Methods and kits to determine the sensitivity of strains of Lactococcus lactis bacteria to phage infection Background to the Invention

In cheese-making, the single most important cause of fermentation failures is caused by bacteriophage infection of the starter culture(s) used. The dairy fermentation industry employs a lot of preventative measures such as starter strain selection and rotation. Traditionally, starter strain classification was performed through the application of phenotypic analysis using, for example, arginine hydrolysis, salt and thermal tolerance. Such analyses are time-consuming and expensive if applied in large scale screens.

It is an object of the invention to overcome at least one of the above-referenced problems. Statements of Invention

The invention relates to kits and methods for determining the sensitivity of bacteria, especially strains of Lactococcus lactis, to phage, particularly phages belonging to the so- called 936 and P335 species. The methods and kits of the invention are based on the discovery that a particular gene cluster/operon, designated here as cwps, which encodes the biosynthetic machinery for the manufacture of the so-called pellicle or cell wall polysaccharide (CWPS) of strains of L. lactis, contains regions that can function as a diagnostic variable of the phage sensitivity of the strain and a tool to determine the phage sensitivity of the strain. Thus, the sensitivity of a strain of Lactococcus lactis to phage, especially phages of the 936 or P335 species, can be determined by analysing the gene content of the cwps operon of the strain to determine the phage sensitivity profile of the strain. In particular, the Applicant has identified three cwps-associated regions that can be used to classify strains into four major phage-related groups having varying phage- sensitivity profiles, and developed multiplex PCR for classifying strains into one of these four phage- related groups. The methods of the invention are therefore based on determining the sensitivity of bacteria to phage, determining whether a mix of starter strains comprises a phage sensitive strain, determining the composite phage sensitivity profile of a mix of starter strains and formulation mixed defined starter cultures that are composed of strains from some or all of the different phage -relates groups. Accordingly, in a first aspect, the invention relates to a kit useful for determining the phage susceptibility of one or more strains of Lactococcus lactis by means of polymerase chain reaction (PCR), ideally multiplex PCR, comprising:

(a) a first primer pair adapted to generate a first amplicon correlating to a region of the cwps operon that is unique to a particular group of L. lactis strains, among which MG1363 and SKI 1, referred to here as the MG-SK type strains;

(b) a second primer pair adapted to generate a second amplicon correlating to a region of the cwps operon that is unique to a particular group of L. lactis strains, among which IL1403 and KF147, referred to here as the IL-KF type strains;

(c) a third primer pair adapted to generate a third amplicon correlating to a region of the cwps operon that is unique to a particular group of L. lactis strains, among which UC509.9 and CV56, referred to here as the UC-CV type strains; and

(d) optionally, a control primer pair adapted to amplify a sequence conserved in strains of L. lactis.

Thus, the four phage-related groups of L lactis are MG-SK type strains, IL-KF type strains, UC-CV type strains, and strains that are not part of any of the above three groups. Typically, the first primer pair is adapted to generate an amplicon comprising or consisting of a sequence of SEQUENCE ID NO: 9. Suitably, the first primer pair comprises a forward primer comprising or consisting of the sequence of SEQUENCE ID NO: 1 and a reverse primer comprising or consisting of the sequence of SEQUENCE ID NO: 2. Typically, the second primer pair is adapted to generate an amplicon comprising or consisting of a sequence of SEQUENCE ID NO: 10. Suitably, the second primer pair comprises a forward primer comprising or consisting of the sequence of SEQUENCE ID NO: 3 and a reverse primer comprising or consisting of the sequence of SEQUENCE ID NO: 4. Typically, the third primer pair is adapted to generate an amplicon comprising or consisting of a sequence of SEQUENCE ID NO: 11. Suitably, the third primer pair comprises a forward primer comprising or consisting of the sequence of SEQUENCE ID NO: 5 and a reverse primer comprising or consisting of the sequence of SEQUENCE ID NO: 6. Preferably, in the kit of the invention: (a) the first primer pair comprises a forward primer comprising the sequence of SEQUENCE ID NO: 1 and a reverse primer comprising the sequence of SEQUENCE ID NO: 2; (b) the second primer pair comprises the sequence of SEQUENCE ID NO: 3 and a reverse primer comprising the sequence of SEQUENCE ID NO: 4; and (c) the third primer pair comprises the sequence of SEQUENCE ID NO: 5 and a reverse primer comprising the sequence of SEQUENCE ID NO: 6.

Typically, the conserved sequence comprises all or part of the rmlB gene. Ideally, the control primer pair is adapted to generate an amplicon comprising or consisting of a sequence of SEQUENCE ID NO: 12. Ideally, the control primer pair comprises a forward primer comprising or consisting of the sequence of SEQUENCE ID NO: 7 and a reverse primer comprising or consisting of the sequence of SEQUENCE ID NO: 8. Preferably, the kit comprises at least two further primer pairs, namely a first further primer pair adapted to generate an amplicon selected from one of SEQUENCE ID NO'S 26 to 30 (for example SEQUENCE ID NO: 26), and a second further primer pair adapted to generate an amplicon selected from another of SEQUENCE ID NO'S 26 to 30 (for example, SEQUENCE ID NO: 27). These further primer pairs may be employed to sub-type bacteria identified as belonging to the MG-SK strain type.

Typically, the kit comprises, 3, 4 or 5 further primer pairs, each of which is adapted to generate a different amplicon selected from SEQUENCE ID NO'S 26 to 30. The invention also relates to a kit useful for determining the phage susceptibility of one or more strains of Lactococcus lactis by means of polymerase chain reaction (PCR), ideally multiplex PCR, comprising:

(a) a first primer pair adapted to generate a first amplicon from a region of the cwps operon that is unique to a particular group of L. lactis strains, among which MG1363 and SKI 1, referred to here as the MG-SK type strains;

(b) a first further primer pair adapted to generate an amplicon selected from one of SEQUENCE ID NO'S 26 to 30; (c) a second further primer pair adapted to generate an amplicon selected from another of SEQUENCE ID NO'S 26 to 30; and

(d) optionally, a control primer pair adapted to amplify a sequence conserved in strains of L. lactis.

Typically, the kit comprises, 3, 4 or 5 further primer pairs, each of which is adapted to generate a different amplicon selected from SEQUENCE ID NO'S 26 to 30.

The invention also relates to a method for classifying a strain of Lactococcus lactis according to phage sensitivity, comprising the steps of:

(i) providing a sample comprising at least one strain of Lactococcus lactis;

(ii) performing a PCR analysis of the sample using at least a first primer pair adapted to generate a test amplicon correlating to a region of the cwps operon that is a biomarker of phage sensitivity profile and, optionally, a control primer pair adapted to generate a second amplicon correlating to a region of the cwps operon that is conserved amongst strains of Lactococcus lactis; and

(iii) correlating the presence or absence of the test amplicon with phage sensitivity profile of the strain of L. lactis. The test amplicon may be, for example, a biomarker of phage sensitivity, phage insensitivity, or intermediate phage sensitivity. Examples of such biomarkers are SEQUENCE ID NOs: 9 to 11 which are regions of the cwps operon on L. lactis strains that correlate with phage sensitivity, intermediate phage sensitivity, and phage insensitivity, respectively. The level of sensitivity referred to here relates to the finding that (a) L. lactis strains of the MG-SK cwps type were observed to be sensitive to more phages than strains possessing the IL-KF or UC- CV cwps type in this study(phage sensitive), (b) L. lactis strains of the UC-CV cwps type were not sensitive to any phages in this study (phage insensitive), while (c) L. lactis strains possessing the IL-KF cwps type were infected by an intermediate number of phages in this study (intermediate phage sensitivity), and these may be representative of Lactococcal strains applied in the dairy industry and in culture collections. Other biomarkers include the sequences of SEQUENCE ID NOs 13, 14 & 15 relating to strains of the MG-SK (phage sensitive), IL-KF (intermediate phage sensitivity) and the UC-CV (phage insensitive) cwps types, respectively. Suitably, the method for classifying a strain of Lactococcus lactis according to phage sensitivity comprises the steps of:

(i) providing a sample comprising at least one strain of Lactococcus lactis;

(ii) performing a multiplex PCR analysis of the sample using

(a) a first primer pair adapted to generate a first amplicon correlating to a region of the cwps operon that is a biomarker of phage sensitivity (L. lactis strains that belong to the so-called MG-SK type);

(b) a second primer pair adapted to generate a second amplicon correlating to a region of the cwps operon that is a biomarker of intermediate phage sensitivity (L. lactis strains that belong to the so-called IL-KF type);

(c) a third primer pair adapted to generate a third amplicon correlating to a region of the cwps operon that is a biomarker of phage insensitivity (L. lactis strains that belong to the so-called UC-CV type); and

(d) optionally, a control primer pair adapted to amplify a sequence conserved in L. lactis; and

(iii) correlating the presence or absence of the amplicons with phage sensitivity profile of the strain of L. lactis. Preferably, the method for classifying a strain of Lactococcus lactis according to phage sensitivity, comprises the steps of:

(i) providing a sample comprising at least one strain of Lactococcus lactis;

(ii) performing a multiplex PCR analysis of the sample using

(a) a first primer pair adapted to generate a first amplicon correlating to a region of the cwps operon that is unique to L. lactis strains MG1363 and SK11 (L. lactis strains that belong to the so-called MG-SK type);

(b) a second primer pair adapted to generate a second amplicon correlating to a region of the cwps operon that is unique to L. lactis strains IL1403 and KF147 (L. lactis strains that belong to the so-called IL-KF type);

(c) a third primer pair adapted to generate a third amplicon correlating to a region of the cwps operon that is unique to L. lactis strains UC509.9 and CV56 (L. lactis strains that belong to the so-called UC-CV type); and (d) optionally, a control primer pair adapted to amplify a sequence conserved in L. lactis; and

(iii) classifying at least one strain according to phage sensitivity, wherein the presence of the first amplicon indicates the presence of a strain that is phage sensitive, the presence of the third amplicon indicates the presence of a strain that is phage insensitive, and the presence of the second amplicon indicates the presence of a strain having intermediate phage sensitivity.

Preferably, the method comprises an additional step of sub-typing strains identified as having a specific phage sensitivity profile according to phage sensitivity. Thus, for example, a strain or strains identified as being phage sensitive (or MG-SK strain type), phage insensitive (or UC-CV strain type), or intermediate phage sensitivity (or IL-KF strain type), can be sub- typed and further classified according to phage sensitivity. Typically, phages identified as phage sensitive or MG-SK strain type, are sub-typed using one or more primer pairs adapted to generate an amplicon selected from SEQUENCE ID NO's 26 to 30, and sub-classified according to phage sensitivity according to the presence or absence of each amplicon. Preferably, the phages are sub-typed using primer pairs adapted to generate one amplicon selected from SEQUENCE ID NO's 26 to 30.

The invention also relates to a method for determining the composite phage sensitivity of a mix of Lactococcus lactis strains, comprising the steps of:

(i) providing a sample comprising a mix of Lactococcus lactis strains;

(ii) performing a multiplex PCR analysis of the sample using

(a) a first primer pair adapted to generate a first amplicon correlating to a region of the cwps operon that is unique to L. lactis strains MG1363 and SKI 1;

(b) a second primer pair adapted to generate a second amplicon correlating to a region of the cwps operon that is unique to L. lactis strains IL1403 and KF147;

(c) a third primer pair adapted to generate a third amplicon correlating to a region of the cwps operon that is unique to L. lactis strains UC509.9 and CV56; and (d) optionally, a control primer pair adapted to amplify a sequence conserved in L.lactis

wherein the PCR analysis provides a plurality of amplicons of different sizes; and (iii) correlating the level of the first, second or third amplicons with phage sensitivity, wherein the first amplicon is indicative of phage sensitivity, the third amplicon is indicative of phage insensitivity, and the second amplicon is indicative of intermediate phage sensitivity.

Examples of the first amplicon include the sequences of SEQUENCE ID NO: 9 & 13.

Examples of the second amplicon include the sequences of SEQUENCE ID NO: 10 & 14.

Examples of the third amplicon include the sequences of SEQUENCE ID NO: 11 & 15. Typically, the first primer pair is adapted to generate an amplicon comprising or consisting of a sequence of SEQUENCE ID NO: 9. Suitably, the first primer pair comprises a forward primer comprising or consisting of the sequence of SEQUENCE ID NO: 1 and a reverse primer comprising or consisting of the sequence of SEQUENCE ID NO: 2. Typically, the second primer pair is adapted to generate an amplicon comprising or consisting of a sequence of SEQUENCE ID NO: 10. Suitably, the second primer pair comprises a forward primer comprising or consisting of the sequence of SEQUENCE ID NO: 3 and a reverse primer comprising or consisting of the sequence of SEQUENCE ID NO: 4. Typically, the third primer pair is adapted to generate an amplicon comprising or consisting of a sequence of SEQUENCE ID NO: 11. Suitably, the third primer pair comprises a forward primer comprising or consisting of the sequence of SEQUENCE ID NO: 5 and a reverse primer comprising or consisting of the sequence of SEQUENCE ID NO: 6. Typically, the conserved sequence comprises all or part of the rmlB gene. Ideally, the control primer pair is adapted to generate an amplicon comprising or consisting of a sequence of SEQUENCE ID NO: 12. Ideally, the control primer pair comprises a forward primer comprising or consisting of the sequence of SEQUENCE ID NO: 7 and a reverse primer comprising or consisting of the sequence of SEQUENCE ID NO: 8.

Preferably, the methods of the invention employ: (a) a first primer pair comprising a forward primer comprising the sequence of SEQUENCE ID NO: 1 and a reverse primer comprising the sequence of SEQUENCE ID NO: 2; (b) a second primer pair comprising the sequence of SEQUENCE ID NO: 3 and a reverse primer comprising the sequence of SEQUENCE ID NO: 4; (c) a third primer pair comprising the sequence of SEQUENCE ID NO: 5 and a reverse primer comprising the sequence of SEQUENCE ID NO: 6; and, optionally, (d) a control primer pair comprising a forward primer comprising or consisting of the sequence of SEQUENCE ID NO: 7 and a reverse primer comprising or consisting of the sequence of SEQUENCE ID NO: 8.

Suitably, the PCR conditions are about 95°C for about 6 minutes followed by about 31 cycles of about 95°C for about 15 seconds, about 57°C for about 30 seconds, and about 72°C for about 1 minute and a final extension step at about 72°C for about 7 minutes. The term "about" should be understand to mean +/- 5%.

The invention also relates to the use of a region of the cwps operon of a strain of Lactococcus lactis as a biomarker of phage sensitivity, ideally 936-type phage sensitivity, wherein the region is selected from the group consisting of:

a region unique among the reference strains to L. lactis strains MG1363 and SKI 1; a region unique among the reference strains to L. lactis strains IL1403 and KF147; and a region unique among the reference strains to L. lactis strains UC509.9 and CV56.

The term "unique" or "unique region" means a region of the cwps operon of a L. lactis strain that is found in both of the reference strains and not found in the other four reference strains of L. lactis. Thus, a region unique to L. lactis strains MG1363 and SKl l is a region of the cwps operon that is found in both strains MG1363 and SKl l but not found in any ofL. lactis strains IL1403, KF147, UC509.9 and SKI 1. Thus, a strain of L. lactis that is found to have a region of the cwps operon that is unique to L. lactis strains MG1363 and SKl l can be classed as part of the MG-SK group of strains (phage sensitive strains). Likewise, a strain of L. lactis that is found to have a region of the cwps operon that is unique to L. lactis strains UC509.9 and CV56 can be classed as part of the UC-CV group of strains (phage insensitive strains). The region is generally of sufficient length to be detected by PCR, including multiplex PCR. The term "phage sensitive" as applied to a strain of L. lactis should be understood to mean a strain belonging to the MG-SK type that is sensitive to infection by phage, typically infection by 936-type phage or P335 group phage

The term "phage insensitive" as applied to a strain of L. lactis should be understood to mean a strain belonging to the UC-CV type that is highly insensitive to infection by phage, typically infection by 936-type phage or P335 group phage

The term "intermediate phage sensitive" as applied to a strain of L. lactis should be understood to mean a strain belonging to the IL-KF type that exhibits a sensitivity to infection by phage, typically infection by 936-type phage or P335 group phage, that is intermediate the sensitivity of phage sensitive and phage insensitive strains.

Thus, for example, the invention also relates to the use of a region of the cwps operon of a strain of L. lactis that is unique to L. lactis strains MG1363 and SK11, for example SEQUENCE ID NO: 9, as a biomarker of phage sensitivity in strains of bacteria, especially strains of Lactococcus lactis. The invention also relates to the use of a region of the cwps operon of a strain of L. lactis that is unique to L. lactis strains UC509.9 and CV56, for example SEQUENCE ID NO: 11, as a biomarker of phage insensitivity in strains of bacteria, especially strains of Lactococcus lactis. The invention also relates to the use of a region of the cwps operon of a strain of L. lactis that is unique to L. lactis strains IL1403 and KF147, for example SEQUENCE ID NO: 10, as a biomarker of intermediate phage sensitivity in strains of bacteria, especially strains of Lactococcus lactis.

The term "composite phage sensitivity" should be understood to mean the phage sensitivity of the starter mix.

The term "phage" as employed herein preferably means phages belonging to the 936 and/or P335 species. The term "936-type phage" means strictly lytic phage, having a well conserved genome organisation in three clusters; early, middle and late expressed regions (9). The term P335 group phage means phages that may be lytic or temperate and having a general genome arrangement of a lysogenic module (if the phage is a temperate one) oriented back-to-back with the replication module and proceeded by the structural module (25).

The invention also relates to the use of a region of the cwps operon of a strain of Lactococcus lactis as a biomarker of phage sensitivity.

The invention also relates to the use of a region of the cwps operon of a MG-SK type strain of Lactococcus lactis as a biomarker of phage sensitivity, ideally P335 or 936-type phage sensitivity, wherein the region is selected from the group consisting of SEQUENCE ID NO'S 26 to 30.

The invention also relates to a mixed defined starter culture comprising a plurality of Lactococcus lactis strains and comprising a first bacterial strain selected from one of an MG- SK type strain, a UC-CV type strain, and an IL-KF type strain, and a second bacterial strain selected from another of an MG-SK type strain, a UC-CV type strain, and an IL-KF type strain. Examples of combinations include a MG-SK type strain plus a UC-CV type strain, or a MG-SK type strain plus an IL-KF type strain, or a UC-CV type strain plus an IL-KF type strain.

The term "defined" means that the strains in the mixed starter culture have been identified as being different strains. Typically, the mixed starter culture comprises an MG-SK type strain, a UC-CV type strain, and an IL-KF type strain. Examples of mixed starter cultures according to the invention include:

L. lactis IL1403 (IL-KF), WM1 (UC-CV) and 3107 (MG-SK); or L. lactis F7/2 (IL-KF), CIO (UC-CV) and FD13 (MG-SK). The term "mixed starter culture" as used herein should be understood to mean a mixture of cultures or a combination of cultures provided in the form of a kit in which the different cultures are not mixed together. The invention also relates to a mixed starter culture comprising a plurality of Lactococcus lactis strains of the MG-SK strain type, and comprising a first bacterial strain selected from one of a Ci to C5 sub-type, and a second bacterial strain selected from another of a Ci to C5 sub-type. Examples of combinations include a Ci plus C2 type strain, or a C2 plus C5 type strain. CWPS sequences that characterise the Ci to C5 sub-types are provided below in Table 6, and primer pairs for use in identification of the sub-types using PCR are provided in Table 5.

The invention also relates to a method of formulating a mixed starter culture comprising the steps of classifying a plurality of strains of Lactococcus lactis according to phage sensitivity according to a method described above to identify a plurality of strains having differing phage sensitivity, and formulating a mixed starter culture using the plurality of strains having differing phage sensitivity.

Typically, the mixed starter culture is formulated into a single composition comprising a mixture of cultures or a kit comprising the plurality of cultures stored separately.

The kits, methods and uses of the invention may be employed to determine or characterise the sensitivity of bacteria, especially L. lactis bacteria, to phage, especially P335 and 936 phage. Brief Description of the Figures

Fig. 1. Comparison of the genomic regions encoding the cwps biosynthesis cluster of six lactococcal strains (UC509.9, CV56, IL1403, KF147, SK11 and MG1363). Homologous protein-encoding genes are joined by grey blocks including the level of identity (aa %). The three sub-groups of cwps are the UC-CV, the IL-KF and the MG-SK sub-groups with unique regions highlighted in the operons of UC509.9, IL1403 and MG1363 in the schematic by stars to indicate the genes upon which the multiplex PCRs are based. The control for the multiplex PCR was based on rmlB, which is conserved in each of the strains. The genes upon which alternative primer sets could be designed are indicated by a black plus sign above the relevant genes, which may be used as alternative biomarkers.

Fig. 2: Multiplex PCR for the detection of conserved (control) regions and differential regions of the CWPS cluster of lactococcal genomes. Strains IL1403 (IL-KF CWPS), MG1614 (MG-SK CWPS) and UC509.9 (UC-CV CWPS) are the controls for each CWPS type tested for in this assay. A negative control was also included without template DNA in the reaction. The control band is observed in all cases with differential amplicons observed for each of the assessed strains with the exception of 184, which does not belong to any of the three known CWPS types.

Fig. 3. Above is a representative phylogenetic tree of the RBP variable C-termini of thirty 936 phages including the eleven sequenced phages of this study. Three major groups of the 936 RBPs are observed: Group 1 includes the majority of phages in this study that infect strains with a MG-SK CWPS (except 936), Group 2 includes those phages in this study that infect strains with an IL-KF CWPS while Group 3 represents a small group with a divergent RBP that predominantly infect strains with an IL-KF CWPS but also infect a strain with the MG-SK CWPS with equal efficiency. Fig. 4. Comparison of the variable regions in MG-SK type CWPS biosynthesis cluster of eight lactococcal strains (MG1363, JM1, 3107, JM2, SK11, JM3, W34 and IO-l). Genes that share a high level of identify (as indicated in % nucleotide identity) are joined by grey blocks compared to the adjoining strain. Five subtypes (Ci to Cs) of the MG-SK genotype are highlighted. Genes marked with * and ** represent genes interrupted in MG1363 derivatives NZ9000-GT1 (LLNZ_0U45) and NZ9000-GT2 (LLNZ_0U50), respectively. Genes with diagonal lines represent 3107 C ₂ subtype genes cloned in pPTPiC2.

Detailed Description of the Invention

Materials & Methods

Lactococcal strains and bacteriophages

Lactococcal strains (Table 1) were grown in M17 broth supplemented with 0.5 % glucose at 30 °C without agitation. Phages were propagated on the relevant strains at 30 °C in M17 broth (Oxoid) supplemented with 0.5 % glucose without agitation as previously described (18). The phages used in this study and relevant details are listed in Table 1. Plaque assays were performed using the double agar method as previously described (15). This method was also applied for the host range analysis performed against a bank of lactococcal strains (Table 1). Frequency of lysogeny assays using the erythromycin-tagged phage TP901erm (a derivative of TP901-1, also designated as TP901-BC1034) was performed as previously described (4).

Phage purification & DNA preparation

Phage purification by Caesium chloride gradient was performed as previously described (22). The generated purified phage suspension (1 ml) was precipitated with 10 % polyethylene glycol 8000 (Sigma-Aldrich) and 0.5 M sodium chloride at 4 °C overnight. Subsequently, the suspension was centrifuged at 17,700 g for 15 minutes and the supernatant removed. Alternatively, phage suspension was dialyzed as described for phage λ. The PEG/salt- induced precipitate was resuspended in 0.5 ml of TE buffer (pH 9.0) and treated with 20 μΐ of 20 mg.ml ^"1 proteinase K for 20 minutes at 56 °C followed by treatment with SDS at a final concentration of 2 % at 65 °C for 20 minutes. This mixture was then phenol/chloroform (25:24: 1 phenol:chloroform:isoamyl alcohol, Sigma Aldrich) treated at least twice and the aqueous phase precipitated with 2.5 volumes of ice cold 96 % ethanol and 0.1 volume of sodium acetate (pH 4.8). Subsequent to centrifugation, the pellet was washed in 70 % ethanol and resuspended in 100 μΐ of TE buffer (pH 8.0).

Genome sequencing, assembly & annotation

5 μg of DNA of phages 645, 340, ViridusJMZ (JM2), PastusJM3 (JM3) and P113g was extracted and verified by nanodrop quantification and confirmatory molecular ID tests were conducted on the DNA extract prior to shipment to the contract sequencing facility (Macrogen Inc., Korea). A 40- to 65-fold sequencing coverage was obtained using pyrosequencing technology on a 454 FLX instrument. The files generated by the 454 FLX instrument were assembled with GS assembler (454, Branford, CT) to generate a consensus sequence. Phages P475, fdl3, φ7. 936. P272 were sequenced using the 454 Roche Titanium platform. These phages were sequenced as part of tagged pools of unrelated phages, built as M ID-tagged Rapid libraries and sequenced in one region (half a picotitre plate) using the GS FLX Titanium Sequencing Kit XLR70. One phage, P680, was sequenced as 96 base reads using the Illumina HighSeq2000 platform, again as part of a pool of unrelated phages, tagged with an index as part of one lane of the flowcell. Custom indexing primers were used to build libraries as described earlier (12). Reads were assembled into contigs using CLC Genomics Workbench 5.0.1 (CLC bio, Aarhus, Denmark). Quality improvement of the genome sequence involved sequencing of 15-25 PCR products across the entire genomes to ensure correct assembly, double stranding and the resolution of any remaining base-conflicts occurring within homopolynucleotide tracts. Protein-encoding open reading frames (ORFs) were predicted using Zcurve_V and Genmark.hmm followed by manual assessment and, where necessary, correction. A preliminary identification and functional annotation of ORFs was performed on the basis of BLASTP analysis against the non-redundant protein database (nr) provided by the National Centre for Biotechnology Information (located at: http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Phage accession numbers

The Genbank accession numbers for the phages sequenced in this study are as follows: 340 KC182542; 645 KC182543; 936 KC182544; fdl3 KC182545; JM2 KC182546; JM3 KC182547; P113G KC182548; P272 KC182549; P475 KC182550; P680 KC182551; φ7 KC182552.

CWPS-typing of lactococcal strains by multiplex PCR

The relevant DNA regions encompassing the cell wall polysaccharide biosynthesis operon in the sequenced lactococcal strains IL1403 (accession number: AE005176.), KF147 (accession number: NC_013656.1), MG1363 (accession number: NC_009004.1), SK11 (accession number: NC_008527.1), UC509.9 (accession number: CP003157.1) and CV56 (accession number: CP002365.1) were analysed and compared using BLASTP analysis as described above. Using this data conserved and unique regions within the operons of these strains were identified (Fig. 1). Primers were designed based on LLKF_205 of IL-KF(Product = 183 bp), llmg_0226 of MG-SK(Product = 686bp) and UC509_0206 of UC-CV CWPS (Product size = 442bp) types as indicated in Fig. 1 (Tables 3 and 4). A control was also included in which primers based on the conserved gene rmlB were used to generate a product of 891bp to verify that the reaction was working in all samples. The multiplex PCR included these four sets of primers and was applied to the strains assessed in the host range analysis (Table 1) under the following conditions: 95 °C for 6 minutes followed by 31 cycles of 95 °C for 15 seconds, 57 °C for 30 seconds and 72 °C for 1 minute and a final extension step at 72 °C for 7 minutes. Electron microscopy

A drop of the purified phage suspension was applied to a Formvar-carbon-coated copper grid for 5 min, then removed with a pipette and immediately replaced with 3 % (vol/vol) uranyl acetate. After 1 min, the liquid was removed with a filter paper. The grids were examined in a Philips CM 12 transmission electron microscope at 80 kV.

Cloning

All recombinant plasmids (Table 1) were generated in Escherichia coli Top 10 (Invitrogen, USA). All primers, except where stated, were ordered from Eurofins MWG (Ebersberg, Germany). The variable section (i.e. variable within C type strains) of the CWPS biosynthesis gene cluster of L. lactis 3107, encompassing genes 3107_003 to 3107_006, was amplified using KOD DNA polymerase (Invitrogen, USA) and cloned into the low copy number, nisin- inducible vector pPTPi. Plasmid constructs were then transformed into the L. lactis MG1363 rasR _^ fif-containing derivative L. lactis NZ9000, in which plasmid pJP005 (27) had been introduced to allow recombineering and nisin-inducible expression.

Recombineering and oligonucleotides

Recombineering was performed in L. lactis NZ9000 or derivatives thereof as previously described (27), with associated modifications as optimized for L. lactis and executing a given transformation with 500 μg of a particular oligonucleotide, which in some cases contained phosphorothioate linkages (Integrated DNA Technologies, Leuven, Belgium).

Bioinformatic analyses

For comparative analysis of the CWPS biosynthesis gene clusters that belong to the MG-SK type, as identified by multiplex PCR, relevant genomic regions encompassing the CWPS biosynthesis gene cluster from lactococcal strains MG1363 (accession number: NC_009004.1), SK11 (accession number: NC_008527.1) and IO-l (accession number: AP012281) were employed. The full genome analyses of L. lactis strains 3107, W34, JM1, JM2 and JM3 are currently in progress and these results will be published elsewhere. The genomic regions responsible for CWPS biosynthesisin the latter five strains were identified based on BLASTN analysis against the reference CWPS biosynthesis gene cluster of L. lactis MG1363 and submitted to GenBank under the following accession numbers; L. lactis 3107 (KF498848), L. lactis W34 (KF498852), L. lactis JM1 (KF498849), L. lactis JM2 (KF498850) and L. lactis JM3 (KF498851). The presumed CWPS region of each genome was analysed and compared in detail using BLASTP and Interpro analysis. Using the genomic data corresponding to the CWPS biosynthesis region of the above mentioned strains, conserved and variable regions were identified.

Results

Selection of phages for this study

Eleven phages were selected for this study in order to assess genome diversity among the 936 phages. The phages represent a broad range of 936 phages isolated across Europe (and one New Zealand phage) during a time period that spannedthe 1980s until 2010 (Table 1). Firstly, phages that have been applied in many studies of 936 phage-host interaction studies over the past decades such as P680, P113g, P272 and 645 were selected. Secondly, phage 936 was selected to serve as the prototype member of the 936 phage species for comparative purposes. Furthermore, its geographical location of isolation was a consideration as the remainder of the phages are of European background (Table 1). Finally, the geographical origin and year of isolation of the selected phages was considered, and therefore phages that had been isolated over the past thirty years in Ireland (ViridusJM2 and PastusJM3), Germany (P680, P113g, P272) and Denmark (fdl3, 645, 340, φ7, P475) and New Zealand (936) were selected. Host range analysis

Thirty four lactococcal strains (Table 1) were assessed for their sensitivity to the eleven phages sequenced in this study. All phages assessed in this study have a relatively narrow host range and are limited to infecting at most six different strains from this panel of thirty four possible hosts. It is also noteworthy that host range convergence was observed for certain members of the sequenced group of phages. For example, 645 and 340 have a similar host range as do P272 and P113g (Table 2). Since these phages are derived from similar geographical locations, it is perhaps unsurprising that these apparent sub-groups of phages possess related host ranges. Viridus JM2, Pastus JM3, fdl3 and 936 display very narrow host specificities infecting only a single strain among those tested.

Multiplex PCR CWPS strain typing

BlastP analysis of the CWPS clusters of the sequenced lactococcal strains identified three major CWPS subgroups based on conserved sequences, allowing classification into the MG- SK, IL-KF and UC-CV CWPS subgroups (Fig. 1). Each subgroup is defined by unique regions that were used to develop a multiplex PCR-based typing method (see Materials and Methods section). This was applied to classify the CWPS type of each of the strains used in this study. Of the 34 strains assessed, six strains were in this way classified as the IL-KF CWPS-type, fourteen belonged to the MG-SK CWPS-type, while thirteen belonged to the UC-CV CWPS-type. One strain (L. lactis subsp. lactis 184) did not generate an amplicon for any of the three CWPS types although the conserved region present in all three subtypes was amplified (Fig. 2), which may be indicative of an as yet unidentified CWPS type not represented by the three sub-types presented in this study.

Correlation of CWPS type & host range

Of the eleven phages, five phages (fdl3, 936, φ7, ViridusJMl and PastusJM3) are largely limited to infecting strains of the MG-SK CWPS type (Table 2). Conversely, the remaining six phages are almost completely limited to infecting hosts with the IL-KF CWPS type. There are exceptions to this generalisation, however, such as phages 645 and 340 which were shown to infect L. lactis subsp. cremoris 3107 (MG-SK cwps) as well as L. lactis subsp. lactis IL1403 (IL-KF cwps) and four other strains of this cwps type. These phages can infect strains of both cwps types with a relative efficiency. The same is true for phage P475, which infects strains of the IL-KF cwps type, but which also infects L. lactis W22 (MG-SK cwps) however at a much lower efficiency (EOP =10 ^"6/7 ). Interestingly, none of the strains possessing the UC-CV cwps-type were infected by any of the 936-type phages assessed in this study.

Correlation ofRBP group & host range

The RBPs of the sequenced phages were used to perform a comparative sequence analysis which also included sequences of previously sequenced 936 phage RBPs. Since the amino- terminal regions of these proteins are well conserved, the first 130 residues were removed from the RBP sequences and a comparison of the much more variable sequences of the RBPs carboxy-terminus was performed. Through this analysis, three sub-groups of RBP are identifyable (Fig. 3): Group 1 corresponds to what was previously termed the ski-like or L. lactis subsp. cremoris-mfectmg phages (17); Group 2 contains the bIL170-like or L. lactis subsp. Zaciis-infecting phages (17); while Group 3 represents a distinct but small group of phages that infect primarily L. lactis subsp. lactis strains, but are also capable of infecting strains of L. lactis subsp. cremoris (Table 2). While there are three main RBP groups, subtle sub-groups within these groups can be distinguished (Fig. 2). The phages in this study of the RBP group 1 exclusively infect strains that possess the MG-SK CWPS type. Similarly, the majority of phages in this study of RBP group 2 infect strains that possess the IL-KF-type CWPS, while those belonging to group 3 appear to preferentially infect strains of the IL-KF CWPS type, though they are occasionally also capable of infecting strains with a MG-SK- type CWPS as mentioned above (the cases of P475, 645 and 340).

Sub-typing of CWPS type MG-SK strains

As determined, three variations of a particular genetic locus present in L. lactis strains, termed the UC-CV, IL-KF, and MG-SK types, can be linked to RBP phylogeny of 936 phages. To determine if additional genetic diversity within the CWPS biosynthesis gene cluster of a given CWPS type exists, we analysed the genetic locus encompassing the presumed CWPS biosynthetic operon of eight lactococcal genomes(three publicly available genomes and the CWPS regions of five strains from our own collection), all belonging to the MG-SK type (as first determined by PCR). This comparative sequence analysis revealed the presence of a variable region within these examined CWPS MG-SK type loci (Fig. 4), allowing the identification of five subtypesamong members of the MG-SK type (designated subtype Ci to C ₅ ; Fig. 4) based on differences/similarities within this variable region within the various type MG-SK CWPS biosynthesis loci. Primers for generating the Ci to Cs subtypes are provided in Table 5 and the generated amplicons are provided in Table 6.

Primers were designed based on the unique regions of each of the 5 sub-types and can be applied in a multiplex PCR approach to perform CWPS MG-SK sub-typing of lactococcal strains (Table 5). The sequence differences between subtypes suggest that C ₂ to Cs subtype strains produce structurally different CWPS compared to the previously determined Ci type structure of L. lactis MG1363 (5). The sub-typing of lactococcal strains of the CWPS MG-SK type permits a deeper insight into the specific relationships between phages and these strains and elucidates the components that phages of the 936 or P335 species recognise and specifically target. For example, the lactococcal P335 phages LC3 and TP901-1 cannot infect L. lactis NZ9000, which possesses the CWPS Ci sub-type. Conversely, these phages can infect another lactococcal strain named 3107, which possesses CWPS C ₂ sub-type. Genetic swapping of the variable region of the CWPS of a MG-SK subtype strain causes a change in phage sensitivities to both 936 and P335 type phages

Due to the high level (99-100 %) of DNA sequence identity observed across conserved regions of the CWPS biosynthesis gene clusters found in the Ci subtype strain L. lactis MG1363 and the C ₂ subtype strain L. lactis 3107 (Fig. 4), it was reasoned that if the variable genes found in the C ₂ subtype CWPS biosynthesis locus of L. lactis 3017 were to be supplied in trans to the L. lactis MG1363 NICE expression system derivative L. lactis NZ9000 (Ci subtype), carrying a mutation in one of its variable CWPS genes, the resulting recombinant strain would produce the structural equivalent of L. lactis 3107 CWPS (which is of the C ₂ subtype), thus effectively causing change of CWPS subtype by this genetic swapping. To test this hypothesis, the variable region of the C ₂ subtype CWPS biosynthesis gene cluster was first cloned from L. lactis 3107 (i.e. genes 3107_003, 3107_004 and 3107_005) into the nisin- inducible plasmid pPTPi, thereby generating plasmid pPTPiC2. The latter plasmid was then introduced into L. lactis NZ9000-GT1, an NZ9000 derivative in which gene LLNZ_01145, which is one of the genes of the variable region within the native CWPS biosynthesis MG-SK type gene cluster, had been mutated by recombineering. This mutant carries an in-frame TGA stop codon in LLNZ_01145, which in turn causes the resulting mutant, designated NZ9000-GT1, to display a phage-resistant (to phages belonging to the 936 species) and sedimenting phenotypes (data not shown), all being consistent with the expected loss of CWPS biosynthesis. Introduction of plasmid pPTPiC2 and induced expression of the variable region of the C ₂ subtype CWPS biosynthesis gene cluster from L. lactis 3107 on this plasmid in L. lactis NZ9000-GT1 restored wild type non- sedimenting cell growth, indicating the production of a functional CWPS of subtype C ₂ . To determine if the presumed C ₂ subtype CWPS produced in L. lactis NZ9000-GT1 pPTPiC2 functions as a bacteriophage-host cell surface receptor, the induced strain was challenged by plaque assay with various P335 species phages (Table 7), whose primary indicator strain is L. lactis 3107. Of the phages tested, only the P335 species phage cpLC3, which is unable to form plaques on WT L. lactis NZ9000 or un-induced NZ9000-GT1 pPTPiC2, was able to infect and form plaques on induced NZ9000-GT1 pPTPiC2 at an EOP of 10 ^"1 and can be propagated to levels of 10 ⁷ - 10 ⁸ pfu/ml (data not shown). This clearly demonstrates that the CWPS of L. lactis 3107 is the host cell-surface receptor of the P335 species phage cpLC3, and that this CWPS, when produced in NZ9000, is sufficient for this strain to become susceptible to cpLC3 infection. Interestingly, another P335 species phage, TP901 erm, which also uses L. lactis 3107 as a host, was not able to form plaques on induced NZ9000-GT1 pPTPiC2. However, the frequency of lysogeny of TP901 erm on induced L. lactis NZ9000-GT1 pPTPiC2 increases 10 ⁴ fold compared to L. lactis NZ9000-GT1 pPTPi (TP901 erm can lysogenize L. lactis NZ9000 at a very low frequency (10 ^~8 )), reaching levels similar to those observed for L. lactis 3107 (10 ^~4 ), thus showing that CWPS from 3107 is also the cell surface receptor for of the P335 species phage TP901erm.

Table 1. Features of the lactococcal strains and phages used in this study

Strain Source/Reference L. lactis subspecies CWPS Phage infected type

IL1403 (1) lactis IL-KF +

F7/2 (24) lactis biovar diacetylactis IL-KF +

Bu2-60 (19) lactis biovar diacetylactis IL-KF +

455 cremoris IL-KF +

UC77 UCC cremoris IL-KF +

229 UCC lactis IL-KF -

WM1 UCC lactis UC-CV -

ML8 (6) cremoris UC-CV -

CIO UCC cremoris UC-CV -

IE-16 (6) cremoris UC-CV -

SMQ-450 (6) cremoris UC-CV -

SMQ-562 (6) cremoris UC-CV -

111 (6) cremoris UC-CV -

UC063 UCC cremoris UC-CV -

UC509.9 UCC cremoris UC-CV -

UL8 UCC lactis UC-CV -

275 UCC lactis UC-CV -

KH (23) cremoris UC-CV -

C3 (10) cremoris UC-CV -

W22 (21) cremoris MG-SK +

W34 (11) cremoris MG-SK +

3107 (3) cremoris MG-SK +

WG2 (24) cremoris MG-SK +

FDD (7) cremoris MG-SK +

H2L (2) cremoris MG-SK +

158 (9) cremoris MG-SK +

V32.2 (7) cremoris MG-SK +

JM3 UCC cremoris MG-SK +

JM2 UCC cremoris MG-SK +

SMQ86 (6) cremoris MG-SK -

US 3 cremoris MG-SK -

E8 cremoris MG-SK -

MG1614 (8) cremoris MG-SK -

901-1 (3) cremoris MG-SK -

184 UCC lactis unknown -

Phage Source Year of first Propagating Genome G+C No. orfs RBP- location report/isolation host length (kb) % group fdl3 Denmark 2004 FD13 30.674 34.7 53 1

P113g Germany 1986 IL1403 30.796 34.1 58 2

P272 Germany 1986 IL1403 30.778 34.1 61 2

936 NZ 1984 158 27.302 34.5 49 2

P475 Europe Unknown 455 30.961 34.3 57 3

P680 Germany 2009 IL1403 29.631 35.1 49 2 φ7 Denmark 2004 V32.2 32.382 34.2 57 1

645 Denmark 2004 IL1403 29.247 35.0 51 3

340 Denmark 2010 IL1403 32.337 34.5 58 3

ViridusJMl Ireland 2010 JM2 31.090 34.3 59 1

PastusJM3 Ireland 2010 JM3 28.674 34.4 52 1 Table 2. Host range of phages assessed in this study

Infected by phage:

Strain subspecies CWPS P680 P113g P272 P475 645 340 φ7 fdl3 936 Viridus Pastus

JM3 cremoris MG-SK

Table 3:Primers for CWPS multiplex PCR

Primer Sequence (5'-3') Product size name (bp)

MG-SKfw AAAGCTCATCTTTCCCCTGTTGT (SEQ ID NO: 1)

MG-SKrv GCACCATAGTCTGGAATAAGACC (SEQ ID NO: 2) 686

IL-KFfw GATTCAGTTGCACGGCCG (SEQ ID NO: 3)

IL-KFrv AGTAAGGGGGCGGATTGTG (SEQ ID NO: 4) 183

UC-CVfw GTGCCTATGCTCCGTTAGTC (SEQ ID NO: 5)

UC-CVrv CGAGGGCCAATCTCTTTACC (SEQ ID NO: 6) 442

CONfw GTACACTATGTTTATAACAATCATCCAG (SEQ ID (Control)

NO: 7)

CONrv GCAAACCAGATTCAAAGTCAGTATG (SEQ ID NO: 8) 891

Table 4: Amplicon (product) sequences

MG-SK

AAAGCTCATCTTTCCCCTGTTGTCTATGTAAACAATGTATCAATTCAACAAGGAACAAGT

GGAATGACGGCGAATGTGATTTCTCAGACAACTGGAACTCAGATTACCTCGTCTGCT CCC AAAGTGAATACGACTGGTATAAATTTTGTTGAAGTGGATGCAAATAAAAATAAGATGGT TAGAGATGCTGTTTATATTTTAGGGAAGAATGTGGGTGGTAAAAAGTATCTATATGATAG TCAAGGGAAATGGAGCGAAATTCAAGATTTGTCTACAGTTTCTCCTACAAGCTATACTTT

TAATACAAGATTCAATTATGATTATGAACGTGATACCAAAATTAATCAATCTTTAAT CAA ATTATTTGGTTTGGGAGAAGGAAAAGATTATTTCCTTTATCAGGTTGCCGCTCCTACTAA CTACTCTGTTGATAAAACACCCATTGATTTTTCTATTTTTTCAGAAAATGTAGTTTCGCC T AATGGTAGCCAACTTACTAAAACTAGTATGAAGACAGCAAGCAACCAATCATTTAAACT AAATGGTCTTATTCCAGACTATGGTGC (SEQ ID NO: 9) IL-KF

GATTCAGTTGCACGGCCGTATGAAACTCATCAAGTTAATTTTGATAGTACGATGACACTT

GCAGTTTATGGTGATGAGCCAACACTTCCTAAGCAGGAAGAGGGCACAATCCGCCCC CT TACT (SEQ ID NO: 10)

UC-CV

GTGCCTATGCTCCGTTAGTCTTTGATCATGAAATCCAAATTTCCCCAACAGCAAGTGATA

CATTGAAAATGATTAATTTTCCTAAAAAACCAGGAATTTATTCTGAAAATCTAACTG GGA

TTTCAAGTTGTCTGGTTGTAAAGAGCGACTTATTAATAGAAATTGGCGGACTGAATA TTG

GGTAGTAGTAATTAATGAAAAAATAAATCACCATCTTTCTATTCAAGAAATAAATCA AAC

CCGGAATTGCTCTTAAATCTTTATAAAAAATATGTTAGAATGATTATTAAAGGAATT CTT GGTAAAGAGATTGGCCCTCG (SEQ ID NO: 11)

CON

GTACACTATGTTTATAACAATCATCCAGACGTGCATATCACAGTTTTGGATAAATTA ACT

TATGCAGGAAATGTTAATAACATTAACATGTTATTTGACAGTGGACGTGTCGAACTT GTT

GTTGGCGACATTGCAGACCCTGAAATTGTTGATCAAGTGGCTTCTAAAGCAGATGCA ATT

GTTCACTATGCAGCTGAGAGCCATAATGATAATTCATTGAAATCTCAAGATGAATTT ATA

CAAACAAACTTTATCGGAACTTATACGTTGATTCAAGCGGCTCGTAAATATGATTTA CGT

TTCCACCATGTTTCTACTGATGAAGTTTATGGTGATTTGCCTTATCGCGAAGATTTG CCAG

GCCATGGCGAAGGTGAAGGTGAAAAATTTACTGACAAAACACCTTACAATCCATCAA GC

CCCTACTCTTCAACTAAAGCGGCTTCTGATTTAATTGTTCGTGCTTGGGTACGTTCA TTTG

AATTTATTCCTCGTCAAATTACTAATATTCTTTCAGGAATCAAACCAAAACTTTACG GTG

ATGGTAAAAACGTTCGAGACTGGATTCATACAGACGACCATTCATCAGGCGTTTGGA CA

ATTTTGAATAAAGGTCGTATGGGTGAAACTTACCTTATCGGTGCAGACGGTGAAAAA AA

TAACAAAGAAGTTCTTGAAGATATTTTGACTCGTATGGGCAAAGATAAGAGCGATTA TG

ATCGTGTAACTGACCGTGCTGGCCATGACCTTCGGTACGCAATTGACAATACAAAAT TGC

GTACCGAACTTGGTTGGGCTCCAAAACATACTGACTTTGAATCTGGTTTGC (SEQ ID NO:

12)

MG-SK

ttgaaagtattaaaaaactatcttctgaatagctcttaccaattattgattgtgatt attccaattatcacgattccttatatttctagggtgcttggcacaacg gctataggtttaaatacatttacctatgcaattattcagtactttgtattagctggttca attgggataacaacttatggaaatcgagagattgcatatcatc aatctgataaagaaaagagaagccaaattttttgggaaatttcatttttgagattttgta ccattgctctctcttttcttattttctgtattttcttagcctttcaaa aacaggattttgagatttatctcttgcagagtatcgctattatcgctgcggcttttgata tttcttggtattttatgggagtggaaaatttcaagcgaacagt agggcgtaattttattgtctctattatttcagttattttcatctttacttttattaagag tccaaaggatttgcccatttatgttttaatcattactggaacatcattg attggaaatctttcactttggccatatttgcgcaaggaaattttcgctcccaaatggaaa gaattagcattaggacatcatttaaaacccacactattactt tttttgccgcaaattgctacacagatttatacaattgcaaacaaaacgatgattgggatt tttgatgggaaaacggcatcgggattttttagtcaatcgga tagtctgattaaagtaacattgagcattgtcacttctttaggtgtagtcatgttgcctca tgtttcaaatcttttttcaaaaggtaaaattaaagaagttcaag agactttaaaaaaatctttcgtccttatgactgggcttgctgtaccgataatgtttggcg ttatgggaatagcactcaattttgcaggattctttttcggccc taaatgggtagctgttggtcctttgctaatgatggaagccccaattattatattcattgc ttggagtaatgttttgggaattcagtaccttcttccattaaatc ggatgagagaattcacaacttcggtaacaattggtgcagttcttaatattttacttaatt ttgctttgattcctcttttggggctcactggtgcaatgattgcg acagtaattgccgaggcttcagtgacgatttatcaattttatattttgagaaatgacttt gagataattccaatgattttatcttgctggaaatactttttatctg gtgcagtgatgtttggagctgttttctatttaaacaattctttgaagatgaatatgcata atcttatatttcaagtattgattggtgctataatatatataattctt aatatcttattgaaatccagtctttttattgaagtcaaaaagattgtaagtaaaaaataa (SEQ. ID. NO. 13).

IL-KF

atgataacaaagagtaacatactaataacaggtggagcaggttttattggctcaagt ttagcgaacgaattactacctcaaaataagattactgttattg ataatctttcgatgggagattttaataatcttcatgaaacaagtaatcttacaaaaattt taggtgatgtcactgataaaaatctcctggtaaaagttttgga agaaaatgattttgattatatttttcatttagcagcaattgcttcggttgccgattcagt tgcacggccgtatgaaactcatcaagttaattttgatagtacga tgacacttctcgagatcttacgtcaaaataaaaaatcattgaaacgatttgttttttctt caagcgcagcagtttatggtgatgagccaacacttcctaagc aggaagagggcacaatccgcccccttactccttatgcaattgataaatttgcatctgaaa aaatgacaatgatttataataatttatatgatgttcctacca gtgcaacacgctttttcaatgtttatggaccaaatcaaaatcccagttc

accctattcaggatttatttccattcttgttgaccgtttgcgagaaaatacagaatt gactatttttggagatggagaacagtctcgagattttgtatatata gaagatgttattcaagcattattactaatagcgacctctgaacaatcctttggagaagtc tataatgtcgggactggggtcaaaaattcaataaacgattt aacgaaatttgctcaaaaatttacaaataaagagttatctatcaaatttgatgatgtgcg acaaggagatatcaaagactcagtttccgatatttcaaaac ttaaggacataggatactcacctaaatttgatttatctaatggtatgaagaagtatctca actacgagtttaaataa (SEQ. ID. NO. 14).

UC-CV

atgaatttaacaatttgcttagtagcttatagtcaaaaatttacagagacagtctca ttttattctttattaaacttgactaagaatctaaaagaaaatattaatt tatatatttttgacaatggaagtgaagatttttcgtcctctcatgaggagttggatactc attcatttcatagcttgaactatatttataataaacaaaaagaa cgtggtactagaattgcttatcaaagtattttagatgttagtcaagatgaatggttaatg tttttagacgatgatacagaaatttcacaaccatatttatcga aaattttatcagaaataaaaaaagaaaaccaaagtgatatttgtgcctatgctccgttag tctttgatcatgaaatccaaatttccccaacagcaagtgat acattgaaaatgattaattttcctaaaaaaccaggaatttattctgaaaatctaactggg atttcaagttgtctggttgtaaagagcgacttattaatagaaa ttggcggactgaatattgaatttcccttagattatcttgaccattggctattttggaaaa tttttaattctaataaaaaggtagtagtaattaatgaaaaaata aatcaccatctttctattcaagaaataaatcaaacaaatgatctgagattttatagtatc ttctctagtgaatatcatttttataagtattataagccggaattg ctcttaaatctttataaaaaatatgttagaatgattattaaaggaattcttggtaaagag attggccctcgttg

gaaaattttattaaaaatattattggagagaaaatga (SEQ. ID. NO. 15).

Table 5. Primers used in the sub-classification of the MG-SK CWPS type lactcococcal strains (CI to

C5)

Primer name Primer sequence (5 '-3') SEQ ID Expected amplicon size (bp)

Clfw gtcatcaaacatactttcgtc 16 650

Clrv aagttttgccattgtttctcc 17

C2fw gaacaatggattatttatgctga 18 450

C2rv attcccattttcagcaacaag 19

C3fw gttgtaattgttactagccag 20 250

C3rv tcaatcgcattatagattacacc 21

C4fw gattttattcgaggcttagca 22 968

C4rv tagcattacaatcaatctgtca 23

C5fw gattatattcggggcttagca 24 1141

C5rv tgtaatatggtattgtctagca 25

Table 6: Sub-typing amplicon (product) sequences gtcatcaaacatactttcgtcatatgtgcgtatatgcaatctccgtatttagaggaaagt ataaaatcaattttagaccaaggatctattaaagaaggaaa tcagaagtggttttatatacttctactcctaatgattatatagaaaatatttgtcataaa tataatatcaaaatatttattggtgaaggtggaggcattggagc agattggaatggagctttagcggcagttcaaactaaatatgctacaattgttcatcaaga cgatctatatgataagaaatatggagaaatgataattaat gattttgagtctcaaaaagactctaatattgtttttactgactattatgaaattgatgaa tactctaaacctagaaaaagaaatattaatttaaaaataaaaag tttaggattaaaactaatgtctttttgggaaaataaaaaatatcaaagaagagtttactc ttttggtaattttatttgttgcccagctgtttcttataatatggaa cgtcttaaagattttagatttaatgaagagatgaaaatggcggtagactgggatgcatgg gaaagaataatgaaaaaatctggacatgtccattatctt ccgctaaaattgatggctcatagaatacatagtgattcggaaacaaccaataatacttta aataaaaacagagaaaaagaagagcacgagatgtttcg aagatattggggagaaacaatggcaaaactt [SEQ ID NO. 26] C ₂

gaacaatggattatttatgctgaaaaattaaagcaaatgtttcctgaaatgaaatta atagggtggctgcatctggatctcaaccattatgaaacttatcat cttgcgaaaagcaaaaatcaatttttaaatggtcttagggtgtgtgatcggttgattgta ctaactcaagaggaaaaaaatgttctagaaaaccgcgga tttgaaaaagttcaagttttgcataatccacttacattaaatgatacaaataaagttgtg gacttgaataaaaaaataatatcatgggtaggacggattgat attttgcataagggcttggactatttaatagagatagcgaaaattttgcctgacgactgg gaaattagagtagctggttccggaagggacaacaaaag atttaaaaaattgattgaaaaaaataggcttacgaataaaattttatgggtaggtccgaa aaatggagaagaactaacatatcattatttaaatagctcca tttttttaatgacatcaagatttgaaagctttggcttagtattggtagaagcaatgaact atggtcttcctataattgcattctcacaaacaggaagtgatga aattcttaaaaatgaacaatatggccttgttgctgaaaatgggaat [SEQ ID NO. 27]

C ₃

gttgtaattgttactagccagcatgataaagacttgcctactaaagaagaatttgat catttgaaaatatatcggttgcctattagaaagatttggaaaaat cggtatccttttccattaaaaaatgaaagatataaacaattaatttctgatattacttct gaaccaattgactattatgtagtgaatacaagatttcaattgcct gctttattaggtgcccagttagcaaaaaaagcaggaaaagaagccttggtattagagcat gggaccacgtacctcacattaaataattctttgttagat agcatacttcacaggatagagcactttttggttaaaaaaatcaaaaaaaatactaagact ttttacggtgtttcaaaagaagctactgaatggttaaaga cttttggaatagaggctaaaggtgtaatctataatgcgattga [SEQ ID NO. 28]

C ₄

gattttattcgaggcttagcaaagaaaattttaacatagaaagagtatttttataaa tgagtgaaaagaaatatgattcggagactttggaacgacttcaa gagatactacgaatgatgttagcagattttgataaaatatgctaagataacgatattgat tattttttaatgggtggaagtttacttggagctgtaaggcac aatgggatgataccttgggatgatgatattgacgttggaatgactggtgagaattatgat aaatttatacaagtcatgaataattcacaaaatgaaaaat atagtcttatgagtagtgagagcgaggaaagctataccactcagattgtactggatattt tagcatttgacaatctagcagataatgaaagacgtgcaa aaattcaaggtataaaatctttcatatatgggaaactttcatatcttacaacaattgaaa accctacagttcatcaagtaggaataaaactattttttattaat actacaataaaaagtctttcaaaaaacattcaaactttttaaagttacccctaaattttt tattaaaaaaggtgaaggagtagctagaaaatataactcttct caaacgcaaagagtaatgtatatgaatcagtcaaaaatgttttcagaaatttttgatcga aatgacctcttaccaactagacgtgttgattttgatggaca aaaaataaaaattccaaaggaaacagataaatatttggctgcacattatggtgattatat gacacttccaccagaagataaaagatataatcattatacg gccattttagattttggaaagtataaataaggtaaaatataatgaaagataaagaaaaaa ttgtattggttgctggaacttttgatattttgcatgagagcc acgttaatatgttgaaaaatgctaaaaatcttggtgacagattgattgtaatgcta [SEQ ID NO. 29]

C ₅

gattatattcggggcttagcaaagaaaattttaacatagaaagagtatttttataaa tgagtgaaaagaaatatgattcggagactttagaaggacttca agagatactacgaatgatgttagcagattttgataaaatatgcgaagataacgatattga ttattttttagtaggtggaagtttacttggagcgataaggc acaatgggatgataccttgggatgatgacattgacattggtatgaccggaaaagattatg ataaatttattcaaatcatgaagaagagggaatctgata aatatacgcttataagtagtgaaacaaataaagaatttccatatttgcgatctgcgggat ttatgctaaatggtacaaaatttataaaagatatttcaataat ggatgaaacctcttctagtatagtagttgacattattgcgttcgataacttagcagatgg taagttaaaaagcatctcacaaggcttaaaaacatttttcta cggtaaatctgccttccttacaactctgaataatcctacgaatcataggaaaggaatttc aaaaattgttacgacagttatgataaaaatgctccatggat tatttaagttatttaaagtaaccccacatttcttcattaacaaaggaaacaaaatagcaa cgaaatataatatgctggaaacaggaagagtcatgtacat gaatgaatctaaaccgtttttagttacaattaagaagaaaaatttatttcccataaagaa aattccttttgatggcttgatgattagtgtcccaaataaccca gaacagtatttattagaaagatatggagattataagacactaccgccagaaagtgagcaa tataatcatttcccagatgtattggactttggagaataca aaaatttaaggagaagagagcaatgaaattagcattattgacggctggaggagttggtag ccgtatgaaacaagaggtacctaaacagtttattcatg taaatgataaacctttaatcatttatgctttagaggcttttcaaaagcatccagatattg atgtaattgctattgcatgcttagaaggatggaaaaatgttttg gaggcatatgctagacaataccatattaca [SEQ ID NO. 30] Table 7. Strains, plasmids and bacteriophage used in sub-typing examples

Strains, plasmids or phage Relevant features Reference/source

Bacterial strains

Lactococcus lactis subsp. L. Lactis MG1363 derivative containing nisRK. (14) cremoris NZ9000 Host to ski

Lactococcus lactis subsp. Host to φΙΧ3, TP901-1E, ViridusJM2 and JW31 (3)

cremoris 3107

Lactococcus lactis subsp. Nisin producing L. lactis strain (14) cremoris NZ9700

Lactococcus lactis subsp. NZ9000 with GAATTC insert in LLNZ_0045, This work cremoris NZ9000-GT1 resulting in an in-frame TGA stop codon

Lactococcus lactis subsp.

cremoris NZ9000-GT2 NZ9000 with GAATTC insert in LLNZ_0050, This work

resulting in an in-frame TGA stop codon

Escherichia coli One Shot® F-racrA Δ (mrr-hsdRMS-mcr C) φδΟ/acZ ΔΜ15 Invitrogen, USA TOP10 MacXI rechl ara Δ139 Δ {ara-leu)l '697 galXJ

galK rpsL (Str ^r ) end Al nupG

Plasmids

pJP005 pNZ8048 containing recA (26, 27) pPTPi E. coli-L. lactis shuttle vector, PnisA, Tet ^r (20) pPTPiC2 pPTPi containing genes 3107_003, 3107_4 and This work

3107 5

Bacteriophages

cpLC3 P335 species, propagated on 3107 (16)

TP901<?rra P335 species, Em', propagated on 3107 (13)

The invention is not limited to the embodiment hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.

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