Cloning and sequence analysis of the gene coding for a Leuconostoe Bacteriocin

Abstract

Previous studies have shown that Leuconostoc (Lc.) carnosum Tal la produces a bacteriocin that has been designated leucocin B-Tal la [Papathanasopoulos, 1993, M.Sc thesis, University of the Witwatersrandl. Leucocin B-Talla is active against Listeria nnnocytogenes and several lactic acid bacteria. An 8.9 MDa plasmid in Leuconostoc carnosum Tal1a hybridised to a 36-meroligonucleotide probe (JF-1) that is homologous to the amino-terminal sequence of the leucocin A-UAL187 structural gene. A library of Lc. carnosum Talla plasmid DNA was constructed by partial digestion of DNA with ^lar3A and ligation into the BamHl site of pUC1l8. A plasmid (pJF8.1), containing a 4.9 kb insert was identified by Southern blotting and hybridisation to JF-l. A subclone of this plasmid, with a 2.3 kb insert (pJF5.5), was generated by internal deletion of a 2.6 kb Xbal fragment and religation of the plasmid. Sequence analysis of pJF8.1 and pJF5.5 revealed the presence of two open reading frames (ORF). ORFI codes for a protein of 61 amino acid residues. This protein product is proposed to be the prepeptide of a 37 amino acid bacteriocin, leucocin B-Ta11a, by virtue of DNA sequence homology to leucocin A-UALI87 [Hastings et al., 1991. J. Bacteriol 173:749L-7500]. The 24 amino acid residue amino-terminal extension, possibly cleaved during processing of the prepeptide may function as a leader peptide. The aminoterminal extension of leucocin B-Talla differed trom the similar region in Ieucocin A-UAL187 by seven residues. The predicted protein of the ORF2 consists of 113 amino acids and is identical to the amino acid sequence of the cognate ORF of the leucocin A-UAL187 operon. Expression of leucocin B-

Talla was attempted in Escheichia coli JMl03 transformed with pJF8.1 and pJF5.5. Results of inhibition studies with various cell fractions of the transformed strains showed that no bacteriocin was produced by these transformants.