Identification of bacteriocin-like inhibitors from rumen Streptococcus spp. and isolation and characterization of bovicin 255

Streptococci obtained from rumen sources were tested for the production of antibacterial compounds using a deferred-antagonism plating assay. Of 35 is olates tested, 7 were identified that inhibited the growth of other strepto cocci, None of the inhibitory activity was due to bacteriophage. Three isol ates, LRC0253, LRC0255, and LRC0476, were selected for further characteriza tion. Analysis of 16S ribosomal DNA indicated that LRC0476 was a strain of Streptococcus bovis, while isolates LRC0253 and LRC0255 are likely strains of Streptococcus gallolyticus. The antibacterial compounds produced by thes e bacteria were protease sensitive, remained active in a pH range from 1 to 12, and did not lose activity after heating at 100 degreesC for 15 min. Th e inhibitory peptide from strain LRC0255 was purified using pH-dependent ad sorption and desorption to bacterial cells, followed by ammonium sulfate pr ecipitation and reversed-phase chromatography and gel filtration. The pepti de was 6 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel el ectrophoresis, An oligonucleotide probe based on the N-terminal sequence of the purified peptide was used to identify the gene encoding the inhibitory peptide. The antibacterial peptide has characteristics that are very simil ar to those described for class II bacteriocins of gram-positive bacteria.