Heterologous expression of the lactacin F peptides by Carnobacterium piscicola LV17.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 and is active against five other Lactobacillus species and Enterococcus faecalis. The genetic determinants encoding the lactacin F complex are organized in a 1-kb polycistronic operon which comprises three genes, lafA, lafX, and ORFZ (encoding the putative immunity protein). The lafA and lafX genes encode the bacteriocin precursors with N-terminal extensions characterized by a Gly-Gly-1*Xaa+1 cleavage site (*). The Gly-Gly motif is conserved in several other bacteriocins, including carnobacteriocins A, BM1, and B2. Carnobacterium piscicola LV17 produces carnobacteriocins which are active against Listeria monocytogenes and other lactic acid bacteria. In this study, the lactacin F operon was introduced into C. piscicola LV17. The transformants produced lactacin F concurrently with the carnobacteriocins. When the lafA and lafX genes were separated and cloned individually into LV17, production of either LafA or LafX by C. piscicola LV17 was detected by complementation with L. johnsonii clones producing LafX or LafA, respectively. Transformants of C. piscicola LV17 which produced lactacin F, LafA, or LafX, in combination with the carnobacteriocins, were assayed for an increased and expanded inhibitory spectrum. The recombinant organisms were only active against lactacin F- and carnobacteriocin-sensitive strains. A plasmidless derivative of LV17 which does not produce the carnobacteriocins failed to produce lactacin F, LafA, or LafX when transformed with the appropriate recombinant plasmids. The ability of C. piscicola LV17 to produce lactacin F demonstrates that the machinery for the carnobacteriocins is capable of processing and exporting bacteriocins from both systems.     

Molecular characterization of genes involved in the production of the bacteriocin leucocin A from Leuconostoc gelidum.

Leucocin A is a small heat-stable bacteriocin produced by Leuconostoc gelidum UAL187. A 2.9-kb fragment of plasmid DNA that contains the leucocin structural gene and a second open reading frame (ORF) in an operon was previously cloned (J. W. Hastings, M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 173:7491-7500, 1991). When a 1-kb DraI-HpaI fragment containing this operon was introduced into a bacteriocin-negative variant (UAL187-13), immunity but no leucocin production was detected. Leucocin production was observed when an 8-kb SacI-HindIII fragment of the leucocin plasmid was introduced into L. gelidum UAL187-13 and Lactococcus lactis IL1403. Nucleotide sequence analysis of this 8-kb fragment revealed the presence of three ORFs in an operon upstream of and on the strand opposite from the leucocin structural gene. The first ORF (lcaE) encodes a putative protein of 149 amino acids with no apparent function in leucocin A production. The second ORF (lcaC) contains 717 codons that encode a protein homologous to members of the HlyB family of ATP-binding cassette transporters. The third ORF (lcaD) contains 457 codons that encode a protein with marked similarity to LcnD, a protein essential for the expression of the lactococcal bacteriocin lactococcin A. Deletion mutations in lcaC and lcaD resulted in loss of leucocin production, indicating that LcaC and LcaD are involved in production and translocation of leucocin A. The secretion apparatus for lactococcin A did not complement mutations in the lcaCD genes to express leucocin A in L. lactis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15

Aim: To characterize novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15. Methods and Results: Leuconostoc pseudomesenteroides QU 15 isolated from Nukadoko (rice bran bed) produced novel bacteriocins. By using three purification steps, four antimicrobial peptides termed leucocin A (ΔC7), leucocin A‐QU 15, leucocin Q and leucocin N were purified from the culture supernatant. The amino acid sequences of leucocin A (ΔC7) and leucocin A‐QU 15 were identical to that of leucocin A‐UAL 187 belonging to class IIa bacteriocins, but leucocin A (ΔC7) was deficient in seven C‐terminal residues. Leucocin Q and leucocin N are novel class IId bacteriocins. Moreover, the DNA sequences encoding three bacteriocins, leucocin A‐QU 15, leucocin Q and leucocin N were obtained. Conclusions: These bacteriocins including two novel bacteriocins were identified from Leuc. pseudomesenteroides QU 15. They showed similar antimicrobial spectra, but their intensities differed. The C‐terminal region of leucocin A‐QU 15 was important for its antimicrobial activity. Leucocins Q and N were encoded by adjacent open reading frames (ORFs) in the same operon, but leucocin A‐QU 15 was not. Significance and Impact of Study: These leucocins were produced concomitantly by the same strain. Although the two novel bacteriocins were encoded by adjacent ORFs, a characteristic of class IIb bacteriocins, they did not show synergistic activity.     

Kinetic studies of the action of lactacin F, a bacteriocin produced by Lactobacillus johnsonii that forms poration complexes in the cytoplasmic membrane.

The bacteriocin lactacin F is bactericidal against Lactobacillus delbrueckii, Lactobacillus helveticus, and Enterococcus faecalis. Activity against L. delbrueckii was recently shown to be dependent on two peptides, LafA and LafX, which are encoded within the lactacin F operon (T. R. Klaenhammer, FEMS Microbiol. Rev. 12:39-87, 1993). It has been proposed that two peptides form an active lactacin F complex. In this study, the action of lactacin F against E. faecalis ATCC 19443 and the effects of various environmental parameters were investigated in detail. Addition of lactacin F induced the loss of K+ from cells of L. delbrueckii, Lactobacillus johnsonii 88-4, and E. faecalis, while the lactacin F producer L. johnsonii VPI 11088 was not affected by the bacteriocin. Lactacin F caused an immediate loss of cellular K+, depolarization of the cytoplasmic membrane, and hydrolysis of internal ATP in E. faecalis. Lactacin F induced loss of K+ in 3,3',4',5-tetrachlorosalicylanilide-treated cells, indicating that pores are formed in the absence of a proton motive force. ATP hydrolysis was not due to dissipation of the proton motive force but was most likely caused by efflux of inorganic phosphate, resulting in a shift of the ATP hydrolysis equilibrium. Action of lactacin F was optimal at acidic pH values and was reduced in the presence of di- and trivalent cations. The lanthanide gadolinium (Gd3+) prevented action of lactacin F completely at a concentration of 0.2 mM. Lactacin F-induced loss of cell K+ was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of the lactacin F operon.

Lactacin F is a nonlantibiotic, heat-stable, peptide bacteriocin produced by Lactobacillus johnsonii VPI11088. Molecular analysis of the lactacin F DNA region characterized a small operon that codes for three open reading frames, designated lafA, lafX, and ORFZ. The peptide encoded by lafA, the lactacin F structural gene, was compared with various peptide bacteriocins from lactic acid bacteria, and similarities were identified in the amino and carboxy termini of the propeptides. Site-directed mutagenesis of the LafA precursor at the two glycine residues in positions -1 and -2 defined an essential motif for processing of mature lactacin F. The involvement of the peptides encoded by lafX and ORFZ in bacteriocin expression was investigated by subcloning various fragments from the lactacin F region into the shuttle vector pGKV210. In addition to lafA, expression of lafX is essential to lactacin F activity. The lactacin F operon resembles the genetic organization of lactococcin M. Although no function has been assigned to ORFZ by genetic analysis, both peptide Z and the lactococcin M immunity protein are predicted to be integral membrane proteins with four putative transmembrane segments. Lactacin F activity, defined by bactericidal action on Lactobacillus delbrueckii, is dependent on the expression of two genes, lafA and lafX.     

Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum.

Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 +/- 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.     

Control of beef spoilage by a sulfide-producing Lactobacillus sake strain with bacteriocinogenic Leuconostoc gelidum UAL187 during anaerobic storage at 2 degrees C.

Chill-stored, vacuum-packaged beef inoculated with sulfide-producing Lactobacillus sake 1218 developed a distinct sulfide odor within 3 weeks of storage at 2 degrees C, at which time the bacteria had reached maximum numbers of 10(6) CFU cm(-2). Coinoculation of the meat with the wild-type, bacteriocinogenic (Bac+) strain of Leuconostoc gelidum UAL187 delayed the spoilage by L. sake 1218 for up to 8 weeks of storage. Coinoculation of meat samples with an isogenic, slowly growing Bac+ variant, UAL187-22, or with the Bac- variant UAL187-13 did not delay the onset of spoilage by L. sake 1218. The study showed that spoilage of chill-stored, vacuum-packaged beef by a susceptible target organism could be dramatically delayed by the Bac+ wild-type strain of L. gelidum UAL187. Inoculation with L. sake 1218 can be used as a model system to determine the efficacy of biopreservation of vacuum-packaged meats.     

Multiple Bacteriocin Production by Leuconostoc mesenteroidesTA33a and Other Leuconostoc/Weissella Strains

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins. Received: 11 April 1997 / Accepted: 10 June 1997     

Double-glycine-type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis

Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors which have N-terminal leader peptides that share similarities in amino acid sequence and contain a conserved processing site of two glycine residues in positions -1 and -2. A dedicated ATP-binding cassette (ABC) transporter is responsible for the proteolytic cleavage of the leader peptides and subsequent translocation of the bacteriocins across the cytoplasmic membrane. To investigate the role that these leader peptides play in the recognition of the precursor by the ABC transporters, the leader peptides of leucocin A, lactococcin A or colicin V were fused to divergicin A, a bacteriocin from Carnobacterlum divergens that is secreted via the cell's general secretion pathway. Production of divergicin was monitored when these fusion constructs were introduced into Leuconostoc gelidum, Lactococcus lactis and Escherichia coli, which carry the secretion apparatus for leucocin A, lactococcins A and B, and colicin V, respectively. The different leader peptides directed the production of divergicin in the homologous hosts. In some cases production of divergicin was also observed when the leader peptides were used in heterologous hosts. For ABC-transporter-dependent secretion in E. coli the outer membrane protein TolC was required. Using this strategy, colicin V was produced in L. lactis by fusing this bacteriocin behind the leader peptide of leucocin A.     

Characterization of leucocin B-Ta11a: A bacteriocin fromLeuconostoc carnosum Ta11a isolated from meat

Leuconostoc (Lc.)carnosum Ta11a, isolated from vacuum-packaged processed meats, produced a bacteriocin designated leucocin B-Ta11a. The crude bacteriocin was heat stable and sensitive to proteolytic enzymes, but not to catalase, lysozyme, or chloroform. It was active againstListeria monocytogenes and several lactic acid bacteria. Leucocin B-Ta11a was optimally produced at 25°C in MRS broth at an initial pH of 6.0 or 6.5 An 8.9-MDa plasmid inLeuconostoc carnosum Ta11a hybridized to a 36-mer oligonucleotide probe (JF-1) that was homologous to leucocin A-UAL187. A 4.9-kbSau3A fragment from a partial digest of the 8.9-MDa plasmid was cloned into pUC118. The 8.1-kb recombinant plasmid (pJF8.1) was used for sequencing and revealed the presence of two open reading frames (ORFs). ORF1 codes for a protein of 61 amino acids comprising a 37-amino-acid bacteriocin that was determined to be the leucocin B-Ta11a structural gene by virtue of its homology to leucocin A-UAL 187 (Hastings et al. 1991. J. Bacteriol 173: 7491–7500). The 24-amino-acid N-terminal extension, however, differs from that of leucocin A-UAL187 by seven residues. The predicted protein of the ORF2 has 113 amino acids and is identical with the amino acid sequence of the cognate ORF of the leucocin A-UAL 187 operon.     

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

Leuconostoc carnosum 4010 is a protective culture for meat products. It kills the foodborne pathogen Listeria monocytogenes by producing two class IIa (pediocin-like) bacteriocins, leucocin A and leucocin C. The genes for leucocin A production have previously been characterised from Leuconostoc gelidum UAL 187, whereas no genetic studies about leucocin C has been published. Here, we characterised the genes for the production of leucocins A and C in L. carnosum 4010. In this strain, leucocin A and leucocin C operons were localised in different plasmids. Unlike in L. gelidum, leucocin A operon in L. carnosum 4010 only contained the structural and the immunity genes lcaAB without transporter genes lcaECD. On the contrary, leucocin C cluster included two intact operons. Novel genes lecCI encode the leucocin C precursor and the 97-aa immunity protein LecI, respectively. LecI shares 48 % homology with the immunity proteins of sakacin P and listeriocin. Another leucocin C operon lecXTS, encoding an ABC transporter and an accessory protein, was 97 % identical with the leucocin A transporter operon lcaECD of L. gelidum. For heterologous expression of leucocin C in Lactococcus lactis, the mature part of the lecC gene was fused with the signal sequence of usp45 in the secretion vector pLEB690. L. lactis secreted leucocin C efficiently, as shown by large halos on lawns of L. monocytogenes and Leuconostoc mesenteroides indicators. The function of LecI was then demonstrated by expressing the gene lecI in L. monocytogenes. LecI-producing Listeria was less sensitive to leucocin C than the vector strain, thus corroborating the immunity function of LecI.     

Primary amino acid and DNA sequences of gassericin T, a lactacin F-family bacteriocin produced by Lactobacillus gasseri SBT2055

A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SBT 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII-HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60% sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Characterization of the amylovorin locus of Lactobacillus amylovorus DCE 471, producer of a bacteriocin active against Pseudomonas aeruginosa, in combination with colistin and pyocins

Lactobacillus amylovorus DCE 471 produces amylovorin L, a bacteriocin with an antibacterial activity against some strains of the Lactobacillus lineage. Based on the sequence of one active peptide, a gene encoding active amylovorin L was cloned and sequenced. Genome walking allowed us to sequence a larger fragment of 7577 bp of genomic DNA, with 12 predicted ORFs. The previously characterized amylovorin L peptide-encoding gene is preceded by another gene encoding a small polypeptide with a typical bacteriocin-processing double-glycine site, suggesting that amylovorin L is a two-component class IIb bacteriocin (amylovorin Lalpha/beta). Lalpha and Lbeta show the highest similarity to gassericin T from Lactobacillus gasseri SBT2055 and BlpN from Streptococcus pneumoniae R6, respectively, and to LafA and LafX, which form the lactacin F bacteriocin of Lactobacillus johnsonii NCC 533. As for other lactic acid bacteria bacteriocins, amylovorin L showed no activity against the Gram-negative opportunistic pathogen Pseudomonas aeruginosa on its own, but showed synergistic inhibitory activity when used in combination with the peptide antibiotic colistin, and, remarkably, with the P. aeruginosa soluble bacteriocins, pyocins S1 and S2.     

Characterization and purification of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides.

A Leuconostoc mesenteroides ssp. mesenteroides was isolated from goat's milk on the basis of its ability to inhibit the growth of Listeria monocytogenes. The antimicrobial effect was due to the presence in the culture medium of a compound, named mesentericin Y105, excreted by the Leuconostoc mesenteroides Y105. The compound displayed known features of bacteriocins from lactic acid bacteria. It appeared as a proteinaceous molecule exhibiting a narrow inhibitory spectrum limited to genus Listeria. The apparent relative molecular mass, as indicated by activity detection after SDS-PAGE, was 2.5-3.0 kDa. The bacteriocin was purified to homogeneity by a simple three-step procedure: a crude supernatant obtained from an early-stationary-phase culture in a defined medium was subjected to affinity chromatography on a blue agarose column, followed by ultrafiltration through a 5 kDa cut-off membrane, and finally by reverse-phase HPLC on a C4 column. Microsequencing of the pure bacteriocin and of tryptic fragments showed that mesentericin Y105 is a 36 amino acid polypeptide whose primary structure is close to that of leucocin A-UAL 187, which contains an extra residue at the C-terminus and displays only two differences in the overlapping sequence. However, unlike leucocin A-UAL 187, mesentericin Y105 displayed a bactericidal mode of action.     

Mutational analysis of mesentericin y105, an anti-Listeria bacteriocin, for determination of impact on bactericidal activity, in vitro secondary structure, and membrane interaction

Mesentericin Y105 is a 37-residue bacteriocin produced by Leuconostoc mesenteroides Y105 that displays antagonistic activity against gram-positive bacteria such as Enterococcus faecalis and Listeria monocytogenes. It is closely related to leucocin A, an antimicrobial peptide containing beta-sheet and alpha-helical structures. To analyze structure-function relationships and the mode of action of this bacteriocin, we generated a collection of mesentericin derivatives. Mutations were obtained mostly by PCR random mutagenesis, and the peptides were produced by an original system of heterologous expression recently described. Ten derivatives were obtained displaying modifications at eight different positions in the mesentericin Y105 sequence. Purified peptides were incorporated into lysophosphatidylcholine micelles and analyzed by circular dichroism. The alpha-helical contents of these peptides were compared and related to their respective bactericidal activities. Moreover, studies of the intrinsic fluorescence of tryptophan residues naturally occurring at positions 18 and 37 revealed information about insertion of the peptides in micelles. A model for the mode of action of mesentericin Y105 and related bacteriocins is proposed.     

Characterization of Leucocin B-KM432Bz from Leuconostoc pseudomesenteroides Isolated from Boza,and Comparison of its Efficiency to Pediocin PA-1

A bacteriocin-producing bacterium was isolated from boza and identified as Leuconostoc pseudomesenteroides KM432Bz. The antimicrobial peptide was purified and shown to be identical to other class IIa bacteriocins: leucocin A from Leuconostoc gelidum UAL-187 and Leuconostoc pseudomesenteroides QU15 and leucocin B from Leuconostoc carnosum Ta11a. The bacteriocin was named leucocin B-KM432Bz. Leucocin B-KM432Bz gene cluster encodes the bacteriocin precursor (lcnB), the immunity protein (lcnI) and the dedicated export machinery (lcnD and lcnE). A gene of unknown and non-essential function (lcnC), which is interrupted by an insertion sequence of the IS30 family, is localized between lcnB and lcnD. The activity of leucocin B-KM432Bz requires subunit C of the EII^t _Man mannose permease, which is the receptor for entry into target cells. The determination of the minimum inhibitory concentrations revealed the lowest values for leucocin B-KM432Bz over Listeria strains, with 4 to 32 fold better efficiency than pediocin PA-1.     

An analysis of bacteriocins produced by lactic acid bacteria isolated from malted barley

AIMS: The aim of this study was to perform a detailed characterization of bacteriocins produced by lactic acid bacteria (LAB) isolated from malted barley. METHODS AND RESULTS: Bacteriocin activities produced by eight LAB, isolated from various types of malted barley, were purified to homogeneity by ammonium sulphate precipitation, cation exchange, hydrophobic interaction and reverse-phase liquid chromatography. Molecular mass analysis and N-terminal amino acid sequencing of the purified bacteriocins showed that four non-identical Lactobacillus sakei strains produced sakacin P, while four Leuconostoc mesenteroides strains were shown to produce bacteriocins highly similar or identical to leucocin A, leucocin C or mesenterocin Y105. Two of these bacteriocin-producing strains, Lb. sakei 5 and Leuc. mesenteroides 6, were shown to produce more than one bacteriocin. Lactobacillus sakei 5 produced sakacin P as well as two novel bacteriocins, which were termed sakacin 5X and sakacin 5T. The inhibitory spectrum of each purified bacteriocin was analysed and demonstrated that sakacin 5X was capable of inhibiting the widest range of beer spoilage organisms. CONCLUSION: All bacteriocins purified in this study were class II bacteriocins. Two of the bacteriocins have not been described previously in the literature while the remaining purified bacteriocins have been isolated from environments other than malted barley. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a thorough analysis of bacteriocin-producing LAB from malt and demonstrates, for the first time, the variety of previously identified and novel inhibitory peptides produced by isolates from this environment. It also highlights the potential of these LAB cultures to be used as biological controlling agents in the brewing industry.     

Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin.