Mode of action of a bacteriocin (J46) produced by Lactococcus lactis subsp. cremoris J46

Abstract

The mode of antibacterial action of bacteriocin J46, a bacteriotin from Lactococcus lactis subsp. cremoris, was studied. Bacteriotin J46 specifically adsorbed on susceptible bacteria. Adsorption on gram negative or gram- positive resistant strains was significantly lower. Bacteriocin J46 has been shown to be bactericidal towards log-phase indicator cells but ineffective against stationary cells. In addition energy-depleted log-phase cells were unaffected. It was shown that both a ΔpH and a ΔΨ were required for bacteriocin J46 activity. Using a continuous fermentation procedure, we were able to confirm the crucial importance of the physiological state of the indicator on its sensitivity. Negligible antibacterial activity was detected against cells of the sensitive strain, L. lactis subsp. cremoris SCL l (μmax: 0.93) growing at dilution rates below about 0.6 h-1 even with high bacteriocin concentrations. When assays were conducted in a growth medium, there was a continuous decrease in bacterial viability as a function of time, suggesting that actively growing cells were sensitive to the bactericidal activity of bacteriocin J46. This antibacterial peptide caused an immediate loss of cellular K+ and a hydrolysis of internal ATP in L. lactis subsp. cremoris SCL l. The initial rate of K+ efflux increased with increasing bacteriocin concentration, being saturated at approximately 10,000 AU/ml. Decreasing the ionic strength of the assay buffer did not result in a significant modification of the K+ efflux rate induced by J46 addition. Bacteriocin activity was pH dependent: at pH 7.0, no adsorption and no antibacterial activity were detected. These results suggest that the bactericidal activity of bacteriocin J46 was due to the formation of pores in the cytoplasmic membrane of log-phase cells, stationary cells remaining unaffected.