Cloned gene encoding for bacteriocin from pediococcus acidilactici

Abstract: Isolation and identification of a gene encoding for a bacteriocin precursor in Pediococcus acidilactici , cloning of the gene in a vector plasmid and transformation to bacteria is described. The bacteriocin is particularly useful for inhibiting Listeria in food products.

Description: The present invention relates to a sequenced gene encoding for a bacteriocin in Pediococcus acidilactici and in particular to a gene that is essential for the production of the functional bacteriocin, called hereafter helper protein, and to the cloned gene in a vector which is transformed into a bacterium. In particular, the present invention relates to a sequenced gene encoding for a bacteriocin derived from a plasmid in Pediococcus acidilactici . The pediococci are a diverse group of Gram-positive homofermentative lactic acid bacteria often found as saphrophytes on vegetable material (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 53 :2534-2538 (1987); and Mundt, J. O., W. G. Beattie, and F. R. Wieland, J. Bacteriol. 98 :938-942 (1969)). Commercially, pediococci are used in the fermentation of vegetables (Pederson, Bacteriol. Rev. 13 :225-232 (1949) and meats (Smith, J. L., and S. A. Palumbo, J. Food Prot. 46 :997-1006 (1983)). Some strains of P . pentosaceus , P . cerevisiae and P . acidilactici have been found to contain resident plasmids although the roles of most of these remain unknown (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 46 :81-89 (1983); Graham, D. C., and L. L. McKay, Appl. Environ. Microbiol. 50 :532-534 (1985); and Raccach, M., CRC Crit. Rev. Microbiol. 14 :291-309 (1987)). The association of raffinose fermentation and plasmid DNA has been reported (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 51 :105-109 (1986)), as has been the ability of P . acidilactici to ferment sucrose (Gonzalez, C. F. and B. S. Kunka, Appl. Environ. Microbiol 53 :2534-2538 (1987)). Moreover, there have been several reports which associate the production of bacteriocins with host plasmid DNA (Daeschel, M. A., and T. R. Klaenhammer, Appl. Environ. Microbiol. 51 :1538-1541 (1985); Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 53 :2534-2538 (1987); Graham, D. C., and L. . McKay, Appl. Environ. Microbiol. 50 :532-534 (l985); and Bhunia et al, J. Applied Bact. 65 :261-268 (1988)). It was shown by Gonzalez, C. F. and B. S. Kunka (Appl. Environ. Microbiol. 53 :2534-2538 (1987)) that bacteriocin production was encoded by a 9.0 kbp plasmid pSRQ11 in P . acidilactici PAC1.0. Further work (Pucci, M. P., E. R. Vedamuthu, B. S. Kunka and P. A. Vandenbergh, Appl. Environ. Microbiol. 54 :2349-2353 (1988)) demonstrated that the bacteriocin of P . acidilactici PAC1.0 was active against a wide spectrum of gram positive lactic acid bacteria, and also against Listeria monocytogenes . This anti-listerial activity was observed in broth and on agar plates, as well as in some dairy products. Inhibition of L . monocytogenes by this bacteriocin, PA-1, has also been noted in fermented semi-dry sausage (Berry, E. D., M. B. Liewen, R. W. Mandigo and R. W. Huthine, J. Food Protection 53 , 194-197 (1990)) and fresh meat (Nielsen, J. W., J. S. Dickson and J. D. Crouse, Appl. Environ. Microbiol. 56 , 2142-2145 (1990)). The cloning of genes for the production of the bacteriocin has not been described and this would be useful for producing bacteriocin in significant quantities in genera unrelated to Pediococcus , or enhancing production in the pediococci. Cloned Gram-positive genes for different unrelated proteins have been shown to express in Escherichia coli (Gilmore, M. S., Curr. Top. Microbiol. Immunol. 118 :219-234 (1985); Rogeson, J. P., R. G. Barletta, and R. Curtiss III, J. Bacteriol. 153 :211-221 (1983); and Smorawinska, M., J. C. Hsu, J. B. Hansen, E. K. Jagusztyn-Krynicka, Y. H. Abiko, and R. Curtiss III, J. Bacteriol. 153 :1095-1097 (1983)). Earlier European patent application 0 406 545 discloses a gene segment of isolated and purified DNA from a Pediococcus which is preferably Pedicoccus acidilactici NRRL-B-18050 (PAC 1.0), encoding for a polypeptide which is a bacteriocin having a molecular mass of between about 19,000 to 20,000 daltons by SDS-PAGE analysis. It is therefore an object of the present invention to provide the sequenced gene for the bacteriocin and its essential helper protein(s), which are used as vectors that can be transferred to other microorganisms that contain the genetic information of these genes in such a way that the functional bacteriocin is produced by these new hosts. Such microorganisms are particularly in the genera Lactococcus, Lactobacillus, Leuconostoc, Streptococcus , Pediococcus , Escherichia , Bacillus and yeasts. These and other objects will become increasingly apparent by reference to the following description and the drawings. Figure 1 shows a restriction endonuclease site map of pSRQ11. P. acidilactici PAC1.0 plasmid pSRQ11 is 9.0 kbp and contains the gene for PA-1 bacteriocin. Figures 2A and 2B show restriction endonuclease site maps of pSRQ11.1 and pSRQ11.2, respectively. Both plasmids are 14.8 kbp and contain erythromycin resistance (ery) genes at the locations indicated. The E . coli origin of replication (ori) and the remaining part of the chloramphenicol resistance (cml) gene are shown. Numbered triangles (Δ) indicate areas of each plasmid which had been subsequently deleted. Figure 3A shows a restriction endonuclease site map of pSRQ220. Plasmid pSRQ220 is 9.3 kbp and is a chimera of Escherichia coli plasmid pBR322 and PAC1.0 plasmid pSRQll digested with Eco RI and Sal I and ligated together. The Escherichia coli origin of replication (ori) and the ampicillin resistance (amp) gene are indicated. The 5.6 kbp Eco RI- Sal I fragment is indicated by the open box. Figure 3B shows a physical map of the 5.6 kbp Eco RI- Sal I fragment from pSRQ220. The horizontal arrows denote open reading frames discussed hereinafter (ORF 1, ORF 2, and ORF 3). The horizontal lines, indicated by numbered triangles (Δ1, Δ2 and Δ3), represent three deletions present in plasmids pUR5204 (Δ1), pSRQ220.2 (Δ2), and pSRQ11.13 (Δ3), respectively. Figure 4 shows the nucleotide sequence of the 5.6 kbp Eco RI- Sal I insert from pSRQ220. The derived amino acid sequences of ORF1, ORF2, and ORF3 are also shown. The arrow indicates the start of the mature PA-1 bacteriocin. The TAG termination codons are denoted with an asterisk (*). Figure 5A shows a coomassie stained 5-22% acrylamide SDS-PAGE gel of purified PA-1. a = 66000, b = 45000, c = 36000, d = 29000, e = 24000, f = 20100, g = 14200, h = 6500 Daltons. Standards a through g are MW-SDS-70L (Sigma), standard h is aprotinin (Sigma). Figure 5B shows an unstained gel overlayed with a lawn of Pediococcus pentosaceus FBB63 indicator cells. Inhibition zone (large arrow) is apparent. 1 = 110000, 2 = 84000, 3 = 47000, 4 = 33000, 5 = 24000, 6 = 16000 Daltons. Prestained standards (Biorad) were used. The present invention relates to a nucleotide sequence corresponding to the nucleotide sequence which can be isolated from a strain belonging to the genus Pediococcus, consisting of genes coding for both a bacteriocin precursor which is ORF1 and a gene for at least one protein selected from the group consisting of ORF2, ORF3 and ORF2 and ORF3 as given in Figure 4 and modifications thereof essential for obtaining a functional active bacteriocin. Preferred embodiments thereof are described in claims 2-5. The invention further relates to a nucleotide sequence encoding only a bacteriocin precursor, said nucleic acid being selected from the group consisting of ORF1 in Figure 4, and modifications thereof that encode a protein still having the capability of being converted into an active bacteriocin. The invention further relates to a vector, that can be stably maintained in a host microorganism, which vector can be maintained as a plasmid or can integrate into a chromosome of the host microorganism, comprising a nucleotide sequence described hereinbefore. Preferred embodiments thereof are described in claims 8-10 and 13. The invention further relates to microorganisms transformed by introducing said vector, capable of producing bacteriocin. Preferably, the microorganism is selected from the group consisting of the genera Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia, and yeasts. The invention further relates to a nucleotide sequence encoding a protein essential for obtaining a functional active bacteriocin, said nucleotide sequence being ORF2 or ORF3, as given in Figure 4, or modifications thereof as described in claims 14 or 15, respectively. The DNA encoding the bacteriocin can be replicated by means of a polymerase chain reaction as described in Chemical Engineering News, pages 36-46, October 1, 1990 and in other references. The appropriate 3' and 5' terminal regions of the DNA encoding the bacteriocin can be used as primers defining the region to be replicated. The gene segment is preferably derived from Pediococcus acidilactici NRRL-B-18050 also known herein as PAC1.0, which is deposited with the Northern Regional Research Laboratory in Peoria, Illinois under the Budapest Treaty. The genes involved in bacteriocin activity are carried on a 9.0 kbp plasmid designated herein as pSRQ11. A DNA segment ( Sal I to Eco RI; 5.6 kbp) is ligated in purified form in a vector plasmid pBR322 and called pSRQ220. This plasmid is transformed to Escherichia coli NRRL-B-18429 and deposited at the same depository under the Budapest Treaty. U.S. Patent No. 4,883,673 which is assigned to a common assignee describes the isolation of a bacteriocin from Pediococcus acidilactici NRRL-B-18050 which inhibits various bacteria. A plasmid in this strain was disclosed to encode for the bacteriocin. The bacteriocin was described to be useful in foods to inhibit bacterial spoilage. U.S. Patent No. 4,929,445, assigned to a common assignee, describes a method of using the bacteriocin to inhibit Listeria monocytogenes which produces a severe illness in humans. The plasmid pSRQ11 was described as the source of the bacteriocin. The usefulness of the bacteriocin is well established. SPECIFIC DESCRIPTION The following Examples show the steps in sequencing the gene encoding for the bacteriocin. Bacterial strains and media. The bacterial strains used are listed in Table 1. Bacterial Strains and Plasmids Strain or plasmid Remarks Reference P. acidilactici PAC1.0 contains 9.0 kbp PA-1 pediocin plasmid, PSRQ11 (4) PAC1.14 PAC1.0 derivative cured of pSRQ11 (4) P . pentosaceus FBB63C Sensitive indicator strain for PA-1 pediocin (4) E. coli V850 Hypersensitivity to macrolide antibiotics (5) V871 Tetracycline sensitive (7) 2g4 Tetracycline sensitive Ampicillin sensitive (8) Plasmids pBR322 Ap r , Tc r (1) pACYC184 Cm r , Tc r (2) pVA891 Em r (6) pSA3 Em r , Cm r , Tc r (3) pSRQ11 9.0 kbp PA-1 pediocin plasmid (4) Pediococcus spp. were routinely maintained on MRS agar (Difco Laboratories, Detroit, MI). Escherichia coli strains were routinely carried on Lennox L agar (Gibco/BRL, Gaithersburg, Md.). Escherichia coli strains were also grown on modified MRS agar (no citrate or acetate) or in M9 medium (Maniatis, T., E. F. Fritsch, and J. Sambrook, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)) supplemented with 1% yeast extract (Oxoid, Ltd., Basingstoke, Hampshire, U.K.) and 1% Hy Case™ (Sheffield Products, Norwich, NY) for bacteriocin assays. Selective antibiotic concentrations were as follows: ampicillin, 25 ug/ml; tetracycline, 10 ug/ml; erythromycin, 50 ug/ml; and chloramphenicol, 25 ug/ml. All antibiotics were purchased from Sigma Chemical Co., St. Louis, MO. Bacteriocin assays . Production of bacteriocin was assayed as previously described (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 53 :2534-2538 (1987)). Strains were patched on MRS agar or modified MRS agar for Escherichia coli and incubated at 35°C for 18 hours. The plates were then overlaid with soft agar (0.8%) seeded with indicator cells. Isolates which produced a clear, defined zone of inhibition were considered as bacteriocin producers. One arbitrary unit (AU) of bacteriocin was defined as 5 microliters of the highest dilution of culture supernatant yielding a definite zone of growth inhibition on the indicator lawn. The titer was expressed as the reciprocal of the highest dilution showing inhibition. Isolation and analysis of plasmid DNA . Covalently closed circular plasmid DNA was isolated from Escherichia coli by the method of Clewell and Helinski (Clewell, D. B., and D. R. Helinski, Biochemistry 9 :4428-4440 (l970)). Escherichia coli strains were screened for plasmid content as previously described (Macrina, F. L., J. A. Tobian, K. R. Jones, R. P. Evans, and D. B. Clewell, Gene 19 :345-353 (1982)). Pediococcus plasmid DNA was obtained by a scaled up modification of the LeBlanc and Lee procedure (LeBlanc, D. J., and L. N. Lee, J. Bacteriol. 140 :1112-1115 (1979)) as described by Gonzalez and Kunka (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 46 :81-89 (1983)). Plasmid DNA and restriction endonuclease digests were analyzed by agarose gel electrophoresis on 0.8% agarose (Bethesda Research Laboratories, Inc., Gaithersburg, MD) slab gels. Size standards were Escherichia coli V517 (Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J. Ayers, and S. McCowen, Plasmid 1 :417-420 (1978)) for undigested plasmid DNA and Hin dIII - digested bacteriophage lambda DNA (Bethesda Research Laboratories) for restriction endonuclease - cleaved plasmid DNA. DNA enzymology . Restriction endonuclease digestions were performed in low-, medium-, or high-salt buffers, as recommended by Maniatis et al. (Maniatis, T., E. F. Fritsch, and J. Sambrook, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)). Restriction enzymes were obtained from Bethesda Research Laboratories. DNA ligation reactions were carried out with T4 DNA ligase (Bethesda Research Laboratories) at 4°C for 18 hours according to conditions recommended by the manufacturer. Bacterial transformations . Escherichia coli was transformed by the CaCl 2 heat shock method (Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)) with cells harvested at an optical density at 660 nm of 0.2 to 0.3. Purification of PA-1 . Cultural supernatant was neutralized to pH 6.0 with sodium hydroxide prior to gel filtration. A 450 ml aliquot of neutralized supernatant was applied to a 5 cm x 55 cm column (Pharmacia) containing one liter of Spectra/Gel AcA 202 (Spectrum) gel filtration resin which had been equilibrated with 0.05 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0. Activity was eluted using the same buffer. Active fractions were pooled and applied to a 2.5 cm x 90 cm CM-Sepharose column equilibrated with .05 M MES, pH 6.0. Activity was eluted with a linear gradient to .05 M MES containing 1 M sodium chloride, pH 6.0. Active fractions were pooled and dialyzed against a 10 fold excess of water using 1000 Da molecular weight cut-off dialysis tubing (Spectra-Por 6, Spectrum). Dialysate volume was reduced 12 fold by applying the dialysis tubing directly to solid 20 KDa polyethylene glycol (Carbowax, Union Carbide) and was then further reduced 3.5 fold by vacuum centrifugation (Speed-Vac, Savant). Concentrated PA-1 was applied to a 1.0 cm x 25 cm C18 reversed-phase column (Vydac) equilibrated with 0.1% aqueous trifluoroacetic acid. Activity was eluted with a linear gradient to 45% acetonitrile over 30 minutes at 1.5 ml/min. Active fractions were determined by directly spotting aliquots of column effluent on MRS plates overlaid with soft agar containing indicator cells. Active fractions were dried by vacuum centrifugation and stored at -20°C. Specific activity is defined as AU per milligram protein. Protein analyses were performed using the BCA protein assay kit (Pierce) using directions supplied with the kit. Example 1 Restriction endonuclease map of pSRQ11 . The genes involved in bacteriocin PA-1 activity were previously shown to be associated with the presence of a 9.0 kilobase plasmid, designated pSRQ11 (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 53 :2534-2538 (1987)). Plasmid pSRQ11 was digested with a number of restriction endonucleases to generate the restriction site map shown in Figure 1. The plasmid contained several unique sites including Eco RI, Nde I, Xba I, Sal I, and Sst I. Other restriction enzymes which cleaved the plasmid were Cla I, Hin dIII, Pvu II, and Eco RV. The following restriction sites were not found on pSRQ11: Ava I, Bam HI, Sph I, Nru I, Pst I, and Bgl II. Expression of PA-1 bacteriocin in E. coli . Plasmid pSRQ11 was digested with Eco RI and cloned into the Eco RI site on plasmid pVA891 (Macrina, F. L., et al., Gene 25 :145-150 (1983)), which contains an erythromycin resistance marker expressed in both Escherichia coli and streptococci. Recombinant plasmids were obtained with pSRQ11 inserted in both orientations and were designated pSRQ11.1 and pSRQ11.2 as shown in Figure 2. These Escherichia coli strains were assayed for expression of the PA-1 bacteriocin as previously described (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 53 :2534-2538 (1987)). The strains were grown on modified MRS medium and overlaid with Pediococcus pentosaceus FBB63 indicator strain. Escherichia coli strains containing pSRQ11.1 and pSRQ11.2 both produced zones of inhibition in the indicator lawn while the control Escherichia coli V850 strains showed no zone of inhibition (Table 2). The plasmid pSRQ11 was also cloned in the unique Eco RI site of the E . coli -Streptococcus shuttle plasmid pSA3. The resulting clone was called pSRQl6l. When the E . coli V850 strain carrying pSRQ161 (Table 2) was grown overnight in M9 medium supplemented with 1% yeast extract and 1% Hy Case, the filter sterilized culture supernatant yielded approximately 400 AU/ml of the bacteriocin PA-1. This observation indicated that E . coli V850 (pSRQ161) was producing and excreting PA-1 into the media. Also, other E . coli strains were transformed with the plasmid pSRQl6l and observed to produce PA-1. From this data, it was concluded that a gene fragment encoding bacteriocin PA-1 from P . acidilactici PAC 1.0 can be expressed and is functional in an E . coli host strain. Example 3 Deletion derivative analysis of pSRQ11 subclones. In order to localize the region encoding the PA-1 gene(s), Sal I and Pvu II deletion derivatives of pSRQ11.1 and pSRQ11.2 were obtained (Figure 2). The Sal I deletion of pSRQ11.1 retained activity while the Pvu II deletion derivatives displayed no zones of inhibition against the indicator strain (Table 2). Both the Pvu II and Sal I deletion derivatives of pSRQ11.2 expressed no PA-1 activity (Table 2). These data suggested that the bacteriocin gene was located on the approximately 5.6 kbp Eco RI- Sal I fragment of pSRQ11.1 as shown in Figure 2A. This 5.6 kbp Eco RI- Sal I fragment then was subcloned into the Eco RI and Sal I restriction sites on the Escherichia coli plasmid pBR322 (Bolivar et al., Gene 2 :95-113 (1977)), and the resulting chimeric plasmid was designated pSRQ220 (Figure 3A). The Escherichia coli strain containing pSRQ220 was assayed and found to express bacteriocin activity. Two additional deletion derivatives of pSRQ220, i.e., a plasmid derivative lacking a 2.7 kbp Hin dIII fragment and a plasmid derivative lacking a 1.3 kbp Hin dIII- Sal I fragment (Figure 3B), were assayed and both found to be negative for PA-1 activity. Also the following deletion derivatives were obtained: pSRQ210, which consisted of the pSRQ11, Xba I- Sal I fragment cloned into E. coli vector pACYC184 (Chang, A. C. Y., et al., J. Bacteriol. 134 :1141-1156 (1978)), and pSRQ211, which consisted of pSRQ11 Hind III fragment c (from map coordinates 1.5 to 4.2, Figure 1) also cloned into pACYC184. Neither of these two strains expressed PA-1 activity. Together with the bacteriocin PA-1 negative Pvu II and Cla I deletion derivatives (Figures 2A, and 3B (Table 2)), these results show that several genes, or one very long gene (or operon), present on the 5.6 kbp Eco RI- Sal I fragment, are responsible for PA-1 activity. Example 4 Insertional inactivation of bacteriocin PA-1 production . Since the Xba I restriction site is unique on both pSRQ11 and pSRQ220 and lies within the region involved in PA-1 production, it was chosen as a site to insert a foreign DNA fragment and interrupt transcription of the bacteriocin gene. Plasmid pACYC184, approximately 4 kbp in size and also containing a single Xba I site, was cloned into the Xba I site on pSRQ220. The strain containing the resulting recombinant plasmid, pSRQ221, was assayed for PA-1 activity and proved negative (Table 2). When the pACYC184 insert was removed by Xba I digestion, followed by religation, resulting in pSRQ 221.1, activity was once again restored. Another construct where the Xba I- Eco RI fragment of pSRQ220 was replaced by the Xba I- Eco RI fragment of pACYC184 also was negative for bacteriocin activity (Table 2). Example 5 Nucleotide sequence analysis of pSRQ220 . The DNA sequence of the 5.6 kbp Sal I- Eco RI DNA fragment, as present on plasmid pSRQ220, was established by the Sanger dideoxy chain termination procedure (Sanger, F., Nicklen, S., and Coulson, A. R., Proc. Natl. Acad. Sci. USA, 74 :5463-3967 (1977)) with the modifications as described by Biggin et al (Biggin, M.D. et al., Proc. Natl. Acad. Sci. USA, 80 :3963-3965 (1983)), using alpha- 35 S-dATP (2000 Ci/mmol) and Klenow enzyme (Amersham), ddNTP's (Pharmacia-PL Biochemicals) and dNTP's (Boehringer). The sequencing reaction products were separated on a denaturing polyacrylamide gel with a buffer gradient as described by Biggin et al. (Biggin, M. D. et al., Proc. Natl. Acad. Sci. USA, 80 :3963-3965 (1983)). Purified, double-stranded plasmid DNA of pSRQ220 served as template in the sequence reaction, following the procedure described by Hattori and Sakaki (Hattori, M., and Sakaki, Y., Anal. Biochem. 152 : 232-238 (l986)). Deoxy-oligonucleotide primers were synthesized on a DNA-synthesizer (Applied Biosystems 380A) using the Phosphoamidit technique (Barone, A. D. et al., Nucleic Acid Research, 12 :4051-4061 (1984)). The DNA sequence when translated in all possible reading frames revealed at least three open reading frames (Fig. 4). The first open reading frame (ORF 1) encodes a protein which consists of 62 amino acid residues followed by a TAG stop codon (Fig. 4). The second open reading frame (ORF 2), positioned just downstream of ORF 1, codes for a protein which consists of 112 amino acid residues followed by a TAG stop codon (Fig. 4). Further downstream the third open reading frame (ORF 3) predicts a protein consisting of 724 amino acid residues with a TAG stop codon (Fig. 4). ORF 1 encodes a protein of 62 amino acids of which amino acid residues 19 to 62 correspond entirely with the amino acid sequence of a protein, which was isolated from P. acidilactici NRRL-B-18050 called bacteriocin PA-1, and which, when separated on a polyacrylamide gel, inhibited P. pentosaceus FBB-63 effectively in an overlay experiment which is the subject of U.S. application Serial No. 514,102 (Fig. 4, and Fig. 5). This proves that ORF 1 encodes a precursor of bacteriocin PA-1, containing an 18 amino acid N-terminal peptide which is cleaved off during the process of synthesis or excretion. Both the Pvu II deletion derivative pSRQ11.13 and the Hin dIII deletion derivative pSRQ220.2 (Table 2; Figure 3B) result in a loss of PA-1 bacteriocin activity. As these deletions disturb both ORF 2 and ORF 3, or ORF 3 only, but not the PA-1 bacteriocin encoding gene (ORF 1), it can be concluded that also the presence of either ORF 2 or ORF 3, or both is necessary for PA-1 bacteriocin activity. Example 6 Site-specific mutagenesis of genes involved in PA-1 bacteriocin production. The specific role in PA-1 bacteriocin production of each of the open reading frames was determined by introduction of frameshift mutations in the various genes. Plasmid pSRQ220 contains two sites for the restriction enzyme Bal I. One is situated in the pBR332-part of the plasmid, whereas the other is positioned within ORF 1 which encodes the PA-1 bacteriocin (Figure 3A, and 3B). A frameshift mutation in ORF 1 was introduced by insertion of a double-stranded oligonucleotide linker fragment with the sequence 5'-TGCATGGATCCTGATC-3' into this Bal I-site. Plasmid pSRQ220 was therefore partially digested with Bal I, generating linear blunt-ended DNA molecules. This was achieved by incubation of the plasmid DNA in a restriction buffer for a short time period using only low amounts of the restriction enzyme. The linker fragment was added and allowed to ligate with the Bal I-treated vector DNA. Insertion of the linker fragment disrupts the Bal I site, but introduces a new and unique Bam HI site into the plasmid, that was used for identification of the desired mutant. After transformation of the ligation mixture, plasmid DNA was isolated from the transformants and screened for the presence of a Bam HI site, concomitant with the loss of a Bal I site. In this way plasmid pUR5217 was identified which carried the desired linker insertion within ORF 1. Introduction of the mutation was confirmed by determination of the nucleotide sequence around the restriction site of the mutant. E . coli cells containing pUR5217 were assayed for PA-1 bacteriocin activity and found to have lost this property. This result is in good agreement with the previous obtained deletion data and it again proves that the presence of ORF 1 is essential for PA-1 activity. Restriction enzyme Hin dIII has only two restriction sites in pSRQ220, one of which is positioned in ORF 2, while the other is positioned in ORF 3 (Figure 3B). These sites were therefore well suited for introduction of mutations in these genes. Plasmid pSRQ220 was partially digested with Hin dIII, as described above. To fill in the 3'-restriction ends Klenow enzyme and a mixture of the four dNTP's (A, T, G, C, 1mM each) were added to the DNA-sample, followed by incubation at 37°C for 30 minutes. After ligation for 16 hours at 15°C the DNA-mixture was transformed to E . coli 294. Plasmid DNA was isolated from the transformants and screened for the loss of the Hin dIII restriction sites by digesting with Hin dIII. Introduction of the mutations was confirmed by determination of the nucleotide sequence around the restriction site of each mutant. In this way plasmid pUR5206 which carried a mutation at the Hin dIII site in ORF 2, and plasmid pUR5205 which carried a mutation at the Hin dIII site in ORF 3 were identified. E. coli cells containing pUR5206 were assayed and found to express PA-1 bacteriocin activity, whereas E. coli cells containing pUR5205 were negative for PA-1 bacteriocin activity. From these data it can be concluded that, besides the presence of the PA-1 bacteriocin gene (ORF 1), also the presence of an intact ORF 3 is needed for PA-1 bacteriocin activity. The function of ORF 2 is not known. Although E. coli cells containing pUR5206 are able to produce bacteriocin PA-1 activity, it cannot be ruled out that ORF 2 is involved in the secretion or processing of bacteriocin PA-1. From the nucleotide sequence analysis some other tentative open reading frames can be deduced (data not shown). Therefore it is possible that other information is present on the 5.6 kbp Eco RI- Sal I fragment which is also needed for PA-1 bacteriocin activity. It is intended that the foregoing description be only illustrative of the present invention and the present invention is limited only by the hereinafter appended claims. APPENDIX 1 Sequence Listing (1) GENERAL INFORMATION: (i) APPLICANT: J.D. Marugg, A.M. Ledeboer, P.A. Vandenbergh and J.T. Henderson (ii) TITLE OF INVENTION: Cloned Gene Encoding for Bacteriocin From Pediococcus acidilactici (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Ian C. McLeod (B) STREET: 2190 Commons Parkway (C) CITY: Okemos (D) STATE: Hichigan (E) COUNTRY: USA (F) ZIP: 48864 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.50 inch, 1.44 Mb storage (B) COMPUTER: IBM AT (C) OPERATING SYSTEM: MS-DOS 4.01 (D) SOFTWARE: ASCII text editor (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: 07/635,965 (B) FILING DATE: December 31, 1990 (C) CLASSIFICATION: Unknown (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Ian C. McLeod (B) REGISTRATION NUMBER: 20,931 (C) REFERENCE/DOCKET NUMBER: MT 4.1-129 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (517) 347-4100 (B) TELEFAX: (517) 347-4103 (C) TELEX: None (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5595 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Plasmid DNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) FRAGMENT TYPE: N-terminal, internal and C-terminal fragments (vi) ORIGINAL SOURCE: (A) ORGANISM: Pediococcus acidilactici (B) STRAIN: NRRL-B-18050 (C) INDIVIDUAL ISOLATE: PAC1.0 (D) DEVELOPMENTAL STAGE: N/A (E) HAPLOTYPE: (F) TISSUE TYPE: (G) CELL TYPE: N/A (H) CELL LINE: N/A (I) ORGANELLE: N/A (vii) IMMEDIATE SOURCE: N/A (viii) POSITION IN GENOME: N/A (ix) FEATURE: (A) NAME/KEY: bacteriocin encoding DNA (B) LOCATION: ECORI to SalI DNA fragment 5.6 kbp. (C) IDENTIFICATION METHOD: sequencing (D) OTHER INFORMATION: DNA needed for bacteriocin expression. (x) PUBLICATION INFORMATION: N/A (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Claims: A nucleotide sequence corresponding to the nucleotide sequence which can be isolated from a strain belonging to the genus Pediococcus, consisting of genes coding for both a bacteriocin precursor which is ORF1 and a gene for at least one protein selected from the group consisting of ORF2, ORF3 and ORF2 and ORF3 as given in Figure 4 and modifications thereof essential for obtaining a functional active bacteriocin. The nucleotide sequence according to Claim 1, in which the Pediococcus is Pediococcus acidilactici. The nucleotide sequence according to Claim 2, in which the Pediococcus is Pediococcus acidilactici NRRL-B-18050. The nucleotide sequence according to Claim 1 containing the three open reading frames, ORF1, ORF2 and ORF3 as given in Figure 4, and derived from the plasmid pSRQ11. The nucleotide sequence according to any preceding Claim 1 to 4, containing in addition transcriptional and translational initiation and termination sequences of open reading frames as given in Figure 4. A nucleotide sequence encoding only a bacteriocin precursor, said nucleic acid being selected from the group consisting of ORF1 in Figure 4, and modifications thereof that encode a protein still having the capability of being converted into an active bacteriocin. A vector, that can be stably maintained in a host microorganism, which vector can be maintained as a plasmid or can integrate into a chromosome of the host microorganism, comprising a nucleotide sequence according to any preceding Claim 1 to 6. The vector according to Claim 7, in which the nucleotide sequence contains open reading frames ORF1 and ORF3, and optionally ORF2 as given in Figure 4. A vector according to Claim 7, which comprises modified versions of any of ORF1, ORF2 and ORF3 as given in Figure 4, that encode a protein still having the capability of being converted into an active bacteriocin. The vector according to Claim 7 containing ORF1, and optionally ORF2 or ORF3 or both, as given in Figure 4, in which the ORFs are under control of one or more promotor systems functional in said host microorganism and at least after the most downstream ORF of ORF1, ORF2 or ORF3 a terminator sequence is present, wherein the ORF1 can have a promotor system, optionally followed by a terminator sequence, or ORF1 can form part of an operon containing ORF2 or ORF3, or ORF2 and ORF3 together. A microorganism transformed by introducing a vector according to any preceding Claim 7 to 10, capable of producing a bacteriocin. A microorganism according to Claim 11 selected from the group consisting of the genera Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia, and yeasts. The vector of claim 7 that replicates or is stably maintained, in the microorganism selected from the group consisting of the genera Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia, and yeasts. A nucleotide sequence encoding a protein essential for obtaining a functional active bacteriocin, said nucleotide sequence being ORF3 in Figure 4, or a modification thereof that encodes a protein still having the capability of assisting in production of an active bacteriocin. A nucleotide sequence encoding a protein, said nucleotide sequence being ORF2 as given in Figure 4, or a modification thereof that encodes a protein still having the function of the protein encoded by ORF2 in producing the functionally active bacteriocin. The nucleotide sequence of Claim 1 derived by a polymerase chain reaction method. A nucleotide sequence encoding a protein having an amino acid sequence selected from the group consisting of ORF1, ORF2 or ORF3 as given in Figure 4 and modifications thereof capable of encoding proteins which function in the manner of ORF1, ORF2 or ORF3.

Inventors: HENDERSON JAMES T, LEDEBOER ADRIANUS M, MARUGG JOHN D, VANDENBERGH PETER A, HENDERSON, JAMES T., LEDEBOER, ADRIANUS M., MARUGG, JOHN D., VANDENBERGH, PETER A.
Applicants: QUEST INT, QUEST INTERNATIONAL B.V.
Application year: 1991
Main classification: C12N15
IPC codes: C12N1, C12N15, C12R1, C07K14, A23L3, C12P21