BACTERIOCfN POLYPEPTIDES AND USES THEREOF

Technical Field

[1 ] The present invention relates to novel bacteriocins with a wide antimicrobial activity against bacteria, including food spoilage and pathogenic bacteria, and the genes that encode said bacteriocins. !n particular, the present invention relates to the isolation, characterization and use of a novel cyclic bacteriocin, Plantacyciin B21AG and biologically active fragments and variants thereof, and the genes associated with Plantacyciin B21AG production and its cyclization and their application as a potential heterologous expression system for the expression of cyciised proteins, and transfer of these genes to other related bacterial strains.

Background of invention

[2] Lactic acid bacteria (LAB) have a long history of application in fermented foods because of their beneficial influence on nutritional, organoleptic, and shelf-life characteristics. They cause rapid acidification of the raw material through the production of organic acids particularly lactic acid. The use of functional starter cultures of LAB to confer functionalities beyond acidification is attracting increasing interest.

[3] LABs also produce a variety of compounds with antimicrobial activities, these include bacteriocins which are proteinaceous substances having antimicrobial activity against species closely related to the producer strain and/or other food spoilage and pathogenic bacteria.

[4] There is a need for more natural and microbiologically safe food products without the use of chemical additives, and for natural preservatives in the food industry to prevent the growth of food spoilage and pathogenic bacteria. [5] The present inventors have characterised a novel cyclic bacteriocin of

LAB, expressed by the Lactobacillus plantarum strain B21 ^* which has a wide antimicrobial activity against Lactobacillus (LAB) strains and food borne pathogens. As demonstrated herein, the bacteriocin exhibits advantageous stability characteristics due to its unexpected cyclic nature. The bacteriocin, Plantacyclin B21AG, is the first cyclic bacteriocin to be discovered in Lactobacillus plantarum. Moreover, the genes responsible for Plantacyclin B21AG production are located on an autonomous plasmid, Plasmid 1 . The present inventors have also demonstrated Lactobacillus plantarum strains B20, B31 and B33 contain a plasmid encoded cyclic bacteriocin with 88% amino acid sequence similarity to mature Plantacyclin B21AG.

[6] In one aspect the invention provides an isolated polynucleotide which encodes a bacteriocin or a biologically active fragment or variant thereof, wherein the bacteriocin is Plantacyclin B21AG.

[7] In another aspect, the isolated polynucleotide which encodes a bacteriocin or a biologically active fragment or variant thereof, wherein the bacteriocin is a cyclic bacteriocin. In one aspect, the polynucleotide described herein comprises: (i) a sequence of nucleotides as provided in SEQ ID NO: 3 or SEQ ID NO: 53 (ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in SEQ ID NO: 6 or SEQ ID NO: 52; (iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 38% identical to SEQ ID NO: 6 or SEQ ID NO: 52; (iv) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 52% identical to SEQ ID NO: 6 or SEQ ID NO: 52; (v) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 64% identical to SEQ ID NO: 6 or SEQ ID NO: 52; or (vi) a sequence complementary to any one of (i) to (v).

[8] In another aspect the invention provides a substantially purified and/or recombinant bacteriocin polypeptide or a biologically active fragment or variant thereof, wherein the bacteriocin is Plantacyclin B21AG, In one embodiment, the bacteriocin is a cyclic bacteriocin. [9] In one embodiment, the invention provides a polypeptide according as described herein wherein the polypeptide comprises: (i) an amino acid sequence as provided in SEQ ID NO: 6 or SEQ ID NO: 52; or (ii) an amino acid sequence encoded by a polynucleotide as described herein.

[10] In another aspect, the invention provides a plasmid or vector comprising at least one polynucleotide as described herein. In one embodiment, the plasmid is a Lactobacillus plantarum Plasmid 1 .

[1 1 ] In another embodiment the invention provides a vector as described herein wherein the vector is an expression vector,

[12] In another aspect the invention provides a host cell comprising at least one polynucleotide according as described herein and/or at least one plasmid or vector as described herein,

[13] In one embodiment the host cell is prokaryotic cell.

[14] In one aspect the invention provides a strain of Lactobacillus comprising a polynucleotide as described herein,

[15] In another aspect the invention provides a strain as described herein, wherein the strain is selected from the group of Lactobacillus plantarum B21 ^* , B20, B31 and B33.

[16] In another aspect the invention provides a probiotic, a medicament or a culture comprising a host cell as described herein or a strain as described herein.

[17] In another aspect the invention provides a composition comprising the bacteriocin polypeptide or biologically active fragment or variant thereof described herein.

[18] In one embodiment the invention further comprises a pharmaceutically acceptable carrier, vehicle or diluent, and/or a second bacteriocin.

[19] In one embodiment the invention provides a use of a bacteriocin polypeptide or a biologically active fragment or variant thereof, a strain, a culture a composition, for a method of food production as described herein. [20] In another aspect the invention provides a method of food production comprising contacting a food with an effective amount of a bacferiocin polypeptide or a bioiogically active fragment or variant thereof, a strain, a culture or a composition as described herein.

[21 ] In another aspect, the invention provides a method of inhibiting the growth of a bacterium in a material comprising contacting the material with an effective amount of a bacteriocin polypeptide or a biologically active fragment or variant thereof, a strain, a culture or a composition as described herein,

[22] In another aspect, the invention provides a method of forming an intramolecular peptide bond in a target polypeptide comprising contacting the polypeptide with one or more Piantacyclin B21AG-cyciisation polypeptide.

[23] In another aspect the invention provides a method of forming a cyclic Piantacyclin B21AG comprising contacting a Piantacyclin B21 AG with one or more Piantacyclin B21AG~cyciisation polypeptide.

[24] In one embodiment, the one or more Piantacyclin B21AG-cyciisation polypeptide is selected from the group consisting of ORF-B, ORF-C, ORF-D, ORF-E, ORF-F, B33-ORF-21 and biologically active fragments or variants thereof.

[25] In one embodiment the target polypeptide is a polypeptide having a biological activity or comprising an antigen.

[26] In another embodiment, the target polypeptide is flanked at the N- terminus and/or the C-terminus by a Piantacyclin B21AG polypeptide or a fragment thereof.

[27] In another aspect the invention provides an isolated polynucleotide which encodes a Piantacyclin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof.

[28] In another embodiment the invention provides an isolated polynucleotide which encodes a Piantacyclin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof, wherein the Piantacyclin B21AG-cyclisation polypeptide selected from the group consisting of ORF-B, ORF-C, ORF-D, ORF-E, ORF-F, B33- ORF-21 and biologically active fragments or variants thereof.

[29] In one aspect the invention provides a polynucleotide as described herein, wherein the polynucleotide comprises: (i) a sequence of nucleotides as provided in SEQ ID NO: 7, 9, 1 1 , 13, 15, 20, 22, 24, 26, 28 or 30; (ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in SEQ ID NO: 8, 10, 12, 14, 16, 21 , 23, 25, 27, 29, 31 or 32; (iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 35% identical to SEQ ID IMG: 8; (iv) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 39% identical to SEQ ID NO: 10; (v) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 52% identical to SEQ ID NO: 12; (vii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 40% identical to SEQ ID NO: 14; (viii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 31 % identical to SEQ ID NO: 16; or (ix) a sequence complementary to any one of (i) to (viii).

[30] In another aspect the invention provides a substantially purified and/or recombinant Piantacyciin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof, wherein the Piantacyciin B21AG-cyclisation polypeptide is selected from the group consisting of ORF-B, ORF-C, ORF-D, ORF-E, ORF-F B33-ORF-21 and biologically active fragments or variants thereof.

[31 ] In one embodiment, the present invention provides a polypeptide as described herein wherein the polypeptide comprises: (i) an amino acid sequence as provided in SEQ ID NO: SEQ ID NO: 8, 10, 12, 14, 16, 21 , 23, 25, 27, 29, 31 or 32; or (ii) an amino acid sequence encoded by a polynucleotide as described herein.

[32] In one aspect, the invention provides a method of modifying a strain of bacteria comprising transferring into the bacteria at least one polynucleotide as described herein and/or at least one plasmid or vector described herein. In one embodiment, the strain of bacteria is a probiotic strain. Brief Description of Drawings

[33] Figure 1 shows fast protein liquid chromatography (FPLC) of the active fractions from a cation exchange column eluted with a linear sodium chloride gradient (green, dotted line). The eluted protein was detected at 280 nm (blue, solid line) and 214 nm (red, dashed line).

[34] Figure 2 shows the purified Plantacyclin B21AG bacteriocin protein resolved on Tris-Tricine-SDS-PAGE analysis. Figure 2A, the SDS-PAGE gel image; lane 1 , the low molecular protein marker; lane 2, the purified Plantacyclin B21AG protein. Figure 2B, the antimicrobial assay of the SDS-PAGE gel protein after renaturation and incubation with a sensitive indicator strain, L piantarum A6. Figure 2C, demonstrates the alignment of the single gel band and with the killing zone on the indicator plate.

[35] Figure 3 shows the IVIALD!-TOF MS spectrum of purified B21 ^* bacteriocin, Plantacyclin B21AG, showing the singly and doubly charged species. The average molecular mass of Plantacyclin B21AG is 5668 Da.

[36] Figure 4 shows RP-HPLC elution profile of the FPLC purified Plantacyclin B21AG; Top figure A220; Bottom figure A ₂ ao; An analytical ZORBAX Eclipse Cis column was fitted to an Aligent 1 100 RP-HPLC system with a flow rate of 1 m Urn in. Plantacyclin B21AG was eluted with approximately 85% water acetonitrile

[37] Figure 5 shows the UVA is absorption spectrum of purified PiantacyciinB21AG measured in a SH!MADZU 1800 UVA is spectrophotometer (solid line) with a 1 nm slit width, with a wavelength range 190 -500 nm). On the absorption spectrum of purified PlantacyclinB21AG; Arrow 1 , absorbance peak at 280 nm; Arrow 2, absorbance peak at 220 nm. The concentrated purified Plantacyclin B21AG sample was buffer exchanged into clean 20 mM sodium phosphate buffer pH 6.0.

[38] Figure 6 shows the CD spectra of purified Plantacyclin B21AG in 20 m!Vl sodium phosphate buffer. Spectra were recorded in a Jasco J-815 CD spectrometer in 1 cm cell at a protein concentration of 5.3 \M with a scan speed of 50 nm/min and temperature control at 25 °C. Spectra were averaged from three scans. [39] Figure 7 shows the partial de novo peptide sequencing of Plantacyciin B21AG using LC-MS/MS techniques. The +4 species centred at 1417.81 was manually selected for fragmentation analysis

[40] Figure 8 shows the partial de novo peptide sequencing of Plantacyciin B21AG using ESI-LC-MS/MS fragmentation techniques. This figure shows the identification of the b ions and the manual first principles extraction of a partial peptide sequence from the MS/MS data, the peptide sequence obtained was PGWAVAAAGALG.

[41 ] Figure 9 shows de novo peptide sequencing of Plantacyciin B21AG using ESI-LC-MS/MS techniques. This figure shows the identification of the late b ions and the manual first principles extraction of a second peptide sequence from the MS/MS data, the peptide sequence obtained was AAVILGV.

[42] Figure 10 shows the identification of the full amino acid and nucleotide sequence of Plantacyciin B21AG. (A) Amino acid sequences were obtained by de novo sequencing using LC-MS/MS. (B) Plantacyciin B21AG mature peptide sequence corresponding to the linear isotopic mass of 5682 Da, confirming the data obtained by MS/MS analysis. (C) The Plantacyciin B21AG full putative peptide sequence deduced from a search of the six frame translation of the total genome using the partial peptide sequences deduced from the MS/MS data. A single combined hit allowed the ORF for plantacyclinB21AG to be identified to a specific gene sequence in scaffold 5. Subsequent completion of a gapless genome and plasmid complement allowed this gene and its associate operon to be assigned to one of three new plasmid sequences.

[43] The hypothetical cleavage site of the leader peptide for Plantacyciin B21AG is indicated by an arrow. (D) The nucleotide sequence obtained from the new B21 ^* genomic data corresponding to the Plantacyciin B21AG amino acid sequence (scaffold 5). The symbol (-) demonstrates the translation stop codon. (E) The Predicted circular structure of Plantacyciin B21AG. The arrow shows the main ion fragmentation site identified by the proteomic analysis. The actual experimental mass was always 18 AMU less than the predicted mass of the mature peptide, and conventional IM-terminal sequencing repeatedly failed, hence its circular nature. [44] Figure 1 1 shows phylogeneiic analysis of Piantacyclin B21AG full protein sequence (a) and protein sequence alignment (gassericin A, acidocin B, pentocin KCA1 and Piantacyclin B21AG) (b) using Phylogeny.fr online tool (2). Figure (A): The protein sequence of cyclic bacteriocins were obtained from NCBI database, accession numbers AS-48, CAA72917.1 , gassericin A, BAH08712.1 , acidocin B, CAA84399.1 , butyrivibriocinARI O, AAC69560.1 , uberolysin, A5H1 G9.1 , circularin A, AAN86036.1 , garvicin ML, ACZ98827.1 , pentocin KCA1 , EIW1 922.1 , lactocyciicin Q, BAH2971 1 .1 , carnocyciin A, ACC93994.1 . Figure (B): similar residues are coloured as the most conserved ones according to BLOSUfV162 alignment score. Average BLOSUM62 score: Maximum: 3.0 (light grey/blue), low: 0.5 (dark grey). Gas-A (gassericin A), Aci-B (acidocin B), KCA1 (pentocin KCA1 ), B21AG (Piantacyclin B21AG).

[45] Figure 12 shows a full genetic map of the putative Piantacyclin B21AG operon from L plantarum B21 ^* , pentocin A from L pentosus KCA1 and gassericin A from L. gasseri LA39 obtained from previously publish work (Anukam et a/. 2013; Ito et al. 2009) for comparison. The bacteriocin related genes are represented by arrows in different colours/shades corresponding to the gene function. Small black arrows represent the predicted promoter sequences. Colour code for the cyclic bacteriocin cluster genes: green for the cyclic bacteriocin structural genes (GaaA, PenA, QRF- A): orange and hatched orange arrows for bacteriocin immunity genes (Gaal, Pen!, ORF-B and ORF-C); blue arrows for ABC-transporter genes (GaaT, GaaE, PenT, PenE, ORF-D and ORF-E): grey for membrane protein genes (PenB, PenR, ORF-F); black for genes that are not specifically related to bacteriocin function (Orf-a2, Offal).

[46] Figure 13 shows purified Piantacyclin B21AG shows activity against L. monocytogenes (A and B) in antimicrobial assays. 1 , purified Piantacyclin B21AG, 2; partially purified Piantacyclin B21AG, 3; sodium phosphate buffer and 4; MRS broth. The zones of inhibition demonstrate the Piantacyclin B21AG shows activity against L. monocytogenes.

[47] Figure 14 shows the nucleotide sequence of Piasmid 1 (SEQ ID NO: 17). [48] Figure 15 shows the predicted open reading frames (ORFs) and annotations of P!asmid 1 .

[49] Figure 16 shows the killing activity of the circular bacteriocin Plantacyciin B21AG can be transferred to a heterologous bacterial strain. This data shows killing of three indicator strains (Panel A: L lactis, Panel B: A6 and Panel C: ATCC 8014) by the probiotic LAB strain WCFS1 when transformed with a shuttle vector comprising the Plantacyciin B21AG operon ("Bac"). Well 1 ; B21 Non-concentrated, Well 2; WCFS1 Non-concentrated, Well 3; WCFS1 + pTRKH2 Concentrated, Weil 4; WCFS1 + pTRKH2 10-fold dilution, Weil 5; WCFS1 + pTRKH2 20-fold dilution, Well 6; WCFS1 + pTRKH2 30-fold dilution, Well 7; WCFS1 +Bac Concentrated, Weil 8; WCFS1 +Bac 10-fold dilution, Well 9; WCFS1 +Bac 20-fold dilution, Well 10; WCFS1 +Bac 30-fold dilution. Killing is shown by a cleared zone in the well diffusion experiment shown. Four different strains are evaluated; B21 ^* as positive control; untransformed LAB WCSFS1 which is an authentic probiotic LAB; LAB WCSFS1 transformed with a shuttle vector pTRKH2 with an erythromycin resistance marker; and LAB WCSFS1 transformed with a shuttle vector containing the full set of plantacyciin B21AG genes previously described (with additional ORF; B33 ORF-21 ), designated "WCFS1 + Bac". There is strong killing in well 7 on all three indicator strains and even when diluted 10- fold (well 8), No killing is seen in the absence of the Plantacyciin B21AG operon, even though the cells were also grown with antibiotic selection and washed in the same way (wells 3 to 6). Some staining of the agar rather than killing is seen, due to coloured media products in the concentrate (e.g. compare 3 and 4 with 7 and 8). This data demonstrates that Plantacyciin B21AG can be recombinantly expressed in heterologous species of bacteria, and confer killing activity on the heterologous bacteria. This data also demonstrates that Plantacyciin B21AG can be recombinantly expressed in probiotic bacteria, such as WCFS1 .

[50] Figure 17 shows the predicted open reading frames (ORFs) and annotations of the 20kb plasm id of strain B33.

[51 ] Figure 18 shows a full genetic map of the putative Plantacyciin B21AG operon from L. piantarum B21 ^* ("Plantacyciin B21AG"), B20, B31 and B33. The bacteriocin related genes are represented by arrows in different colours/shades corresponding to the gene function. Small black arrows represent the predicted promoter sequences. Colour code for the cyclic bacteriocin cluster genes: green for the cyclic bacteriocin structural genes (GaaA, Pen A, QRF-A); orange and hatched orange arrows for bacteriocin immunity genes (Gaa!, Peril, ORF-B and ORF-C); blue arrows for ABC-transporter genes (GaaT, GaaE, PenT, PenE, QRF-D and ORF-E); grey for membrane protein genes (PenB, PenR, ORF-F, B20 ORF-19, B31 ORF-15, B33 ORF-21 {"New Or ' )); black for genes that are not specifically related to bacteriocin function (Orf-a2, Or†-a1).

[52] Figure 19 shows the killing activity of the circular bacteriocin Plantacyciin B21AG can be transferred to a probiotic bacterial strain and killing is associated with the cyclic bacteriocin Plantacyciin B21 AG. A. Weil 1 ; B21 Non-concentrated, Weil 2; WCFS1 Non-concentrated, Well 3; WCFS1 + pTRKH2 Concentrated, Weil 4; WCFS1 + pTRKH2 10-fold dilution, Weil 5; WCFS1 + pTRKH2 20-fold dilution, Well 6; WCFS1 + pTRKH2 30-fold dilution, Well 7; WCFS1 +Bac Concentrated, Weil 8; WCFS1 +Bac 10-fold dilution, Well 9; WCFS1 +Bac 20-fold dilution, Weil 10; WCFS1 +Bac 30~fold dilution. This data shows killing of A6 by the probiotic LAB strain WCFS1 when transformed with a shuttle vector comprising the Plantacyciin B21AG operon ("Bac"). Killing is shown by a cleared zone in the well diffusion experiment shown. Four different strains are evaluated; B21 ^* as positive control; untransformed LAB WCSFS1 which is an authentic probiotic LAB; LAB WCSFS1 transformed with a shuttle vector pTRKH2 with an erythromycin resistance marker; and LAB WCSFS1 transformed with a shuttle vector containing the full set of plantacyciin B21 AG genes previously described (with additional ORF; B33 ORF-21 ), designated "WCFS1 + Bac". There is strong killing in well 7. No killing is seen in the absence of the Plantacyciin B21AG operon, even though the cells were also grown with antibiotic selection and washed in the same way (wells 3 to 6). Brown staining around well 3 results from concentration of the WCFS1 + pTRKH2 ceil free supernatant; some staining of the agar rather than killing is seen, due to coloured media products in the concentrate (e.g. compare 3 and 4 with 7 and 8). This data demonstrates that Plantacyciin B21AG can be recombinantly expressed in heterologous species of bacteria, and confer killing activity on the heterologous bacteria. This data also demonstrates that Plantacyciin B21AG can be recombinantly expressed in probiotic bacteria. This data also demonstrates a target polypeptide can be cyciised by contacting the target polypeptide with one or more Plantacyclin B21AG-cydisation polypeptides encoded by the plantacyclin B21AG operon. B, MALDI MS of culture supernatants shows the killing activity in Well "1 " (B21 ) and Weil "7" (WCFS1 expressing Plantacyclin B21AG) of Figure 19A is associated with circular Plantacyclin B21AG, This data demonstrates that Plantacyclin B21AG can be recombinantly expressed in heterologous species of bacteria, and confer killing activity on the heterologous bacteria. This data also demonstrates a target polypeptide, Plantacyclin B21AG, can be cyclised by contacting the target polypeptide with one or more Plantacyclin B21AG-cyclisation polypeptides encoded by the plantacyclin B21AG operon. MALDI MS of culture supernatant WCFS1 with shuttle vector pTRKH2 (Weil "3" of Figure 19A) demonstrates this strain does not express plantacyclin B21AG, and that strains comprising Plantacyclin B21AG operon produce a single Butanol extractabie bacteriocin polypeptide.

Detailed Description

[53] The present invention is based in part on the characterisation of new wide antimicrobial activity against Lactobacillus (LAB) strains and food borne pathogens such as Listeria monocytogenes and Clostridium by ceil free supernatants of B21 ^* a strain of Lactobacillus plantarum. The present inventors demonstrated by genome sequencing and conventional gene mapping that the function of the classical bacteriocin (pin) locus is impaired at the genetic level in B21 ^* but that surprisingly, B21 ^* expresses a novel cyclic bacteriocin, referred to herein as Plantacyclin B21AG. Importantly, the present inventors have demonstrated the Plantacyclin B21AG has antimicrobial activity, and can be transferred to other species of bacteria. Accordingly, in one aspect the present invention provides an isolated polynucleotide which encodes a novel cyclic bacteriocin or a biologically active fragment or variant thereof, wherein the bacteriocin is Plantacyclin B21AG. This is the first such report from a Lactobacillus plantarum strain, many of which are in commercial use as probiotic organisms. Moreover, the associated genes located in the associated operon encode the post transiationai ability to catalyse cyciisation of the immature protein and concomitant secretion by a nonstandard export route that offers potential for the expression, cyclisation and secretion of other therapeutic bioactive proteins that have been appropriately engineered.

[54] Similarly, cyclic bacteriocin production has also been confirmed for strains B20, B31 and B33 where the central pin locus is also down regulated; these additional strains produce the cyclic bacteriocin Piantacyclin B21AG.

[55] The overall bacteriocin yield of the purification processes described herein was approximately 19% and the overall purification factor was more than 8, 000-foid. Without wishing to be bound by theory, a simple back calculation suggests that LAB B21 ^* produces approximately 1 mg/L bacteriocin in its cell free supernatant (CFS). The bacteriocin protein showed great stability during eight weeks of storage at 4 °C. Tris-Tricine-SDS-PAGE suggested a molecular weight of approx. 5.5 kDa and MALDI-TOF MS measurements of 5868 Da. The purity of the protein sample was confirmed by a single major peak on a RP-HPLC Cia column and single peak on MALDI. The UV-Vis absorbance spectrum of the bacteriocin gave evidence of multiple Trp residues and little or no Tyr residues. The UV~Vis data and the peptide sequence were then used to calculate a molar extinction coefficient at 280 nm and this was used to calculate a theoretical protein concentration of 16 μΜ which agreed well with that determined from conventional protein assays. CD spectroscopy was used to investigate the secondary structure of the protein and showed that the protein structure consisted of 69% a-heiix. Initial attempts to determine the N- termina! amino acid sequence of the B21 ^* bacteriocin protein by Edman-degradation failed, suggesting that the peptide was potentially N-terminaily blocked. A de novo sequencing approach was then taken to identify the peptide sequence of the purified bacteriocin protein using ES!-LC-IV1S/MS techniques. Two short sequences were manually obtained from the MS/MS data and were searched against a six frame translation of the entire B21 ^* genome. A strong hit was found which correctly predicted the remaining B ions in the MS/MS data. Furthermore, a mass discrepancy between the theoretical and experimental masses of 18 Da, strongly suggested that the bacteriocin was in fact cyclic. Piantacyclin B21AG has 67% identity to the pentocin KCA1 , and was shown to be stable and partially resistant to proteolysis with a wide spectrum of bacterial killing activity, including inhibitory activity against food borne pathogens Clostridium perfringens and Listeria monocytogenes in addition to other food spoilage LABs,

[56] Without wishing to be bound by theory, the present inventors consider the new cyclic bacteriocin possesses properties desirable for food preservation in terms of pH and temperature stability.

[57] In another aspect, the isolated polynucleotide which encodes a bacteriocin or a biologically active fragment or variant thereof, wherein the bacteriocin is a cyclic bacteriocin.

[58] As used herein, the term "isolated," when referred to a molecule, refers to a molecule that has been identified and separated and/or recovered from a component of its natural environment.

[59] As used herein the term "bacteriocin" includes proteins such as a polypeptide produced by a microbial cell (e.g. a bacteria), wherein such polypeptide possesses microbicidal (e.g. bactericidal) activity. The microbicidal (e.g. bactericidal) activity of a bacteriocin can be measured using several techniques known by those skilled in the art, such as those described herein in the Examples. The presence of a bacteriocin can also be assessed with other standard methods such as Western blotting, 2D electrophoresis, capillary electrophoresis, imaging techniques (e.g. specific antibodies coupled to immunofluorescent compounds or an enzyme that enables co!orimetric visualisation), ELISA, RIA and protein micro-array.

[60] The present inventors have demonstrated that Plantacyclin B21AG has bactericidal activity against Lactobacillus, Clostridium and/or Listeria spp.

[61 ] In one embodiment, the Listeria species is selected from the group consisting of L fleischmannii, L. grayi, L. innocua, L. ivanovii, L. marthii, L. monocytogenes, L. rocourtiae, L. seeiigeri, L, weihenstephanensis, and L. welshimeri.

[62] Accordingly, in one embodiment the bacteriocin Plantacyclin B21AG possesses bactericidal activity against Lactobacillus plantarum A6, Lactobacillus plantarum ATCC 8014, Lactobacillus arabinosus 17-5, Lactococcus iactis 345-18, Lactobacillus brevis 19012, Listeria monocytogenes 192/1-2 ACM 3173 and/or Clostridium perfringens 52/6-1,

[63] As used herein, the term "lactic acid bacterium" or "LAB" refers to a gram- positive, microaerophiiic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found within the order "Lactobacillaies" which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp,, Brevi bade um spp,, Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria, bifidobacteria, i.e. Bifidobacterium spp., are generally included in the group of lactic acid bacteria. These are frequently used as food cultures alone or in combination with other lactic acid bacteria. Lactic acid bacteria, including bacteria of the species Lactobacillus sp. and Streptococcus thermophilus,, are normally supplied to the dairy industry either as frozen or freeze-dried cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product.

[64] The term "starter culture" as used herein, refers to a microbiological culture which performs fermentation.

[65] The term "strain" as used herein, refers to a stock of bacteria obtained from a specific source and maintained in successive cultures or animal inoculations. Strain is used interchangeably with the term "isolate".

[86] The present inventors have also demonstrated the bacteriocin Piantacyciin B21AG is a cyclic bacteriocin. The putative Piantacyciin B21AG peptide has 91 amino acids (pre-peptide) consisting of a 33-amino-acid leader peptide and a 58-amino-acid pro-peptide. The putative cleavage site for removing the leader peptide is located between asparagine and isoieucine. Cleavage at this site would remove a 33-amino-acid leader sequence and produce a mature bacteriocin containing 58 amino acids. The 58-amino-acid peptide is predicted to undergo a post-translationai modification that results in the linking of the N-terminal asparagine to the C-terminal isoieucine, with the elimination of a water molecule, resulting in the active and mature Piantacyciin B21AG molecule that is actually secreted.

[67] The present inventors have also demonstrated Lactobacillus plantarum strain B20, Lactobacillus plantarum strain B31 , and Lactobacillus plantarum strain B33 contain the bacteriocin Piantacyciin B21AG.

[68] In another aspect the invention provides a substantially purified and/or recombinant bacteriocin polypeptide or a biologically active fragment or variant thereof, wherein the bacteriocin is Piantacyciin B21AG. !n one embodiment, the bacteriocin is a cyclic bacteriocin.

[69] The substantially purified and/or recombinant bacteriocin polypeptide or a biologically active fragment or variant thereof is an excellent candidate for directed evolution selection for new peptide-based antibiotics with increased antimicrobial potency towards conventionally antibiotic resistant organisms.

[70] Circular bacteriocins constitute a group of ribosomaSiy synthesized antimicrobial peptides characterized by their N-to-C-terminal covaient linkage, forming a structurally conserved circular peptide backbone. Circular bacteriocins are synthesized as linear precursors, containing a leader sequence of variable size which is cleaved off during maturation.

[71 ] As will be discussed in more detail below, circularization requires a post- translational modification catalyzed by an ABC transporter that has cyclisation activity. The present inventors have shown this activity is encoded in the same operon as the Piantacyciin B21 AG and is predicted to involve the QRF-D and QRF-E genes.

[72] The circular bacteriocins are distinct from other Gram-positive bacteriocins and are considered a separate group, following debate about which class in the classification scheme of Gram-positive bacteriocins should be ascribed to. Few circular bacteriocins (around 10) have been characterized to date and none from Lactobacillus plantarum. [73] The leader peptides in cyclic bacteriocins vary in length, from 3 to more than 30 amino acids. Without wishing to be bound by theory, the present inventors have characterised mature Plantacyclin B21AG (e.g. SEQ ID NO: 6 or SEQ ID NO: 52), and propose that the mature Plantacyclin B21AG is formed by cleavage of a leader peptide (SEQ ID NO: 5) from the full predicted peptide sequence (e.g. SEQ ID NO: 4 or SEQ ID NO: 19).

[74] Accordingly, in one embodiment, the present invention provides an isolated polynucleotide which encodes a bacteriocin or a biologically active fragment or variant thereof, wherein the bacteriocin is a cyclic bacteriocin, referred to herein as Plantacyclin B21AG.

[75] As used herein, the term "cyclic" refers to a cyclic or circular peptide, including those formed by circuiarisation, for example in the bacteria. Circuiarisation typically occurs during or shortly after a leader peptide sequence (typically at least at about 3 amino acids long) is cleaved off N-terminaliy where after the C-terminal peptide generally joins in a peptide bond with the N-terminal part of the peptide.

[76] In one embodiment the present invention provides an isolated polynucleotide which encodes a cyciisation polypeptide or a biologically active fragment or variant thereof.

[77] In one embodiment the present invention provides an isolated polynucleotide which encodes a Plantacyclin B21AG-cyciisation polypeptide or a biologically active fragment or variant thereof.

[78] "Polynucleotide", or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length, and include, but are not limited to, DNA and RNA, The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. The invention includes nucleic acid comprising sequences complementary to those described above (e.g. for antisense or probing purposes).

[79] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, piy-L-iysine, etc.), those with intercalafors (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxy! groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphoryiated or substituted with amines or organic capping groups moieties of from 1 to 20 carbon atoms. Other hydroxyis may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl- 2'-0~ailyl, 2'-fluoro- or 2'~azido~ribose, carbocyclic sugar analogs, alpha. -anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiesfer linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S("thioate"), P(S)S ("dithioate"), "(0)NR.sub.2 ("amidate"), P(0)R, P(0)0R, CO or CH ₂ ("formacetai"), in which each R or R is independently H or substituted or unsubstituted alkyl (1 -20 C) optionally containing an ether (— O— ) linkage, aryi, alkenyl, cycioalkyl, cycioalkenyl or araidyi. Not ail linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA. [80] "Oligonucleotide," as used herein, generally refers to short, generally single stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides,

[81 ] The inventors have characterised nucleic acids and isolated proteins encoded by the isolated nucleic acids and variants thereof, that can function as a bacteriocin. The present inventors have also characterised the nucleic acids and proteins encoded by the isolated nucleic acids that can confer a biological function on a ceil or other protein.

[82] Accordingly, in one aspect, the invention provides isolated polynucleotides which encode a bacteriocin or a biologically active fragment or variant thereof, wherein the bacteriocin is Plantacyclin B21AG.

[83] The nucleotide sequence of Plantacyclin B21AG is provided below (SEQ ID NO: 1 ):

AT G C T T T C AG CAT AT C G TAG C AG G T TAG GAT T G AAC AAG T T T GAG G T T G C T AT T T T AAT GAT CAT CTCTCT T T T TAT T T TAT TAT T T GCCACAG T T AAT AT T G T AT GGAT T GC AAGACAAT T T G GT GT GCAT T T G AC A AC A A AG C T T AC A C AAA AG G C T T TAGAT T TAT TAT C T T C T GGAGCAT C T T T G G G T AC C G TAG C AG C T G T TAT C T TAG G C G T T AC A T T G C C AG GAT G G G C AG T T G C AG C A G C TGGGGCTCTCGGGGGTACCGCAGCT TAA

[84] As a matter of routine, the skilled person will be able to identify the regions of the above nucleic acid molecule that encode the specific regions of Plantacyclin B21AG proteins, including the IM-terminai leader peptide and the point at which the bacteriocin is circularised, as described herein.

[85] For example, the nucleotide sequence encoding the N-terminai leader peptide of Plantacyclin B21AG is provided below (SEQ ID NO: 2):

AT G C T T T C AG C A TAT C G TAG C AG G T TAG GAT T GAAC AAG T T T GAG G T T G C T T T T T AAT GAT CAT C T C T C T T T T TAT T T TAT TAT T T GCCAC G T AAT

[86] The nucleotide sequence of mature Plantacyclin B21AG is provided below (SEQ ID NO 3): AT TGTATGGAT T GCAAGACAAT T T GGTGTGCAT T T GACAAC AAAGC T T ACACAAAAGGC T T T AG AT T TAT T A T C T T C T G GAG C A T C T T T G G G TAG C G TAG C AG C T G T TAT C T T AG G C G T TAG AT TGCCAGGATGGGCAGT TGCAGCAGCTGGGGCTCTCGGGGGTACCGCAGCT TAA

[87] The present invention includes those specific nucleotide subsequences, and any alterations that are available by virtue of the degeneracy of the genetic code. Furthermore, the invention provides nucleic acid which can hybridise to these nucleic acid molecules, preferably under "high stringency" conditions (e.g. 65°C in a 0.1 x SSC, 0.5% SDS solution). Nucleic acid according to the invention can be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself, etc.) and can take various forms (e.g. single stranded, double stranded, vectors, probes, etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other mammalian or host ceil nucleic acids).

[88] In one embodiment an isolated nucleic acid of the invention is a complementary DNA (cDNA) copy of a corresponding mRNA. An mRNA may be transcribed in vivo or in vitro encoding the encoded isolated proteins of the invention. Also provided are deduced amino acid sequences of a cognate peptide/protein of the cDNAs provided by the invention.

[89] The present inventors have characterised Plantacyclin B21AG polypeptides and polynucleotides from other strains of Lactobacillus planiarum, including strains B20, B31 and B33 as described herein.

[90] In one embodiment the polynucleotide described herein comprises: (i) a sequence of nucleotides as provided in (i) a sequence of nucleotides as provided in SEQ ID NO: 3 or SEQ ID NO: 53; (ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in SEQ ID NO: 6 or SEQ ID NO: 52; (iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 38% identical to SEQ ID NO: 6 or SEQ ID NO: 52; (iv) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 52% identical to SEQ ID NO: 6 or SEQ ID NO: 52; (v) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 64% identical to SEQ ID NO: 6 or SEQ ID NO: 52; or (vi) a sequence complementary to any one of (i) to (v). [91 ] In another embodiment, the present invention provides a substantially purified bacteriocin polypeptide or a biologically active fragment or variant thereof, wherein the bacteriocin is Plantacyclin B21AG

[92] In another embodiment, the present invention provides a recombinant bacteriocin polypeptide or a biologically active fragment or variant thereof, wherein the bacteriocin is Plantacyclin B21 AG.

[93] As used herein, the term "substantially purified" means that the polypeptide has been separated from its in vivo cellular environment.

[94] The amino acid sequence of Plantacyclin B21AG is provided below (SEQ ID NO: 4)

MLSAYRSRLGLNKFEVAILMI ISLFILLFATVNIV IARQFGVHLTTKLTQKALDLLSSGAS LGTVAAVILGVTLPGWAVAAAGALGG AA

[95] The amino acid sequence of the N-terminal leader peptide of Plantacyclin B21AG is provided below (SEQ ID NO: 5):

MLSAYRSRLGLNKFEVAILMI ISLFILLFATVN

[96] The amino acid sequence of mature Plantacyclin B21AG is provided below (SEQ ID NO: 6):

IVWIARQFGVHLTTKLTOKALDLLSSGASLGTVAAVILGVTLPGWAVAAAGALGGTA A

[97] The present inventors have demonstrated the mature 58-amino-acid peptide is predicted to undergo a post-transiationai modification that results in the linking of the N-terminal asparagine to the C-terminai isoleucine, with the elimination of a water molecule, resulting in the active and mature Plantacyclin B21AG molecule that is actually secreted.

[98] Accordingly, in one embodiment, the bactenocin is a cyclic bacteriocin.

[99] In one embodiment the present invention provides a polypeptide as described herein, wherein the polypeptide comprises: (i) an amino acid sequence as provided in SEQ !D NO: 6 or SEQ !D NO: 52; or (ii) an amino acid sequence encoded by a nucleic acid as described herein.

[100] The present inventors have shown the amino acid composition of the mature cyclic Plantacyclin B21AG consists of a very high proportion (59%) of hydrophobic amino acid residues (Ala, Val, Leu, lie, Phe, Trp and Pro) and also uncharged hydrophilic amino acid residues (32%) (Gly, Ser, Thr and Gin). There is also high ratio of basic (Lys, Arg and His) relative to acidic amino acids (Asp) suggesting a strong basic protein character.

[101 ] Without wishing to be bound by theory, the present inventors propose that the basic residues such as Lys present a highly Iocalised positive charge on the surface of the cyclic bacteriocin structure which is responsible for attracting the peptide to the surface of the negatively charged membrane. Unlike some other cyclic bacteriocins, Plantacyclin B21AG does not contain any Cys residues, so there are no di-sulphide bridges. The Plantacyclin B21 AG also contains three aromatic residues (two Trp and one Phe).

[102] Polypeptide derivatives and analogs of the present invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of the peptide including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in conservative amino acid substitution.

[103] For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpoiar (hydrophobic) amino acids include alanine, leucine, isoieucine, valine, proline, phenylalanine, tryptophan, and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and giutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to significantly affect apparent molecular weight as determined by po!yacry!amide gel electrophoresis or isoelectric point. Non-conservative amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, Cys may be introduced at a potential site for disulfide bridges with another Cys. Pro may be introduced because of its particularly planar structure.

[104] The peptides of the present invention can be chemically synthesized. Synthetic peptides can be prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, and can include natural and/or synthetic amino acids. Amino acids used for peptide synthesis may be standard Boc(N ^a -amino protected N ^a -t-butyioxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling, and wash protocols of the original solid phase procedure of Merrifield (J. Am. Chem. Soc, Volume 85, 2149-2154, 1963), or the base-labile N ^a -amino protected 9- fluorenyimethoxycarbonyl (Fmoc) amino acid (Carpino and Han, J. Org. Chem., Volume 37, 3403-3409, 1972). In addition, the method of the present invention can be used with other N-protecting groups that are familiar to those skilled in the art. Solid phase peptide synthesis may be accomplished by techniques within the ordinary skill in the art (See for example Stewart and Young, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, III., 1984; Fields and Noble, Int. J. Pept. Protein Res., Volume 35, 161 -214, 1990), or by using automated synthesizers.

[105] The skilled person understands that strict compliance with any amino acid sequence disclosed herein is not necessarily required, and he or she could decide by a matter of routine whether any mutation is deleterious or preferred. For example, where the protein has a given biological activity that can be assayed (such as a microbicidal or microbiostatic activity) the effect of any mutation on that biological activity may be directly observed.

[106] Thus, the polypeptides of the present invention can include sequences with identity to a polypeptide described herein. The term "identity" is meant to include nucleic acid or protein sequence homology or three-dimensional homology. Several techniques exist to determine nucleic acid or polypeptide sequence homology and/or three-dimensional homology to polypeptides. These methods are routinely employed to discover the extent of identity that one sequence, domain, or model has to a target sequence, domain, or model,

[107] In some embodiments, a polypeptide has at least about 50% identity, for example, at least about 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the polypeptides described herein. Percent identity may be determined using the BLAST software (Altschul, S.F., et a/. (1990) "Basic local alignment search tool." J, Mol. Biol. 215:403-410, accessible on the World Wide Web at blast.ncbi.nlm.nih.gov) with the default parameters.

[108] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 59% identity to a Piantacyciin B21AG polypeptide comprising SEQ ID NO: 4:

[109] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 69% identity to a Piantacyciin B21AG polypeptide comprising SEQ ID NO: 6.

[1 10] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 35% identify to a Piantacyciin B21AG- cyclisafion polypeptide comprising SEQ ID NO: 8 (ORF-B).

[1 1 1 ] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 39% identity to a Piantacyciin B21AG- cyclisation polypeptide comprising SEQ ID NO: 10 (ORF-C).

[1 12] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 52% identify to a Piantacyciin B21AG- cyclisation polypeptide comprising SEQ ID NO: 12 (ORF-D).

[1 13] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 40% identity to a Piantacyciin B21AG- cyclisation polypeptide comprising SEQ ID NO: 14 (ORF-E). [1 14] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 31 % identity to a Piantacyclin B21AG- cyclisation polypeptide comprising SEQ ID NO: 16 (ORF-F).

[1 15] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 88% identity to a Piantacyclin B21AG polypeptide comprising SEQ ID NO: 4:

[1 16] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 94% identity to a Piantacyclin B21AG- cyclisation polypeptide comprising SEQ ID NO: 8 (ORF-B),

[1 17] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 89% identity to a Piantacyclin B21AG- cyclisation polypeptide comprising SEQ ID NO: 10 (ORF-C).

[1 18] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 95% identity to a Piantacyclin B21AG- cyclisation polypeptide comprising SEQ ID NO: 12 (ORF-D).

[1 19] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 94% identity to a Piantacyclin B21AG- cyclisafion polypeptide comprising SEQ ID NO: 14 (ORF-E).

[120] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 90% identity to a Piantacyclin B21AG- cyclisation polypeptide comprising SEQ ID NO: 16 (ORF-F).

[121 ] In one embodiment, the polypeptide, including a biologically active fragment or variant thereof has at least 95% identity to a Piantacyclin B21AG- cyclisation polypeptide comprising SEQ ID NO: 32.

[122] The polypeptides also include variants (e.g. allelic variants, homoiogs, orthoiogs, paraiogs, mutants, etc.). The molecules may lack one or more amino acids from the C-terminus and/or one or more amino acids from the N-terminus, or have one or more deletions in the polypeptide sequence. In some embodiments, a variant has enhanced or decreased levels of cytotoxic or growth inhibition activity on the same or a different microorganism or species of microorganism relative to the wild type protein.

[123] The present inventors have demonstrated Plantacyclin B21AG has a biological activity against a wide range of bacteria. Accordingly, the present invention also relates to a bacteriocin as described herein or a biologically active fragment or variant thereof.

[124] The term "biologically active" refers to fragments or variants of a polypeptide having a biological activity of the polypeptide from which they are derived. For example, a biologically active fragment or variant of a bacteriocin of the present invention will possess a biological activity of the bacteriocin.

[125] A biologically active fragment or variant of a cyciisation polypeptide of the will possess a biological activity to circularise a bacteriocin polypeptide.

[126] A "biological activity" of the bacteriocin, as used herein, includes a microbicidal or microbiostatic effect. In one aspect the microbicidal or microbiostatic effect is a bactericidal or bacteriostatic effect.

[127] The term "microbiostatic effect" includes inhibiting the growth or reproduction of a microbe, and includes decrease or reduction in growth or reproduction when compared to the growth or reproduction in a control, for example by arresting the cell cycle. In one embodiment, the growth or reproduction may be decreased or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to an untreated control. In a further embodiment, the growth or reproduction may be decreased or reduced by 100% relative to an untreated control.

[128] The term "bacteriostatic effect" includes inhibiting the growth or reproduction of a bacterium, and includes decrease or reduction in growth or reproduction when compared to the growth or reproduction in a control. In one embodiment, the growth or reproduction may be decreased or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to an untreated control. In a further embodiment, the growth or reproduction may be decreased or reduced by 100% relative to an untreated control. [129] The term "microbicidal effect" includes killing microbial organisms, for example bacteria, yeast, algae, synthetic microorganisms, and the like.

[130] The term "bactericidal effect" includes killing bacteria.

[131 ] It is advantageous for the bactericidal or bacteriostatic effect to be in respect of Gram-positive bacteria and/or Gram-negative bacteria. Preferably, the bactericidal or bacteriostatic effect is in respect of Gram -positive bacteria.

[132] The present inventors have demonstrated Plantacyclin B21AG has a biological activity against Lactobacillus _: Lactococcus, Clostridium and Listeria spp.

[133] Accordingly, in a preferred aspect the bactericidal or bacteriostatic effect is in respect of a Gram -positive bacteria seiected from species of Bacillus, Brochothrix, Carnobacterium, Clostridium, Enterococcus, Listeria, Lactobacillus, Leuocostoc, Micrococcus, Pediococcus, and Streptococcus.

[134] In another aspect the bactericidal or bacteriostatic effect is in respect of an organism selected from species of Clostridium, Bacillus, Listeria, Staphylococcus,, lactic acid bacteria, Pseudomonas, Escherichia coii, Salmonella, Campylobacter, Yersinia.

[135] In a preferred aspect the bactericidal or bacteriostatic effect is in respect of an organism seiected from Gram -positive bacteria associated with food spoilage or foodborne disease including Bacillus species, Bacillus subiiiis, Bacillus cereus, Listeria species, Listeria monocytogenes, lactic acid bacteria, lactic acid spoilage bacteria, Lactobacillus species, Staphylococcus aureus, Clostridium species, C. sporogenes, C. tyrobutyncum and C. botuiinum.

[136] In a preferred aspect the bactericidal or bacteriostatic effect is in respect of LAB such as Lactobacillus, Leuconostoc, Carnobacterium, and Enterococcus; Listeria monocytogenes, spore forming heat resistant bacteria such as Bacillus and Clostridium; and Brochothrix thermosphacta.

[137] In a preferred aspect the bactericidal or bacteriostatic effect is in respect of Lactobacillus, Leuconostoc, Carnobacterium, Enterococcus, Listeria monocytogenes, Bacillus, Clostridium, and Brochothrix thermosphacta. [138] In a preferred aspect the bactericidal or bacteriostatic effect is in respect of an organism selected from Listeria monocytogenes and/or Clostridium perfringens.

[139] In one aspect the antimicrobial material is present in an amount effective to provide a microbicidal or microbiostatic effect.

[140] The inventors have demonstrated Piantacyciin B21AG is circular, and have characterised the polypeptides implicated in Piantacyciin B21AG-cyclisation.

[141 ] Accordingly, the present invention provides a method of forming a cyclic Piantacyciin B21AG comprising contacting a Piantacyciin B21 AG with one or more Piantacyciin B21AG-cyciisation polypeptide or a biologically active variant or fragment thereof.

[142] A "biological activity" of a Piantacyciin B21AG-cyclisation polypeptide, as used herein, includes cleavage of the leader sequence, ATPase function, membrane association and polypeptide cyciisation.

[143] The present inventors have demonstrated the gene encoding Piantacyciin B21AG is present on a plasm id in L. piantarum B21 ^* and on a plasm id in L plantarum B20, B31 and B33. Accordingly, in one embodiment the present invention provides a piasmid or vector comprising at least one polynucleotide as described herein.

[144] In another aspect the invention provides a host cell comprising at least one polynucleotide according as described herein and/or at least one piasmid or vector as described herein.

[145] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host ceil into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operativeiy linked. Such vectors are referred to herein as "expression vectors". [146] The term "plasm id" as used herein, refers to an extra-chromosomal genetic element and includes naturally occurring plasm ids isolated from bacteria, such as Lactobacillus spp and/or other LAB. The term also includes plasmids which could potentially be engineered for heterologous protein production without the necessity of antibiotic selection. Plasmids are DNA molecules that can vary in size from 1 kb to more than 200 kb. Most plasmids consist as double-stranded, covalentiy closed, circular (CCC) DNA molecules that can be isolated from bacterial cells in superheiical form. Plasmids are found to naturally occur in a wide variety of bacterial species. Most plasmids have a narrow host range and can be maintained only in a limited set of closely related species. Other plasmids have a broader host range and are stably maintained in a wider variety of bacterial genera. Plasmids are extra- chromosomal elements that behave as accessory genetic units that autonomously replicate and are inherited independently from the bacterial chromosome. Plasmids have evolved a variety of mechanisms to maintain a stable copy number of the plasm id in the bacterial host ceil and to partition plasm id molecules accurately to daughter ceils. Plasmids usually depend, to a greater or lesser extent, on chromosomally encoded enzymes and proteins for their replication, transcription and translation. Frequently, plasmids contain genes that are advantageous to the bacterial host cell. Phenotypes that are conferred by naturally plasmids include resistance to and production of antibiotics, resistance to heavy metals, virulence factors, the ability to metabolize specific carbohydrates, and restriction and modification enzymes. Although plasmids are mostly extra-chromosomal, the term plasm id does not exclude molecules that integrate into the bacterial chromosome, at varying frequency.

[147] In one embodiment the p!asmid is a Lactobacillus plasmid.

[148] In another embodiment, the plasmid is Lactobacillus plantation plasmid.

[149] The inventors have demonstrated that the Piantacyciin B21AG operon is encoded by the Lactobacillus piantarum B21 ^* plasmid, Plasmid 1 . Plasmid 1 comprises the nucleotide sequence of scaffold 5 (SEQ ID NO: 17).

[150] Accordingly, in a further embodiment, the plasmid is Lactobacillus piantarum Plasmid 1. [151 ] The inventors have also demonstrated that the Plantacyclin B21AG operon is encoded by the 20kb Lactobacillus plantarum B33 piasmid (annotated in Figure 17), and by B31 and B20.

[152] Accordingly, in a further embodiment, the piasmid is the 20kB piasmid of Lactobacillus plantarum B20, B31 or B33,

[153] In one embodiment, the piasmid comprises a repiicon which confers on the piasmid the ability to replicate autonomously. In one embodiment, the repiicon confers on the piasmid the ability to maintain normal copy number.

[154] In one embodiment the present invention provides a host cell comprising at least one polynucleotide as described herein, and/or at least one piasmid or vector as described herein.

[155] In a preferred embodiment, the piasmid is a natural piasmid.

[156] In another embodiment, the natural piasmid is Piasmid 1 of B21 ^* .

[157] In another embodiment, the natural piasmid is the 20kb piasmid of B20, B31 or B33. ln one embodiment the host cell is prokaryotic ceil.

[158] In one aspect the invention provides a strain of Lactobacillus comprising a polynucleotide as described herein.

[159] In another aspect the invention provides a strain as described herein, wherein the strain is Lactobacillus plantarum B21 ^* .

[160] In another aspect the invention provides a strain as described herein, wherein the strain is Lactobacillus plantarum B20.

[161 ] In another aspect the invention provides a strain as described herein, wherein the strain is Lactobacillus plantarum B31 .

[162] In another aspect the invention provides a strain as described herein, wherein the strain is Lactobacillus plantarum B33. [163] In another aspect the invention provides a probiotic, a medicament or a culture comprising a host cell as described herein or a strain as described herein.

[164] The present inventors have demonstrated that strains of bacteria, including probiotic bacteria, can be modified to express Piantacyclin B21AG.

[165] Accordingly, in one embodiment the present invention provides a method of modifying a strain of bacteria comprising transferring into the bacteria at least one polynucleotide according as described herein, and/or at least one plasrnid or vector as described herein, !n one embodiment, the strain of bacteria is a probiotic strain.

[166] In one embodiment the replicon preferably comprises ail the sequence elements that are necessary in c/ ^' s for replication of a plasrnid containing the replicon as sole replicon, for example in a Lactobacillus host cell, preferably a L plantarum host ceil. In as far as necessary for replication, the various cis-acting sequence elements of the replicons are operably linked.

[167] Preferably such sequences are homologous to the host ceil.

[168] The use of plasm ids, replicons and/or sequences homologous to the host ceil or which and/or plasm ids, replicons and/or sequences that are not recombinant/geneticaiiy modified, allows for the generation of strains, including probiotic strains, having the bacteriocin activity, that are not genetically modified.

[169] The vectors and plasmids of the invention may further comprise sequences for promoting conjugation or other means of transfer of genetic material. Preferably such sequences are homologous to the host ceil.

[170] Examples of such sequences include e.g. the mobilisation gene cluster of the Lactococcus lactis plasrnid pMRCOI . This L. lactis Tra region comprises an origin of transfer (or/7) sequence flanked by a gene cluster of 18 genes (traA, orf6, orf7, traB, traC, traD, traE, traF, or†13, orf14, orf15. tra , traK, orf18, traL. tral, orf21, ItrC), and a gene cluster of two genes in the opposite orientation (or†4 and or†3) (Dougherty et a/., 1998). A preferred mobilisation gene cluster is actually found in pWCFS103 of the present invention. The Tra region of pWCFS103 shares homology with the L lactis Tra and runs from orf37 (orf3 in pMRCOI ) to orf17 (ItrC in pMRCOI ) and is flanked by two transposase genes. The FS103 Tra region has an additional ORF, orf20, of unknown function in between traL and tral. The pWCFS103 oriT region contains the CGAAG sequence, which is conserved among several conjugative plasm ids (Dougherty et ai, 1998, Wang and Macrina, 1995) and which is preceded by a TTAAG sequence. Thus in a preferred embodiment, the vectors and plasmids of the invention comprise a mobilisation gene cluster from pWCFS103.

[171 ] The vectors and plasmids of the invention may further comprise a second repiicon different from the repiicons of the invention as described above. The purpose of the second repiicon is to expand the host-range for the vectors and plasmids of the invention.

[172] The terms "host cell," "host cell line," and "host ceil culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[173] Examples of host cells include a bacterial host cell such as Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia and yeasts.

[174] The bacterial host cell preferably is a Gram-positive bacterium, more preferably a Gram -positive bacterium that belongs to a genus selected from the group consisting of Lactobacillus, Lactococcus, Leuconostoc, Carnobacterium, Bifidobacterium, Bacillus, Streptococcus. Most preferably the bacterial host ceil is a bacterium that belongs to a species selected from the group consisting of L acidophilus, L. amyiovorus, L, bavaricus, L. brevis, L. caseii, L, crispatus, L. curvatus, L. deibrueckii, L delbrueckii subsp, buigaricus, L. fermentum, L gaiiinarum, L. gasseri, L. helveticus, L. jensenii, L. johnsonii, L. minutis, L. murinus, L. paracasei, L. plantarum, L. pontis, L. reuteri, L. sacei, L. salivarius, L. sanfrancisco, Lactobacillus ssp., C. piscicola, B. subtiiis, Leuconostoc mesenteroides, Leuconoctoc iactis, Leuconostoc ssp, B. bifidum, B, iongum, B. infantis, B. breve, B, ado!escente, B. animalis, B. gal!inarum, B. magnum, and B. thermophilum,

[175] In another embodiment, the bacteria! host eel! is of the same species as the species from which the replicons are derived, i.e. L. plantarum.

[176] Preferably all further sequences that may be present in the nucleic acid molecule or vector as defined above are also derived from the species L. plantarum, i.e. are homologous to L. plantarum. Such further sequences may thus include selection markers, promoters and genes of interest known in the art. In case a strain is constructed through genetic engineering such that the resulting recombinant strain comprises only sequences derived from the same species as the strain is, albeit in recombined form, the strain is said to be obtained through "self -cloning". Strains obtained through self-cloning have the advantage that there application in food (or pharmaceuticals) is more readily accepted by the public and regulatory authorities as compared strains comprising foreign nucleic acid sequences. The present invention includes the construction of self-cloned L. plantarum strain for food, pharmaceutical or nutraceutical applications.

[177] One embodiment of such vectors or plasmids comprises the polynucleotide sequence SEQ ID NO: 3 or SEQ ID NO: 53 or a biologically active fragment or variant thereof. Each construct (vector or plasm id) of the invention being capable of expressing the bacteriocin of the invention encoded therein in ceils transformed with said construct of the invention.

[178] A polynucleotide can be contained in a vector, which can facilitate manipulation of the polynucleotide, including introduction of the polynucleotide into a target ceil. The vector can be a cloning vector, which is useful for maintaining the polynucleotide, or can be an expression vector, which contains, in addition to the polynucleotide, regulatory elements useful for expressing the polynucleotide and, where the polynucleotide encodes a peptide, for expressing the encoded peptide in a particular cell. An expression vector can contain the expression elements necessary to achieve, for example, sustained transcription of the encoding polynucleotide, or the regulatory elements can be operativeiy linked to the polynucleotide prior to its being cloned into the vector. [179] In another embodiment the invention provides a vector as described herein wherein the vector is an expression vector,

[180] An expression vector (or the polynucleotide) generally contains or encodes a promoter sequence, which can provide constitutive or, if desired, inducible or stage specific expression of the encoding polynucleotide, a poiy~A recognition sequence, and a ribosome recognition site or internal ribosome entry site, or other regulatory elements such as an enhancer. The vector also can contain elements required for replication in a prokaryotic or eukaryotic host system or both, as desired. Such vectors, which include plasm id vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semiiki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison Wl; Stratagene, La Joiia CA; G!BCO/BRL, Gaithersburg MD) or can be constructed by one skilled in the art (see, for example, Meth. Enzymoi. Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Cane. Gene Ther, 1 :51 -64, 1994; Fiotte, J. Bioenerg, Biomemb. 25:37- 42, 1993; Kirshenbaum et a!., J. Clin. Invest. 92:381 -387, 1993; each of which is incorporated herein by reference).

[181 ] A nucleic acid is "operably-iinked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operabiy-linked to a coding sequence if it affects the transcription of the sequence, or a ribosome-binding site is operabiy-linked to a coding sequence if positioned to facilitate translation. Generally, "operabiy-linked" means that the DMA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

[182] A polynucleotide of the invention can be inserted into a vector, which can be a cloning vector or a recombinant expression vector. The term "expression vector" refers to a plasm id, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of a polynucleotide encoding Plantacyciin B21 AG or a polynucleotide encoding a Plantacyciin B21AG-cyciisation polypeptide or a biologically active fragment or variant thereof. [183] Such expression vectors contain a promoter sequence, which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector generally contains an origin of replication, a promoter, as well as specific genes which allow phenofypic selection of the transformed ceils. Vectors suitable for use in the present invention include, but are not limited to, the T7-based expression vector for expression in bacteria, the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J, Biol. Chem. 263:3521 , 1988) and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operabiy linked to regulatory elements, for example, a promoter, which can be a T7 promoter, metallothionein I promoter, polyhedrin promoter, or other promoter as desired, particularly stage specific promoters or inducible promoters.

[184] Bacterial expression vectors are discussed in more detail below.

[185] Viral expression vectors can be particularly useful for introducing a polynucleotide useful in a method of the invention into a mammalian cell.

[186] A polynucleotide of the present invention, which can be contained in a vector, can be introduced into a cell by any of a variety of methods known in the art (Sambrook et a/., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989); Ausubel et a/,, Current Protocols in Molecular Biology. John Wiley and Sons, Baltimore, MD (1987, and supplements through 1995), each of which is incorporated herein by reference). Such methods include, for example, transfection, lipofection, microinjection, electroporation and, with viral vectors, infection; and can include the use of liposomes, microemulsions or the like, which can facilitate introduction of the polynucleotide into the ceil and can protect the polynucleotide from degradation prior to its introduction into the ceil. The selection of a particular method will depend, for example, on the ceil into which the polynucleotide is to be introduced, for example, whether the cell is isolated in culture.

[187] The present invention also includes a culture of ceils transformed with a construct of the invention, each such culture being capable of expressing a Piantacyciin B21AG or a biologically active fragment or variant thereof, or a polynucleotide encoding a Plantacyclin B21AG~cyciisation polypeptide or a biologically active fragment or variant thereof.

[188] A polynucleotide sequence encoding a Plantacyclin B21AG or a biologically active fragment or variant thereof or a polynucleotide encoding a Plantacyclin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing polynucleotides having eukaryotic or viral sequences in prokaryotes are well known in the art, as are biologically functional viral and plasmid DNA vectors capable of expression and replication in a host. Methods for constructing an expression vector containing a polynucleotide of the invention are well known, as are factors to be considered in selecting transcriptional or trans!ationai control signals, including, for example, whether the polynucleotide is to be expressed preferentially in a particular ceil type or under particular conditions (see, for example, Sambrook et a/., supra, 1989).

[189] A variety of host cell/expression vector systems can be utilized to express a Plantacyclin B21AG or a biologically active fragment or variant thereof or a polynucleotide encoding a Plantacyclin B21AG-cyciisation polypeptide or a biologically active fragment or variant thereof, including, but not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeast cells transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors such as a cauliflower mosaic virus or tobacco mosaic virus, or transformed with recombinant plasmid expression vector such as a Ti plasmid; insect ceils infected with recombinant virus expression vectors such as a bacuiovirus; animal ceil systems infected with recombinant virus expression vectors such as a retrovirus, adenovirus or vaccinia virus vector; and transformed animal ceil systems genetically engineered for stable expression.

[190] A selectable marker may be used to discriminate host ceils containing a vector or plasmid of the present invention from those that do not. [191 ] The gene encoding a selectable marker may encode a dominant, a recessive or a bidirectional selectable marker as described above. Preferred selectable marker genes are homologous to the host cell. Suitable dominant selectable markers are genes providing antibiotic resistance, heavy metal resistance or the capability to utilise certain sugars. Examples thereof include e.g. genes conferring resistance to antibiotics such as kanamycins, rifampicin, erythromycin, actinomycin, chloramphenicol, tetracyclines, nisin and lactacin F or genes conferring resistance to a heavy metal such as arsenite, arsenate, antimony, cadmium or organo-mercurial compounds. Such genes are generally known to the skilled person. Preferred dominant markers are food-grade markers such as the sugar utilisation genes xylRAB from L. pentosus, levA from L. plantarum or L case/ and scrA/scrB from P. peniosaceus.

[192] Recessive selectable markers for use in the present invention include e.g. genes which confers prototrophy to an auxotrophic strain, so-called auxotrophic markers. Examples of suitable food grade auxotrophic markers include e.g. tRNA (gin) of L. lactis (Dickely et ai , 1995, Mol. Microbiol. 15: 839-847) and air of L. lactis or L. plantarum (Bron et ai , 2002, Appl, Environ. Microbiol. 68: 5663-5670),

[193] An example of a suitable bidirectional marker for use in the present invention is a gene encoding an orotidine-5'-decaboxylases which is to be used in host lacking endogenous orotidine-S'-decaboxyiase. Host cells containing a vector encoded orotidine-5'-decarboxylase can then be selected in a (mineral) medium lacking uracil, whereas such host cells can be cured from vectors encoding a orotidine-S'-decarboxylase by counter-selection on a medium containing S-fiuoro- orothic acid. Similarly, a gene encoding an acetamidase can be used as bidirectional marker by selection on a medium containing acetamide as sole carbon and or nitrogen source and by counter-selection on a medium containing fiuoro-acetamide. An additional advantage of acetamidase markers is that with respect to most bacterial host cells they are dominant selectable markers, which do not require a mutant (auxotrophic) strain, but which can be used directly in wild type strains.

[194] In one embodiment the selectable marker is HRPT. For example, the hrpt gene can be used with hrpt negative lines containing a stably integrated gene encoding a conditionally active ere recombinase to allow efficient manipulation of targeted loci by site-specific recombination,

[195] The capacity for conjugal transfer is an important characteristic for plasm ids. Accordingly, the vectors and plasm ids of the invention may further comprise sequences for promoting conjugation or other means of transfer of genetic material. Preferably such sequences are homologous to the host cell.

[196] The vectors and plasmids of the invention may further comprise a second repiicon different from the replicons of the invention as described above. The purpose of the second repiicon is to expand the host-range for the vectors and plasmids of the invention. Such a vector or plasm id containing two different replicons may thus be shuttled between two or more different types of host ceils. A particularly preferred second repiicon is a repiicon that is capable of replication in Escherichia coii, so as to facilitate multiplication and manipulation in molecular cloning. Suitable replicons for E. coli are well known to the skilled molecular biologist and include e.g. pMB1 or a modified form thereof, pKN402, p15A, pSC1 Q1 , and coiE1 (see Sambrook and Russell (2001 ) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York).

[197] Vectors of the invention further preferably comprise one or more restriction sites that are suitable for insertion of desired DNA fragments. Restriction sites that are suitable for this purpose preferably are unique in the vector, allowing linearization of the vector into a single DNA fragment corresponding to the complete vector. Such restriction sites further preferably are not present in DNA sequences that are required for replication, maintenance or selection. The vectors may further comprise a multiple cloning site, as is known in the art. Such multiple cloning site contains several different unique restriction sites that may conveniently be used for insertion of fragments into the vectors,

[198] In a further embodiment, the vector comprises a promoter that, when the vector is present in a suitable host ceil, is capable of driving transcription of a nucleotide sequence of interest operably linked to the promoter. In principle, any suitable constitutive or inducible promoter that is active in the host ceil may be used for this purpose. Preferred promoters are homologous to the host ceil. Examples of suitable constitutive promoters are the usp45 promoter, the pepN promoter and promoters mentioned in de Vos and Simons in "Genetics and Biotechnology of Lactic Acid Bacteria", Gasson and de Vos, eds. , pp. 52-106, Chapman and Hall, 1994. Examples of suitable regulated promoters are the bacteriophage 31 middle promoter and or/ ^' -based expression system, the xylA promoter, the repressor/operator system of bacteriophage rlt, the acid-inducib!e or acid-responsive F1 FO-ATPase promoter of L. acidophilus, the promoters of the lactose operon of S, thermophilus and lac ABCDFEGX operon of L lactis, and the sait-inducibie promoters of L. lactis.

[199] A highly suitable class of promoters are the auto-inducibie promoters. Non-limiting examples are the promoters from the bacteriocin gene cluster of Carnobacterium piscicola, the Sakaricin gene cluster from L. sake, as well as (presumably) the Subtiiin gene cluster from Bacillus subtilis. According to the invention these also include promoters for similar, non-bactericidal signal proteins involved in the quorum sensing process in microorganisms, such as the promoter for Plantaricin A from the bacteriocin gene cluster of L. piantarum. The preferred promoters used are the auto-inducibie promoters from the nisin gene cluster of L lactis, especially the nisA and nisF promoters. These promoters have already been used for the controlled (over-) expression of homologous and heterologous polypeptides (such as the gusA reporter gene from E, coli, pepN, and the lytic genes iytH and lytA) in lactic acid bacteria and for the nisin-induced expression in heterologous hosts such as L heiveticus and Leuconostoc lactis. Preferably, in the vectors of the invention a unique restriction site or a multiple cloning site is located immediately downstream of the promoter in the direction of transcription from the promoter. Further suitable promoters for use in the nucleic acid molecules of the invention are known to persons skilled in the art.

[200] Vectors of the invention that comprise a promoter as described above for the expression of a coding sequence of interest preferably comprise additional sequence elements to regulate the expression of the coding sequence, i.e. sequences for initiation of translation, secretion, termination of transcription and the like. Such additional sequence elements for regulation of expression are preferably homologous to the host ceil. In the vectors, the promoter may be operabiy linked to more than one coding sequence, i.e. several coding sequences may be present in a single multi-cistronic transcription unit regulated by the promoter. The one or more coding sequences operably linked to the promoter are preferably each provided with the appropriate signals for initiation of translation such as e.g. a Shine-Daigamo sequence and a translation initiation codon. Optionally, a coding sequence in the vector comprises a signal sequence encoding a signal peptide causing translocation of the polypeptide encoded by the coding sequence across the cell membrane, and preferably secretion of the polypeptide from the host cell. Signal sequences are well known to the skilled person and a variety of suitable signal sequences is available in the art. Usually the skilled person will select a signal sequence known to effect secretion of polypeptides from a given host cell of choice. Preferably, the signal sequence to be used in the invention will be homologous to the host organism or will originate from an organism closely related to the host organism. Signal sequences that are preferably used in the context of the present invention include signal sequences from lactic acid bacteria of which preferably Lactobacillus. Examples of such signal sequences include e.g. signal sequences from a~amylase, slpA, and prtP. Further suitable signal sequences may be identified using dedicated genetic screening methods known in the art. In the vectors of the invention, the transcription unit comprising the one or more coding sequences linked to the promoter is preferably terminated by a suitable transcription terminator, such e.g. ildH or islP. Suitable bacterial transcription terminator sequences may be identified using known methods.

[201 ] In a further embodiment, the vectors of the invention comprise one or more nucleotide sequences of interest. Preferably the nucleotide sequence of interest is a coding sequence coding for a polypeptide of interest. Preferably the nucleotide sequence of interest is homologous to the host ceil.

[202] The present inventors have demonstrated that the killing activity of the circular bacteriocin Plantacyclin B21AG can be transferred to a heterologous bacterial strain.

[203] For example, the present inventors have demonstrated in Example 13 and Figure 16 that the Plantacyclin B21AG operon can be expressed in the probiotic strain WCSFS1 and confer killing activity on the probiotic strain. Accordingly, in one embodiment the present invention provides a bacterium comprising the Plantacyciin B21AG operon described herein.

[204] The bacterium may be any bacterium, in particular a Gram -positive bacterium. Preferably, the bacterium is a lactic acid bacterium (LAB), preferably a probiotic LAB. The probiotic LAB is preferably a live microorganism which when administered in adequate amounts confer a health benefit on the host. Lactic acid bacteria (LAB) are the most common type of microbes used as probiotics.

[205] LAB are able to convert sugars (including lactose) and other carbohydrates into lactic acid. This not only provides the characteristic sour taste of fermented dairy foods such as yogurt, but also by lowering the pH may create fewer opportunities for spoilage organisms to grow. Without wishing to be bound by theory, this may create possible health benefits on preventing gastrointestinal infections. Strains of the genera Lactobacillus and Bifidobacterium, are the most widely used probiotic bacteria.

[206] In one embodiment, the bacterium is a recombinant bacterium. The term "recombinant bacterium", as used herein, refers to a bacterium whose genetic makeup has been altered by deliberate introduction of new genetic elements. Such recombinant bacterium may be prepared by methods well known in the art.

[207] The term "recombinant bacterium" may also include so-called "clean deletion mutants", i.e. deletion mutants that do not contain any foreign DNA. Such clean deletion mutants may be constructed using approaches involving suicide vectors such as pUC19. Procedures for obtaining clean deletion mutants have been described by Lambert et ai. (Lambert JM, Bongers RS, Kleerebezem M.Appl Environ Microbiol. 2007 Feb; 73(4): i 128-35). Such (clean) deletion mutants may be distinguished from a naturally occurring bacterium using a PGR approach involved PGR primers in the flanking region of the mutagenised gene, as the resulting ampiicon will be distinctly smaller for the mutant compared to the wild type strain.

[208] In another embodiment, the bacterium a non-recombinant bacterium. The term "non-recombinant bacterium", as used herein, refers to a bacterium whose genetic makeup has not been altered by deliberate introduction of genetic elements other than a genetic element such as a plasmid or vector comprising a Plantacyclin B21 AG operon or portion thereof as described herein.

[209] If circularisation of the expressed Plantacyclin B21AG or a biologicaily active fragment or variant thereof is desired, it can be particularly advantageous to select a host cell/expression vector system that can affect the desired circularisation, for example, a host cell/expression vector system also expressing the Plantacyclin B21AG operon described herein.

[210] Depending on the host ceil/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like can be used in the expression vector (Bitter et a/., Meth. Enzymol. 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters can be used. When cloning in mammalian ceil systems, promoters derived from the genome of mammalian cells, for example, a human or mouse metallothionein promoter, or from mammalian viruses, for example, a retrovirus long terminal repeat, an adenovirus late promoter or a vaccinia virus promoter, can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the inserted sequence encoding a Plantacyclin B21AG or a biologically active fragment or variant thereof.

[21 1 ] In yeast ceils, a number of vectors containing constitutive or inducible promoters can be used (see Ausubel et a/., supra, 1987, see chapter 13; Grant et ai., Meth. Enzymol. 153:516-544, 1987; Glover, DNA Cloning Vol. II (IRL Press, 1986), see chapter 3; Bitter, Meth. Enzymol. 152:673-684, 1987; see also, The Molecular Biology of the Yeast Saccharomyces (Eds., Strathern et a/., Cold Spring Harbor Laboratory Press, 1982), Vols. I and II). A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL can be used (Rothstein, DNA Cloning Vol. II (supra, 1986), chapter 3). Alternatively, vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.

[212] Eukaryotic systems, particularly mammalian expression systems, allow for proper post-translationai modifications of expressed mammalian proteins. Eukaryotic ceils which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and advantageously, plasma membrane insertion of the gene product can be used as host cells for the expression of a Plantacyclin B21AG or a biologically active fragment or variant thereof or a polynucleotide encoding a Plantacyclin B21AG-cyciisation polypeptide or a biologically active fragment or variant thereof.

[213] Mammalian ceil systems which utilize recombinant viruses or viral elements to direct expression can be engineered. For example, when using adenovirus expression vectors, the sequence encoding a Plantacyclin B21AG or a biologically active fragment or variant thereof or a polynucleotide encoding a Plantacyclin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. Alternatively, the vaccinia virus 7.5K promoter can be used (Mackett et a/., Proc. Natl. Acad. Sci. USA 79:7415-7419, 1982; Mackett et a/., J. Virol. 49:857-864, 1984; Panicali et a!., Proc. Natl. Acad. Sci.. USA 79:4927-4931 , 1982). Particularly useful are bovine papilloma virus vectors, which can replicate as extrachromosomal elements (Sarver et a/., Moi. Cell. Biol. 1 :486, 1981 ). Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host cell chromosome, thereby yielding a high level of expression. These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene. Alternatively, the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of sequence encoding a Plantacyclin B21AG polypeptide or a biologically active fragment or variant thereof in host ceils (Cone and Mulligan, Proc. Natl. Acad. Sci. USA 81 :6349-6353, 1984). High level expression can also be achieved using inducible promoters, including, but not limited to, the metailothionein MA promoter and heat shock promoters.

[214] The invention provides a method for producing a Plantacyclin B21AG polypeptide or a biologically active fragment or variant thereof protein as described herein or a polynucleotide encoding a Plantacyclin B21AG-cyciisation polypeptide or a biologically active fragment or variant thereof described herein. The invention also provides a method for producing a polypeptide encoded by the nucleotide sequence of described herein or fragments thereof, including culturing the host cell under conditions suitable for the expression of the polypeptide and recovering the polypeptide from the host ceil culture.

[215] A polypeptide or a fragment thereof, can be encoded by a recombinant or non-recombinant nucleic acid molecule and expressed in a ceil. Preparation of a Piantacyclin B21 AG or a biologically active fragment or variant thereof or Piantacyclin B21AG~cyclisation polypeptide or a biologically active fragment or variant thereof of the present invention by recombinant methods provides several advantages. In particular, the nucleic acid sequence encoding Piantacyclin B21 AG or a biologically active fragment or variant thereof or a polynucleotide encoding a Piantacyclin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof can include additional nucleotide sequences encoding, for example, peptides useful for recovering the polypeptide from the host ceil. A polypeptide can be recovered using well known methods, including, for example, precipitation, gel filtration, ion exchange, reverse-phase, or affinity chromatography (see, for example, Deutscher et a/. , "Guide to Protein Purification" in Meth. Enzymol. Vol. 182, (Academic Press, 1990)). Such methods also can be used to purify a fragment of a polypeptide described herein, for example, a particular binding sequence, from a ceil in which it is naturally expressed.

[216] A recombinant nucleic acid molecule encoding a Piantacyclin B21 AG polypeptide or a biologically active fragment or variant thereof or a polynucleotide encoding a Piantacyclin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof can include, for example, a protease site, which can facilitate cleavage of a Piantacyclin B21 AG or a biologically active fragment or variant thereof from a non Piantacyclin B21 AG sequence, or a polynucleotide encoding a Piantacyclin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof from a non Piantacyclin B21 AG-cyclisafion polypeptide or a biologically active fragment or variant thereof for example, a tag peptide, secretory peptide, or the like.

[217] Without wishing to be bound by theory, specific features in the mature bacteriocin polypeptide sequence are essential for circuiarization to occur. The island C~terminai ends of the circular bacteriocins consist mainly of stretches of hydrophobic residues. The circuiarization point in appears to be located internal to an alpha helix in the structure of the peptides, requiring the circuiarization process to occur in a largely hydrophobic and stericaily hindered region. Many circular bacteriocins contain several aromatic and/or small hydrophobic residues in the N terminus, which may indicate that the hydrophobic nature of these residues is important for biosynthesis (including circuiarization) and/or antimicrobial activity.

[218] A recombinant nucleic acid molecule also can encode a tag peptide such as a polyhistidine sequence, a FLAG peptide (Hopp et a/., Biotechnology 6: 1204 (1988)), a glutathione S-transferase polypeptide or the like, which can be bound by divalent metal ions, a specific antibody (U.S. Patent No. 5,01 1 ,912), or glutathione, respectively, thus facilitating recovery and purification of the Plantacyclin B21 AG or a biologically active fragment or variant thereof comprising the peptide tag. Such tag peptides also can facilitate identification of the Plantacyclin B21AG or a biologically active fragment or variant thereof through stages of synthesis, chemical or enzymatic modification, linkage, or the like. Methods for purifying polypeptides comprising such tags are well known in the art and the reagents for performing such methods are commercially available.

[219] A polynucleotide encoding a Plantacyclin B21AG or a biologically active fragment or variant thereof or a polynucleotide encoding a Plantacyclin B21AG- cyclisation polypeptide or a biologically active fragment or variant thereof can be engineered to contain one or more restriction endonuclease recognition and cleavage sites, which can facilitate, for example, substitution of an element of the Plantacyclin B21 AG or a biologically active fragment or variant thereof or the Plantacyclin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof. As such, related Plantacyclin B21AGs or a biologically active fragments or variants thereof can be prepared, each having a similar activity, but having different specificity, or a Plantacyclin B21AG-cyclisafion polypeptide or a biologically active fragment or variant thereof can be prepared, each having a similar activity, but having different specificity.

[220] It will be understood by the skilled person that the biologically active fragments or variants thereof may be made using mutagenesis. [221 ] Polypeptides of the invention and their variants (e.g. variants generated by deletion, domain shuffling or duplication, insertion, substitution, or combinations thereof) are also useful for characterising biologically active variants, and determining structure-function relationships (e.g., alanine scanning, internal deletion, N-terminal or C-terminal truncation, conservative or non-conservative amino acid substitution). For example, folding and processing of protein, secretion of the protein, circuiarisation of the bacteriocin, inhibition by or resistance to bacteriocin, or combinations thereof. Directed evolution by random mutagenesis or gene shuffling may be used to acquire new and improved functions using selection for a biological activity. Mutant and polymorphic polypeptides are encoded by suitable mutant and polymorphic polynucleotides.

[222] The present inventors have demonstrated probiotic strains can be generated which comprise a bacteriocin encoding polynucleotide as described herein. Accordingly, in one aspect, the present invention provides strains of LAB comprising a bacteriocin encoding polynucleotide as described herein. For example, in in one aspect the present invention provides a LAB strain selected from the group consisting of L. plantarum strain B21 ^* L, piantarum strain B20, L, piantarum strain B31 , L. piantarum strain B33, or a LAB strain comprising a vector comprising a bacteriocin encoding polynucleotide as described herein or a piasmid comprising a bacteriocin encoding polynucleotide as described herein.

[223] In one aspect the present invention provides a probiotic comprising Piantacyciin B21AG.

[224] In one embodiment the present invention provides a probiotic capable of secreting Piantacyciin B21 AG.

[225] The present inventors have demonstrated L, piantarum B21 ^* expresses a novel bacteriocin having a wide range of activity against bacteria, and L. piantarum strain B20, L. piantarum strain B31 , and L piantarum strain B33 have the novel bacteriocin, and that the novel bacteriocin can be transferred to other strains, thereby transferring the antimicrobial activity. Accordingly, in one aspect the present invention provides the means to transfer the antimicrobial activity of Piantacyciin B21AG ίο other commercial probiotic strains without the use of genetic modification technology.

[226] As used herein the term "probiotic" as used herein refers to a strain which has a positive effect on the health and well-being of a host when ingested by said host, for example by improving intestinal microbial balance in a host.

[227] In one embodiment the probiotic strain is L plantarum WCFS1.

[228] In another embodiment, the strain is a strain (including a probiotic strain) which lacks a functional pin locus. For example, a strain in which the classical bacteriocin (pin) locus is impaired at the genetic level.

[229] In another embodiment, the strain has no endogenous bacteriocin.

[230] In another aspect the, the host cell is L aigilis, L. lactus or L. plantarum A17.

[231 ] L plantarum WCFS1 is a probiotic strain that is well characterized. L plantarum is a common inhabitant of the human Gl tract. L. plantarum WCFS1 is a single colony isolate of the esophageal L. plantarum strain NCIMB8826, which was shown to survive stomach passage in an active form. Its genome has been sequenced and appears to be one of the largest genomes known among lactic acid bacteria (3.3 Mb) (Kleerebezem et al. (2003) PNAS 100:4; 1990-1995 and Siezen et al. (2012) J. Bacteriol.194 no. 1 195-196).

[232] Preferably, a probiotic strain should possess one or more of the following abilities; the ability to inhabit or survive the gastrointestinal (Gl) tract in vivo, the ability to suppress or inhibit growth of microorganisms unwanted in the Gl tract, the ability to adhere to the Gl tract (GIT) and the ability to strengthen the resistance of the epithelial cell layer to pathogenic microorganisms,

[233] Appropriate tests to determine the presence or absence of these abilities of a strain could be readily carried out by a person skilled in the art. [234] The present inventors have demonstrated culture supernatants from LAB strains expressing Piantacyclin B21AG are able to inhibit the growth of susceptible bacteria.

[235] Accordingly, in another aspect the invention provides a composition comprising the bacteriocin polypeptide or bioiogically active fragment or variant thereof described herein,

[236] In one embodiment, the present invention provides a composition comprising Piantacyclin B21 AG.

[237] The Piantacyclin B21AG and/or a Piantacyclin B21AG-cyciisation polypeptide may be isolated and used. Otherwise, a culture containing bacteriocin Piantacyclin B21AG and/or a Piantacyclin B21AG-cyclisafion polypeptide may be used as it is, with no isolation of the bacteriocin from the host ceil, e.g. LAB. Because purification procedures such as isolation are generally laborious, preferably, a LAB culture itself is added in a process. For example a LAB culture itself is added in a method of producing various types of fermented food products.

[238] To obtain a LAB culture containing Piantacyclin B21AG and/or a Piantacyclin B21AG-cyclisation polypeptide, the LAB culture containing Piantacyclin B21AG and/or a Piantacyclin B21AG-cyciisation polypeptide should be cultivated. Methods for cultivation are known in the art and are also described herein.

[239] Methods of culturing and harvesting cell free supernatant are known in the field. One method of preparing culture supernatant described herein is based on Schiiiinger & Lucke (1989), In brief, LAB strains from -80°C stock preparation were resuscitated in 5 mL of MRS broth at 30°C for 24 h. A 2% (v/v) 24 h culture was used to inoculate 10 mL MRS broth and incubated at 30°C for 24 h. The ceil free supernatant (CPS) was harvested by centrifugafion at 5000 χ g for 20 min at 4°C (3K10, Sigma, UK). The effect of acid was eliminated by adjusting the pH of the CPS to 8.0 - 6.5 using sodium hydroxide (10 N). To rule out any possible inhibitory activity from hydrogen peroxide, catalase (Sigma-Aidrich, USA) was added at level of 5 mg/mL to the LAB CPS, followed by filtration through 0.2 μητι microfilter (Sarstedt, Germany). [240] The present inventors have demonstrated that purified Plantacyciin B21AG has bacteriocin activity, !n one aspect the present invention provides a composition comprising a purified bacteriocin polypeptide or biologically active fragment or variant thereof as described herein.

[241 ] Another embodiment of the present invention includes a method of treating bacterial infections in animals or humans using a composition comprising a host bacteria transformed with a piasmid or expression vector described herein, a bacteriocin produced by a bacterium as described herein, a composition comprising a bacteriocin polypeptide or biologically active fragment or variant thereof as described herein, or combinations thereof.

[242] Another aspect of the present invention is to provide therapeutic compositions. The compositions may be for oral, nasal, pulmonary administration, injection, etc. The therapeutic compositions include effective amounts of at least one bacteriocin of the present invention and their derivatives and/or at least one novel strain to at least reduce the levels of colonization by at least one target bacteria together with acceptable diluents, preservatives, solubilizers, emuisifiers, adjuvants, and/or carriers. Diluents can include buffers such as Tris-HCi, acetate, phosphate, for example; additives can include detergents and solubiiizing agents such as Tween 80, Polysorbate 80, etc., for example; antioxidants include, for example, ascorbic acid, sodium metabisulfite, etc.; preservatives can include, for example, Thimersol, benzyl alcohol, etc.; and bulking substances such as lactose, mannitol, etc.

[243] A bacteriocin or a composition comprising a bacteriocin as described herein and/or host cells comprising a bacteriocin as described herein may be used in a broad range of application areas.

[244] Compositions of the present invention may be (used) for inhibiting microbial growth, for modulating the gut flora in a mammal, for preventing and/or treating a microbial infection in a mammal and/or for preventing and/or reducing the level of microbial colonization in a food product. [245] In one embodiment the invention provides a use of a bacteriocin polypeptide or a biologically active fragment or variant thereof, a strain, a culture a composition, for a method of food production as described herein.

[246] In another aspect the invention provides a method of food production comprising contacting a food with an effective amount of a bacteriocin polypeptide or a biologically active fragment or variant thereof, a strain, a culture or a composition as described herein.

[247] In another aspect, the invention provides a method of inhibiting the growth of bacteria in a material comprising contacting the material with an effective amount of a bacteriocin polypeptide or a biologically active fragment or variant thereof, a strain, a culture or a composition as described herein.

[248] Encompassed in the present invention are compositions that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the ceil-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Piantacyciin B21AG) of the invention and/or combination thereof and a carrier for use in the prevention and/or treatment of a microbial infection.

[249] In one embodiment the invention further comprises a pharmaceutically acceptable carrier, vehicle or diluent, and/or a second bacteriocin.

[250] A "carrier" in a composition of the invention may be used, for example and without limitation, for solubilization, preservation, stabilization, emuisification, filling, coloring, adoring and/or antioxidative purposes. Carriers of the present invention may be aqueous and/or nonaqueous solutions. When compositions of the invention are administered to a mammal, the carriers in such compositions may be any type of carrier that have little or no negative and/or toxic side effects. In general, any carrier used in a composition of the present invention should not impact negatively on the function and/or use of an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Piantacyciin B21AG) of the invention and/or combination thereof. A carrier in a composition of the invention may also encompass a nutritionally acceptable carrier such as any liquid and/or solid form of nourishment that a mammal may assimilate.

[251 ] In a further aspect thereof, the present invention relates to a food product that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyciin B21 AG) of the invention and/or combination thereof.

[252] According to the present invention, a "food product" refers to any substance that may be ingested by a mammal. Such food product may be, for example and without limitation, meat, dairy, fruit, vegetable, grain, cereal, alcohol, water and/or beverage products. The food product of the present invention may be fermented and/or non-fermented food products.

[253] In one embodiment of the present invention, a food product may be a fermented food product, for example a fermented dairy food product (for e.g. milk and/or cheese), a fermented soy food product, a fermented vegetable food product and/or a fermented meat food product (for e.g. salami, chorizo, dry and semi-dry sausages, pancetta, prosciufto, bacon, etc). The isolated lactic acid bacteria of the present invention may be constitutively present in the food product. For example, a lactic acid bacteria of the present invention may be used to ferment a dairy, a soy, a vegetable and/or a meat food product. An isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyciin B21AG) of the invention and/or combination thereof may also be added to food products. For example, ceil- free culture supernatant may be added to a food product. A food product that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the ceil-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyciin B21AG) of the invention and/or combination thereof for use in the prevention and/or treatment of a microbial infection is also encompassed herein. [254] In yet a further aspect thereof, the present invention relates to a method for inhibiting microbial growth. The method may comprise the step of contacting a microbe with an isolated LAB strain of the present invention (such as B21 ^* , B20, B31 or B33), contacting a microbe with a pure culture of isolated LAB of the invention, contacting a microbe with the ceil-free culture supernatant obtained from culture of isolated LAB of the invention, contacting a microbe with the isolated bacteriocin (e.g. Piantacyciin B21AG) of the invention and/or contacting a microbe with combination thereof, contacting a microbe with the food product that may comprise the isolated LAB strain of the present invention and/or combination thereof. The contact may occur in a food product and/or in a mammal (in need thereof) and/or samples derived therefrom.

[255] It is to be understood herein that by "inhibiting" microbial growth it is meant a process by which the microbial growth may be reduced, decreased, lowered and/or impaired.

[256] Inhibition may be partial and/or complete. Inhibition may occur at any time following contact,

[257] "Contact" of a microbe in a food product may involve combining the of contacting a microbe with an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Piantacyciin B21AG) of the invention and/or combination thereof with the food product and/or a sample derived therefrom wherein the food product and/or food product sample may comprise and/or may be suspected of comprising microbes.

[258] Contacting a microbe in a sample derived from a mammal may include combining the isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Piantacyciin B21AG) of the invention, a combination thereof isolated lactic acid bacteria of the present invention, the food product of the invention and/or combination thereof in vitro or ex vivo in a biological sample. As used herein, a biological sample refers to a sample obtained from biological fluids or tissues of a mammal; it is also meant to encompass derivatives and fractions of such samples (e.g., cell iysates). The biological sample may be suspected of comprising and/or may comprise microbes.

[259] Contacting a microbe in a mammal may include administering an effective amount of an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the ceil-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Piantacyciin B21AG) of the invention, a combination thereof, the composition of the invention, a food product of the invention and/or combination thereof in vivo to a mammal in need thereof.

[260] As such, the present invention also relates to a method for inhibiting microbial growth that may comprise the step of administering an effective amount of a composition that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the ceil-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Piantacyciin B21AG) of the invention and/or combination thereof to a mammal in need thereof. The method may also comprise the step of administering the food product of the invention to a mammal in need thereof.

[261 ] The use of a composition that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the ceil-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Piantacyciin B21AG) of the invention and/or combination thereof and/or the use of a food product of the invention for the manufacture of a medicament for inhibiting (reducing, decreasing, lowering, impairing) microbial growth is encompassed within the present invention.

[262] A composition that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Piantacyciin B21AG) of the invention and/or combination thereof and/or the use of a food product of the invention for use in inhibiting microbial growth is also encompassed within the present invention.

[263] It is to be understood that the term "microbe" as used herein, for example in microbial growth, microbial infection and/or microbial colonization, is meant to include any organisms comprised in the phyiogenetic domains bacteria and archea. The term microbe may encompass, without limitation, pathogenic microbes (microbes that may cause a deleterious effect in a mammal such as, for example, eliciting a disease response), foodborne microbes (microbes that may grow in food products and/or may colonize and/or infect a mammal following food ingestion), antibiotic-resistant microbes (microbes that may have partially or completely reduced susceptibility to one or more antibiotics) and/or spoilage microbes (microbes that may cause food to deteriorate),

[264] Microbes responsible for microbial growth, microbial infection and/or microbial colonization may encompass any gram -positive bacteria and/or gram- negative bacteria, including those discussed above.

[265] In an additional aspect thereof, the present invention relates to a method for modulating the gut flora in a mammal in need thereof. The method may comprise the step of administering an effective amount of a composition that may comprise an isolated LAB strain of the present invention (such as B21 ^* , B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof to a mammal in need thereof. The method may comprise the step of administering a food product that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof to a mammal in need thereof.

[266] "Gut flora" is meant to refer to the microbial flora (microbiota) which normally inhabits the human gut. "Modulating" the gut flora encompasses either an increase and/or a decrease in the development (such as the growth) of the gut flora, whichever is advantageous to the host. For example, modulation may involve decreasing, suppressing, attenuating, diminishing and/or arresting the development of deleterious gut flora.

[267] Modulation may also comprise promoting, increasing, intensifying and/or augmenting the development of beneficial flora. In an exemplary embodiment of the present invention, the beneficial flora may comprise Lactobacilli and/or lactic acid bacteria.

[268] As used herein, an "effective amount" is the necessary quantity to obtain a desired result. For example, the necessary quantity to obtain positive results without causing excessively negative effects in the host to which an isolated LAB strain of the present invention (such as B21 ^* , B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyciin B21AG) of the invention and/or combination thereof, the composition of the invention, the food product of the invention and/or combination thereof, may be administered.

[269] An effective amount of a Plantacyciin B21AG~cyciisation polypeptide is a quantity sufficient to result in cyciisation of a Plantacyciin B21A polypeptide or a fragment or variant thereof.

[270] An exemplary effective amount encompassed in the present invention relate to a quantity which may be sufficient to inhibit microbial growth. An effective amount may also encompass an amount sufficient to prevent the establishment of an infection and/or substantially improve some symptoms associated with an infection. An effective amount may also encompass an amount sufficient to modulate the gut flora. An effective amount may also encompass a quantity which may be sufficient to prevent and/or reduce in any manner the growth and/or colonization of microbes in a food product.

[271 ] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as the prevention or treatment of a disease, an infection or modulation of gut flora. [272] A "prophylacticaily effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting the rate of disease onset or progression, infection onset or progression or modulation of gut flora. A prophylactica!ly effective amount can be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.

[273] An effective amount may be administered in one or more administrations, according to a regimen. The privileged method of administration and the quantity that may be administered is function of many factors. Among the factors that may influence this choice are, for example, the condition, the age and the weight of the host to which a composition is to be administered. An exemplary form of administration of the present invention may be oral administration. Oral administration may comprise any food forms (food products) and/or any food suppiements including, but not limited to, capsules, tablets, liquid bacterial suspensions, dried oral supplements, wet oral supplements, dry tube feeding and/or wet tube feeding.

[274] Isolated lactic acid bacteria of the present invention may be administered in a lyophiiised form.

[275] The present invention also encompasses the use of a composition that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof for modulating the gut flora (in a mammal; for a mammal). The use of a food product that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof for modulating the gut flora (in a mammal; for a mammal) is also encompassed by the present invention. [276] In yet an additional aspect thereof, the present invention relates to a method for preventing and/or treating a microbial infection in a mammal in need thereof.

[277] The method may comprise administering an effective amount of a composition that comprises an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof and a carrier to a mammal in need thereof. The method may also comprise administering a food product that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof to a mammal in need thereof.

[278] A "microbial infection" refers to the multiplication and/or colonization of a microbe in an individual's body tissues. In an exemplary embodiment, an infection may be caused by a pathogenic microbe but an "infection" is meant to encompass the multiplication and/or colonization of non-pathogenic microbes as well. An exemplary infection encompassed herein may be a bacteremia (infection caused by bacteria; a bacterial infection). An infection may be asymptomatic (clinically unapparent) or symptomatic. An exemplary infection may be a gastrointestinal infection. Exemplary infections of the invention may be, for example and without limitation, infections caused by Listeria bacterial species (listeriosis),

[279] By "preventing" an infection, it is meant a process by which an infection may be prevented from establishing itself (occurring) within a mammal. For example, arresting/inhibiting the colonization and/or development of microbes, an infection may be prevented.

[280] By "treating" an infection it is meant a process by which the development and/or colonization of microbes causing the infection is reduced either partially or totally. Treating an infection also encompasses a process by which the symptoms of an infection may not worsen, may remain stable, may be reduced and/or may be completely eliminated.

[281 ] The present invention also encompasses the use of a composition that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyciin B21AG) of the invention and/or combination thereof for preventing and/or treating a microbial infection (in a mammal). The use of a food product that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyciin B21AG) of the invention and/or combination thereof for preventing and/or treating a microbial infection (in a mammal) is also encompassed by the present invention.

[282] The present invention also encompasses the use of a composition that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyciin B21AG) of the invention and/or combination thereof in the manufacture of a medicament for preventing and/or treating a microbial infection (in a mammal).

[283] The use of a food product that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the ceil-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyciin B21AG) of the invention and/or combination thereof in the manufacture of a medicament for preventing and/or treating a microbial infection (in a mammal) is also encompassed by the present invention.

[284] In a further aspect thereof, the present invention relates to a method for preventing and/or reducing the level of microbial colonization in a food product. The method may comprise contacting the food product with an effective amount of a composition that may comprise an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell- free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof.

[285] As used herein, the term "contacting," includes bringing into physical contact or exposing an isolated LAB strain of the present invention (such as B21 ^* B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof with a material, such as food product, culture of bacteria, or bringing into physical contact or exposing a polypeptide such as a Plantacyclin B21AG polypeptide with a Plantacyclin B21 AG-cyclisation polypeptide.

[286] "Microbial colonization" (contamination) in a food product refers to the growth of microbes that may be deleterious to the food product or to a mammal that may ingest the food product. A deleterious microbe in a food product may be a microbe that prematurely leads to spoilage of the product. A deleterious microbe for a mammal may be a microbe that upon ingestion of the food product will lead to a deleterious effect in a mammal (such as a food pathogen).

[287] By "preventing colonization" it is meant preventing the growth of microbes (for example, pathogenic microbes, food-borne microbes, antibiotic-resistant microbes and/or spoilage microbes) within a food product. By "reducing colonization" is meant to reduce, decrease, lower and/or impair the growth of microbes within a food product. By preventing and/or reducing colonization in a food product, the shelf- life of a food product may be increased.

[288] The use of a composition that comprising an isolated LAB strain of the present invention (such as B21 * B20, B31 or B33), a pure culture of isolated LAB of the invention, the ceil-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof for preventing and/or reducing the level of microbial colonization in a food product is also encompassed in the present invention. [289] In an embodiment of the present invention, a mammal may be a human being. A mamma! in need of preventing and/or treating a microbial infection may be a mammal having or suspected of having a microbial infection. Such mammal may or may not present symptoms of a microbial infection. A mamma! in need of modulating its gut flora may be a mammal for which an increase and/or a decrease in the development (such as the growth) of its gut flora, whichever is advantageous, would be beneficial.

[290] In another aspect, the present invention relates to a kit. The kit may comprise at least one container that contains an isolated LAB strain of the present invention (such as B21 ^* , B20, B31 or B33), a pure culture of isolated LAB of the invention, the cell-free culture supernatant obtained from culture of isolated LAB of the invention, the isolated bacteriocin (e.g. Plantacyclin B21AG) of the invention and/or combination thereof, a composition of the invention, a food product of the invention and/or combination thereof. A kit of the present invention may also comprise instructions for its use in the form of a pamphlet or of any other support, indicating, for example, the instructions for the administration of the product contained therein and/or the instructions to mix given components.

[291 ] As discussed herein the present invention may prevent and/or inhibit the growth of, and/or kill a microorganism in a material. This may be slowing or arresting a microorganism, such a bacteria, or by killing the microorganism present on contact with the present composition,

[292] The present inventors have demonstrated Plantacyclin B21AG has bacteriocin activity against a wide range of microorganisms.

[293] The present inventors have also demonstrated probiotic bacteria into which Plantacyclin B21AG has been transferred have bacteriocin activity.

[294] Accordingly, in one aspect the present invention provides a method of inhibiting the growth of a bacteria in an environment which may or may not be a food environment and which is susceptible to contamination by the bacteria.

[295] The present inventors have demonstrated mature Plantacyclin B21AG can be circularised by the formation of an intramolecular cyclic bond. The present inventors have also demonstrated the Plantacyciin B21AG operon includes a number of ORFs implicated in Plantacyciin B21AG transport and the formation of an intramolecular cyclic bond.

[296] Importantly, the present inventors have also demonstrated that the Plantacyciin B21AG operon can confer on a host cell lacking the ability to cyclise a target polypeptide the ability to cyclise a target polypeptide. For example, Figure 19 demonstrates that WCFS1 containing the plantacyciin B21AG operon is able to cyclise a target polypeptide, Plantacyciin B21AG.

[297] The presence of the cyclic target polypeptide, Plantacyciin B21AG, in the culture supernatant of the heterologous bacteria comprising the plantacyciin B21AG operon also demonstrates the plantacyciin B21AG operon confers the ability to cyclise immature protein and concomitant secretion by a nonstandard export route.

[298] This offers potential for the expression, cyciisation and secretion of therapeutic bioactive proteins that have been appropriately engineered.

[299] Accordingly, the present invention provides methods of forming an intramolecular cyclic bond in a polypeptide. In one aspect, the invention provides a method of forming an intramolecular peptide bond in a target polypeptide comprising contacting the polypeptide with one or more Plantacyciin B21AG-cyciisation polypeptide. In another aspect the invention provides a method of forming a cyclic Plantacyciin B21AG comprising contacting a Plantacyciin B21AG with one or more Plantacyciin B21 AG-cyciisation polypeptide.

[300] Cyciisation of proteins improves their thermodynamic stability, while chemical methods exist for short peptides co translational or co secretionai cyciisation in vivo is rare and offers potential advantages for the production and heterologous delivery of bioactive peptides and proteins.

[301 ] Cyclic polypeptides are known for their high pH and thermal stability, as well as for resistance to many proteolytic enzymes, properties which make such polypeptides highly promising for potential industrial applications. [302] Cyclic peptides are valuable pharmaceuticals, biotechnoiogical products, and tools for scientific research. Cyclic peptides in general have advantages over their linear relatives in that they sample a more constricted conformational and configurational space. Stemming from this basic property, cyclic peptides often have stronger binding constants and favorable pharmacological properties such as resistance to proteases, such as the resistance described herein. There is a great need for new methods for making cyclic peptides, particularly for the manufacture of synthetic cyclic peptides for clinical investigations and therapeutic use, and for the production of cyclic peptide libraries that can be screened to identify cyclic peptides with a desired activity. The present inventors have characterised novel cyciisation polypeptides.

[303] The biosynthesis of such cyclic polypeptides is also of particular interest as a possible model system for the synthesis of highly stable bioactive peptides.

[304] As used herein, the term "circular" is used interchangeably with "cyclic", and the term includes ribosomai head~to-taii-iigated polypeptides.

[305] The term "intramolecular cyclic bond" as used herein includes an intramolecular peptide bond.

[306] Without wishing to be bound by theory, the present inventors propose the ORFs in the Piantacyciin B21 AG operon characterised herein are involved in the biosynthesis of intramolecular cyclic bonds.

[307] Biosynthesis of circular bacteriocins requires three steps: cleavage of the leader sequence, circularization, and export out of the cell. The removal of the leader sequence is considered to be the first step in biosynthesis and a requirement for further processing into the mature peptide.

[308] Specific features in the mature peptide sequence are considered to be essential for circularization to occur. The N- and C-terminal ends of the circular bacteriocins, which are likely to contribute to this process, consist mainly of stretches of hydrophobic residues. The circularization point in is typically located internal to an alpha helix in the structure of the peptides, requiring the circularization process to occur in a largely hydrophobic and stericaliy hindered region, and the nature of both the N- and the C~terminai residues is considered to be critical to the efficiency of the circularization process,

[309] In contrast to other types of head-to-tail-linked circular peptides, circular bacteriocins stand out in that they contain no C-terminal extension and that the N- terminal leader appears to have no role in the circularization reaction.

[310] The enzymes responsible for performing the circularization reaction of circular bacteriocins are encoded within the bacteriocin gene cluster. The Piantacyciin B21AG operon includes 6 orfs implicated in Plantacyclin B21AG circuiarisation; orf-B, orf~C, orf-D, orf-E, orf-F and a new orf (corresponding to S33- ORF-21). Accordingly, in one embodiment, the one or more Plantacyclin B21AG- cyclisation polypeptide is selected from the group consisting of ORF-B, ORF-C, ORF-D, ORF-E, ORF-F, B33-ORF-21 and biologically active fragments or variants thereof.

[31 1 ] BLASTp analysis of the ORF-B putative protein sequence (157 amino acids) (Figure 10) detected a putative conserved domain from the DUF95 superfamiiy; members of this family have several predicted transmembrane regions. Without wishing to be bound by theory, the present inventors consider ORF-B gene could be involved in the production of or immunity to the Piantacyciin B21 AG.

[312] The nucleotide sequence of orf-B of the Piantacyciin B21AG operon is provided below (SEQ ID NO 7):

G T GAATAAAT CACGGAG TAATATACC T GC T T TAT T GAT AG T AT T TAT AAT T T T T C TAAT T T T GGGTGCCT TAT T TAG C AAAT AT T T C G C AAC T G C AAG C T C T C AAC AT T CAT T G GAT T C G T T T A AAG T AAT T C AT T T AAAC T G G T T T AC T GAAAT T AT C T T T AGAAAT AC AAT AG C AT T T T T AG T A T T AAG C T C T AC TCT T T T T T TAG GAAAT A TAG T GAG T G T T A T AT T C T T T T G C G T T AAT G G C T T T AAC GT TGGTCTGATATGCGGT C AAT T AC C AAT C T T T CAAAG T AT C G T T T T AC T C T TAG C AC AT G G T AT T AT T GAAATAAC T T CAT AT G T AT G G T TAG T AT AT G C T G T AAC AC AT G T TAA C AC AT GAAAAT T AAT AT AAT AAGAG CT TACTGTCT T T TGT TCCT TG C AG C AAT AAT T GAAG T T T T T G T T AC T C CAGAAT TAGC C T T AC GG T T T T T AGGAGAT ΤΑΆ

[313] The amino acid sequence of ORF-B encoded by the Piantacyciin B21 AG operon is provided below (SEQ ID NO: 8)

82 MNKSRSNIPALLIVFI T FLILGALFSKYFATASSQHSLDSFKVIHLNWFTEI I FRNTIAFLV LSSTLFLGNIVSVI FFCVNGFNVGLICGQLPI FQS IVLLLPHGI IEITSYV LVYAVTHVNH MKINI IRAYCLLFLAAI IEVFVTPELALRFLGD

[314] BLASTn analysis of ORF-C sequence did not find any significant homology from NCBI database at the DMA level. Similarly BLASTp analysis of ORF- C putative protein sequence (54 amino acids) (Figure 10) did not detect any putative conserved domains, however it did show 38% identity (query cover 83%) to the Gaal protein from L gasseri which is involved in self immunity to its cyclic bacteriocin (accession number WP__012621085.1 ) (Ito et al. 2009). Without wishing to be bound by theory, these results suggest that the ORF-C gene is involved in immunity of L plantarum B21 ^* to Plantacyclin B21AG.

[315] The nucleotide sequence of orf-C of the Plantacyclin B21AG operon is provided below (SEQ ID NO 9):

ATGAAAAA TTAGATATGTTAGTAGGTGTCATTACAATTATTGTATTGTTAGCGACAATAAT TGCATTTTTCTTCCAGGGACTCAGTAGTATAACTTATGTATGTGCAATAATTAGAGTAAT AT TGTCTTTTGTATATCAACTAATTAAACGGCATACAGAT AA

[316] The amino acid sequence of ORF-C encoded by the Plantacyclin B21AG operon is provided below (SEQ ID NO: 10)

MKNLDMLVRVIT11VLLATI IAFFFQGLSTITYVCAI ITVILSFVYQLIKRHTD

[317] The ORF-D putative protein sequence (220 amino acids) (Figure 10) BLASTp analysis showed specific hits to the conserved domain of ABC_DR_subfamily_A (ABC-transporter ATP-binding proteins); this family of ATP- binding proteins belongs to a multi- subunit transporter involved in drug resistance, lipid transport, and bacteriocin immunity (such as lantibiotics). The BLASTp analysis of this ORF also detected hits to ABC-ATPase superfamily (Marchler-Bauer et al. 2009; Marchler-Bauer & Bryant 2004; Marchler-Bauer et al. 201 1 ) which are a large family of proteins involved in the transport of a wide variety of different compounds such as sugars, ions and peptides, !t has been reported that dedicated transmembrane translocators belonging to the ATP-binding cassette (ABC) transporter superfamily are involved in the cleavage of the ieader-peptide from Class II bacteriocins and in the transportation of the mature bacteriocin molecule across the cytoplasmic membrane. Without wishing to be bound by theory, the present inventors consider ORF-D is involved in the cleavage of the leader peptide and transport of the Plantacyclin B21AG bacteriocin protein.

[318] The nucleotide sequence of orf-D of the Plantacyclin B21AG operon is provided below (SEQ ID NO: 11):

AT G T G TAT GAC T AAG G T GAG TAT GAG T CAC G T CAAGAAAAAAT AC GAT AAT T T TAT AT T GAG C GAC AT AAAT T T T C AAG C AAAAGAAAAAG AAA CATTGGTTTAATTGGT GAAAAC G G T G C C G GAAAAAC TACAC TAT TAAAAT CAAT T GGAGGAAT CAATAAGATAGAT T T T GGGAC TAT TAAA AAAGAT T T TAAGGAAT TGGGAT TTTGCTTT GACAGCAT T CCAT T T CC T GAAGAAC TAAATAT TCTT C AG T T G G A AC AC G TAT T T CAAAACAT T GGAAT AAAC T G G GAT AC C C AAG CTTTTTGGC C T TAT AT T AAAGCAC T T CAAT T AC C T AC T AAAACAC C GAT AT C T AAT T T T T C CAAAGGAAT G ΑΑΑΆΤ GCAAC T AAAC T TAT GCAT T T C TAT T T CACAT CAT C CAGAT T TAT T G T T G T T AGAT GA AAT AAC T AG T G G C C T T GAT C C T C T T AT G C G C C GAAAAG TGTTGCGCC T AAT C ΑΑΑΑΑΆΤ A T G T AGAT C AAAAT GAC TGTT CAG T AAT AAT T AC CAC C CAT AAT T T GAAT GAT G T T GTAGAAAT C T G TAG T C G T T T T GAT T T GC T AGAT CAC GGAAAAA CAT T T T AGAAAAAAGC AT GCAAAAC T T T G G G C C AGAAAAC C T T GAGAAAC T AT T T GAAGAAAC AG T AC AAAAG G C GAAG T TAG G T GAAT AA

[319] The amino acid sequence of ORF-D encoded by the Plantacyclin B21AG operon is provided below (SEQ ID NO: 12)

MCMTKVSMSHVKKKYDNFILSDINFQAKEKEI IGLIGENGAGK LLKS IGGINKIDFG IK KDFKELGFCFDS IPFPEELNILQLEHVFQNIGINWDTQAFWPYIf ALQLPTKTPISNFSKGM KMQLNLC I S I SHHPDLLLLDE I TSGLDPLMRRKYLRL IKKYVDQNDCSVI I TTHNLNDWE I CTRFDLLDHGKI ILEKSMQNFGPENLEKLFEETVQKAKLGE

[320] ORF-E did not find any significant homology from NCBI database at DNA level. The BLASTp analysis of ORF-E putative protein sequence (214 amino acids) (Figure 10) detected specific hits to the ABC_membrane_5 putative conserved domain from the database which related to the ABC_2 membrane transporter family. These domains are from superfamiiy of Acyl_transfer_3 including a range of acyltransferase enzymes. Without wishing to be bound by theory, the present inventors consider these results suggest ORF-E is involved in the involved in transport of the Plantacyclin B21AG.

84 [321 ] The nucleotide sequence of orf-E of the Plantacyclin B21AG operon is provided below (SEQ ID NO: 13):

ATGCTTGGAT AATGTTAAAAGATTATTATCAGCTGTGTGATAAGTGGTTCAAAA GATATA TTTGTTAGGTGTATCTTTTTCTTTAATTATAGCCACAA TTTTCTAAAAAGTGATAGTTGGA TAGTAACAACATTGGTATCAATGATAATGATTAATAGCATTCAATCACTCTTTCTATCCG AT AATAAAAACAACTGGATAAATTTTTTAACAACTTTATCTATTAAAAAATCTATAAGTGTT CT TGCAAGGTATCTATTTGTTATCATTATCTGTGCTGTAACTGCTATACTGAGTGGACTATT TT TTCTAGTGATATCATTATTTTTCAAAGGTATTACTATTGAAAGTATCATGATTGTTCCTA TT TGCTTATTTACGGTTTCAATAATTTATATATCCTTTATTCTTCCTTTTTTATATGCTTTT CA GCAAAACGGACTAACTGTTGGTGTGTTAATGATTTTAGGGATTGCTTTTGTAAGTATACG TT TTTTGGGTGTTTTGTCTAAAATAAAAAAATTAATTTTACTAGATTCTAAAA.TTG.AA.T TAATA TTTCTTGTTGCTCTTACTTTAATTAT ACTGTTGTTTTGTCATACAGTATTGCCTACG AAT TAGTTTAGTTAGAGGAGAAGACTAA

[322] The amino acid sequence of ORF-E encoded by the Plantacyclin B21AG operon is provided below (SEQ ID NO: 14):

MLGLMLKDYYQLCDKWFKKIYLLGVSFSLI IA I FLKSDSWIVTTLVSMIMINS IQSLFLSD NKNNWINFLT LS IKKS ISVLARYLFVI I ICAVTAILSGLFFLVISLFFKGI IES IMTVPI CLFTVS I IYISFILPFLYAFQQNGLTVGVLMILGIAFVS IRFLGVLSKIKKLILLDSKIELI FLVALTL11 TWLSYS IAYVISLVRGED

[323] BLASTn analysis of ORF-F did not find any significant homology from NCBI database at DMA level. The BLASTp analysis of this ORF putative protein sequence (173 amino acids) (Figure 10) did not detect any putative conserved domains corresponding to the amino acid level. The putative protein sequence showed 31 % (query cover 91 %) to PenB protein from L penfosus (accession number WP__003637686.1 ) and 30% identity (93% query cover) to a membrane protein with 5 predicted transmembrane segments (TMS) from L gasseri which is involved in production of the cyclic bacteriocin, gassericin A (accession number WP_01262 081 .1 ). Without wishing to be bound by theory, the present inventors consider these results suggest ORF-F is involved in the production of Plantacyclin B21AG.

[324] The nucleotide sequence of orf-F of the Plantacyclin B21AG operon is provided below (SEQ ID NO: 15): AT GAAGT T TAGAGATAGAAT AC TATAT T GGAG T T T GGGAT GGGGAGGAT TAG T TAGAT TAGC GAT CC T AAGAATAT T TAAAACAT T CAAAAT T C TGCCTAAT T C TAAC CGTAT T T TAAAGGAAA T T C C T AT T G G T AT AG GAG C AC C TAG C T T T G GAAT C T T G T T AT G T T T G G T AG T T T T T AT AT C A G C T T T G G GAAC T TAG C TAG TAT TCT T TG CAT T T AAT AAAAT C AAAC G G T T GAG C C T TAGAT T C T TAG C T C T C T T T AAAGC AAAGG TATAT GACAT T TAT CTGTCT TCG TATAT T G TAT AT AAT T TAG TATAT G T CAT T TATAT C TAG T TATACGAAAAAACAGC AACC AAC T T T CAAATAAATAT T T T TAGC T TGCTAC T T GGAAG T T T TAT CAGC T T T C TAATAT T TAAC TAT C T TAGAAAGCAAAA AAT AT C T T T AAAAAACAAT AT AGAAT T T T CAAG T ACAAT T C T T G T AAT AAAT AT T ATAAC AC CCATATATAGCT ΤΑΆΤ T T T T T TAT AG

[325] The amino acid sequence of ORF-F encoded by the Plantacyclin B21AG operon is provided below (SEQ ID NO: 16)

MK FRH I L Y W FAW G G L L LA I L R I FK FK I L P S NR I L KE I P I G I ΑΆΡ T FG I L L C L W F I S ALGT YLVFFAFNKI KRLS LT FLPL FKAKVYD I YLS S Y IVYNLLYVI Y I YLYEKTATN FQ IN I FS LLLGT FI S FL I FNYLRKQKI S LKNN I E FS S I LVINI I T P I YS L I FL

[326] The present inventors have aiso demonstrated L. plantarum strains B20, B31 and B33 each contain Plantacyclin B21AG, and the Plantacyclin B21AG operon, including the 6 oris implicated in Plantacyclin B21AG circularisation; orf-B, orf-C, orf- D, orf-E, orf-F and an orf corresponding to B33-ORF-21). Accordingly, in one embodiment, the one or more Plantacyclin B21AG-cyciisation polypeptide is selected from the group consisting of ORF-B, ORF-C, ORF-D, ORF-E, ORF-F, B33-ORF-21 and biologically active fragments or variants thereof.

[327] For example, Figure 17 shows Plantacyclin B21AG and the Plantacyclin B21AG operon-encoded ORFs of L. plantarum strains B33 aligned with those of B21 ^* Plantacyclin B21AG and the Plantacyclin B21AG operon-encoded ORFs of L plantarum strains B20 and B31 are identical in amino acid sequence to those of B33 described herein.

[328] The nucleotide sequence of orf-A (Plantacyclin B21AG) of the Plantacyclin B21AG operon of B33 is provided below (SEQ ID NO: 18):

AT G C T T T GAG CAT A T C G T AG T AAAT TAG GA T T GAAC AAAT T T GAG G T TAG T G T T T T AAT GAT CAT C TCTC T T T T T AT T T TAT T AT T T GC C ACAG T T AAT AT T G TAT G GAT T GC AAAACAAT T T G GT G TGCAT T T GACAAC TAG T C T AC AC AAAAAG C T T TAGAT C TAT TAT C T G C TG GAT CAT C T TTGGGCACCGTGGCAGCTGCTGTC AGG G TAGA TGCCAGCATGGGCAGTTGCAGCAGC TGGGGCTCTCGGGGGTACTGCAGCTTAA

[329] The amino acid sequence of ORF-A (Plantacyciin B21 AG) encoded by the P!antacyc!in B21AG operon of B33 is provided below (SEQ !D NO: 19)

MLSAYRSKLGLNKFEVTVLMI ISLFILLFATVNIV IAKQFGVHLTTSLTQKALDLLSAGSS LGTVAAAVLGVTLPAWAVAAAGALGG AA

[330] The amino acid sequence of mature ORF-A (Plantacyciin B21AG) encoded by the Plantacyciin B21AG operon of B33 is provided below (SEQ ID NO:

52) :

IV I KQFGVHLTTSLTQKALDLLSAGSSLGTVAAAVLGVTLPAWAVAAAGALGGTAA

[331 ] The nucleotide sequence of mature ORF-A (Plantacyciin B21AG) encoded by the Plantacyciin B21AG operon of B33 is provided below (SEQ ID NO:

53) :

ATTGTATGGATTGCAAAACAATTTGGTGTGCATTTGACAACTAGTCTTACACAAAAA GCTTT AGATCTATTATCTGCTGGATCATCTTTGGGCACCGTGGC GCTGCTGTCTT GGTGTTACAT TGCCAGCATGGGCAGTTGCAGCAGCTGGGGCTCTCGGGGGT CTGCAGCT AA

[332] The nucleotide sequence of orf-B of the Plantacyciin B21AG operon of B33 is provided below (SEQ ID NO: 20):

GTGAATAAACCGCGGAGTA TATCCCTGCTTTATTGATAGTATTTAT ATTTTTTTAATTTT GGGTGTCTTGTT AGCAAATATTT GCAACTGCAAGCTCTCAACATTCATTAGATTCATTTA AAGTAATTCATTTAAACTGGTTTACTGAAATTATCTTTAGAAATACAATAGCATTTTTAG TA TTAAGCTC ACTCTTTTTTTGGGAAA A AG AAGTGTC TATTCTTTTGCGTTAATGGCTT AACGTTGGACTAATATGCGGTCAATTACCAA ATTTCAAAGTATCGTTCTTCTCTCACCAC ATGGTGTTATTGAAATAACTTCGTATATATGGTTAGTATATGCTGTAACACATGTTAA CAC ATGAAAATTAATATAATAAGATCCTACTGTCTTTTGTTTCTTGCAGCAATAATTGAAGTT TT TGTTACCCCAGGA AGCC TATGGTTGTTAGGAGAT AA

[333] The amino acid sequence of ORF-B encoded by the Plantacyciin B21 AG operon of B33 is provided below (SEQ ID NO: 21 )

87 MNKPRSNIPALLIVFI T FLILGVLFSKYFATASSQHSLDSFKVIHLNWFTEI I FRN IAFLV LSSTLFLGNIVSVI FFCVNGFNVGLICGQLPI FQS IVLLSPHGVIEITSYIWLVYAVTHVNH MKINI IRSYCLLFLAAI IEVFVTPGLALWLLGD

[334] The nucleotide sequence of orf-C of the P!antacyc!in B21AG operon of B33 is provided below (SEQ ID NO: 22):

ATGAAGAA TTAGATATGTTAGTACGTGTCATTACAATTATTTTATTGTTAGCGACAATAAC TGCCTTTTTCTTTAAGGGTCTCAGTACTATAACTTATATATGTGCAATAATTACAGTGGT AT TGGCTTTTGTATATCAGCTAATTAAACGGCATACAGATTAA

[335] The amino acid sequence of ORF-C encoded by the Piantacyciin B21AG operon of B33 is provided below (SEQ ID NO: 23)

MKNLDMLVRVITilLLLATITAFFFKGLSTITYICAI ITWLAFVYQLIKRH D

[336] The nucleotide sequence of or†~D of the Piantacyciin B21AG operon of B33 is provided below (SEQ ID NO: 24):

GAAATCATTGGTTTAATTGGCGAAAATGGTGCTGGAAAAACCACACTATTAAAATCT ATTGG AGGAATCAATAAGA AGATTTTGGAACTATTAAAAAAGATTTTAAAGAATTAGGGTTTTGCT TTGACAGCATTCCATTCCCTGAAGAACTAAACATACTTCAGTTGGAACATATATTTCAAA AC ATTGGAATAAACTGGGATACTCAAGCTTTTTGGCCTTATATTAAAGCACTTCAGTTACCT AT AAAAT CCGATATCTAATTTTTCCAAAGGAATGAAAATGCAACTAAACTTATGCATTTCTA TTTCACATCATCCGGACTTATTGTTGTTAGATGAAATAACTAGTGGCCTTGATCCACTTA TG CGACGAAAAGTATTGCGACTAATCΑΑΑΑΑΆΤATGTAGATCAAAATGACTGTGC AGTAATAAT TACCACTCATAATTTGAATGATGTTGTAGAAATCTGTACTCGTTTTGACTTGCTAGATCA TG GAAAAATCATTTTGGAAAAAAACATGCAAAAATTTGGGGCAGAAAACCTTGAGAAACTAT TT GAAGAGACAGTAAAAAAAGCGAATTTAGGTGAAT A

[337] The amino acid sequence of ORF-D encoded by the Piantacyciin B21 AG operon of B33 is provided below (SEQ ID NO: 25)

MCMTKVSMSQVRKKYDNFILGDINFQAKEKEI IGLIGENGAGKTTLLKS IGGINKIDFGTIK KDFKELGFCFDSIPFPEELNILQLEHIFQNIGI WDTQAF PYIKALQLPIKIPISNFSKGM KMQLNLCIS ISHHPDLLLLDEITSGLDPLMRRKVLRLIKKYVDQNDCAVI ITTHNLNDWEI CTRFDLLDHGKI ILEKNMQKFGAENLEKLFEE VKKANLGE

[338] The nucleotide sequence of orf-E of the Piantacyciin B21AG operon of B33 is provided below (SEQ ID NO: 26): ATGCTTGGATTGATGT AAAAGATTAT ATCAGCTGTGTGA AAGTGGTTCAAAAAAATATA TTTGTTAGGTGTATCTTTTTCTTTAATTATAGCCACAATTTTTCTAAAAAGTGATAGTTG GA TAGTAGCAACATTGATATCAATGATAATGATTAA AGCATTCAATCACTCTTTCTATCCGAT ΑΆΤAAAAACAACTGGATAAATTTTTTAACAACTTTATCTATAAAAAAATCTATAAGT GTTCT TGCAAGGTATCTATTTGTTATCATTGTCTGTGCTGTAACTGCTATACTGAATGGGCTATT TT TTCTAGTGATATCATTATTTTTCAAAGGTATTACTATTGAAAGTATAATGATTGTTCCTA TT TGCTTGTTTAGAGTTTCAATAATTTATATATCCTTTATTCTCCCTTTTTTATATGCTTTT CA GCAAAACGGATTAACTGTTGGTGTGTTACTGATTTTAGGGATAGCTTTTGTAAGTATACG TT TTTTTGG TCTGTCTAAAATAAAAAAATTAATTTTAGTAGATTCTAAAACTGAATTAATA TTTCTTGTCGCTCTTGCTTTAATTATAACTGTTGCTTTGTCATACAGTATTGCCTATGTA AT TAGTTTAATTAGAGGAGAAGAATAA

[339] The amino acid sequence of ORF-E encoded by the Piantacyclin B21 AG operon of B33 is provided below (SEQ ID NO: 27)

MLGLMLKDYYQLCDKWFKKIYLLGVSFSLI IA I FLKSDSWIVATLISMIMINS IQSLFLSD NKNNWINFLTTLS IKKS ISVLARYLFVI IVCAVTAILNGLFFLVISLFFKGITIES IMIVPI CLFTVS I IYISFILPFLYAFQQNGLTVGVLLILGIAFVS IRFFGILSKIKKLILLDSKTELI FLVALAL I ITVALSYSIAYVISLIRGEE

[340] The nucleotide sequence of orf-F of the Piantacyclin B21AG operon of B33 is provided below (SEQ ID NO: 28):

ATGAAA TTAGACATACAATACTATATTGGATTTTCGCATGGGGGGCATTGTTTGTATTAGC GATCTTAAGATTATTTAAAACATTCAAAATTCTGCCTAATTCTAATCGCATTTTAAAAGG GA TTCCTGTTGATATAGTAGCACCTAGCTTTGGAATCTTGCTATGTTTAGTAGTTTTTATAT CA GCTTTAGGAAGTTACCTAGTATTCTTTGTATTTAATAAAATCAAACGGTTGAACCTTACA TT CTTATCTCGCTTTAAAACAAAGGTATATGACATTTATCTATCTTCATACATTGTATATAA T TACTATATGTCATTTATATCTACTTATATAAAAAAACAGCAACCAACTTTCAAATAAATA TT TTTAGCTTAGTGCTTGGAACTTTTATCAGCTTTCTAATATTTAACTATCTTAGAAAGCAA AA AATATCTTTAAAAAATAATATGGAATTTTCAAGTACCATTCTTTTAATAAATATTATCAC AC CTATATATAGCTTGATTTTTTTATAG

[341 ] The amino acid sequence of ORF-F encoded by the Piantacyclin B21AG operon of B33 is provided below (SEQ ID NO: 29) KFRHTILYWI FAWGALFVLAILRLFKTFKILPNSNRILKGIPVDIVAPTFGILLCLWFIS ALGSYLVFFVFNKIKRLNLTFLSRFKTKVYDIYLSSYIVYNLLYVIYIYLYKKTATNFQI NI FSLLLGTFTSFLT FNYLRKQKISLKNNMEFSST ILLTNI ITP IYSL I FL

89 [342] The nucleotide sequence of orf~21 of the Plantacyclin B21AG operon of B33 is provided below (SEQ !D NO: 30):

ATGAGTAAATTTTCAAAAGTATCAATCGGGGTTATCTATGTTATATGTACAATAGTG CCTGC A AATTACGATATTTGAACGGAAATTATTTTGGATTGGACTAG AGC CTAGGGTATTTTT GTTATATTGGCTGGTTTATTTTTATΑΑΑΆΤCGCATGATAACCTATAA

[343] The amino acid sequence of ORF-21 encoded by the Plantacyclin B21AG operon of B33 (B33-ORF-21 ) is provided below (SEQ ID NO: 31 ):

MSKFSKVS TGVIYVICTIVPAI ITT FERKLFWTGLVALGYFCYIGWFI FIKSHDNL

[344] The amino acid sequence of ORF-21 encoded by the Plantacyclin B21 AG operon of B21 ^* is provided below (SEQ ID NO: 32):

MSRFSKVS IGVIYVICTIVPAI TMI FERGLFWIGLVALGYFCYIGWFI FIKSHDNL

[345] In another aspect the invention provides an isolated polynucleotide which encodes a Plantacyclin B21AG-cydisation polypeptide or a biologically active fragment or variant thereof.

[346] In another embodiment the invention provides an isolated polynucleotide which encodes a Plantacyclin B21AG-cyciisation polypeptide or a biologically active fragment or variant thereof, wherein the Plantacyclin B21AG-cyclisation polypeptide selected from the group consisting of ORF-B, ORF-C, GRF-D, ORF-E, QRF-F, B33- ORF-21 and biologically active fragments or variants thereof.

[347] In one aspect the invention provides a polynucleotide as described herein, wherein the polynucleotide comprises: (i) a sequence of nucleotides as provided in SEQ ID NO: 7, 9, 1 1 , 13, 15, 20, 22, 24, 26, 28 or 30; (ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in SEQ ID NO: 8, 10, 12, 14, 16, 21 , 23, 25, 27, 29, 31 or 32; (iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 35% identical to SEQ ID NO: 8; (iv) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 39% identical to SEQ ID NO: 10; (v) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 52% identical to SEQ ID NO: 12; (vii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 40% identical to SEQ ID NO: 14; (viii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 31 % identical to SEQ ID NO: 16; or (ix) a sequence complementary to any one of (i) to (viii).

[348] In one aspect the invention provides a polynucleotide as described herein, wherein the polynucleotide comprises: (i) a sequence of nucleotides as provided in SEQ ID NO: 7, 9, 1 1 , 13, 15, 20, 22, 24, 26, 28 or 30; (ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in SEQ ID NO: 8, 10, 12, 14, 16, 21 , 23, 25, 27, 29, 31 or 32; (iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 94% identical to SEQ ID NO: 8; (iv) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 89% identical to SEQ ID NO: 10; (v) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO: 12; (vii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 94% identical to SEQ ID NO: 14; (viii) a sequence of nucieotides encoding a polypeptide comprising an amino acid sequence which is at least 90% identical to SEQ ID NO: 16; (ix) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO: 30 or (x) a sequence complementary to any one of (i) to (ix).

[349] In another aspect the invention provides a substantially purified and/or recombinant Piantacyciin B21AG-cyclisation polypeptide or a biologically active fragment or variant thereof, wherein the Piantacyciin B21AG-cyclisation polypeptide is selected from the group consisting of ORF-B, ORF-C, ORF-D, ORF-E, ORF-F, B33-ORF-21 and biologically active fragments or variants thereof.

[350] In one embodiment, the cyciisation polypeptide is ORF-D and/or ORF-E

[351 ] In one embodiment, the present invention provides a polypeptide as described herein wherein the polypeptide comprises: (i) an amino acid sequence as provided in SEQ ID NO: SEQ ID NO: 8, 10, 12, 14,16, 21 , 23, 25, 27, 29, 31 or 32; or (ii) an amino acid sequence encoded by a polynucleotide as described herein. [352] The present invention provides methods that may be used to generate cyclic polypeptides, which can be used for a variety of purposes. For example, the cyclic polypeptides can be used as drugs to inhibit disease-causing targets. They can also be used as affinity reagents for validating the therapeutic potential of targets or in general applications that require affinity reagents. Many naturally occurring cyclical peptides are known and the methods of the present invention can be used to readily produce such polypeptides. Other suitable uses will of course be apparent to one of skill in the art.

[353] Accordingly, in one method the present invention provides a method of forming an intramolecular peptide bond in a target polypeptide of interest comprising contacting the polypeptide with one or more cyclisation polypeptide(s).

[354] In another method the present invention provides a method of forming an intramolecular peptide bond in a polypeptide comprising contacting the polypeptide with one or more Plantacyclin B21AG-cyclisation poiypeptide(s).

[355] In one embodiment, a polypeptide of interest may comprise a cyclisation signal, for example a Plantacyclin B21AG polypeptide or a fragment thereof. The presence of the Plantacyclin B21AG polypeptide or a fragment thereof in the peptide of interest results in the peptide of interest being circularised on contact with the one or more cyclisation polypepfide(s).

[356] Accordingly, in one embodiment, the target polypeptide is flanked at the N-terminus and/or the C-terminus by a Plantacyclin B21AG polypeptide or a fragment thereof. The Plantacyclin B21AG polypeptide or a fragment thereof should be sufficient to allow circularisation of the target polypeptide.

[357] For example, portions of a Plantacyclin B21AG polypeptide can be fused to the N- and C-terminal ends of a target polypeptide so that the N- and C-terminai protein fusion molecules can be circularised by the one or more Plantacyclin B21AG- cyclisation poiypeptide(s).

[358] In one embodiment, the target polypeptide is a polypeptide having a biological activity or a polypeptide comprising an antigen. [359] The target polypeptide may have industrial or medical (pharmaceutical) applications. A target polypeptide for use in the present invention includes antagonists, agonists, hormones, cytokines, hematopoietic factors, growth factors, polypeptides that bind other molecules, and other bioactive molecules. A target polypeptide for use in the present invention also includes antigenic polypeptides.

[360] In one embodiment, the target polypeptide is a polypeptide that binds molecules of interest, wherein the molecules of interest include biomarkers or antibodies

[361 ] A target polypeptide can be any polypeptide for which a cyclic conformation is expected to impart advantageous biologic properties, such as those described herein, including favourable pharmacological properties such as resistance to proteases.

[362] In one embodiment, the invention provides a method of forming a cyclic Piantacyciin B21AG comprising contacting a Piantacyciin B21AG with one or more Piantacyciin B21 AG-cyclisation polypeptide.

[363] In another embodiment, the one or more Piantacyciin B21AG-cyciisation polypeptide is selected from the group consisting of ORF-B, ORF-C, ORF-D, ORF-E, ORF-F, B33-ORF-21 and biologically active fragments or variants thereof.

[364] The present invention also provides methods for the manufacture of synthetic cyclic peptides for clinical investigations and therapeutic use, and for the production of cyclic peptide libraries that can be screened to identify cyclic peptides with a desired activity. The present inventors have characterised novel cyclisation polypeptides.

[365] In another aspect, the invention provides a method of making a library of circular polypeptides using the methods described above, and methods of using the library to screen for cyclic polypeptides with a biological activity of interest.

[366] For example, a library of polypeptides of interest may be formed which comprise a cyclisation signal, for example a Piantacyciin B21AG polypeptide or a fragment thereof. The presence of the Piantacyciin B21 AG polypeptide or a fragment thereof in the peptide of interest results in the peptide of interest being circularised on contact with the one or more Piantacyciin B21AG cyclisation polypeptide, thereby forming a library of cyclic polypeptides.

[367] In one embodiment, a library of target polypeptides is flanked at either or both of the N and C termini by a Piantacyciin B21AG polypeptide or a fragment thereof. For example, portions of a Piantacyciin B21AG polypeptide can be fused to the N~ and C~terminal ends of a library of target polypeptide so that the N- and C- terminal protein fusion molecules can be circularised by the one or more Piantacyciin B21AG-cyciisation polypeptide.

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!XAMPLES

[452] Example 1 : Bacteria! strains and culture conditions.

[453] L. p!antarum B21 , was isolated from Vietnamese fermented sausage nem chua in Vietnam, and identified by 18s rRNA gene sequence analysis.

[454] The strain designated herein as L plantarum B21 ^* was derived in Australia from the Vietnamese isolate L. plantarum B21 following laboratory culture. B21 ^* differs in antimicrobial activity when compared to B21 .

[455] The indicator strain in bacteriocin activity assays, L. plantarum A6, was isolated from the same source (34). The remaining indicator strains, L. plantarum ATCC 8014, Lactobacillus arabinosus 17-5, Lactococcus lactis 345-18, Lactobacillus brevis 19012, Listeria monocytogenes 192/1 -2 ACM 3173 and Clostridium perfringens 52/6-1 were purchased from the American Type Culture Collection (ATCC) or obtained from the RM!T University culture collection as indicated in Table 1.

[456] Example 2: B21* cell free supernatants have antimicrobial activity,

[457] Ail LAB strains were grown in MRS broth (Oxoid, England) at 30°C for 24 h without shaking. For growth of the LAB strains on solid media MRS agar (Oxoid, England) was used and the plates were incubated at 30°C for 48 h. The Clostridium perfringens and Listeria monocytogenes were grown in Brain-heart infusion (BHI, Oxoid, England) broth. Listeria monocytogenes was incubated at 30°C. Clostridium perfringens was incubated at 37°C anaerobicaily. Stock cultures of bacterial strains were stored at -80°C in MRS/LB broth supplemented with 30% (v/v) glycerol (Sigma, Germany),

[458] The cell free supernatants (CFS) of strain B21 ^* and B21 were prepared using 2% (v/v) inoculum of a 24 h culture in MRS broth at 30°C (36). The well diffusion agar (WDA) assay was modified from Tagg & McGiven (37) for the evaluation of bacteriocin activity from B21 and B21 ^* . Sterilised semi-solid MRS agar (0.8% agar) was mixed with approximately 10 ⁶ CFU/mL of the overnight culture of the indicator strain at 47°C. After agar solidification, wells of 8 mm in diameter were perforated and 100 μί_ of the prepared CFS was placed into each well. The agar plates were held at 4°C for 2 h to allow for the diffusion of the culture supernatant into the semi-solid agar and then incubated at 30°C for 18-24 h before being examined for a clear zone of inhibition. The diameters of the inhibition zones were measured and the diameter of the well (8 mm) was subtracted from the total zone diameter. To confirm the presence of antimicrobial activity the inhibitory activity was directly related to the zone of inhibition diameter in millimetre. Inhibition was recorded as negative if no zone was observed around the agar well. For quantitative bacteriocin activity assay, two-fold serial dilutions of the CFS were prepared using saline water solution. The antimicrobial activity was expressed as arbitrary units (AU/mL) as described previously (28).

[459] The ceil free supernatant (CFS) from L plantarum B21 demonstrated a wide antimicrobial activity against the LAB strains tested and food borne pathogens Clostridium perfringens and Listeria monocytogenes,

[460] In contrast, the ceil free supernatant (CFS) from L plantarum B21 ^* demonstrated a wide antimicrobial activity against the LAB strains tested and food borne pathogens Clostridium perfringens and Listeria monocytogenes but not Staphylococcus aureus (Table 1 and Figure 13),

[461 ] This data demonstrates strain B21 ^* cell free supernatant exhibits a wide antimicrobial activity.

ible 1 : Evaluation of L plantarum B21* antimicrobial activity by WDA assay

Indicator strain Source Antimicrobial activity*

Lactobacillus plantarum A8 Isolated from nem chua (1) +++ ^■ +

Lactobacillus plantarum ATCC American Type Culture ++÷

8014 Collection (ATCC)

Lactobacillus arabinosus 17-5 RM!T University Culture ++++

Collection

Lactococcus lactis 345-18 R IT University Culture +++

Collection

Lactobacillus brevis 19012 RMIT University Culture +

Collection

Listeria monocytogenes 192/1 RMIT University Culture ++

2 ACM 3173 Collection

Clostridium perfringens 52/6-1 RMIT University Culture ++

Collection

^ ^' Positive inhibitory activity [+], No inhibitory activity [-]

[462] Example 3: B21* antimicrobial activity is sensitive to proteolytic enzymes.

[463] Antimicrobial substance(s) produced by B21 ^* had interesting characteristics, such as good pH (3 to 10) and thermo-stability (up to 90°C for 20 min. To confirm the proteinaceous nature of the LAB B21 ^* antimicrobial activity, the neutralised CFS was treated with proteolytic (proteinase K, trypsin and pepsin) and non-proteolytic (cataiase) enzymes. L plantarum A6 and ATCC 8014 were used as the indicator strains in two separate experiments.

[464] The proteinaceous nature of the B21 ^* antimicrobial activity was confirmed using a modified method from Omar et al. (38). Proteolytic enzymes, proteinase K, trypsin, pepsin (Sigma-Aldrich, USA) and cataiase (Sigma-Aldrich, USA), as control, were prepared in sterilised Milli-Q® water at concentration of 10 mg/mL. Each enzyme solution (10 μΙ_) was mixed with 100 μΙ_ of the B21 ^* CFS and incubated at 37°C for 3 h, to allow for digestion. This was followed by WDA assay to examine the loss of antimicrobial activity. The clear zone of inhibition shown in Figure 2 was compared with that produced by an untreated CFS or catalase treated. The loss or reduction in the size of the inhibition zone was taken as indication of the sensitivity of the antimicrobial activity to proteolytic digestion. The culture supernatant treated with catalase was used as a negative control.

[465] The killing activity of the LAB strain B21 ^* CFS showed sensitivity to proteinase K, trypsin and pepsin (data not shown). The bacteriocin activity was completely eliminated with proteinase K but only partly eliminated by trypsin and pepsin. This shows that the B21 ^* antimicrobial activity is due to a protein, is not due to hydrogen peroxide, but that it may be more resistant to trypsin and pepsin than for other bacteriocins reported (26, 38, 40). The bacteriocin from L. piantarum B21 ^* shows a differential sensitivity to different proteolytic enzymes.

[466] This data indicates that one or more proteins is responsible for the B21 ^* antimicrobial activity.

[467] Example 4: Genome Sequencing of B21* to characterise bacteriocin (pin) related genes.

[468] To examine the characterise bacteriocin (pin) related genes, the B21 ^* genome was sequenced using the lllumina HiSeq 2000 sequencing platform (BGI, China). A total of 527 Mb of data was produced with next -generation lllumina paired- end sequencing technology. A genome library containing different fragment length insertions (500 bp and 6 kb) was constructed, and 6,354,788 of paired end 90-bp reads, were generated with 164 fold coverage. The cfe novo assembly was performed using SOAPdenovo (6, 7) and consisted of 29 contigs and 20 scaffolds. Coniigs were further assembled using multiple assembly software. Finally the SOAPdenovo software was used in combination with a PGR approach to close the gaps and correct the mis-assemblies. This resulted in an optimised assembly consisting of one final super-scaffold and one contig. The Glimmer 3.0 programme was used to conduct gene prediction and the obtained gene sequences were compared using the IMCB! prokaryotic genome annotation pipeline, for functional gene annotation. The rRNAmmer, tRNAscan and Rfam software were used to predict rRNA, tRNA and sRNA respectively (8-10).

[469] The complete genome sequence of L. planiarum B21 ^* showed one circular chromosome of 3,284,260 bp with a GC content of 44.47%. The chromosome contains 3, 1 17 genes (including 2,930 coding sequences, 51 pseudo genes and 18 frame shifted genes) with a total length of 2,766,912 bp, which made up 84.3% of the genome. The average gene length was 887 bp and the GC content in the gene region was 45.5%. The number of rRNA, tRNA and sRNA genes was 17, 65 and 2 respectively. There is ambiguous evidence for the presence of a number of natural plasmids in this strain, which are currently under further investigation.

[470] Comparison of the B21 ^* genome with the L. plantarum WCFS1 reference strain (Genbank accession number AL935263.2) revealed that 2668 out of 31 17 genes (80.17%) of B21 ^* aligned well with those of WCFS1 with a mean identity of 99%. There were 449 unique genes in the B21 ^* genome that did not align to WCFS1 .

[471 ] The significant number of transpositions relative to its nearest neighbour WCFS1 , confirm the well-known plasticity of the LAB genome and the B21 ^* strain derived from the Vietnamese isolate B21 .

[472] Example 5: Functional impairment of the classical pin locus at the genetic level in B21*

[473] Genome sequencing revealed five operons were identified in the pin locus of strain B21 ^* {plnABCD, plnJKLR, plnMNOP, plnEFI and plnGHSTUVW) by genome sequencing, and a novel transposon was located between the plnC and plnD genes. This new transposon (1319 bp), located between the plnC and pinD genes, is likely to impair the plnABCD regulatory operon.

[474] A part of the ORF-5 amino acid sequence of the transposon showed one hit at 93% identity to part of an antimicrobial peptide ABC transporter ATPase from Lactobacillus fermentum (accession number WP__014562300.1 ). A search of the new B21 ^* genome data did not identify the rest of the complete ABC transporter gene from Lactobacillus fermentum. This suggested that a part of this gene may have been transferred to the B21 ^* genome with the transposable element from L. fermentum or a similar strain and this may have been a relatively recent insertion event,

[475] Previously, other transposons have been reported in the pin locus of L, plantarum strains (Cho et al. 2010; Diep et al. 2009). Diep et al. (2009) reported a 1414 bp transposon upstream of the plnQ gene in L. plantarum V90. Similarly, Cho et al. (2010) demonstrated the insertion of a transposon disrupting the pINCSHK gene in L. plantarum PCS20 which had 92% identity to the transposon reported in V90, Searches in the NCBI database revealed that the B21 ^* transposon (1319 bp) is the first of its kind found in L plantarum. The B21 ^* transposon protein sequence showed high similarity (up to 99% identity) to the transposons commonly found in Lactobacillus fermentum strains (accession numbers WPJ324626250.1 and WP_012391422.1 ). The new B21 ^* genome was searched to identify any repeats of the new transposon gene. The full transposon gene sequence (1319 bp) is repeated three times in the rest of B21 ^* genome.

[476] The locus was shown to be impaired by a novel transposon insertion, interrupting the bacteriocin regulatory operon plnABCD. A number of other pin genes, most notably plnH, were also subjected to mutation which was predicted to impair function. A PCR-based approach was used to identify the same transposon gene in two other LAB strains. The transposon appeared to be unique, and had not been reported previously in L. plantarum strains.

[477] B21 ^* plnH showed 99% identity to that of strain WCFS1 (Table 2); however the deletion of nucleotide A (Scaffold 1_ between 1779520 and 1779521 ) from B21 ^* plnGH operon, resulted in a frame shift of the ORF. Therefore, the first

130 translated amino acids are 100% identical to the PlnH protein from strain

WCFS1 followed by a stop codon in strain B21 ^* The PlnH protein contains 457 amino acids in L. plantarum WCFS1 . Diep et al. (1996) demonstrated that PInG and

PlnH are members of the so-called ABC transporters family and their accessory proteins, respectively. They are believed to constitute the dedicated processing and secretion machinery for the production of bacteriocin-iike peptides with doubie- glycine-type leaders. The plnH nucleotide deletion and the consequent frame shift in the translated protein results in a major change in the length of the protein (from 457 to 130 amino acids) suggesting that the functionality is lost.

Table 2: Comparative sequence analysis (alignment) and summary of function for the pin locus genes from L. piantarum B21* with reference to the L, piantarum WCFS1. Clone manager software was used for alignment analysis,

[478] The loss of functionality for the PinH protein suggests a major impairment in the maturation and secretion of classical bacteriocin peptides in B21 ^*

[479] Since the genome data showed that the function of the classical pin locus was impaired at the genetic level, the identity of the bacteriocin produced by B21 ^* responsible for the killing activity in B21 ^* culture supernatant remained unknown.

[480] Example 6: Purification of B21* bacteriocin.

[481 ] Numerous classical LAB bacteriocin proteins have been purified efficiently using ammonium sulphate precipitation (Anderssen et al. 1998; Gonzalez et al. 1994; Hata et al. 2009; Hata, Tanaka & Ohmomo 2010; Hoick et al. 1992; Jimenez- Diaz, Rufino et al. 1995; aidonado, Ruiz-Barba & Jimenez-Diaz 2003; Nissen- Meyer et al. 1992; Smaoui et al. 2010). Concentrated supernatant was subject to a variety of ammonium sulphate saturation levels (data not shown). As obvious visible precipitate was obtained only when the saturation level of ammonium sulphate was taken to 80%. After centrifugation the resultant protein precipitate and supernatant were examined for antimicrobial activity. In all trials the bacteriocin activity was only

[482] partly precipitated in the 80% ammonium sulphate fraction (-80%), but was also present in the supernatant (-40%). The overall recovery in the precipitate did not exceed 60%. These results appeared to show that the ammonium sulphate precipitation was not an efficient method for the recovery of the B21 ^* bacteriocin protein, resulting in little or no purification.

[483] Classical bacteriocins are generally hydrophobic in nature (Atrih et al. 2001 ; Ennahar et al. 2000; Hoick et al. 1992; Maidonado, Ruiz-Barba & Jimenez- Diaz 2003) and are precipitated efficiently at a 40 - 80% ammonium sulphate saturation level (Deraz et al. 2005; Hoick et al. 1992; Jimenez-Diaz, Rufino et al. 1995; Metivier et al. 1998). This was not achieved for the putative bacteriocin in this study.

[484] The B21 ^* bacteriocin protein was purified from the culture supernatant by a four-step protocol consisting of a CFS harvesting and concentration step, extraction into butanoi, a size exclusion/desalting/buffer-exchange step and a final cation exchange chromatography by FPLC. [485] In brief, the B21 ^* bacteriocin protein was purified from the culture supernatant by a four-step protocol consisting of a CFS harvesting and concentration step, extraction into butanol, a size exclusion/desalting/buffer-exchange step and a final cation exchange chromatography by Fast Protein Liquid Chromatography (FPLC). All the purification steps were carried out at room temperature, LAB strain B21 ^* was used as an inoculum 2% (v/v) into 165 mL of MRS broth with an initial pH of 6,0 and was incubated at 30 °C for 24 h without shaking. The neutralised culture supernatant was prepared as described previously. The CFS obtained was concentrated to 7.2 mL using an Am icon UF cell (Model 8200, Millipore, USA) and a 10 kDa cut off membrane (poiyethersulphone, PES-10 kDa ultrafiltration membrane discs, Generon, UK). The method for extraction of bacteriocin protein from 70 mL of concentrated B21 * culture supernatant using n-butanol was modified from Abo-Amer (26). The concentrated B21 ^* CFS was extracted twice in ½ volume of water saturated butanol and centrifuged at 10,000 ^χ g for 10 min. The butanol fraction containing the bacteriocin protein was freeze dried (FDU-8612, Operon Co. Ltd, Korea) to remove the solvent. The butanol (redissoived in 20 mM sodium phosphate buffer, pH 6.0) and aqueous fractions were examined for antimicrobial activity. The B21 ^* dried butanol extraction was resuspended in 1 mL of 20 mM sodium phosphate buffer (pH 6.0). The redissoived protein sample was desalted using a NAP10 desalting column pre-packed with Sephadex G-25 resin (NAP™-10 Columns, Sephadex™ G25, GE Healthcare Life Sciences, UK) and eiuted in 20 mM sodium phosphate buffer (pH 6.0). Purification of the desalted bacteriocin protein was performed using a Uno S-6 prepacked monolith cation exchange column (12x55 mm, Bio-Rad, USA) on a fast protein liquid chromatography (FPLC) system (BioLogic DuoFlow System, Bio-Rad, USA). The Uno S-6 column was equilibrated with 20 mM sodium phosphate buffer, pH 6.0 (buffer A) and the desalted bacteriocin extract was eiuted from the column with a linear NaCI gradient (0-1 M) in buffer A. A total of 33 fractions of 3 mL were collected and assayed for the antimicrobial activity. The two FPLC-fractions showing the strongest bacteriocin activity (F14 and F15) were pooled together (6 mL) and concentrated/buffer exchanged using an Am icon® Ultra~4 Centrifugal Filter Units (3 kDa, Millipore, Ireland). The final volume of the purified bacteriocin protein fraction was reduced to 200 μί. The concentration of the bacteriocin protein was measured after each step of protein purification using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA).

[486] The neutralised culture supernatant was prepared from a 165 mL culture and the bacteriocin activity was calculated as 800 (AU/mL) against L. piantarum A6. The concentration of B21 ^* CPS using a 10 kDa cut off membrane resulted in 16-fold concentration of the bacteriocin, increasing the inhibitory activity from 800 (AU/mL) to 12,800 (AU/mL), while no inhibitory activity was detected in the filtrate. The concentrated bacteriocin was extracted into n-butanoi and the organic phase was freeze dried. The WDA assay of the dried butanol phase (resuspended in 20 mM sodium phosphate buffer, pH 6) showed strong bacteriocin activity against L. piantarum A6 while no activity was observed in the aqueous phase. Butanol extraction exhibited complete recovery of the bacteriocin activity. The extraction and extent of recovery seemed to be even more efficient than for some other bacteriocins where some residual activity is always observed in aqueous phase (26). The size exclusion and desalting of the bacteriocin using the NAP10 desalting column prepacked with Sephadex G~25 resin served to help remove excessive lipopoiysaccharides and any residual salt in preparation for further chromatography. Approximately 1 mL of extract was adsorbed to a 6 mL column of Uno S-6 equilibrated with buffer A (20 m!Vl sodium phosphate buffer, pH 6.0) and the bacteriocin was eluted from the column with a linear NaCI gradient (0-1 M) in 66 mL (Figure 1 ). A single peak in absorbance at 214 nm and a small peak at 280 nm corresponding to eluted protein were observed (Figure 1 ), the former arising from the peptide bond and the latter from the aromatic residues of the bacteriocin peptide. The presence of one major peak is because of the selectivity of the butanol extraction and the fact that the protein is strongly basic, exhibiting an overall positive charge at pH 6.0 (probably more than +2). Thirty three collected fractions were examined for antimicrobial activity. The fractions taken across the single absorbance peak at 214 nm and 280 nm (F14, F15 and E15) showed the strongest bacteriocin activity, while the flow through fractions (F3 and F4) also showed some bacteriocin activity (data not shown). The fractions showing the strongest bacteriocin activity (F14 and F15) were pooled together (6 mL) and were subjected to concentration and buffer exchange using a 3 kDa Amicon Ultra Centrifugal Filter and concentrated to 200 ί for further protein analysis. A summary of the bacteriocin purification is presented in Table 3. The overall yield, using the G25 fractions was 19% and the overall purification factor was more than 8000.

[487] Butanol extraction was highly effective and appeared very selective, far more selective than for other classical bacteriocins (26, 46), where residual activity was present in the aqueous phase. A simple back calculation would suggest that LAB B21 ^* produces 1 g/mL bacteriocin in its CFS. This is consistent with yields reported in the literature of -5.5 mg/L (47). During the ion exchange purification step approximately 20-30% of the activity did not bind to the column and later mass spectrometry indicated the same bacteriocin polypeptide mass in the flow through. Slowing column and reducing the loading did not change this. One possible explanation could be that a proportion of the bacteriocin was bound to another protein or is highly aggregated masking its charge properties in some way.

Table 3. Purification of B21* bacteriocin protein.

³ Total activity was determined by multiplying the volume and the activity. Protein concentration was determined by BCA assay. ^'' Total protein was determined by multiplying volume and the protein. " Specific activity is the activity units divided by protein concentration, ^e The yield is the total activity as the percentage of the initial total activity, ' Purification fold was calculated based on specific activity. [488] The purified B21 ^* bacteriocin (F14 and F15) from the cation exchange column that showed the strongest antimicrobial activity were stored at 4 °C and examined for bacteriocin activity by WDA assay over an eight weeks period. The B21 ^* purified bacteriocin remained active against L. plantarum A6 and showed no reduction in activity over the period (data not shown). This excellent stability is in contrast to that reported for classical bacteriocins. It has been widely reported that purified classical bacteriocins from LAB are unstable after being purified. Hechard et ai. (48) has reported that bacteriocin activity of purified mesentericin Y105 dropped 80% after being stored at 4°C for 8 h. Hastings et ai (49) also showed that leucocin A-UAL 187 antimicrobial activity was lost rapidly after purification. Similar results are reported for carnobacteriocins A (50), while Davey and Richardson (51 ) demonstrated that Streptococcus cremoris 346 bacteriocin was completely inactive after storage at 4°C for 1 week. Similarly Pasteris et ai (52) and Banerjee et ai, (53) reported a decrease in bacteriocin stability during storage at 4 C.

[489] Example 7: A single protein band is responsible for B21* bacteriocin.

[490] The molecular weight and purity of bacteriocin preparations was determined using tris-fricine-sodium doedecyl sulfate-polyacrylamide gel electrophoresis (Tris-Tricine-SDS-PAGE, 16.5% resolving gel) as previously described (39). After electrophoresis at 60 V for 30 min and at 100 V for 1 .5 h, half of the gel was stained using a coomassie based staining solution (Expedeon, UK), while the other half was fixed (20% isopropanol, 10% acetic acid in Milli Q® water) and used to examine the antimicrobial activity of the protein as described previously (40). L. plantarum A6 was used an indicator strain. Precision Plus Protein™ Dual Xtra Standards (Bio-Rad, USA) was used as molecular weight marker. The average molecular mass of FPLC-purified bacteriocin was investigated by matrix-assisted laser desorption/ionization time-of-fiight mass spectrometry (MALDI-TOF MS) (41 ) using an Autofiex Speed MALDI-TOF instrument (Bruker, Germany) containing a 355-nm Smartbeam Π laser for desorption and ionization. The acceleration and reflector voltages were 20 and 23.4 kV in pulsed ion extraction mode. A molecular mass gate of 450 Da improved the measurement by filtering out most matrix ions. The purity of the 10-fold diluted purified bacteriocin protein (in 0.1 % TFA in water) was evaluated using an Aligent 1 100 analytical reverse-phase high-performance liquid chromatography (RP-HPLC) (42) fitted with a ZORBAX Eclipse XDB analytical Ci8 column (4,6x150 mm, 5 μηη particle size, Agilent Technologies, USA). The bacteriocin protein was eiuted using a 40-min linear water-ACN gradient (increasing the gradient to 95%),

[491 ] Figure 3 shows MALD!-TOF MS spectrum of purified B21 ^* bacteriocin, showing the singly and doubly charged species. The average molecular mass of 5668 Da is observed for B21 ^* bacteriocin.

[492] A size estimation of B21 ^* FPLC-purified bacteriocin protein was made using a Tris-Tricine-SDS-PAGE gel. The gel was cut into two halves and the first half was used to estimate the bacteriocin peptide molecular mass after staining. The SDS-PAGE result showed a single protein band of approximately 5 kDa (Figure 2A), No other bands were observed on the gel, providing evidence of the bacteriocin protein purity. A large number of LAB bacteriocin proteins have been reported to be low molecular weight proteins of less than 10 kDa (10, 40, 54-57). The second half of the Tris-Tricine-SDS-PAGE gel was used to confirm that 5kDa bacteriocin protein gave an inhibitory zone around the protein band, confirming that the single protein band is responsible for bacteriocin activity (Figure 2C).

[493] Many classical bacteriocins have been analysed by RP-HPLC and are reported to be eiuted from a C ₁₈ RP-HPLC column using a water-ACN gradient from 0% to 60% (8, 29, 58, 59). The purified bacteriocin protein of strain B21 ^* was injected onto a ZORBAX Eclipse analytical Cis column (4.6x150 mm). Surprisingly, no protein was eiuted using an ACN-water gradient up to 60% (ACN), no peak was observed at A220 and Α ₂ βο· The ACN concentration gradient was increased to 95% and the bacteriocin protein was successfully eiuted from the column using a 40-min linear water-ACN gradient. The only major peak obtained at 37.5 min, corresponded to the B21 ^* purified bacteriocin protein (Figure 4). This experiment was repeated four times and the peaks from all runs overlapped.

[494] Failure to elute the bacteriocin protein from a C ₁₈ column by 60% water- ACN strongly suggested that the B21 ^* bacteriocin protein molecule was more hydrophobic than the common classical bacteriocins. The combined electrophoresis, RP-HPLC and MLADI-TOF MS analysis confirm a very high level of purity and homogeneity of the final B21 ^* purified bacteriocin sample.

[495] Example 8: UV/Vis absorbance spectrum and secondary structure content indicate B21* bacteriocin adopts an a-helix structure.

[496] The optical absorbance spectrum of purified B21 ^* bacteriocin protein was scanned using a SHIMADZU 1800 UV/Vis spectrophotometer.

[497] In brief, the optical absorbance spectrum (from 500 to 190 nm) of diluted purified B21 ^* bacteriocin protein in 20 mM sodium phosphate buffer was recorded using a Shimadzu 1800 UV/Vis spectrophotometer (Shimadzu, China). The spectra were recorded versus the 20 mM sodium phosphate buffer (pH 6.0) blank. The protein concentration was correlated with the A ₂ BO values recorded. The bacteriocin polypeptide sequence was used in an online tool to determine a theoretical molar extinction coefficient for the protein (43). The absorbance at 280 nm and the molar extinction coefficient value were used to calculate the protein concentration (44). The circular dichroism spectra of purified bacteriocin protein were recorded from 280 to 200 nm using a Jasco J-8 5 CD Spectropolarimeter (Jasco International Co., Ltd., Japan). The purified bacteriocin was diluted 6 fold in 20 m!Vl sodium phosphate buffer (pH 8.0) resulting in the final concentration of -25 g/mL in the solution. The spectral measurements were repeated three times at 25 °C and the average spectrum was reported. The instrument parameters were as follows; scanning speed 50 nm/min, data pitch 0.1 nm, response time one second and bandwith 1 .00 nm. The spectra were recorded in standard CD mdeg units and were converted to standard unit of molar ellipticity using the theoretical protein concentration. The CD data was used to predict the overall secondary structure of the bacteriocin by the K2D3 online tool (45).

[498] Figure 5 shows UV spectrum of the bacteriocin protein with a broad absorbance peak (0.176) at 280 nm and a pronounced shoulder closer to 290 nm. This peak resembled tryptophan. The broad absorbance near 280 nm and the shoulder at longer wavelength is consistent with a strong tryptophan content (60-62). A lower wavelength peak at 251 nm (0.132) is more likely to arise from phenylalanine residue(s). Based on Schmid (62), phenylalanine residues contribute fine structure to the spectrum between 250 and 260 nm. In the near-UV (240-300 nm) the molar absorbance of phenylalanine is much smaller than that of tyrosine and tryptophan, and the spectrum of a protein in this wavelength range is dominated by the tyrosine and tryptophan contributions. It is important to note that since the absorption of tryptophan is about four times that of tyrosine, at their respective peaks (~ 275 nm and 280 nm respectively), the precision in the determination of tryptophan will naturally exceed that of tyrosine (60). The absorbance observed at 220 nm is due to the bacteriocin peptides bonds. The bacteriocin polypeptide sequence was used in an online tool to determine a theoretical molar extinction coefficient for the protein (43). The value calculated for the bacteriocin was 1 1 ,000 M ^" cm ^"1 which gave a protein concentration for the spectrum in Figure 5 of 16 μΜ, corresponding to 0.091 mg/mL The experimentally determined value using the BCA protein assay of 0.072 mg/mL was in good agreement.

[499] The CD spectrum of B21 ^* bacteriocin is presented in Figure 6. The theoretical protein concentration was used to convert the spectral data to standard units of molar eilipticity. A strong Θ positive maximum around 190 nm and negative Θ maxima around 208 and 222 nm shows that the bacteriocin peptide consisted mainly of a-helical structure (63). The CD data was used to predict the overall secondary structure of the bacteriocin by the K2D3 online tool (45). The bacteriocin was approximately 69% a-heiix structure, no β-sheet and 31 % extended confirmation. Again features that distinguish B21 ^* bacteriocin from other classical bacteriocins, that tend to be more unstructured in aqueous solution. Papathanasopoulos et ai. (64) showed that three bacteriocins (leucocin A, B and C) produced by Leuconostoc rnesenteroides TA33a seem to have a less ordered structure in water as compare to a membrane-mimicking environment. They reported all three bacteriocins to have CD spectra more typical of β-sheet than a-helix. In another study Gaussier (65) demonstrated that classical bacteriocin pediocin PA-1 (from class I la) is mainly composed of flexible and disordered polypeptide chains. Similarly, Netz et al. (66) showed that the aureocin A53 secondary structure is mixed consists of a-helix and β-sheet, the latter stabilised by disuiphide bridges. Like the B21 ^* bacteriocin studied here, gassericin A, reutericin 6 and carnocyclin have been shown to adopt a defined a-heiix structure (63, 67). These bacteriocins are notable in having a cyclic molecular structure and adopt a very defined rigid structure. [500] As will be described below, the B21 * bacteriocin studied here appeared to have stronger similarity to cyclic bacteriocin class.

[501 ] Example 9: De novo sequencing and analysis shows B21* bacteriocin is cyclic,

[502] Initial attempts to determine the N-terminal amino acid sequence of the purified B21 * bacteriocin protein by Edman-degradation failed, suggesting that the peptide may be N-terminally blocked, A de novo sequencing approach was then taken to identify the peptide sequence of the purified bacteriocin peptide using an ESi-LC-MS/MS technique.

[503] In brief, B21 * purified bacteriocin was analysed using an LTQ™ Orbitrap Elite ETD (Thermo Scientific, USA) coupled to an UltiMate 3000 RSLCnano System (Dionex, Thermo Scientific, USA). The nanoLC system was equipped with an Acclaim™ Pepmap™ 100 Cis nano-trap column (1 ^χ 2 cm, 5 m particle size, Thermo Scientific, USA) and an Acclaim™ Pepmap™ 100 Cia analytical column (length 15 cm, 5 μπΊ particle size, Thermo Scientific, USA). 2 μ!_ of the bacteriocin sample was loaded onto the trap column at 3% (v/v) ACIM (Merck, USA) containing 0.1 % (v/v) formic acid (Sigma-Aldrich, USA) for 5 min before the enrichment column is switched in-line with the analytical column. An initial run on the LTQ™ Orbitrap Elite ETD mass spectrometer (Thermo Scientific, USA) was set to operate in data-dependent mode, where subsequent MS/MS spectra were acquired for the top five peaks by collision induced dissociation (C!D) activation, followed by higher-energy collision dissociation (HCD). This experiment identified a strong precursor ion at m/z 1417.0630 (monoisotopic peak) corresponding to an intact mass of 5664.252. This was consistent with the average molecular mass of the bacteriocin protein obtained from MALDI-TOF MS and also the band size observed on SDS-PAGE gel.

[504] Examination of the ion fragmentation spectra for this precursor indicated that both CID and HCD methods provided some practical fragment information, however ETD failed to provide any useful data (data not shown). To improve the MS/MS information a targeted run was setup in which the m/z 1417 precursor (with an isolation width of 4 Da) was subject to both CID and HCD at three different energy levels (CID: 26, 32, 36, and HCD: 24, 26, 30) and activation Q of 0.25 (CID) or activation time of 0.1 ms (HCD). The spectrum produced from HCD at 26EV was interrogated through the MASCOT package but failed to identify any candidate sequence in the existing databases. Given the spectrum appeared to contain significant sequence information, sequence tags were extracted manually (PGWAVAAAGALG, Figure 8) and BLAST was used to search a six frame translation of the L. piantarum B21 ^* lllumina genome (Example 5).

[505] These amino acid peptide sequences showed 100% similarity to a part of the translated amino acid sequence of one of the three identified plasm ids (scaffold 5) of the B21 ^* genome data (unpublished). A putative bacteriocin mature protein sequence was obtained from the predicted ORF (Figure 7). The precursor mass of the peptide was found to be 18Da less than the apparent mass of the predicted mature amino acid sequence, deduced from the DMA sequence. The apparent N- terminal sequence of the peptide, based on mass spectrum data started with the sequence PGWAVAAAGALG (Figure 8). From the same fragment spectrum the late b ions produced a sequence of AAVILGV (Figure 9) indicating that this sequence was close to the C-Terminus of the peptide. When compared to the predicted amino acid from the genome data, this made no sense as the sequence AAVILGV occurs just before the PGWAVAAAGALG sequence in the predicted ORF and it is not separated by more than 4000Da of mass (Figure 8). The only logical explanation of this data is that the bacteriocin peptide is in fact cyclic. The formation of a peptide bond between the N and C-Terminus and resulting loss of water would also explain the mass discrepancy of 18 Da between the gene sequence (5682 Da) and the experimental mass of 5864 Da.

[506] After the full putative peptide sequence was deduced from the genome data, the remainder of the peptide sequence could be traced and confirmed in the MS/MS data (Figure 8 and 9). The B21 ^* cyclic bacteriocin peptide was named Plantacyciin B21AG.

[507] The proteomic analysis of Plantacyciin B21AG presented in this study were consistent with a cyclic bacteriocin, Plantacyciin B21 AG had been shown to demonstrate good stability both in culture and during storage at 4 °C (up to 8 weeks).

Cyclic bacteriocins have been reported to be generally more stable than classical bacteriocins (68, 69). Maqueda et a/. (2004) (70) demonstrated that cyclic bacteriocin enterocin AS-48 was a very stable peptide. They suggested that substantial part of this increased stability was due to the entropic constraints induced by the cyclic nature of the polypeptide chain. Piantacyclin B21AG showed some level of resistance to proteolytic enzymes; cyclic bacteriocins are less susceptible to digestion by endoproteinases and this probably increases their spectrum of activity. Borrero ef ai. (201 1 ) (71 ) suggested that the resistance of garvicin ML to proteolytic enzymes was not due to the absence of digestion sites but to the inaccessibility of the recognition sites due to a tightly folded three-dimensional structure. In a similar manner, cactocyciicin Q was shown to have a high resistance to proteases (72). The Piantacyclin B21AG peptide sequence could not be sequenced by N-terminal sequencing in agreement with Borrero et ai. (201 1 ) (71 ) who reported that circular bacteriocin garvicin ML was resistant to IM-terminai Edman degradation sequencing. Similar results have been reported for other cyclic bacteriocins, enterocin 62-6 (68), lactocyciicin Q (72) and butyrivibriocin AR10 (73). The B21 ^* bacteriocin peptide was shown to behave more hydrophobicaiiy on RP-HPLC column requiring a high concentration (~80%) of ACN for eiution. This behaviour has been observed for other cyclic bacteriocins. Maqueda et ai. (2004) (70) reported that high concentrations of solvent were necessary to purify enterocin AS-48 on RP-HPLC which allowed efficient separation of the circular bacteriocin from hydrophilic compounds. Dezwaan ef ai. (2007) (68) used RP-HPLC on Cis column to purify cyclic bacteriocin enterocin 62-6 but was unable to recover the bacteriocin from the column due to bacteriocins high hydrophobicity. The CD spectroscopy analysis of Piantacyclin B21AG revealed that the secondary structure of the bacteriocin appeared to be substantially a-helical, again consistent with secondary structure measurements reported for other cyclic bacteriocins. Maqueda et ai, (2004) (70) demonstrated the three-dimensional structure of enterocin AS-48 in solution by high resolution nuclear magnetic resonance (NMR), consisting of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Similar results were reported for lactocyciicin Q (72), gassericin A, reutericin 6 (63) and carnocyclin A (67).

[508] Example 10: Sequence analysis of Piantacyclin B21AG

[509] The DMA sequence of Piantacyclin B21AG putative pre-peptide (273 bp) and the mature bacteriocin (174 bp) were analysed by nucleotide BLAST (BLASTn) search in the IMCB! database (74) and no significant similarities were found at the DNA level. This result showed that the Plantacyclin B21AG gene was unique. The Piantacyciin B21AG putative protein sequence (91 amino acids) was analysed by protein BLAST (BLASTp) search (74) and compared to all available sequences in the NCBI database. The highest hit from the BLASTp search showed only 58% identity (100% query cover) to a cyclic bacteriocin produced by Lactobacillus pentosus (accession number WP_003637681 .1 ). The second highest similarly showed 53% identity (83% query cover) to the gassericin A and acidocin B, cyciic bacteriocins produced by Lactobacillus gasseri and Lactobacillus acidophilus respectively (accession numbers WP_012821083.1 and CAA84399.1 ). The BLASTp analysis of the mature bacteriocin peptide (58 amino acids) showed 67% identity (100% query cover) to the cyclic bacteriocin from Lactobacillus pentosus (accession number WP_003637681 .1 ) and 65% identity (74% query cover) to gassericin A (accession numbers WP_012621083.1 ). These results demonstrate that Plantacyclin B21AG is a new member of cyciic bacteriocin class produced by L plantarum.

[510] The MS/MS data was used for de novo assembly of two short sequences from Piantacyciin B21AG (Figure 7-9). The sequence of the putative full and mature bacteriocin peptide was then deduced from the predicted ORF from the L plantarum B21 ^* genome data (Figure 9D). The putative bacteriocin peptide has 91 amino acids (pre-peptide) consisting of a 33-amino-acid leader peptide and a 58-amino-acid propeptide (Figure 10c). The putative cleavage site for removing the leader peptide is located between asparagine and isoleucine, and has homology to the leader peptide cleavage site in gassericin A and acidocin B (75, 76). Cleavage at this site (Figure 10c) would remove a 33-amino-acid leader sequence and produce a mature bacteriocin containing 58 amino acids. The 58-amino-acid peptide is predicted to undergo a post-translationai modification that results in the linking of the !M-termina! asparagine to the C-terminal isoleucine, with the elimination of a water molecule, resulting in the active and mature B21AG molecule that is actually secreted. The leader peptides in cyclic bacteriocins vary in length, from 3 to more than 30 amino acids (67, 71 , 77). The amino acid composition of the mature cyclic bacteriocin consists of a very high proportion (59%) of hydrophobic amino acid residues (Ala, Vai, Leu, lie, Phe, Trp and Pro) and also uncharged hydrophilic amino acid residues (32%) (Giy, Ser, Thr and Gin). There is also high ratio of basic (Lys, Arg and His) relative to acidic amino acids (Asp) suggesting a strong basic protein character. It has been suggested that the basic residues such as Lys present a highly localised positive charge on the surface of the cyclic bacteriocins structure which is responsible for attracting the peptide to the surface of the negatively charged membrane (71 , 78). Unlike some other cyclic bacteriocins (47, 79, 80) Plantacyclin B21AG does not contain any Cys residues, so there are no di~sulphide bridges. The bacteriocin peptide also contains three aromatic residues (two Trp and one Phe). The characteristic influence of Trp is seen in the UV~Vis observations (Figure 5).

[51 1 ] Example 11 : Phylogenetic analysis of Plantacyclin B21AG protein and sequence alignment.

[512] The phylogenetic analysis of the full protein sequence of Plantacyclin B21AG (91 amino acids) was performed to investigate the homology of this bacteriocin in comparison to other cyclic bacteriocins from Gram-positive bacteria available in the NCBI database. A phylogenetic tree was constructed using the Phylogeny.fr online tool (81 ) (Figure 1 1 ). Plantacyclin B21AG and pentocin KCA1 (82) cluster together as expected from with the BLASTp analysis. The other two bacteriocins in the same clade as Plantacyclin B21AG were gassericin A (75) and acidocin B (76, 83), both bacteriocins from Class IV. The phylogeny shows that Plantacyclin B21AG, pentocin KCA1 , gassericin A and acidocin B are closely related. Ail four cyclic bacteriocins have 33 amino acids leader peptides and 58 amino acids mature bacteriocin peptides of the active protein. The conserved regions with a maximum BLOSUM62 score are highlighted in light gre/blue (Figure 1 1 ). The butyrivibriocin AR10 (84) and lactocyclicin Q (72) bacteriocins were also clustered in clade I, but Plantacyclin B21AG did not share high similarity with these two bacteriocins. The other five cyclic bacteriocins (carnocyclicin A, uberolysin, garvicin ML, AS-48 and circularin A) were clustered in a separate clade (clade Π) and the peptide sequence, number of amino acids and the peptide cleavage site is different for this group of cyclic bacteriocins in comparison to Plantacyclin B21 AG.

[513] Example 12: Genetic analysis of Plantacyclin B21AG gene cluster.

[514] The b21 ag gene (ORF-A) (Figure 12) responsible for Plantacyclin B21AG production was located on the bacterial chromosome (B21 ^* genome data, scaffold 5_2899_2624 reverse strand). The b21 ag structural gene encodes a 91 ~amino~acid pre-peptide consisting of a 33-amino-acid leader peptide and a 58-aniino-acid bacteriocin peptide. The DNA sequence of Plantacyclin B21AG was searched against L. p!antarum B21 ^* whole genome using BLAST and no other similar genes were observed in the other parts of the genome. The DNA sequence of Plantacyclin B21AG putative pre-peptide (273 bp) and the mature bacteriocin (174 bp) were analysed by nucleotide BLAST (BLASTn) search in the NCBI database (Aitschul et ai. 1990) and no significant similarities were found at the DNA level. This result showed that the Plantacyclin B21AG gene was unique. The Plantacyclin B21AG putative protein sequence (ORF-A, 91 amino acids) was analysed by protein BLAST (BLASTp) (Aitschul et ai 1990) search and compared to all available sequences in the NCBI database. The highest hit from the BLASTp search showed only 58% identity (100% query cover) to a cyclic bacteriocin produced by Lactobacillus pentosus (accession number WP_003637681 .1 ). The second highest similarly showed 53% identity (83% query cover) to the gassericin A and acidocin B, cyclic bacteriocins produced by Lactobacillus gasseri and Lactobacillus acidophilus respectively (accession numbers WP_012621083.1 and CAA84399.1 ). The BLASTp analysis of the mature bacteriocin peptide (58 amino acids) showed 67% identity (100% query cover) to the cyclic bacteriocin from Lactobacillus pentosus (accession number WP_003637681 .1 ) and 65% identity (74% query cover) to gassericin A (accession numbers WP__012621083,1 ). These results demonstrate that Plantacyclin B21AG is a new member of cyclic bacteriocin class produced by L. piantarum. Interestingly, this also shows that the mature sequence is more conserved than the leader sequences.

[515] Five additional ORFs (Figure 12, ORF-B to ORF-F) were predicted from the new L. piantarum B21 ^* genome data (scaffold 5) to form a potential transcriptional unit which contains the Plantacyclin B21AG gene (Figure 12). The putative proteins encoded by each predicted ORF were analysed and annotated using NCBI BLAST tools in order to identify the functional gene cluster involved in the production of Plantacyclin B21AG.

[516] The Plantacyclin B21AG gene operon is encoded on a previously unknown and uncharacterized piasmid, herein referred to as Piasmid 1 . Figure 14 shows the nucleotide sequence of Piasmid 1 , and Figure 15 shows the predicted ORFs and their annotations.

[517] Without wishing to be bound by theory, the present inventors propose Piasmid 1 is transmissible to other bacteria strains, including probiotic strains, thereby conferring the activity of Plantacyclin B21AG to the other strains, without the use of genetic modification technology.

[518] Without wishing to be bound by theory, the present inventors propose the 20 kb piasmid of stain B20, B31 and B33 is also transmissible to other bacteria strains, including probiotic strains, thereby conferring the activity of Plantacyclin B21 AG to other strains, without the use of genetic modification technology.

[519] The BLASTn analysis of ORF-B sequence (location: scaffold5_2532_2059 reverse strand) (Figure 10) did not show any significant homology to NCBI database sequences at DNA level. The BLASTp analysis of the ORF-B putative protein sequence (157 amino acids) (Figure 12) detected a putative conserved domain from the DUF95 superfamiiy. The members of this family have several predicted transmembrane regions (Marchler-Bauer ef a/. 2009; Marchier- Bauer & Bryant 2004; Marchler-Bauer ef a/. 201 1 ). The ORF-B putative protein sequence has 39% identity (query cover 75%) to the PenD gene from L. pentosus (accession number WP__003637682), 33% identity (query cover 94%) to the GaaD gene (a membrane protein involved in production of gassericin A, accession number WP_012621084.1 ) (Ito ef a/. 2009) and 30% identity (query cover 88%) to the BviE protein sequence from Butyrivibrio fibrisolvens (accession number AAC69561 .2), Kaimokoff ef a/. (2003) suggested that bviE gene is involved in self immunity to the cyclic bacteriocin, butyrivibriocin. Based on the report of Nes ef a/. (1996), the dedicated immunity gene is always located next to and downstream from bacteriocin structural gene. This analysis strongly suggests that the ORF-B gene could be involved in the production of or immunity to the Plantacyclin B21 AG.

[520] The BLASTn analysis of ORF-C sequence (location: scaffold5_2056_1892 reverse strand) (Figure 12) did not find any significant homology from NCBI database at the DNA level. Similarly BLASTp analysis of ORF- C putative protein sequence (54 amino acids) (Figure 12) did not detect any putative conserved domains, however it did show 38% identity (query cover 83%) to the Gaa! protein from L. gasseri which is again involved in self immunity to its cyclic bacteriocin (accession number WP__012621085.1 ) (Ito et al. 2009). These results suggest that the ORF-C gene is involved in immunity of L. plantarum B21 ^* to Plantacyclin B21AG.

[521 ] The BLASTn analysis of the ORF-D sequence (location: scaffoid5__1851__1 189 reverse strand) (Figure 12) did not find any significant homology from NCBI database at DNA level. The ORF-D putative protein sequence (220 amino acids) (Figure 12) BLASTp analysis showed specific hits to the conserved domain of ABC_DR_subfamily_A (ABC-transporter ATP-binding proteins). This family of ATP-binding proteins belongs to a multi- subunit transporter involved in drug resistance, lipid transport, and bacteriocin immunity (such as lantibiotics) (Marchler-Bauer et al. 2009; Marchler-Bauer & Bryant 2004; Marcbler- Bauer et al. 201 1 ). The BLASTp analysis of this ORF also detected hits to ABC- ATPase superfami!y (Marchler-Bauer et al. 2009; Marchler-Bauer & Bryant 2004; Marchler-Bauer et al. 201 1 ) which are a large family of proteins involved in the transport of a wide variety of different compounds such as sugars, ions and peptides. If has been reported that dedicated transmembrane transiocators belonging to the ATP-binding cassette (ABC) transporter superfamily are involved in the cleavage of the leader-peptide from Class II bacteriocins and in the transportation of the mature bacteriocin molecule across the cytoplasmic membrane (Nes et al. 1996). The ORF- D (Figure 12) showed 49% identity (query cover 98%) to the PenT protein from L. pentosus (accession number WP__003637684.1 ) and 46% identity (query cover 96%) to GaaT protein from L. gasseri which is involved in the transportation of gassericin A (accession number WP_012621086.1 ) corresponding to amino acid level. These results suggested that ORF-D gene should be involved in the cleavage of the leader peptide and transport of Plantacyclin B21AG bacteriocin protein.

[522] The BLASTn analysis of ORF-E sequence (location: scaffold5_1 186_542 reverse strand) (Figure 12) did not find any significant homology from NCB! database at DNA level. The BLASTp analysis of ORF-E putative protein sequence (214 amino acids) (Figure 12) detected specific hits to the ABC_membrane_5 putative conserved domain from the database which related to the ABC__2 membrane transporter family. These domains are from superfamily of Acyl_transfer_3 including a range of acyltransferase enzymes (Marchler-Bauer et al. 2009; Marchler-Bauer & Bryant 2004; Marchler-Bauer et al. 201 1 ). The ORF-E putative protein sequence BLASTp showed only 39% (query cover 96%) to PenE protein from L. pentosus (accession number WP_003637685.1 ) and 37% identity (query cover 96%) to membrane protein, GaaE from L. gasseri which is involved in the production and transportation of gassericin A (accession number WP_012621087.1 ). These analysis results suggest that ORF-E gene could be involved in transportation of the Plantacyclin B21AG.

[523] The BLASTn analysis of ORF-F sequence (location: scaffold5__539__18 reverse strand) (Figure 12) did not find any significant homology from NCBI database at DNA level. The BLASTp analysis of this ORF putative protein sequence (173 amino acids) (Figure 10) did not detect any putative conserved domains corresponding to the amino acid level. The putative protein sequence showed only 31 % (query cover 91 %) to PenB protein from L. pentosus (accession number WP_003637686.1 ) and 30% identity (93% query cover) to a membrane protein with 5 predicted transmembrane segments (TMS) from L. gasseri which is involved in production of the cyclic bacteriocin, gassericin A (accession number WP_012621081 .1 ). These results suggested that the ORF-F gene could be possibly involved in the production of Plantacyclin B21AG.

[524] The Orf-a1 and Orf-a2 genes are located next to and upstream of the Plantacyclin B21AG structural gene (locations: scaffold5_3 48_3441 and scaffold5_3512_4471 ) (Figure 12). BLASTn and BLASTp analysis of their sequences did not show any significant shared homology to any bacteriocin related genes from the NCB! database at the DNA or amino acid levels. The BLASTp analysis of the Orf-a2 putative protein sequence (319 amino acids) (Figure 612) revealed homology to the conserved domain of TpcC superfamily. This family of proteins is annotated as conjugative transposons TcpC (Marchler-Bauer et al. 2009; Marchler-Bauer & Bryant 2004; Marchler-Bauer et al. 201 1 ). The Orf-a2 BLASTp analysis showed high similarity (91 %) to a hypothetical transposon protein from Lactobacillus maii (accession number WP_003688364.1 ). [525] The possibility of further bacteriocin relating genes downstream from ORF-F cannot be excluded, data from Pentocin KCA1 (Anukam et al. 2013) suggest the likelihood of an ORF- G homologous to the Pen R gene from L. penfosus KCA1 (Figure 12).

[526] Example 13: Killing activity of the circular bacteriocin P!antacyclin B21AG can be transferred to a heterologous bacteria! strain,

[527] Preparation of test samples:

[528] Day-1

[529] Grow Lactobacillus plantarum WCFS1 transformed with the bacteriocin construct (WCFS1 + Bac) and the base plasmid pTRKH2 (WCFS1 + pTRKH2) in 15m L MRS broth supplemented with 15 g/mL of Erythromycin.

[530] Grow L. plantarum B21 and WCFS1 in 10 mL of MRS broth without antibiotic,

[531 ] Incubate WCFS1 and B21 at 37 °C and 30 °C, respectively for 16-18 hours.

[532] Day-2

[533] Spin overnight culture at 4000 x g for 20 min at 4 ^C C.

[534] Wash Amicon® Ultra-15 Centrifugal Filter Devices (3K)/spin column with 15 mL of sterile Milli-Q water. Perform centrifugation as step 1 . It will take approximately 40 min.

[535] After centrifugation, collect supernatant from B21 and WCFS1 and store at 4 °C until they are ready to use.

[536] Discard Milli-Q water from the spin column.

[537] Add 15 mL of cell free supernatant (CFS) of WCFS1 + pTRKH2 and WCFS1 + Bac onto two separate spin columns. [538] Spin the columns at 4000 x g and 4 °C until the CFS are concentrated to 500 μΙ_. It will take approximately 1 ,5 hr.

[539] Discard filtrate from the spin column.

[540] Wash the concentrated CFS with sterile 1 X PBS.

[541 ] Spin the columns at 4000 x g and 4 °C until the CFS are concentrated to 500 μ!_. It will take approximately 1 hr.

[542] Repeat previous as necessary.

[543] Collect concentrates from the filter devices and store in 1.5 mL microcentrifuge tubes.

[544] Original volume of CFS: 15 mL

30 x concentrated

[545] Concentrated volume of CFS: 500 ί

[546] Perform a 10-, 20- and 30-fold dilution from the concentrated CFS. [547]

[548] Day-1

[549] Grow L. plantarum A6, L. plantarum ATCC 8014 and Lactococcus lactis 345-18 in 10 mL MRS broth without antibiotic.

[550] Incubate the culture at 30 °C for 16-18 hrs.

[551 ] Day-2

[552] Dilute indicator strains at 100-fold. e.g. 100 μί of overnight culture + 9900 pL of MRS broth without antibiotic.

[553] Incubate the diluted culture at 30 °C for 1 hr.

[554] Flood MRS plates (without antibiotic) with the indicator strains, e.g.

Pipette 2 mL of diluted culture onto MRS plate and move the plate around until the surface of the agar is fully covered by the indicator strain. Remove excess culture by pipetting.

[555] Dry MRS plates at 30 °C. It will take approximately 45 min. [556] Make wells on MRS agar. [557] Killing assay

[558] Pipette 100 μί of both concentrated and diluted CFS into each well. The volume of CFS may be reduced depending on the size of the wells.

[559] Incubate the plates at 30 °C for 16-18 hrs.

[560] Figure 16 shows killing of three indicator strains (ATCC 8014, A6 and L. lactis) by the probiotic LAB strain WCFS1 when transformed with a shuttle vector comprising the Piantacyclin B21AG operon. Killing is shown by a cleared zone in the well diffusion experiment shown. Four different strains are evaluated; B21 ^* as positive control; untransformed LAB WCSFS1 which is an authentic probiotic LAB; LAB WCSFS1 transformed with a shuttle vector pTRKH12 with an erythromycin resistance marker; and LAB WCSFS1 transformed with a shuttle vector containing the full set on piantacyclin B21AG genes previously described (with additional ORF; B33 ORF-21 ), designated "WCFS1 + Bac".

[561 ] WCFS1 + pTRKH2, and WCFS + Bac, were grown with antibiotic to maintain selection pressure for retention of the shuttle vector. B21 ^* has good killing activity on two out of three indicator strains in wells 1 and 2, and does not require concentration of the protein fractions on a 3000 Mrw spin column for detection.

[562] Owing to the presence of antibiotic selection, the supernatants from

WCFS1 transformed with the full set of Piantacyclin B21AG genes were concentrated on spin columns and extensively washed to remove the antibiotic.

There is strong killing in well 7 on ail three indicator strains and even when diluted

10- fold (well 8). No killing is seen in the absence of the Piantacyclin B21AG operon, even though the ceils were also grown with antibiotic selection and washed in the same way (wells 3 to 6). Some staining of the agar rather than killing is seen, due to coloured media products in the concentrate (e.g. compare 3 and 4 with 7 and 8). [563] This data demonstrates that Plantacyciin B21AG can be recombinant^ expressed in heterologous species of bacteria, and confer killing activity on the heterologous bacteria. This data also demonstrates that Plantacyciin B21AG can be recombinantly expressed in probiofic bacteria, such as WCFS1 .

[564] Example 14: Characterisation of LAB strain B33 plasmid sequence and the Plantacyciin B21AG operon of B33

[565] The 20 kb plasmid of strain B33 was sequenced, annotated and the predicted ORFs searched against L p!antarum B21 ^* whole genome using BLAST.

[566] The BLASTp analysis is presented in Figure 17, which shows the predicted ORFs and their annotations.

[567] The predicted Plantacyciin B21AG of B33 showed 88% identity to Plantacyciin B21AG of B21 ^*

[568] The BLASTp analysis of ORF-B sequence shows the predicted ORF-B of B33 showed 94% identity to ORF-B of B21 ^*

[569] The BLASTp analysis of ORF-C sequence shows the predicted ORF-C of B33 showed 89% identity to ORF-C of B21 ^*

[570] The BLASTp analysis of ORF-C sequence shows the predicted ORF-C of B33 showed 89% identity to ORF-C of B21 ^*

[571 ] The BLASTp analysis of ORF-D sequence shows the predicted ORF-D of B33 showed 95% identity to ORF-D of B21 ^*

[572] The BLASTp analysis of ORF-E sequence shows the predicted ORF-E of B33 showed 94% identity to ORF-E of B21 ^* .

[573] The BLASTp analysis of ORF-F sequence shows the predicted ORF-F of B33 showed 89% identity to ORF-F of B21 ^* .

[574] The BLASTp analysis of ORF-G sequence shows the predicted ORF-G of B33 showed 76% identity to ORF-G of B21 ^* . [575] The BLASTp analysis of ORF-21 sequence shows the predicted ORF-21 of B33 ("B33-ORF21 ") showed 95% identity to ORF-21 of B21 ^* .

[5/6] The predicted P!antacyclin B21AG operon orfs of B20 and B31 are identical to those of B33 (data not shown).

[577] Example 15: eying activity of the circular bacteriocsn Plantacyclsn B21AG can be transferred to a heterologous bacterial strain.

[578] Samples and killing assays were performed as per Example 13.

[579] For MASLDI MS, supernatants were concentrated on a spin column and washed to remove antibiotic, and then butanoi extracted according to Example 6, and then examined using MALDI MS using the methods described herein.

[580] Figure 19A shows killing of A8 by the probiotic LAB strain WCFS1 when transformed with a shuttle vector comprising the Plantacyclin B21AG operon. Killing is shown by a cleared zone in the well diffusion experiment shown. Four different strains are evaluated; B21 ^* as positive control; untransformed LAB WCSFS1 which is an authentic probiotic LAB; LAB WCSFS1 transformed with a shuttle vector pTRKH2 with an erythromycin resistance marker; and LAB WCSFS1 transformed with a shuttle vector containing the full set of plantacyclin B21AG genes previously described (with additional ORF; B33 ORF-21 ), designated "WCFS1 + Bac". There is strong killing in well 7. No killing is seen in the absence of the Plantacyclin B21AG operon, even though the ceils were also grown with antibiotic selection and washed in the same way (wells 3 to 6). Brown staining around well 3 results from concentration of the WCFS1 + pTRKH2 cell free supernatant; some staining of the agar rather than killing is seen, due to coloured media products in the concentrate (e.g. compare 3 and 4 with 7 and 8). This data demonstrates that Plantacyclin B21AG can be recombinantiy expressed in heterologous species of bacteria, and confer killing activity on the heterologous bacteria. This data also demonstrates that Plantacyclin B21AG can be recombinantiy expressed in probiotic bacteria.

[581 ] This data also demonstrates the one or more Plantacyclin B21AG- cyclisation polypeptides encoded by the plantacyclin B21AG operon can be used to cyclise a target polypeptide by expressing the plantacyclin B21AG operon in a host cell.

[582] This data also demonstrates a target polypeptide can be cyclised by contacting the target polypeptide with one or more Plantacyclin B21AG-cyciisation polypeptides encoded by the plantacyclin B21AG operon.

[583] Figure 19B shows MALDI MS of culture supernatants shows the killing activity in Weil "1" (B21 ) and Well "7" (WCFS1 expressing Plantacyclin B21AG) of Figure 19A is associated with circular Plantacyclin B21AG. This data demonstrates that Plantacyclin B21AG can be recombinant!y expressed in heterologous species of bacteria, and confer killing activity on the heterologous bacteria. This data also demonstrates a target polypeptide, Plantacyclin B21AG, can be cyclised by contacting the target polypeptide with one or more Plantacyclin B21AG-cyclisation polypeptides encoded by the plantacyclin B21AG operon. MALDI MS of culture supernatant WCFS1 with shuttle vector pTRKH2 (Well "3" of Figure 19A) demonstrates this strain not express plantacyclin B21AG.

[584] The presence of the cyclic target polypeptide, Plantacyclin B21AG, in the culture supernatant of the heterologous bacteria comprising the plantacyclin B21AG operon the plantacyclin B21AG operon is able to cyclise the immature protein and confer the ability for concomitant secretion by a nonstandard export route.