Bacteriocin leucocin-B

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.

Leuconostoc carnosum 4010 is an antimicrobial strain used as a protective culture in vacuum-packed meats. In this study, we showed that, in addition to antilisterial class IIa bacteriocins leucocin A and C, the strain also produces class IId bacteriocin leucocin B, the antimicrobial activity of which is limited to the genera Leuconostoc and Weissella. Two novel genes, lebBI encoding the leucocin B precursor with a double-glycine-type leader and putative immunity protein LebI, were identified on L. carnosum 4010 plasmid pLC4010-1. LebI contains three transmembrane spans and shares 55% identity with the mesentericin B105 immunity protein. Genes lebBI were shown to be transcribed in 4010 by RT-PCR analysis. The secretion of leucocin B in L. carnosum 4010 was shown by spot-on-lawn and SDS-gel overlay methods with a Leuconostoc strain sensitive to leucocin B but resistant to leucocins A and C. In addition, leucocins A and B from L. carnosum 4010 were cloned as SSusp45 fusions in heterologous host Lactococcus lactis and the secretion of active bacteriocins was detected on indicator plates.

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 against Listeria monocytogenes and several lactic acid bacteria. Leucocin B-Ta11a was optimally produced at 25 degrees C in MRS broth at an initial pH of 6.0 or 6.5. An 8.9-MDa plasmid in Leuconostoc carnosum Ta11a hybridized to a 36-mer oligonucleotide probe (JF-1) that was homologous to leucocin A-UAL187. A 4.9-kb Sau3A 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.