CHEMICAL AND GENETIC-CHARACTERIZATION OF BACTERIOCINS PRODUCED BY CARNOBACTERIUM-PISCICOLA LV17B

Carnobacteriocins BM1 and B2 are thermostable class II bacteriocins pr oduced by Carnobacterium piscicola LV17B. These bacteriocins were puri fied by a three-step procedure that included hydrophobic interaction, size exclusion, and reversed-phase high performance liquid chromatogra phy. The purified peptides and fragments derived by enzymatic digestio n were analyzed by Edman degradation, amino acid analysis, and mass sp ectrometry. An oxidized form of carnobacteriocin BM1 (carnobacteriocin B1) was also purified and characterized. Probes synthesized using inf ormation from the N-terminal amino acid sequences for the purified bac teriocins were used to locate structural genes for the carnobacterioci ns. A 1.9-kilobase (kb) HindIII fragment from a 61-kb plasmid (pCP40) containing the carnobacteriocin B2 structural gene and a 4.0-kb EcoRI- PstI genomic fragment containing the carnobacteriocin BM1 structural g ene were cloned and fully or partially sequenced, respectively. Expres sion of the chromosomal bacteriocin and its immunity function requires the presence of the 61-kb plasmid. The results indicate that both bac teriocins are synthesized as prebacteriocins. Post-translational cleav age of an 18-amino acid N-terminal extension at a Gly-Gly (positions - 2 and -1) site takes place in each prepeptide to yield the mature 43-a mino acid carnobacteriocin BM1 (molecular mass 4524.6) and the mature 48-amino acid carnobacteriocin B2 (molecular mass 4969.9). These two p eptides showed significant amino acid homology to each other and with those class II bacteriocins which contain the YGNGV amino acid motif n ear the N terminus.