Characterization of two bacteriocins and a food grade plasmid from carnobacteria

Carnobacterium piscicola LV17A and Carnobacterium divergens LV13 are lactic acid bacteria that were isolated from vacuum packaged meat. C. piscicola LV17 was studied for its ability to produce plasmid-mediated bacteriocins, and C. divergens LV13 was used as an indicator strain. The plasmids for bacteriocin production were separated into different strains, and this study was done on C. piscicola LV17A, the producer organism for carnobacteriocin A. The structural gene responsible for carnobacteriocin A (cbnA) was identified on a 2-kb EcoRI fragment of pCP49, the plasmid containing the genetic information for carnobacteriocin A production. The cbnA gene encodes a 71 amino acid preprotein that is comprised of a 53 amino acid mature bacteriocin and an 18 amino acid N-teminal extension. Comparison of the molecular mass of carnobacteriocin A (5050.8 Da) determined by mass spectrum analysis to that predicted from the nucleotide sequence (5052.8 Da) indicated that the bacteriocin contains a disulfide bridge between the cysteine residues at positions 22 and 51. Downstream of cbnA there are 6 additional genes (cbaI, cbaX, cbaK, cbaR, cbaT, cbaC) that, based on homology studies, encode proteins similar to bacterial two-component regulatory systems and ATP binding cassette transporters. C. divergens contains a small (3470 bp) plasmid that encodes a narrow spectrum bacteriocin, divergicin A. The bacteriocin gene (dvnA) was identified and contains a classical signal peptide that is responsible for secreting the 46 amino acid divergicin A. A 514-bp EcoRV-AccI fragment containing dvnA and dviA was cloned behind a P59 promotor and was shown to be capable of producing divergicin A in heterologous hosts, that including Carnobacterium spp., Lactococcus lactis and Escherichia coli. Divergicin A signal peptide was fused in front of the mature portion of alkaline phosphatase and produced active and mature alkaline phosphatase in E. coli.