Characterization and purification of helveticin J and evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481

Abstract

Lactobacillus helveticus 481 produced an antimicrobial agent active against five closely related species. The sensitive indicators included L. helveticus 1846 and 1244, L. bulgaricus 1373 and 1489, and L. lactis 970. The antimicrobial compound was active at neutral pH under aerobic or anaerobic conditions, was sensitive to proteolytic enzymes and heat (30 min at 100 degrees C), and demonstrated a bactericidal mode of action against sensitive indicators. These data confirmed that antimicrobial activity of L. helveticus 481 was mediated by a bacteriocin, designated helveticin J. Production of helveticin J was maximized in an anaerobic fermentor held at a constant pH of 5.5. Ultrafiltration experiments on culture supernatants containing the bacteriocin revealed that helveticin J was present as an aggregate with a molecular weight in excess of 300,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of helveticin J purified through Sephadex chromatography resolved a 37,000-dalton protein band with bacteriocin activity. L. helveticus 481 was shown to harbor a single 8-megadalton plasmid (pMJ1008). Isolates cured of pMJ1008 were phenotypically identical to plasmid-bearing cells in fermentation patterns, helveticin J activity, and immunity spectra. The data provided evidence for a chromosomal location of helveticin J and host immunity determinants.