Production, purification and characterization of a Leuconostoc bacteriocin and analysis of its genetic determinants

heterofermentative lactic acid bacterium, strain UAL 187, isolated from meat packaged under elevated CO;Initial evidence suggested that the genetic information determining production of, and resistance to, the bacteriocin is plasmid mediated. Of the three plasmids found in this organism, loss of the 7.6 MDa plasmid resulted in loss of production of and immunity to the bacteriocin. Loss of the 5.0 MDa plasmid did not result in a detectable phenotypic change in the organism.;The first thirteen amino acids at the N-terminus of leucocin A-UAL 187 were determined by Edman degradation. A mixed oligonucleotide probe (240-mer) homologous to the degenerate sequence of the first eight residues at the N-terminus hybridized to a 2.9 kb HpaII fragment of the 7.6 MDa plasmid from the producer strain, Leuconostoc gelidum UAL 187. The fragment was cloned into the AccI site of pUC118 and then subcloned as a PsiI-SacI fragment into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of two open reading frames (ORF) flanked by a putative upstream promoter and downstream terminator. The first ORF downstream of the promoter contains 61 amino acids and was identified as the leucocin structural gene, consisting of a 37 amino acid bacteriocin and a 24 residue N-terminal extension. The second ORF contains 113 amino acids and may produce an immunity protein. Phenotypic expression of the bacteriocin was not achieved in several lactic acid bacteria that were electrotransformed with the hybrid plasmid, pNZ19, containing the 2.9 kb cloned fragment of the leucocin A plasmid.;The bacteriocin was purified by ammonium sulphate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration and RP-HPLC with a yield of 58% of the original activity. Leucocin A-UAL 187 is stable at low pH, heat resistant and the pure form is stabilized by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry as 3930.4...