme synthesized) products and their use in the prevention and treatment of disease is now well established. In contrast, industrial and medical use of proteinaceous (ribosomally synthesized) inhibitory substances has been much more limited. Recently however, this situation has changed and in 1988 nisin was granted GRAS (Generally recognized as safe) status by the U.S.

Food and Drug Administration (Federal Register 1988) in recognition of the fact that nisin was produced by Lactococcus lactis strains naturally associated with certain foods during processing and that it has no apparent adverse effects when ingested.

Zoocin A is a unique domain-structured bacteriolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881, which specifically attacks the cell walls of some closely related streptococcal species including the principal causative agents of group A streptococcal sore throat and dental caries respectively (Simmonds et al (1995); Simmonds et al (1996)). It was shown that zoocin A could suppress the growth of S. mutans in a triple species plaque model and that the initiation of the killing sequence occured very quickly. A 6.8 kb EcoR I fragment containing the gene encoding zoocin A (zooA) was cloned into Escherichia coli using the pBluescript® II phagemid vector and the sequence of zooA determined (Simmonds et al (1997)). The N-terminal catalytic domain of zoocin A has a high degree of homology with the N-terminal catalytic domain of a similar bacteriolytic WO 99/26969 PCT/NZ98/00171 2 enzyme lysostaphin, produced by Staphylococcus simulans biovar staphylolyticus, which specifically attacks the cell walls of other staphylococcal species. The Cterminal substrate-binding domain of lysostaphin is known to have a high degree of homology to at least one other staphylococcal cell wall binding enzyme, a Staph.

aureus amidase. By contrast, the substrate-binding domain of zoocin A has homology to no other known sequence. Both enzymes appear to lyse cell walls by cleaving the peptide cross-links within the peptidoglycan (Simmonds et al (1996)).

The bacteriocidal nature of their mode of action and the high degree of species and strain specificity exhibited by these enzymes are characteristics of that group of proteinaceous inhibitory agents referred to as bacteriocin-like inhibitory substances

(BLIS).

Zoocin A targets only a very limited range of bacteria, restricted to some species of Streptococcus only. This species-specific anti-bacterial action is useful. For example, it is active against two groups of medically significant human pathogens and at least one significant animal pathogen.

S. mutans and S. sobrinus are two of twenty or more species of bacteria present in dental plaque. Although not numerically dominant, these two species are considered to be the major aetiological agents of dental caries and their suppression in the oral cavity has been shown to reduce caries incidence (Loesche (1976); Loesche et al (1989)). Group A streptococci (GAS) infect via the upper respiratory tract where the tonsillar region in particular is believed to be the primary site of colonization. GAS carriage in humans is relatively common and GAS pharyngitis left untreated can progress to more serious disease including rheumatic fever and nephritis (Bronze and Dale (1996)). Vaccines are not available to prevent these infections and although it has been shown that these groups of microorganisms can be suppressed in the oral cavity by administration of antibacterial agents such as chlorhexidine (Loesche (1976)), polyvalent cations (Jones et al (1988)) and classical antibiotics (Loesche et al (1989)), the broad spectrum nature of these agents means that many commensal organisms are also suppressed, a condition which is known to pre-dispose the patient to superinfection by resistant microoganisms including gram-negative bacteria and yeasts. In each case the prolonged and widespread use of these agents has not been considered acceptable (Marsh (1991)). In contrast, zoocin A, while having significant bacteriocidal activity against these groups of WO 99/26969 PCT/NZ98/00171 3 microorganisms has little or no activity against many other plaque species such as S. oralis (Simmonds et al (1996)), S. sanguis or non-streptococcal species (Simmonds et al (1995)), or against the major groups colonizing the mucosal surfaces of the oral cavity such as S. salivarius (Simmonds et al (1995)). Therefore, administration of zoocin A to the oral cavity is unlikely to result in the complications seen with the previously mentioned broad spectrum anti-microbial agents, yet should lead to a decrease in the incidence of dental caries and carriage of GAS.

Before zoocin A can be used for its desirable anti-bacterial properties, there is a need for it to be provided in a form that can be administered to a human or an animal safely. For many antibiotics this is achieved by batch fermentation of the organism producing the antibiotic and purifying the antibiotic molecule and adding it to a suitable carrier. This method would be very expensive for zoocin A which has a molecular weight of 28,000. For that reason, the more commercially attractive option is to produce the zoocin A in situ in a naturally fermented food such as yoghurt.

However, zoocin A is produced by S. equi subsp. zooepidemicus, a recognized animal and occasional human pathogen. Serious human disease has been shown to result from the ingestion of S. equi subsp. zooepidemicus contaminated unpasteurized milk (Francis et al (1993)). Therefore, use of the natural producer organism to incorporate zoocin A in a food product as part of a food fermentation process is unlikely to be acceptable, but one solution would be to move the genes required for zoocin A production from the natural host to an organism suitable for use in food fermentation processes. However, this approach presents some difficulties when zoocin A is lethal to the genetically transformed organism.

One solution to these difficulties is to render the organism which is to express zoocin A resistant (immune) to the activity of this enzyme. This solution requires a factor to be identified which protects otherwise susceptible organisms against zoocin A activity.

The applicants have now identified such a factor, which is generally referred to hereinafter as zoocin A immunity factor. It is towards this factor and to its use that the present invention is broadly directed.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

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*e *ooo WO 99/26969 PCT/NZ98/00171 4 SUMMARY OF THE INVENTION In one aspect, the invention provides zoocin A immunity factor, which is a protein which is capable of protecting a host cell expressing zoocin A against the potentially damaging activity of zoocin A.

In a further aspect the invention provides an isolated DNA molecule which has a nucleotide sequence which encodes zoocin A immunity factor (zif).

Preferably the DNA molecule is selected from the group comprising molecules having one or more of: the zif sequence shown in Figure 3, a sequence comprising that sequence, a sequence comprising a part of that sequence active in protecting an organism .from zoocin A, a sequence encoding the same protein as the zif sequence of Figure 3 but differing in nucleic acid sequence by virtue of degeneracy of the genetic code and a sequence which is a functionally equivalent variant of the zif sequence shown in Figure 3.

In still a further aspect of the invention, there is provided a vector comprising the zif encoding molecule defined above, optionally together with a gene encoding the zoocin A active protein or variant defined above.

In yet a further aspect, the invention provides a non-pathogenic organism containing the zif encoding molecule defined above, optionally together with a gene encoding a polypeptide sequence selected from the sequence for zoocin A or a functionally equivalent variant of that sequence.

Preferably, the organism is a food-grade organism.

As another aspect of the invention, there is provided an antibacterial composition comprising a non-pathogenic organism as defined above.

Preferably, the composition is suitable for ingestion, particularly human ingestion, and is a foodstuff, nutriceutical or confectionery.

In yet a further aspect, the invention provides a method of preventing or inhibiting the growth of undesirable organisms susceptible to zoocin A which comprises the step of contacting said organisms or the environment thereof with a composition as defined above.

Preferably, the organisms inhibited are S. mutans, S. sobrinus or S. pyogenes and the composition is administered to the oral cavity of a patient.

Other aspects of the invention will be apparent from the description provided, and from the claims.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

DESCRIPTION OF THE DRAWINGS While the invention is broadly as defined above, it further includes embodiments of which the following description provides examples. It will also be better understood with reference to the accompanying drawings in which: Figure 1 shows a map of pBluescript® II phagemid vector and pVA838.

Figure 2 is a restriction map of PDN488L showing ORFs and subclones. The nucleotides are numbered from the first nucleotide of the EcoR I restriction site located proximal to the Sac I restriction site in the pBluescript® II phagemid vector Sac I Kpn I MCS of pDN488L. The translation is in the direction indicated by the bold arrows.

Figure 3 shows the DNA sequence of 6.8 kb base EcoR I fragment showing the leotide and amino acid sequences for both zooA and zif. It will be appreciated tha strand of nucleic acid coding for zif is complementary to the non-coding t_ strand hown expressly in Figure 3.

WO 99/26969 PCT/NYZ98/00171 6 DESCRIPTION OF THE INVENTION The focus of the invention is on the applicants identification of the gene encoding zoocin A immunity factor (zif). This gene is capable of protecting cells which express zoocin A against the effects of that enzyme.

The zifgene has been identified from S. equi subsp. zooepidemicus 4881 and has the sequence given in Figure 3. This sequence is of the non-coding strand, with the coding strand being complementary. The sequence of the coding strand is recited as SEQ ID NO. 2.

However, it will be appreciated that the sequence need not always be that shown in Figure 3 but can instead be a functionally-equivalent variant of that sequence.

Such variants are in no way intended to be excluded and the resultant molecules are referred to herein as "zif-like genes".

The amino acid sequence of zif (which is coded for by the nucleotides of the coding strand) is also shown in Figure 3. Again, variations are possible while retaining functional equivalency.

The phase "functionally equivalent variants" recognises that it is possible to vary the amino acid/nucleotide sequence of a protein while retaining substantially equivalent functionality. For example, a protein can be considered a functional equivalent of another protein for a specific function if the equivalent protein is immunologically cross-reactive with and has at least substantially the same function as, the original protein. The equivalent can be, for example, a fragment of the protein, a fusion of the protein with another protein or carrier, or a fusion of a fragment which additional amino acids. For example, it is possible to substitute amino acids in a sequence with equivalent amino acids using conventional techniques. Groups of amino acids normally held to be equivalent are: Ala, Ser, Thr, Pro, Gly; Asn, Asp, Glu, Gin; His, Arg, Lys; Met, Leu, Ile, Val; and WO 99/26969 PCT/NZ98/00171 7 Phe, Tyr, Trp.

Equally, DNA sequences encoding a particular produce can vary significantly simply due to the degeneracy of the nucleic acid code.

The probability of one sequence being functionally equivalent to another can be measured by the computer algorithms BLASTP (Altschul, S. F. et al (1990)) and FASTA (Pearson, W. R. et al (1988)) for proteins and DNA respectively.

The zifgene or zif-like gene of the invention can be inserted into organisms which are to be transformed with the zooA gene (which encodes zoocin A) so that a recipient organism which is zoocin A sensitive is protected by expression of the zif gene. The action of zif in protecting a zoocin A producer cell from the.otherwise lethal action of its own product is believed to involve the modification of the cells peptidoglycan cross-links to a chemical form non-hydrolysed by zoocin A.

Organisms which may be usefully transformed with the zif gene include any foodacceptable or pharmaceutically acceptable non-pathogenic organism. When the gene is inserted into zoocin A susceptible organisms, these organisms can be subsequently or simultaneously transformed with zooA in a manner which allows production of zoocin A. The zif gene protects the transformed organism from the lethal effects of zoocin A produced.

It will of course be appreciated that the terms "transformed" or "transformation" are used herein in their broadest possible sense. While normally a recombinant transformation process will be employed, any so-called "natural transfer" approach can also be used. "Natural transfer" approaches involve the placement of an organism including DNA encoding both zif and zoocin A in the proximity of the organism to which the DNA is to be transferred, and allowing exchange to occur naturally.

Both recombinant and natural transfer of DNA from one host organism to another is now routine in the art. It will therefore be appreciated that any conventional approach can be employed, so long as the desired transformation occurs.

WO 99/26969 PCT/NZ98/00171 8 It will however be more usual to effect transformation by recombinant means. This is the preferred approach taken for this invention and normally will involve the use of transformation vectors/gene constructs.

While it is conceivable that separate vectors/constructs could be employed to separately transfer the zif and zoo A genes to a recipient organism, it would be more usual for both genes to be contained in the same vector/construct.

The vector pSB 1131 is a preferred vector for this purpose.

Preferred non-pathogenic organisms for use in the invention include yeasts and bacteria. In particular, organisms having a genus selected from non-pathogenic strains of streptococcus are particularly useful. Especially preferred are nonpathogenic strains of Streptococcus gordonii.

Organisms transformed with the gene of the invention may be used as preservatives in processed cheese, various pasteurised dairy products, canned vegetables, hot baked flour products and pasteurised liquid egg. They may also be used in preservation of naturally fermented foods such as beer, wine, yoghurt and cheeses.

The transformed organisms and/or extracts of the organisms may also be used to prepare pharmaceutical compositions for use topically to prevent establishment of infectious diseases of humans and animals. Such topical compositions are useful in treatment of skin conditions, such as ulcers, in which streptococci are significant pathogens and where poor blood supply limits the effectiveness of systemically administered antibiotics.

Group C streptococci are serious animal pathogens, particularly of horses and are responsible for considerable economic loss to the bloodstock industry. As with GAS in humans, the primary route of infection for these organisms is believed to be the respiratory tract and it is contemplated that the incorporation of organisms according to the invention which express zoocin A with animal feeds may reduce colonization rates in these animals, and hence the rate of serious disease.

WO 99/26969 PCT/NZ98/00171 9 It is however presently preferred that the transformed organisms and/or their zoocin A-containing culture fluid be included in a composition intended for human ingestion (such as a foodstuff, nutriceutical or confectionery). This is particularly the case where the intention is to treat or prevent problems associated with the organisms S. mutans and/or S. sobrinus. These organisms inhabit the oral cavity and, as stated previously, are considered to be the major aetiological agents of dental caries. Their suppression in the oral cavity reduces the incidence of dental caries.

Further, this is particularly the case where the intention is to treat or prevent problems associated with S. pyogenes. These organisms colonise the tonsillar region of the oral cavity and, as stated previously, are the major aetiological agents of GAS associated disease.

Foodstuffs such as processed cheeses and yoghurts are particularly appropriate for such applications. Confectioneries such as wine gums and chewing gums are also contemplated.

The transformed organism of the invention may be admixed with food products, confectioneries and pharmaceutical carriers by conventional means. For fermented products such as yoghurts, conventional methods may also be used including the step of adding the transformed microorganism at the time of culturing the product.

Preferably the transformed microorganism is of the same species as conventionally used for the preparation of the fermented product thus allowing the preparation of the zoocin A and the fermented product to occur simultaneously.

The invention will now be described with reference to the following non-limiting examples.

WO 99/26969 PCT/NZ98/00171 EXAMPLE 1 Materials and Methods.

i) Bacterial strains and plasmids.

Stock cultures of all strains were stored in skim milk at -70°C. Strains in regular use were maintained as plate cultures and subcultured every two weeks. E. coli (Woodcock et al (1989), Raleigh et al (1989)) was grown routinely at 37°C in air and S. equi subsp. zooepidemicus 4881 (Schofield and Tagg (1983)) and S.gordonii DL1 (Macrina et al (1982)) in 5% CO 2 in air atmosphere at 37C.

E. coli DH5aF' was routinely cultured in 2xYT medium (16 g bacto-tryptone (Difco Laboratories, Detroit, MI, USA), 10 g bacto-yeast extract (Difco), and 5 g NaCI (Riedel-de Haen AG, Seeize, Germany) to one litre of distilled water, purified with a Milli-Q system (Millipore Inc., France) (MQ water), Luria-Bertani (LB) medium (10 g bacto-tryptone (Difco), 5 g bacto-yeast extract (Difco), and 10 g NaCl (Riedel-de Haen AG) to one litre of MQ water) or on LB agar (LBA) plates. LBA was prepared by supplementing LB medium with 1.5% bacto-agar (Difco). Plates containing antibiotics were prepared by supplementing LBA with either 100 mg/ml ampicillin (LBA+Ap), 250 mg/ml erythromycin (LBA+Em250), 500 mg/ml erythromycin (LBA+EmS00) or 25 mg/ml chloramphenicol (LBA+Cm). All antibiotics were manufactured by Sigma (Sigma Chemical Co., St. Louis, MO, USA). LBA containing antibiotics was stored at 40C for periods of up to two weeks.

Streptococcus gordonii DL1 strains were routinely cultured in Todd Hewitt broth (THB) (Difco), on Columbia Agar Base (CAB) (GIBCO BRL, Life Tec. Ltd., Paisly UK) plates or on blood agar (BA) (CAB supplemented with 5% whole human blood (Dunedin Public Hospital, Dunedin, Antibiotic containing agar plates were prepared by supplementing CAB with 10 mg/ml erythromycin (CAB+Em). Prior to transformation S. gordonii DL1 were grown in Brain Heart Infusion (BHI) (Difco) supplemented with 0.5% bacto-yeast extract (Difco), 1% membrane filtered horse serum (GIBCO BRL) and 0.1% glucose (Serva Feinbiochemica GmbH Co. KG, Heidelberg, Germany) (BHS broth). CAB containing antibiotics was stored at 4oC for periods of up to two weeks.

WO 99/26969 PCT/NZ98/00171 11 Bacterial strains and their plasmids used in this study are described in Table 1.

Maps of pBluescript® II phagemid vector (Stratagene, La Jolla, CA, USA) and pVA838 (Macrina et al (1982)) are given in Figure 1.

ii) Genetic manipulations.

Restriction enzyme digestion, ligation, and electrophoresis procedures.

Unless otherwise stated, cloning methods were carried out as previously described (Sambrook et al (1989)). Restriction digests were performed according to the manufacturers instructions; EcoR I, Pst I, Hind III, Xba I and Pvu II (Boehringer Mannheim Gmbh, Mannheim, Germany); ClaI and EcoRV (Amersham International plc, Amersham, UK); and Sma I (New England Biolabs, Beverly, MA, USA). Calf Intestinal Phosphatase (CIP) (New England Biolabs) was used to treat vector digests prior to ligation as per the manufacturers instructions. Ligations were performed at temperatures between 120C and 15°C overnight using T4 DNA ligase (Boehringer Mannheim Gmbh) as per the manufacturers instructions. Prior to use in transformations, ligation mixtures were ethanol precipitated with 1 il glycogen (Boehringer Mannheim Gmbh) and resuspended in 10 pl Milli-Q water.

Unless otherwise stated, gel electrophoresis was performed using 1% agarose (Sigma) gels prepared and run with Tris-acetate EDTA (TAE) buffer (per litre: 4.84 g Tris base (Serva), 1.142 ml glacial acetic acid (Rh6ne-Poulenc Chemicals Ltd., Bristol, UK), and 0.8 ml 0.5 M ethylenediaminetetra-acetate (BDH Laboratory Supplies, Poole, UK) (EDTA) at 75 100 V. Electrophoresis was performed using a Pharmacia Electrophoresis Constant Power Supply ECPS 2000/300 (Pharmacia Fine Chemicals AB, Uppsala, Sweden), and gel electrophoresis apparatus including a range of submarine gel tanks: 20 cm x 24 cm Model H4 (Betheseda Research Laboratories, Gaithersburg, MD, USA), 11 cm x 14 cm HORIZON 11*14 (GIBCO BRL), 8 cm x 6 cm minigel tank (Bio-rad).

E. coli DH5aF' electro-transformation.

Unless otherwise stated, preparation of electo-competent E. coli DH5cF' cells and electro-transformation of electro-competent E. coli DH5acF' cells was performed as previously described (Dower (1988)). E. coli DH5cF' electro-transformations were performed with a Biotechnologies and Experimental Research Inc. (BTX) BTX® E.

WO 99/26969 PCT/NZ98/00171 12 coli TransPorator T M (BTX, SanDiego, CA, USA), a Pharmacia LKB 2197 Power Supply (Pharmacia LKB, Broma, Sweden), and 0.1 cm electrode gap Gene PulserTM Cuvettes (Bio-rad Laboratories, Hercules, CA, USA). 40 pl aliquots of E. coli DH5aoF' electrocompetent cells were maintained at -700C until required. Following electroporation, 1 ml of 2xYT broth was immediately added to the transformation mixture and the cells resuspended and transferred to a glass vial. Resuspended cells were incubated at 370C with shaking at 200 rpm for 1 hour to enable the plasmid encoded antibiotic resistance genes to be expressed. Dilutions of the mixture were spread plated on appropriate antibiotic-containing media and incubated at 370C overnight.

Characterisation of E. coli DH5aF' transformants carrying recombinant pBluescript® II phagemid vectors.

Colonies growing on LBA+Ap were patched with a sterile toothpick onto LBA+Ap screening plates spread with 4 pl of 200 mg/ml Isopropyl-b-D-thiogalactoside (IPTG) (Boehringer Mannheim GmbH) and 40 ll of 20 mg/ml 5'-Bromo-4-chloro-3-indoyl-b- D-galactopyranoside (X-gal) (Boehringer Mannheim GmbH). After overnight incubation E. coli DH5aF' transformants containing Bluescript® II phagemid vectors (Stratagene) (Alting-Mees et al, 1989; Short et al, 1988) with inserts were identified as white patches amongst a background of blue patches. A small amount of culture was picked from each white patch with a toothpick and resuspended in pl of cracking solution (In one ml: 835 pl MQ water, 100 pl glycerol (BDH), 25 pl Sodium Dodecyl Sulphate (SDS) (BDH), 25 pl 2 M NaOH (BDH), 10 pI 0.5 M EDTA (BDH) and 5 pl 2% bromocresol green Baker Co., Phillipsburg, NJ, USA)) and incubated at 650C for 30 minutes. After incubation each sample was carefully loaded into dry wells in an agarose gel and electrophoresed at 40 V for approximately 15 minutes until each sample had completely entered the gel. TAE buffer was then added to cover the gel and electrophoresis continued at 75 100 V until completion. DNA bands were visualized by staining the gel for 10 minutes in 0.5 pg/ml ethidium bromide (Sigma) solution. Supercoiled plasmids were clearly visible after ethidium bromide staining. Recombinants were initially characterized by comparing their plasmid size with the plasmid size of supercoiled pBluescript® II phagemid vector carrying no insert.

WO 99/26969 PCT/NZ98/00171 13 E. coli DH5aF' transformants yielding appropriately sized plasmids were used to inoculate 2.5 ml 2xYT broth supplemented with 100 pg/ml ampicillin. Following overnight incubation at 370C plasmid DNA was extracted from 1.5 ml of each culture using the Quantum prepTM plasmid miniprep kit (miniprep) (Bio-rad) and the plasmid DNA eluted from the miniprep matrix in 100 ml of MQ water according to the manufacturers instructions. The eluted DNA was stored at -20°C. The remaining culture was centrifuged and the pellet resuspended in 10% skim milk and stored at -700C.

Those transformants carrying pBluescript® II phagemid vector with an insert were characterized by restriction digestion of miniprep plasmid DNA. Plasmid DNA was digested with restriction enzymes chosen to linearise the plasmid. EcoR I was used to linearise plasmid DNA from pSB1006, pSB1291, pSB1205, and pSB1014 transformants. Sac I was used to linearise plasmid DNA from pSB10313 and pSB1047 transformants, Hind III to linearise plasmid DNA from pSB1083 transformants, and Pst I to linearise plasmid DNA from pSB961 and pSB981 transformants. The digested plasmid DNA was electrophoresed and the size of the plasmid determined relative to known DNA sizing standards (either Pst I or Hind III digested 1 DNA (New England Biolabs)). DNA bands were visualized by staining the gel for 10 minutes in 0.5 pg/ml ethidium bromide (Sigma) solution. The size estimate obtained for each plasmid was compared with the predicted size determined from the previously published restriction map of pDN488L (Simmonds et al (1997)).

Characterisation of E. coli DH5aF' transformants carrying recombinant pVA838 vectors.

E. coli DH5xF' colonies visible on LBA+Em250 after 12 16 hours incubation were streaked onto LBA+Em500 and LBA+Cm plates and incubated overnight at 37oC.

Transformants able to grow overnight on LBA+Em500 but not on LBA+Cm were initially characterized as previously described (Characterisation of E. coli transformants carrying recombinant pBluescript® II phagemid vectors) and the size of their supercoiled plasmids compared with the size of supercoiled pVA838 (Macrina et al (1982)).

WO 99/26969 PCT/NZ98/00171 14 E. coli DH5aF' isolates identified as carrying plasmids of the appropriate size were grown overnight at 370C in 5 ml 2xYT broth supplemented with 500 pg/ml Em.

Plasmid DNA was extracted from 3 ml of each culture using the Quantum prepTM plasmid miniprep kit (Bio-rad) and the plasmid DNA eluted from the miniprep matrix in 100 ml of MQ water according to the manufacturers instructions. The eluted DNA was stored at -20oC. The remaining culture was centrifuged and the pellet resuspended in 10% skim milk and stored at Transformants carrying pVA838 vector with an insert were characterized by restriction digestion of miniprep plasmid DNA essentially as described previously (Characterisation of E. coli DH5aF' transformants carrying recombinant pBluescript® II phagemid vectors). Eco R I was used to linearise plasmid DNA from pSB1847 transformants whereas Eco R I digestion of plasmid DNA from pSB 1311 transformants yielded two fragments (ie. 6.8 kb insert and 9.2 kb vector).

Construction of subclones using pBluescript® II phagemid vector.

Plasmids were constructed using a subcloning strategy based on the previously published restriction map of pDN488L (Simmonds et al (1997)). The cloning of pDN488L, pDN2.2, and pDNO.8 has been previously described. Unless otherwise stated the following method was used to construct all pBluescript® II SK(+) phagemid vector subclones.

At least 1 pg pBluescript® II phagemid vector miniprep DNA was digested with the appropriate restriction enzyme(s), treated with CIP and electrophoresed. Unless otherwise stated, at least 1 pg of the appropriate parent plasmid miniprep DNA was digested with the appropriate restriction enzyme(s), treated with CIP and electrophoresed. Bands corresponding to the 2.9 kb linearised pBluescript® II SK(+) phagemid vector, and the desired insert fragment (Table 1) were extracted from the gel using a Prep-A-Gene T M DNA purification kit (Bio-rad), eluted with 30 pi MQ water according to the manufacturers instructions and ligated. Following ligation of the vector and insert, electro-competent E. coli DH5aF' were transformed as previously described coli electro-transformation) and transformants isolated and characterized as previously described (Characterisation of E. coli transformants carrying recombinant pBluescript® II phagemid vectors).

WO 99/26969 PCT/NZ98/00171 An alternative method was used to construct pSB1006, pSB1014, and pSB1025. A restriction enzyme was chosen that cut once within the 6.8 kb insert of pDN488L and once within the pDN488L multi-cloning site (MCS). Restriction digestion produced two fragments, one corresponded to linearised pBluescript® II SK(+) phagemid vector incorporating a section of pDN488L, and the other corresponded to the remaining region of pDN488L and a short segment of the MCS. The digest was electrophoresed and the band corresponding to linearised pBluescript® II SK(+) phagemid vector incorporating pDN488L DNA was extracted from the gel using a Prep-A-GeneT DNA purification kit (Bio-rad), eluted with 30 pl MQ water according to the manufacturers instructions and self-ligated. Following self-ligation, electrocompetent E. coli DH5aF' were transformed as previously described coli electrotransformation) and transformants isolated and characterized as previously described (Characterisation of E. coli transformants carrying pBluescript® II SK(+) phagemid vectors). pSB1083 was constructed similarly, differing in that the parent plasmid was pSB1014. pSB1047 was constructed similarly, differing in that the parent plasmid was pSB1006 and that two enzymes with unique but compatible restriction sites were used to digest pSB1006.

pSB961 was pBluescript® II phagemid vector incorporating the 0.7 kb Eco RV Pst I fragment of pDN2.2.

pSB981 was pBluescript® II phagemid vector incorporating the 1.5 kb Eco RV Pst I fragment of pDN2.2.

pSB 1006 was pBluescript® II phagemid vector incorporating the 3.7 kb Cla I EcoR I fragment of pDN488L. A Cla I digestion of pDN488L was electrophoresed and the 6.6 kb band was extracted from the gel and self-ligated as described previously (Construction of clones using pBluescript® II phagemid vectors).

pSB1014 was pBluescript® II phagemid vector incorporating the 3.1 kb Hind III EcoR I fragment of pDN488L. A Hind III digestion of pDN488L was electrophoresed and the 6.0 kb band extracted from the gel and self-ligated as WO 99/26969 PCT/NZ98/00171 16 described previously (Construction of clones using pBluescript® II phagemid vectors).

pSB1025 was pBluescript® II phagemid vector incorporating the 3.4 kb Eco RV EcoR I fragment of pDN488L. An Eco RV digestion of pDN488L was electrophoresed and the 6.3 kb band extracted from the gel and self-ligated as described previously (Construction of clones using pBluescript® II phagemid vectors).

pSB1083 was pBluescript® II phagemid vector incorporating the 2.3 kb Hind III Xba I fragment of pSB1014. A Xba I digestion of pSB1014 was electrophoresed and the 5.2 kb band extracted from the gel and self-ligated as described previously (Construction of clones using pBluescript® II phagemid vectors).

pSB 10313 was pBluescript® II phagemid vector incorporating the 0.8 kb Xba I EcoR I fragment of pSB1014.

pSB1047 was pBluescript® II phagemid vector incorporating the 0.2 kb Cla I Eco RV fragment of pSB1006. An Eco RV/Sma I digestion of pSB1006 was electrophoresed and the 3.1 kb band extracted from the gel and self-ligated as described previously (Construction of clones using pBluescript® II phagemid vectors).

pSB1097 was pBluescript® II phagemid vector incorporating the 0.3 kb Hind III EcoR I fragment of pSB 1025.

pSB1291 was pBluescript® II phagemid vector incorporating the 4.0 kb Pst I EcoR I fragment of pDN488L.

Construction of clones using pVA838 vector.

The following procedure was used to construct pSB1311 in E. coli DH5aF'. pVA838 miniprep DNA (at least 1 jg) was digested with EcoR I, treated with CIP and electrophoresed. pDN488L miniprep DNA (at least 1 pg) was digested with EcoR I, WO 99/26969 PCT/NZ98/00171 17 treated with CIP and electrophoresed. Bands corresponding to the 9.2 kb EcoR I digested pVA838 vector and the 6.8 kb EcoR I digested pDN488L insert were extracted from the gel using the Prep-A-GeneTM DNA purification kit (Bio-rad) and eluted with 30 pl MQ water according to the manufacturers instructions. Following ligation of the vector and insert, electro-competent E. coli DH5aF' were transformed as previously described coli electro-transformation) and transformants isolated and characterized as previously described (Characterisation of E. coli transformants carrying recombinant pVA838 vectors).

The following procedure was used to construct pSB1847 in E. coli DH5aF'. pVA838 miniprep DNA (at least 1 jig) was digested with EcoR I and Pvu II, treated with CIP and electrophoresed. pSB1291 miniprep DNA (at least 1 pg) was digested with EcoR I and Sma I and electrophoresed. Bands corresponding to the 8.9 kb EcoR I/Pvu II digested pVA838 vector and the 4 kb EcoRI/Sma I pSB1291 insert were extracted using the Bio-rad Gel Extraction Kit (Bio-rad) and eluted with 30 pl MQ water according to the manufacturers instructions. Following ligation of the vector and insert, electrocompetent E. coli DH5aF' were transformed as previously described (E.

coli electro-transformation) and transformants isolated and characterized as previously described (Characterisation of E. coli transformants carrying recombinant pVA838 vectors).

Transformation of S. gordonii DL1 with pSB1311 and pSB1847.

S. gordonii DL1 was freshly subcultured on CAB prior to each transformation. 50 pl of an overnight culture of S. gordonii DL1 in BHS broth was used to inoculate 5 ml of pre-warmed BHS broth and the culture incubated (with a loosened cap) at 370C in C02 in air for 3 hours. 50 pl of this was used to inoculate 5 ml of pre-warmed BHS broth and the culture incubated (with a loosened cap) at 370C in 5% C02 in air for a further one hour. After one hour the culture was dispensed in 0.8 ml volumes into glass vials and mixed with 10 50 pl (containing a minimum of 1 pg of DNA) of pSB1311 and pSB1847 miniprep DNA obtained from E. coli DH5aF' (pSB1311) and (pSB1847). Vials containing S. gordonii DL1 cells and pVA838 with no insert or S.gordonii DL1 cells and no DNA were included in each experiment as positive and negative controls respectively. Transformation mixtures were incubated for 3 4 WO 99/26969 PCT/NZ98/00171 18 hours at 37oC in 5% C02 in air before dilutions of each mixture were spread plated on CAB+Em and the plates incubated for 24 hours at 37oC in 5% C02 in air.

After incubation colonies were picked from the transformation plates, streaked onto CAB+Em and incubated overnight at 370C in 5% C02 in air. Plasmid DNA was extracted from each isolate as previously described (Vriesema et al, 1996) and resuspended in 30 pl MQ water. S. gordonii DL1 plasmid DNA obtained in this way was characterized by restriction analysis as previously described (Characterisation of E. coli DH5aF' transformants carrying recombinant pVA838 vectors). Plasmid DNA extracted from S. gordonii DL1 (pSB1311) and (pSB1847) transformants was similarly compared with plasmid DNA extracted from E. coli DH5aF' (pSB1311) and (pSB1847) transformants respectively. The E. coli DH5aF' plasmid DNA used for comparison with the S. gordonii DL1 plasmid DNA originated from the same miniprep sample used in the respective S. gordonii DL1 transformation.

Transformants were stored in 10% skim milk at iii) Phenotypic characterization of DL1 transformants.

Testing for BLIS production by deferred antagonism.

BLIS production was assessed using the deferred antagonism procedure (Tagg Bannister (1979)). Briefly, a 1-cm wide streak of the test strain was inoculated diametrically across the surface of CAB plates using a cotton swab heavily charged with cells from a freshly grown THB culture. The inoculated plates were incubated at 37oC for 18 hour in air plus 5% CO 2 after which the visible growth was removed by scraping with the edge of a glass slide. The surface of the medium was sterilized by exposure to chloroform vapour for 30 minutes, aired for 30 minutes and the nine standard indicator strains (I1, Micrococcus luteus; 12, S. pyogenes; 13, S. anginosus; 14, S. uberis; 15, S. pyogenes; 16, Lactococcus lactis subsp. lactis; 17, S. pyogenes; 18, S. pyogenes and 19, S. equisimilis) (Tagg et al, 1979) inoculated from 18 hour THB cultures across the line of the original producer strain with use of cotton swabs.

After incubation for 18 hours in 5% CO 2 at 37 oC the extent of inhibition of each indicator strain was recorded as: for no inhibition and if the zone was wider than each edge of the producer streak.

WO 99/26969 PCT/NZ98/00171 19 Testing for BLIS production by the surface spot method.

BLIS activity in liquid samples was quantitated using the surface spot method (SSM) described by Jack (1991). Briefly, a 20 pl droplet of the sample to be tested was spotted out on the surface of a CAB plate and left to soak into the agar plate. The plate surface was then sterilized by exposure to choloroform vapour for 30 minutes, aired for 30 minutes and standard indicator 12 (overnight culture in THB broth) swabbed evenly onto the surface of the plate. Following overnight incubation at 37oC for 18 hours in air plus 5% CO2, the presence of inhibitory activity was visualized as a circular zone of inhibition in the 12 lawn at the site of droplet deposition. The titre of inhibitory activity in the samples were determined by making doubling dilutions of the test samples and plating out 20 ml drops of each dilution. The reciprocal of the highest doubling dilution at which inhibitory action was observed is given as the titre.

Testing for Zoocin A production.

S. gordonii DL1, S. gordonii DL1 (pVA838) and S. gordonii DL1 (pSB1311) and (pSB 1847) were tested for zoocin A production by the deferred antagonism method.

Testing for sensitivity to Zoocin A.

S. gordonii DL1, S. gordonii DL1 (pVA838) and S. gordonii DL1 (pSB1311) and (pSB1847) were tested for sensitivity to zoocin A by both a modification of the deferred antagonism method, and a modification of the SSM. In the modified deferred antagonism method, the zoocin A producer strain, S. equi subsp.

zooepidemicus 4881 was used as the test strain and S. gordonii DL1, S. gordonii DL1 (pVA838) and S. gordonii DL1 (pSB1311) and (pSB1847) standard indicators 11 and 12 and S. equi subsp. zooepidemicus 4881 used as the indicator strains. In the modified SSM, a partially purified preparation of zoocin A was diluted two-fold and ml drops spotted onto the surface of CAB plates. The presence of inhibitory activity was visualized by swabbing onto the surface of each plate a lawn of either S.

gordonii DL1, S. gordonii DL1 (pVA838) and S. gordonii DL1 (pSB1311) or (pSB1847), standard indicator II or 12 or S. equi subsp. zooepidemicus 4881.

iv) Sequencing the regions flanking zooA.

Subcloning and primer selection.

WO 99/26969 PCT/NZ98/00171 Plasmid DNA used for double stranded DNA sequencing was obtained from E. coli or E. coli XL1 blue pBluescript® II phagemid vector subclones by miniprep. E. coli DH5aF' and XL1 blue pBluescript® II phagemid vector subclones have been previously described (See Figure 2 and section; Construction of subclones using pBluescript® II phagemid vectors).

Table 2 contains a description of the primers used in this study. Universal M13 forward and reverse primers were synthesized by the Oligonucleotide Unit (Department of Biochemistry, University of Otago, Dunedin, NZ) and all other primers were synthesized by GIBCO BRL Custom Primers (GIBCO BRL). Universal M13 forward and reverse primers were used in sequencing reactions with pDNO.8, pSB961, pSB981, pSB1006, pSB1025, pSB10313, pSB1047, pSB1083 and pSB1291 plasmid DNA. SB108.3F2 and SB 108.3R2 primers were designed from the sequence data obtained from sequencing pSB1083 using universal M13 forward and reverse primers respectively. Primers SB108.3F2 and SB108.3R2 were used in sequencing reactions with pSB1083 plasmid DNA. 6.8kbcontigl to 6.8kbcontigl2 primers were designed from contiguous sequence data obtained from sequencing pDNO.8, pSB961, pSB981, pSB1006, pSB1025, pSB10313, pSB1047, pSB1083 and pSB1291 using universal M13 forward, universal M13 reverse, SB108.3F2 and SB108.3R2 primers. 6.8kbcontigl 6.8kbcontigl2 primers were used in sequencing reactions with pDN488L plasmid DNA. ZooA SBD primer 1 was designed from the previously reported zooA sequence (Simmonds et al (1997)). ZooA SBD primer 1 was used in sequencing reactions with pSB981 plasmid DNA. Sequencing reactions were performed by the Centre for Gene Research (University of Otago, Dunedin, NZ) using an Applied Biosystems (ABI) 373 Version 3.0 DNA sequencer and the manufacturers' procedures and specifications.

Sequence analysis.

DNA sequence analysis was performed using an series 6100/66 Power Macintosh Apple computer. The sequence chromatographs were viewed and trimmed using the SeqEd (ABI) application. DNA sequences were compiled and a contiguous sequence was constructed using the DNAstar Seqman application. Open reading frames and putative amino acid sequences were determined using the DNAstar EditSeq application and visualized using either the DNAstar MapDraw or GeneJockey WO 99/26969 PCT/NZ98/0071 21 (Biosoft, Cambridge, England) applications. DNA and amino acid sequence homology searches were performed using the non-redundant protein and nucleotide databases and the gapped basic local alignment search tool (BLAST) program of the National Centre for Biotechnology Information (NCBI) (NCBI, Bethesda, MD, USA).

Sequence alignments and sequence similarity calculations were performed using the DNAstar Megalign application.

Results and Technical Discussion Transformation of E. coli DH5aF' and characterization of transformants.

E. coli DH5aF' were transformed by electro-poration with Bluescript® II SK(+) phagemid vector with a transformation efficiency of approximately 106 transformants per pg plasmid DNA. Transformation efficiency for the electrotransformations of pSB1006, pSB1014, pSB1025, pSB10313, pSB1083, and pSB1097 were less than 20 transformants per pg plasmid DNA. All other recombinant Bluescript® II phagemid vectors gave transformation efficiencies of between 103 10 4 transformants per pg plasmid DNA. 2 50% of E. coli pBluescript® II phagemid vector transformants screened on LBA+Ap containing IPTG and X-gal produced white colonies. 5 100% of white transformants were initially characterized as containing the predicted recombinant pBluescript® II phagemid vector. All pBluescript® II phagemid vectors characterized by restriction analysis yielded banding patterns consistent with those predicted by the cloning strategy. The discrepancies observed between E. coli transformation efficiency and the number of isolates characterized as possessing plasmids with inserts were considered to be the result of minor variations in miniprep preparations, restriction digestion, gel extraction, ligation, and/or electro-poration.

pBluescript® II phagemid vector subclones that involved self-ligation were the simplest to characterize. Although all arose from low efficiency transformations almost 100% of white colonies were shown to carry plasmids with an appropriate insert. In contrast, many of the isolates obtained from higher efficiency transformations were difficult to characterize because of the high background of blue colonies, and the lower proportion (as few as of white colonies that were WO 99/26969 PCT/NZ98/00171 22 subsequently shown to possess plasmids with an appropriate insert. The high background of blue colonies most likely arose as vectors cleaved with a single restriction enzyme recircularised due to incomplete phosphatase treatment. The high proportion of white colonies that did not harbour inserts was probably related to the use of LBA+Ap containing IPTG and X-gal plates unevenly spread with IPTG or X-gal, or the use of plates not prepared on the day of transformation.

E. coli DH5coF' were transformed by electro-poration with pVA838 with an efficiency of 10 4 10 5 transformants per pg plasmid DNA. Electro-competent E. coli were transformed with pSB1311 and pSB1847 with an efficiency of less than transformants per pg plasmid DNA. 100% of E. coli DH5aF' transformants that grew overnight on LBA+Em500, but not on LBA+Cm and were characterized by restriction analysis of plasmid DNA were shown to contain the predicted recombinant pVA838 vector. E. coli DH5F' were naturally partially resistant to erythromycin and very high concentrations were required to enable selection of pVA838 transformants expressing erythromycin resistance genes. It was noted that colonies that grew rapidly (within 12 16 hours) on LBA+250Em transformation plates were far more likely to contain pVA838 or recombinant pVA838 than those that grew after 16 hours. Only pVA838 or recombinant pVA838 transformants were subsequently able to grow on LBA+500Em overnight.

The genetic techniques used in the production of pSB1311 and pSB1847 transformants were essentially the same as those used to produce pBluescript® II phagemid vector subclones. Presumably due to the low copy of pVA838, plasmid miniprep yields were only 25% of those obtained from minipreps of pBluescript® II phagemid vector subclones. Doubling the amount of culture used to 3 ml increased yields, but increasing the volume of culture beyond 3 ml did not significantly enhance yield. Quantum prepTM uses an adaptation of the standard alkaline lysis miniprep method (Sambrook et al (1989)) so there is a limit to the amount of cells that can effectively be lysed without increasing the volume of lysis buffer that is added at the same time. It is most likely that inefficient ligation due to their larger size caused the low transformation efficiencies observed with pSB1311 and pSB1847.

WO 99/26969 PCT/NZ98/00171 23 Construction of E. coZiDH5a.F' subclones.

All E. cohDH5aF' subclones were constructed without difficulty. pVA838 has two restriction sites within the chloramphenicol resistance determinant that are suitable for shuttle cloning between E. coli DH5aF' and S. gordonii DL1 ie. EcoR I and Pvu II.

Use of the EcoR I site enabled pSB1311 to be constructed without difficulty. In contrast it was more difficult to decide the best strategy to use in constructing pSB1847. Although it was possible to use the Pvu II restriction sites flanking the pSB1291 MCS to directly transfer the 4.0 kb insert into pVA838 cleaved with Pvu II, this strategy was not favoured for a number of reasons. It has been reported that ligating fragments with two blunt termini, as opposed to one blunt and one overhanging terminus, is less efficient. Also, pSB1311 did not contain the lac promoter region and there was uncertainty about the effect that its inclusion into the new construct would have on the expression of zif. By using only streptococcal DNA to construct pSB1847 there was little doubt that any observed gene expression was initiated from a streptococcal promoter carried on the 4.0 kb insert and that any phenotypic differences observed between S. gordonii DL1 (pSB1311) and (pSB1847) transformants were a consequence of the additional 2.8 kb of DNA carried by pSB1311.

Transformation of S. gordonii DL1 and characterization of transformants.

Transformation of S. gordonii DL1 with pVA838 gave a transformation efficiency of 103 transformants per pg plasmid DNA. Transformation of S. gordonii DL1 with pSB1311 or pSB1847 gave an efficiency of less than 10 transformants per ig plasmid DNA. Because of the low efficiency of transformation all transformants suspected of carrying a recombinant pVA838 plasmid were phenotypically characterized. Restriction analysis showed plasmid DNA extracted from transformed S. gordonii DL1 to be identical to that obtained from the respective E.

coli DH5aF' strain.

The low transformation efficiency obtained with pSB1311 and pSB1847, but not with pVA838 transformations of S. gordonii DL1 is unlikely to be due to genes carried on the respective inserts as transformants appeared normal in all respects other than their zoocin A resistant zoocin A producer phenotype. pVA838 in S.

gordonii DL1 was very stable, and pSB1311 and pSB1847 were also able to be WO 99/26969 PCT/NZ98/00171 24 maintained without antibiotic selection. It is more likely that the larger size of pSB1311 and pSB1847 made DNA uptake by competent S. gordonii DL1 cells less efficient.

Phenotypic characterization of strains.

The results of the testing of strains for production of and sensitivity to zoocin A by deferred antagonism are given in Table 3. That the inhibitory profile produced by S.

equi subsp. zooepidemicus 4881 was the same as that produced by S. gordonii DL1 carrying pSB1311 but not S. gordonii DL1 carrying pSB1847 confirming that zooA is essential for zoocin A production. A partially purified preparation of zoocin A produced endpoint titres of 2048, 128, 128, 0, 0, 0 and 0 when tested by SSM against standard indicator 12, S. gordonii DL1, S. gordonii DL1 (pVA838), S. gordonii DL1 (pSB1847), S. gordonii DL1 (pSB1311), standard indicator II and S. equi subsp.

zooepidemicus 4881 respectively.

A summary of the results of the phenotypic testing of S. gordonii DL1 transformants is given in Table 4.

Sequence data and sequence analysis.

The subcloning strategy used enabled much of the 6.8 kb EcoR I fragment sequence to be established by sequencing from both ends of each subclone from M13 universal forward and reverse primers. Three internal primers were required to complete the single stranded contiguous sequence of the entire 6.8 kb fragment.

Fragments carried by pSB1083 and pSB981 were too large to be sequenced completely with the M13 universal primers, consequently SB1083R2 and SB1083F2 primers were designed to enable sequencing of the remaining undetermined region within the 2.3 kb pSB1083 insert. SBD primer 1 was used to complete the sequencing of pSB981. To obtain a double stranded contiguous sequence the 6.8kbcontigl 12 primers were designed and used in sequencing reactions with pDN488L.

The nucleotide sequence of the 6.8 kb EcoR I fragment is given in Figure 3 and the identified open reading frames (ORF) are given in Figure 2. Sequence analysis indicated the prescence of an ORF encoding a 411 amino acid protein (including the "stop" residue) which we have called zif. (zoocin A immunity factor). That zif is WO 99/26969 PCT/NZ98/00171 essential for zoocin A immunity is supported by the observation that zoocin A inhibited S. gordonii DL1 and S. gordonii DL1 pVA838, but not S. gordonii DL1 carrying pSB1311 or pSB1847. zifis located on the 4.0 kb EcoR I Pst I fragment of pDN488L that is common to both pSB1311 and pSB1847.

Three further ORFs were identified (Figure ORF 1 encodes a 142 amino acid sequence with homology to the 5' region of rgg which regulates expression of glucosyltransferase in S. gordonii CH1. ORF 2 encodes a 244 amino acid sequence with homology to insertion sequence IS200 found in a range of bacteria including Clostridium perfringens, E. coli, and Yersinia pestis. However, ORF 2 is most closely related to an IS200 sequence identified in S. pneumoniae. ORF 3 encodes a 394 amino acid sequence with homology to a transposase/insertion sequence also identified in S. pneumoniae.

WO 99/26969 WO 9926969PCT/NZ98/OO1 71 Table 1. Bacterial strains and plasmids used in this study.

Species, strain, Size (kb) of Selective Strain and plasmid and (plasmid) Plasinid Insert antibioticc references E. coli XLl-blue (pDNO.8)a 3.5 0.6 AplCO Simmonds et al (1997) XL1-blue (pDN2.2)a 5.1 2.2 AplOC Simmonds et al (1997) XLI-blue (pDN488L)a 9.7 6.8 ApiQO Simmonds et al (1997) (pSK®IIj(+))a 2.9 No insert Ap100 Woodcock et al (1989); Raleigh et al (1989); Alting-Mees and Short (1989); Short et al (1988) (pSB961)a 3.6 0.7 AplQO herein (pSB981)a 4.4 1.5 Apl0O herein (pSBlOO6)a 6.6 3.7 ApIO0 herein (pSB1025)a 6.3 3.4 AplQO herein (pSBl0l4)a 6.0 3.1 ApiQO herein DH~aF' (pSB10313)a 3.7 0.8 AplOO herein (pSB1047)a 3.1 0.2 ApIQO herein (pSB1O83)a 5.2 2.3 AplQO herein DHSaF (pSB1097)a 3.2 0.3 ApiQO herein (pSB1291)a 6.9 4.0 Apl00 herein et al (1982) (pVA838)b 9.2 No insert Cm.25, Em500 herein (pSB131l)b 16.0 6.8 Cm25, Em500 herein (pSB1847)b 13.2 4.0 Cm25, Em500 herein S. gordonii DL1 (pVA838)b 9.2 No insert EmlO Macrina et al (1982) DL1 (pSBl3ll)b 16.0 6.8 EmlO herein DL1 (pSB1847)b 13.2 4 EmlO herein a b

C

Parent vector, pBluescriptO 11 phagemid vector (Stratagene).

Parent vector, pVA838 (kindly donated by Dr H. Jenkinson, Dept. of Oral Biology, University of Otago, Dunedin, NZ).

Antibiotic abbreviations; ApiQO0, 100 mg/mi ampicillin; cm2S, 25 mg/mi chioramphenicol; Em500, 500 mg/nil erythromycin and EmlO, 10 mg/nil erythromycin.

WO 99/26969 WO 9926969PCTNZ98/O1 71 27 Primers used in this study. Table 2.

Primer position Designation sequence a orientation Universal M 13 reverse GGAAACAGCTATGACCATG 806 )b Universal M 13 forward GTAAAACGACGGCCAGT 579g SB 108.3R2 TGAGTGAAGCAACTG 1214 SB 108.3F2 TTATGCTCCAGCACT 2680 ZooA SBD primer 1 GGGTI'GATAATGG 4547 )C 6.8kbcontigl AGTCTGTAGG'ITCGTATTCT 1375 6.8kbcontig2 TGTGGC TCA'ITAGGTCCAA 1754 )c 6.8kbcontig3 AGTACTGTrGGACCTAATGA 1780 6.Skbcontig4 TGCGGGTGCGCGACGAAGGT 2212 6.8kbcontig5 'ITGGGTATAACCTI'CGTCGC 2184 )c 6.8kbcontig6 TTCCCAGTAATACCTAACAT 2592 )c 6.8kbcontig7 TCATAATACTCAAGTCC TfT 3024 )c 6.8kbcontig8 AATATCAAG'ITCTAATACAT 3375 )c 6.8kbcontig9 TCAATC'F1GTCTCTGTCC'IT 5050 )c 6.8kbcontiglO CGTCTTTGAGCTACTCTGA 5231 6.8kbcontig 11 GGCGAATCAAAGTC ITGTAG 5910 )c 6.8kbcontigl2 'ITCTCGA ITGCGCAGGCTAC 5945 a Primer sequence is presented 5' to 3'.

b Primer position is given as the first nucleotide of the primer relative to the sequence of the pBluescript® Il SK phagemid vector as previously described (Short et al, 1988; Alting-Mees et al, 1989).

c Primer position is given as the first nucleotide of the primer relative to the sequence of the 6.8 kb fragment of pDN488L as designated in Figure 3.

WO 99/26969 Table 3.

PCT/NZ98/OO1 71 28 Production and sensitivity to BLIS of strains tested by deferred antagonism.

Indicator Producer strain strains 4 8 8 1 a DLjb pVA838c pSBl3lld pSB1847e 11-- 12 14- 17 18 19 4881---- DU1 pVA838 pSB1311 rpSB1847 a b

C

d

C

S. equi subsp. zooepidemicus 488 1.

S. gordonii DLL1 S. gordonii DL 1 (pVA83 8).

S. gordonii DLl1 (pSB 1311).

S. gordonii DL 1 (pSB 1847).

WO 99/26969 PCT/NZ98/00171 29 Phenotypic characterization of S. gordonii DL1 clones. Table 4.

Phenotype Strain and Zoocin A Zoocin A Em plasmid Genotype a production immunity resistanceb S. gordonii DL1 zooA zif- Em S DL1 (pVA838) zooA zif- E R DL1 (pSB1311) zooA zif+ EmR DL1 (pSB1847) zooA zif+ EmR S. equi subsp.zooepidemicus 4881 zooA zif+ Em

S

a zooA denotes the prescence or absence of the gene encoding zoocin A, zif denotes the prescence or absence of the gene encoding zoocin A immunity, EmR denotes the presence of the erythromycin resistance gene located on pVA838 and Em S indicates no erythromycin resistance gene.

b Denotes sensitivity or resistance to 10 pg/ml erythromycin.

The foregoing examples are illustrations of the invention. The invention may be carried out with numerous variations and modifications as will be apparent to those skilled in the art. For example, the native zif gene need not be used in the transformation. Deletions, insertions and substitutions relative in the zif gene may be used provided that the zif-type activity is retained. Similarly the gene may be incorporated into species other than used in Example 1. Likewise there are many variations in the way in which the invention can be used in pharmaceuticals and food products.

WO 99/26969 PCT/NZ98/00171

REFERENCES

Alting-Mees, M.A. and J.M. Short. 1989. pBluescript II: gene mapping vectors.

Nucleic Acids Res. 17: 9494.

Altschul, S.F. et al. 1990. J. Mol. Biol., 215:403-410.

Bronze, M.S. and J.B. Dale. 1996. The reemergence of serious group A streptococcal infections and acute rheumatic fever. Am. J. Med. Sci. 311: 41-54.

Dower, W.J. 1988. Transformation of E. coli to extremely high efficiency by electroporation. Mol. Biol. Rep. 6: 3-4.

Federal Register. 1988. Nisin preparation: affirmation of GRAS status as a direct human food ingredient. Fed. Regist. 54: 11247-11251.

Francis, G.R. Nimmo, A. Efstratiou, V. Galanis and N. Nuttall. 1993.

Investigation of milk-borne Streptococcus zooepidemicus infection associated with glomerulonephritis in Australia. J. Infect. 27: 317-323.

Jack, R.W. 1991. Production, purification and characterisation of the streptococcal lantibiotic streptococcin A-FF22. A Thesis. University of Otago, Dunedin, NewtZealand.

Jones J.A. Ritchie, P.D. Marsh and F.J.G. van der Ouderaa. 1988. The effect of long-term use of a dentifice containing zinc citrate and a non-ionic agent on the oral flora. J. Dent. Res. 67: 46-50.

Loesche, W.J. 1976. Chemotherapy of dental plaque infections. Oral Sci. Rev. 9: 65-107.

Loesche, S.A. Eklund, D.F. Mehlisch and B. Burt. 1989. Possible effects of medically administered antibiotics on the mutans streptococci, implications for reduction in decay. Oral Microbiol. Immunol. 4: 77-81.

WO 99/26969 PCT/NZ98/00171 31 Macrina, J.A. Tobian, K.R. Jones, R.P. Evans and D.B. Clewell. 1982. A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis. Gene.

19: 345-353.

Marsh, P.D. 1991. Dentifrices containing new agents for the control of plaque and gingivitis: microbiological aspects. Clin. Periodontol. 18: 462-467.

Pearson, W.R. et al., 1988. Proc. Natl. Acad. Sci., 85:2444-2448.

Raleigh, K. Lech and R. Brent. 1989. Selected topics from classical bacterial genetics. In. Current protocols in molecular biology. eds. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl. Publishing Associates and Wiley Interscience, New York. Unit 1.4.

Rodriguez, J.M. and H.M. Dodd. 1996. Genetic determinants for the biosynthesis of nisin, a bacteriocin produced by Lactococcus lactis. Microbiologia. 12: 61-74.

Sambrook, Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning. A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY,

USA.

Schofield, C.R. and J.R. Tagg. 1983. Bacteriocin-like activity of group B and group C streptococci of human and of animal origin. J. Hyg. 90: 7-18.

Short, J.M. Fernandez, J.A. Sorge and W.D. Huse. 1988. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 16: 7583-7600.

Simmonds, J. Naidoo, C.L. Jones and J.R. Tagg. 1995. The streptococcal bacteriocin-like inhibitory substance, zoocin A, reduces the proportion of Streptococcus mutans in an artificial plaque. Microb. Ecol. Health Dis. 8: 281-292.

Simmonds, Simpson, W.J. and Tagg J.R. 1997. Cloning and sequence analysis of zooA, a Streptococcus zooepidemicus gene encoding a bacteriocin-like inhibitory WO 99/26969 PCT/NZ98/00171 32 substance having a domain structure similar to that of lysostaphin. Gene. 189: 255-261.

Simmonds L. Pearson, R.C. Kennedy and J.R. Tagg. 1996. Mode of action of a lysostaphin-like bacteriolytic agent produced by Streptococcus zooepidemicus 4881.

Appl. Environ. Microbiol. 62: 4536-4541.

Tagg, J.R. and L.V. Bannister. 1979. "Fingerprinting" b-haemolytic streptococci by their production of and sensitivity to bacteriocine-like inhibitors. J. Med. Microbiol.

12: 397-411.

Thumm, G. and F. Gotz. 1997. Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus. Mol. Microbiol. 23: 1251-1265.

Vriesema, S.A.J. Zaat and J. Dankert. 1996. A simple procedure for isolation of cloning vectors and endogenous plasmids from viridans group streptococci and Staphylococcus aureus. Appl. Environ. Microbiol. 62: 3527-3529.

Woodcock, P.J. Crowther, J. Doherty, S. Jefferson, E. DeCruz, M. Noyer- Weidner, S.S. Smith, M.Z. Michael and M.W. Graham. 1989. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 17: 3469-3478.

WO 99/26969 PCT/NZ98/00171 33 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: University of Otago New Zealand Pastoral Agriculture Research Institute Limited (ii) TITLE OF INVENTION: Zoocin A Immunity Factor (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: ADDRESS: Russell McVeagh West-Walker STREET: The Todd Building, Cnr Brandon Street and Lambton Quay CITY: Wellington COUNTRY: New Zealand COMPUTER READABLE FORM MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: Windows (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: NZ 329227 FILING DATE: 21 November 1997 (vii) ATTORNEY/AGENT INFORMATION: NAME: Bennett, Michael Roy REFERENCE/DOCKET NUMBER: 23804 MRB (viii) TELECOMMUNICATION INFORMATION: TELEPHONE: 64 4 499 9058 TELEFAX: 64 4 499 9306 INFORMATION FOR SEQ ID NO. 1: SEQUENCE CHARACTERISTICS: LENGTH: 410 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 99/26969 34 (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1: PCTNZ98/0O1 71 Met Lys Phe Gin Glu Ile Asp Ala Leu LYS Phe Ala Asn Thr Gin Lys Arg Arg Ser Thr Phe Arg Glu Ile Ile Glu Leu Asp Arg Asp 140 Tyr Leu Lys Lys Thr 205 Tyr Giu Gly Ile Ser 270 Gin Ser Phe Giu Asn 32 u Phe Phe Leu Asp Giu 115 Asp Giu Val Asn Ser 180 Asp Al a Tyr Phe Asn 245 Ser Glu Arg Phe Glu Gin Phe Ile Gly Ser Lys Il.e Gly Ile Val Lys 155 Ser Asn Giu Thr Giu 220 Thr Ile Ile Lys Giu 285 Val Thr Ile ASP Val Lys Val Gly Leu Glu Giu Lys Tyr Phe Pro 1015 Arg Leu Phe Giu 130 Gly Phe Asp Leu Phe Ser 170 Thr Phe Leu Gin 195 Arg Arg Lys Phe Ile Ala 235 Leu Asp Leu Gly 260 Ile Lys Thr Phe Lys Lys 300 15 Giu Lys Val Asn Arg 80 Thr Tyr lie Lys Asn 145 Thr Lys Gly Ile Gly 210 Phe Thr Gly Thr Asn 275 Leu Tyr Met Tyr Ala 55 Met Tyr Lys Asp Gin 120 Ala Ser Asn Lys Ile 185 Phe Tyr Asp Leu Arg 250 Arg Gin Ile Gly Tyr 315 Gly 30 Phe Leu Gly Leu Lys 95 Asp Asp Gly Giu Leu 160 Gly Lys Ala Asn Ala 225 Asn His Leu Leu Arg 290 Asp Asn Ala Thr Ile Ala Asn Tyr Gly Phe 135 Gin Thr Arg Val Asn 200 Asp Phe Phe Arg Asp 265 Arg Glu Glu Leu Leu Tyr Tyr His Asn Gin 110 Asn Thr Val Ser Pro 175 Arg Ile Lys Lys Arg 240 Leu Lys Giu Giu Asp 305 A~rg Phe Thr Tyr Phe Val Tyr Ile Tyr Thr 150 Giu Leu Lys Thr Gly 215 Asp Giu Glu Asn Leu 280 Glu Val Lys His Gin Gly Leu Leu Ty~r Glu 125 Gin Trp Asn Val Leu 190 Asn Leu Lys Tyr Asn 255 Pro Asn Ala Val Ser Leu Lys Pro Ile Giu 100 Asp Leu Gly His Leu 165 Lys Asn Asp Glu Ser 230 Leu Lys Asn Ser Lys 295 Leu Ala Gly 310 Ser Leu Phe Val Thr Gin Gin Giu Leu Val 320 WO 99/26969 WO 9926969PCTNZ98/OO1 71 Tyr Tyr 335 Ala Ie Phe Ser Val 400 Leu Ala Leu Thr Lys 375 Asn Ile Tyr Pro Lys 350 Gly Gin Phe Gin Ser 325 Ala Lys Lys Asn Ilie 390 Leu Gly Leu Gly Phe 365 Phe Tyr Ile Ser Leu 340 Ile Asp Lys Tyr Lys 405 Tyr Gin Lys Asn Giy 380 Pro Ser Vai Giu Phe 355 Ser Tyr Asn Ile Giu 330 Tyr Tyr Asp Ile Pro 395 Leu Phe Asn Ala Met 345 Asn Met Giy Val 370 Val Arg Arg Lys Arg Arg 410 Lys Leu Leu Leu Lys 385 Leu Phe Asn Giy 360 Cys Phe Lys INFORMATION FOR SEQ ID NO. 2: SEQUENCE CHARACTERISTICS: LENGTH: 1230 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 2:

ATGAAATTTC

TCAGAAAAGA

AGAGTCGAAA

ATAAAGGTTG

TATGGGTATT

ATTTTTTGAT

CTTGATATTT

GTTAATTCAA

GTTTTACATA

TGGCATTATG

AAGAAATCGA

CGTTCTTTTG

TTTTGATGTT

TCGCACTTAC

TATTATGGAC

TGAATTAAAA

TTCCATATGA

GATGGTAATA

TCAGGGGGAT

TTAAAGATTT

TGCACTTACT

AGCAAACCAT

AAATATTTTG

ATATACCCAA

CTATTTTTAG

AAATATACGA

TGATTATCAA

TTGAATTAAG

GAAGTTGGTT

AACTAATCTT

TTTGAAAAAT

TGAAATGGGA

CTCTTTTTCA

AAAATATTTG

TGAAGAAAGA

AAAAAAATAA

TATTATGATG

AGATATTTTT

TTAATAGTGA

ACATCAGAAA

TTGCAAATAC

AATTTAAGAA

TTTGGAGGAA

GTGGCTTGAA

TATCTTGCAC

TGTATTAGAA

ATGAAGGTAG

GAAAAAGCTG

GCAAGTAACT

ATCTACTAAA

i00 150 200 250 300 350 400 450 500 550 TTCATTTTCA AAAAAAGGAC GTCCGTTAGT AAAAAAATCT AATACTTTTG WO 99/26969 PCT/NZ98/00171 36 TAGAAAGCTT AATAAAGATG AACTTCAAAT ATTTGCAAAT ATACAGCCAC TCGTCGAGGT TATAATGACA AAGGACTTGA

GAATAAAAGT

ATAACAAATG

G TATTATGAA

TTGCAACTTT

CATAGGCTTG

TCCAAACTCT

GAGAAACATT

TATGGTGATG

GCAAGAATTA

TTTATGCTCC

AAAGGAATAA

TTCAGATGGT

GTAAGTTTTC

ATACAACTAA

600 650

AAATTTTTCG

GAATTTCCGT

AGAATAAAAT

GAAAAAATAA

TTTAATTAGA

AGGATGTCGT

GTATATCTTT

AGCACTTTTA

AATTTTATAA

GTTCTATGTT

AAATTTTATT

TTAAAAGCAT

ATGCATTTAA AGATAAGTCA GAATTTACTA GAGTATTTAG GCAATATATT GGATGGTCGA

TTCAATTTTA

AAAATCAACT

GAAGAAGAAG

TCTTGCGGGA

ATTCAGGCTC

CAAGAATATG

TATGTTAGGT

TTAAkACAGAA

TACTACCCAA

TTTGAGAAGG

GGCACTAGGT TAGATAAAAA TAGAGAGTTA AATAGTCAAC CGAAATCTTT TGTTAAGAAG AGCCTTTTTG TATATACTCA ATATGTGGAG TTTAACAAGT CTATGTTAAA TGCATTAAAA ATTACTGGGA AATTTGATAA CTTTAAGGGG TATATAGTTC ACCCTAGAAA ATTAAAAGTT 700 750 800 850 900 950 1000 1050 1100 1150 1200 1230 INFORMATION FOR SEQ ID NO. 3: SEQUENCE CHARACTERISTICS: LENGTH: 285 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 99/26969 PCT/NZ98/00171 37 (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 3: Met Lys Arg Ile Phe Phe Ala Phe Leu Ser Leu Cys Leu Phe Ile Phe Gly Thr Gin Thr Vai Gly Asn Val Gly Arg Ala Gly Ala Cys Val Tyr Ala Thr Val Thr Gly 125 Leu Pro Arg Ile 150 Phe Asn Thr Asn Ie Asn 190 Pro Tlir Asp Asp 215 Ser Asp Asn Pro Gly Ser 255 Thr Gly Ser Ile Val Val Asn Leu His 100 Lys Gin Ala Asp Gly 165 Leu Gly Asp Vai Gin 230 Asn Gly Gly Ala Thr Asp Ala His Ile Leu Gin Val Asn 140 Pro Thr Lys Ile Phe 205 Ile Val Asn Gly Asn 270 Ala Thr Tyr Asn Pro Gin Ser Gly 115 Thr Pro Thr Thr Ile 180 Trp Thr Glu Leu Val 245 Leu Val Thr 25 Gly Ala Gly Trp His 90 Lys Gin Gly Asn Gly 155 Pro Tyr Gin Trp Val 220 Gin Lys Ser Trp Tyr Phe Val Thr 65 Met Ala Ile Ile Pro 130 Trp Tyr Thr Lys Val 195 Val Thr Lys Ser Trp 260 Leu Thr Asn 40 Pro Val Leu Asp Ser 105 Ile His Gin Ile Glu 170 Val Arg Asp Ser Gly 235 Val Ala Asn Arg Gly Val Lys Trp Gly Val Gly Leu Asn 145 Ala Pro

ASP

Asn Asn 210 Asn Gly Gly Gin Thr 275 Pro Tyr Gly Phe Met Met Ser Tyr 120 His Gly Asn Thr Asp 185 Asn Gly Gly Tyr Thr 250 Val Thr Leu Pro Thr Ala Ala His Thr Thr Phe Phe Ala 160 Thr Leu Ile Ile Thr 225 Phe Pro Asn Ser

ASP

Gly Pro Gly Gly Thr Asp Gly Giu 135 Ser Pro Pro Gin Leu 200 Al a Arg Val Met Phe 265 Lys Thr His Val Asn Asn Gly Ser 110 Ala Met Gly Val Thr 175 Lys Val Ala Thr Ile 240 Lys Thr Asp Asn Leu Leu Tyr Gly Lys 280 285 WO 99/26969 38 INFORMATION FOR SEQ ID NO. 4: SEQUENCE CHARACTERISTICS: LENGTH: 855 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ, ID NO. 4: PCT/NZ98/0O1 71

TTTCTTAAGT

CTACTTATAC

GGATACCCTG

GGTTAGAGCA

ATCACCCATG

GCTGACGGGA

CACAGATAGT

CCGGCCAAGT

CCTAACTGGC

CGCTAATGCC

CTACAACAAA

GGTATTTGGC

GGTTGATAAT

GAACAAGAAC

AATCCTAATA

TCTATCTTGG

TTATGCTTAT

TCGGCCATTA

GTCATGTTGG

GTTGCAAATG

GATGCTTTGG

TGCATACTGG

ACAGTTAAAC

TACCGGTCCA

AAAATGGTTT

CCTGTATTTA

TTTAAAAATC

AAGTAAGAAA

GGAATTGCAG

CTCTGACCAA

ATGTTAAAAG

GCTCAAGTAA

TTATATTCGG

GATACGGGAA

AGTCGATTAT

GTACAGTCAA

ATGGCTGGAA

ATATGCACAC

AAGGACAAAT

CATTTGCATT

TTCTGGAAGA

ATGGAACAAC

TATAAAGTTG

TAACATACTT

CAGATGATGT

GTTCTTCAAA

TGTTGGAACT

ACTTTACAAC

ATGAAACGTA

AACACAAACG

ATATCACTAC

GCAGTACCCG

ATTTGCAGGT

ACTGTGTTCT

TTATCAAAAA

CATAGGTTAT

TTGAAATGTT

ATAGATCCAA

ACCTACAGAA

ATGATTTACA

GTACCAACTG

AATTGAAGTA

AAGGTGGTTA

CCGATGAAAG

AGGTGGAAAT

TATTTTTTGC

GTATCTGCAG

AGGGTTTAAC

TTGGAACTCC

AATGGGGCTA

AATTCAACAT

TTTCAGTTAG

ACTGGTGCCA

GCCAGCAAAT

CCGGATACAT

CCTACTACTC

AAAAATTAAT

ATTTCACATG

ACTAGCAATG

TTTTGTCATC

GTAGTGGTGG

GTCTGGTTAA

120 170 220 270 320 370 420 470 520 570 620 670 720 770 820 855 ATACTACTAG CAAAGACAAC TTACTTTACG GAAAA

Claims (24)

1. A protein in substantially pure form which comprises the amino acid sequence of SEQ ID NO. 1 and which inhibits zoocin A activity, or a functionally equivalent variant thereof.

2. A protein as claimed in claim 1 which has the amino acid sequence of SEQ ID NO. 1.

3. An isolated DNA molecule having a nucleotide sequence which encodes a protein as claimed in claim 1, but which does not encode a protein having zoocin A activity.

4. An isolated DNA molecule having a nucleotide sequence which comprises SEQ ID NO. 2, or a functionally equivalent variant thereof, but which does not encode a protein having zoocin A activity.

5. A vector which includes a DNA molecule having a nucleotide sequence as claimed in claim 3 or claim 4. 15 6. A vector which includes a DNA molecule which has a nucleotide sequence which encodes a protein as claimed in claim 1 and a DNA molecule which has a nucleotide sequence encoding a protein having zoocin A activity, wherein said vector is capable of expressing said protein as claimed in claim 1 in a host cell such that said host cell is protected against zoocin A activity. 20 7. A vector as claimed in claim 6 wherein said protein having zoocin A activity has or includes the amino acid sequence of SEQ ID NO. 3, or a functionally equivalent variant thereof.

8. A vector as claimed in claim 6 wherein said DNA encoding said protein having zoocin A activity has or includes the nucleotide sequence of SEQ ID NO. 4, or a functionally equivalent variant thereof.

9. A method of protecting an organism susceptible to the bacteriolytic activity of zoocin A against such activity which comprises the step of introducing into said >fga a DNA molecule having a nucleotide sequence according to claim 3 or PCT/NZ98/00171 Received 28 January 2000 83258 WGN A method as claimed in claim 9 wherein said DNA molecule is introduced into said organism in the form of a vector as claimed in claim

11. An organism which has been rendered resistant to zoocin A activity by a method as claimed in claim 9 or claim

12. A method of genetically modifying a non-pathogenic organism to express a protein having zoocin A activity without said organism being itself at risk from said activity which comprises the step of introducing a DNA molecule encoding said protein into an organism as claimed in claim 11.

13. A method of genetically modifying a non-pathogenic organism to express a protein having zoocin A activity without said organism being itself at risk from said activity which comprises the step of introducing into said organism a DNA molecule encoding said protein together with a DNA molecule having a nucleotide sequence according to claim 3 or claim 4.

14. A method as claimed in claim 13 wherein said DNA molecules are introduced into said organism in the form of a vector as claimed in any one of claims 6 to 8. A non-pathogenic organism which has been genetically modified in accordance with a method as claimed in any one of claims 12 to 14.

16. A non-pathogenic organism which is resistant against zoocin A activity and wherein said resistance is due to the presence in said organism of a DNA molecule having a nucleotide sequence as claimed in claim 3 or claim 4.

17. A non-pathogenic organism which expresses a protein having zoocin A activity but which is itself resistant to said activity, wherein said resistance is due to the presence in said organism of a DNA molecule having a nucleotide sequence as claimed in claim 3 or claim 4.

18. A non-pathogenic organism as claimed in claim 17 which is a food grade organism.

19. A non-pathogenic organism as claimed in claim 18 which is a food grade Lji Streptococcus. AM"- .3HEF' PEAAU PCT/NZ98/00171 SReceived 28 January 2000 83258 WGN 41 A non-pathogenic organism as claimed in claim 19 wherein the food grade Streptococcus is S. gordonii.

21. An antibacterial composition which comprises a non-pathogenic organism according to any one of claims 15 and 17 to

22. An antibacterial composition as claimed in claim 21 which is suitable for human ingestion.

23. An antibacterial composition as claimed in claim 21 which is suitable for ingestion by a non-human animal.

24. An antibacterial composition as claimed in claim 22 or claim 23 which is, or is part of, a foodstuff. An antibacterial composition as claimed in claim 22 which is, or is part of, a nutriceutical.

26. An antibacterial composition as claimed in claim 24 or claim 25 which is or contains a dairy product.

27. An antibacterial composition as claimed in claim 22 which is, or is part of, a confectionery.

28. An antibacterial composition as claimed in claim 27 which is a wine gum or chewing gum.

29. A method of preventing or inhibiting the growth of undesirable organisms susceptible to zoocin A which comprises the step of contacting said undesirable organisms or the environment thereof with a composition as claimed in claim 21. A method as claimed in claim 29 wherein said composition is administered to the oral cavity of a patient to prevent or inhibit the growth of S. mutans, S. sobrinus and/or S. pyogenes.

31. A method of treating or preventing Streptococcal sore throat or dental caries in a susceptible patient which comprises the step of orally administering to said patient a composition as claimed in claim 22. SAMr IHE IPEA/AU