Evidence for production of a new lantibiotic (butyrivibriocin OR79A) by the ruminal anaerobe Butyrivibrio fibrisolvens OR79: Characterization of the structural gene encoding butyrivibriocin OR79A

The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin- like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resoluti on gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different a mino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides, A gene with significant homology to one combination of the determined N-terminal sequen ce was cloned, and expression of the gene was confirmed by Northern blottin g. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature pe ptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homol ogy was found between this peptide and previously reported lantibiotics con taining the double-glycine leader peptidase processing site. Immediately do wnstream of bvi79A was a second, partial open reading frame encoding a pept ide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second pepti de was identical to butyrivibriocin OR79A except for amino acid substitutio ns in positions 2 and 3 of the mature lantibiotic, Only a single coding reg ion was detected when restriction enzyme digests of total DNA were probed e ither with an oligonucleotide based on the 5' region of bvi79A or with dege nerate oligonucleotides based on the predicted sequence of the second pepti de.