Bacteriocin J46, a new bacteriocin produced by Lactococcus lactis subsp cremoris J46: Isolation and characterization of the protein and its gene

Lactococcus lactis subsp. cremoris strain J46, isolated from fermented milk, produced a new bacteriocin designated as bacteriocin J46. The bacteriocin demonstrated inhibitory activity towards some strains of lactobacilli, lactococci, bacilli, clostridia and Streptococcus thermophilus, and toward outgrowth of Clostridium tyrobutyricum spores. The antimicrobial substance was resistant to treatment with papain and pepsin but sensitive to pronase, trypsin and alpha-chymotrypsin. Bacteriocin J46 was heat resistant (60 min at 100 degrees C), stable over a wide range of pH (3-9) and retained the bacteriocin activity after ultrafiltration and dialysis. Sensitive Gram-positive cells, but not resistant Gram-positive and Gram-negative cells adsorbed bacteriocin J46. Bacteriocin J46 was bactericidal but not bacteriolytic to sensitive cells. Purification by ammonium sulphate precipitation, DEAE-Sephacel chromatography and gel filtration resulted in a 120-fold increase in specific activity. The purified bacteriocin was identified as a 3.0 kDa peptide by SDS-PAGE. The N-terminal portion was identical to the sequence described for the lantibiotic lacticin 481. However, contrary to lacticin 481, bacteriocin J46 is not a lantibiotic since no modified amino acids such as lanthionine or its derivatives were detected in J46. By use of a synthetic DNA probe, the structural gene (lcp J) was found to be located on a 60-kb plasmid. The lcp J gene was cloned and sequenced. The sequence data revealed that bacteriocin J46 is ribosomally synthesized as a 51-amino-acid precursor including a 24-amino-acid N-terminal extension. It also confirmed that bacteriocin J46 is a new lactococcal bacteriocin. (C) 1996 Academic Press