CHARACTERISTICS AND GENETIC DETERMINANT OF A HYDROPHOBIC PEPTIDE BACTERIOCIN, CARNOBACTERIOCIN-A, PRODUCED BY CARNOBACTERIUM-PISCICOLA-LV17A

Carnobacteriocin A is a hydrophobic nonlantibiotic bacteriocin that is detected early in the growth cycle of Carnobacterium piscicola LV17A and encoded by a 49 MDa plasmid. The bacteriocin was purified using hy drophobic interaction and gel filtration chromatography. and reversed- phase HPLC. Three different active peaks (A1, A2 and A3) were detected , but the purified samples had identical N-terminal amino acid sequenc es for the first 15 amino acids as determined by Edman degradation ana lysis. Only a 2.4 kb fragment of the EcoRI digest of the plasmid pCP49 hybridized with a 23-mer oligonucleotide probe derived from amino aci ds 5 to 13 of the amino acid sequence. The structural gene for carnoba cteriocin A is located 600 base pairs into the 2.4 kb EcoRI fragment, but no other genetic information was detected on this unit. The struct ural gene includes an 18 amino acid N-terminal extension of the bacter iocin, ending with Gly-Gly residues in the -2, -1 positions with respe ct to the cleavage site. The bacteriocin consists of 53 amino acids th at differ markedly from the majority of hydrophobic peptide bacterioci ns characterized to date. Based on the amino acid sequence derived fro m the nucleotide sequence a molecular mass of 5052.85 Da was calculate d. Mass spectrometric analysis showed that the molecular mass of the m ajor component (A3) was 2 Da lower, thereby indicating the presence of a disulphide bridge between Cys 22 and Cys 51. Carnobacteriocin A2 ha s a similar structure except that Met 52 is oxidized to a sulphoxide, whereas A1 appears to be a mixture of peptides derived proteolytically from A3 or A2.