Enterocin P Selectively Dissipates the Membrane Potential of Enterococcus faecium T136

Abstract

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (⌬⌿) and the intracellular ATP pool of the indicator organism, the pH gradient (⌬pH) component of the proton motive force (⌬p) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.Bacteriocins produced by bacteria are a heterogeneous group of ribosomally synthesized antimicrobial proteins, which display antimicrobial activity against other bacteria (16,18,29). The genus Enterococcus is among the lactic acid bacteria (LAB) associated with foods that produce bacteriocins (2,4,6,11,12). Enterococcus faecium P13 and other E. faecium strains isolated from dry-fermented sausages produce enterocin P, a bacteriocin with strong inhibitory activity against Listeria monocytogenes (11,14). Enterocin P is a pediocin-like bacteriocin (27) with an N-terminal signal peptide which may allow it to be secreted via the sec pathway (for a review, see reference 30). The enterocin P operon has been previously characterized (11), and it consists of the bacteriocin structural gene (entP) encoding a 71-amino-acid precursor with a 27-amino-acid signal peptide and has a second open reading frame (orf2) located immediately downstream of entP and potentially encoding the immunity protein.The increasing interest of consumers in minimally processed, naturally preserved foods has prompted the proposal to use bacteriocinogenic LAB or their bacteriocins as biopreservatives to increase microbiological safety (19,25,31). However, rational application of the bacteriocins requires an understanding of the mechanisms underlying their specificity and activity (24, 26), as well as knowledge of the structure-function relationships, to develop new compounds with an improved efficacy. In this context, the objective of this study was to examine the mechanistic action of enterocin P on the sensitive strain E. faecium T136. MATERIALS AND METHODSBacterial strains and growth conditions. E. faecium P13 (11) and E. faecium T136 (6) were propagated in MRS broth (Difco Laboratories, Detroit, Mich.) at 30°C and used as the enterocin P producer and indicator microorganisms for bacteriocin activity, respectively.Purification of enterocin P. The bacteriocin was purified from the supernatant of a 16-h E. faecium P13 culture by hydrophobic interaction and cation-exchange chromatographies, basically as described by Casaus et al. (6). Next, the sample was dialyzed against distilled water in dialysis tubing (Spectrum, Houston, Tex.; molecular weight cutoff, 1,000) and concentrated with polyethylene glycol to half of its original volume. This sample was further purified by preparative isoelectric focusing using a Rotofor cel...

Antilisterial activity of lactic acid bacteria against Listeria monocytogenes strains originating from different sources

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Antibacterial mode of action of <i>Ginkgo biloba</i> leaf essential oil: Effect on morphology and membrane permeability

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