Lactococcin G is a potassium ion-conducting, two-component bacteriocin

Abstract

Lactococcin G is a novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides, termed ␣ and ␤. Peptide synthesis of the ␣ and ␤ peptides yielded biologically active lactococcin G, which was used in mode-of-action studies on sensitive cells of Lactococcus lactis. Approximately equivalent amounts of both peptides were required for optimal bactericidal effect. No effect was observed with either the ␣ or ␤ peptide in the absence of the complementary peptide. The combination of ␣ and ␤ peptides (lactococcin G) dissipates the membrane potential (⌬), and as a consequence cells release ␣-aminoisobutyrate, a nonmetabolizable alanine analog that is accumulated through a proton motive-force dependent mechanism. In addition, the cellular ATP level is dramatically reduced, which results in a drastic decrease of the ATP-driven glutamate uptake. Lactococcin G does not form a proton-conducting pore, as it has no effect on the transmembrane pH gradient. Dissipation of the membrane potential by uncouplers causes a slow release of potassium (rubidium) ions. However, rapid release of potassium was observed in the presence of lactococcin G. These data suggest that the bactericidal effect of lactococcin G is due to the formation of potassium-selective channels by the ␣ and ␤ peptides in the target bacterial membrane.Bacteriocins produced by lactic acid bacteria are peptides displaying bactericidal activity against gram-positive bacteria, particularly closely related species. The study of such antibacterial agents is of interest because of their potential application as food additives. Most bacteriocins produced by lactic acid bacteria are small peptides with sizes of 35 to 60 amino acid residues. The antimicrobial activities of most bacteriocins studied so far require the action of a single peptide, which is thought to form nonselective pores according to the ''barrel stave '' mechanism (19). Bacteriocin activity of lactococcin G is associated with the complementary action of two peptides termed ␣ and ␤ (17). The ␣ and ␤ peptides have molecular masses of 4,346 and 4,110 Da, consist of 39 and 35 amino acids, and have isoelectric points of 10.9 and 10.4, respectively. The amino-terminal halves of both peptides may form amphiphilic ␣ helices and may oligomerize in such a way that the nonpolar side of the amphiphilic ␣-helix region faces the membrane lipids, while the polar side faces the center of the pore, as described for the barrel stave mechanism.Peptide synthesis yielded biologically active lactococcin G which was used to study the impact of lactococcin G on the energy-transducing properties of sensitive cells of Lactococcus lactis. Our data suggest that lactococcin G has a novel bactericidal activity in forming potassium-selective channels in the target cells rather than nonselective pores. MATERIALS AND METHODSBacteriocin assay. Bacteriocin activity was measured as previously described (17), using a microtiter plate assay system. Briefly, 200 l of culture medium (supplemented with 0.1% [vol/v...

Assessment of viability of microorganisms employing fluorescence techniques

“…The properties of the ideal membrane potential probe are discussed by Lolkema (66) and summarized here: (i) the probe should pass rapidly the membrane, (ii) it should not bind to the membrane or other constituents, (iii) it should be detectable at very low concentrations, and (iv) it should be biologically inert. Distributional fluorescent probes applied in microbiology are Rhodamine 123, positively charged carbocyanines such as 3,3-dihexyloxacarbocyanine iodide (DiOC 6 (3)), 3,3-diethyloxacarbocyanine iodide (DiOC 2 (3)), and 3,3'-dipropylthiadicarbocyanine iodide (DiS 3 (5), and the negatively charged bis-(l,3-dibutylbarbituric acid)trimethine oxonol (DiBAC 4 (3)) (23,51,55,71,72,73,83,94,97,142,145,154). Rhodamine 123, DiOC 6 (3) and DiBAC 4 (3) can all be excited by the 488 nm line and emit green fluorescence.…”

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“…19 Another hypothesis is that very small amounts of contaminating brochocin B or another enterocin NKR-5-3A enhancing substance, which could not be purified and detected by our purification procedure, are present in the purified enterocin NKR-5-3A sample, perhaps due to the hydrophobic nature of the peptide. The synergistic phenomenon has been reported on many two-peptide bacteriocins such as brochocin C, 11 enterocin L50A and enterocin L50B, 14 thermophilin 13, 15 plantaricins EF and JK, 16 anti-Staphylococcus aureus lantibiotic, 17 plantaricin W 20 and lactococcin G. 21 Although there is a firm understanding of the molecular recognition responsible in some two-peptide bacteriocins, the synergistic mechanism in those twopeptide is far from understood.…”

Structure analysis of the two-peptide bacteriocin lactococcin G by introducing d-amino acid residues

“…It was the first two-peptide bacteriocin to be isolated and is perhaps the one that at present is best characterized (Hauge et al, 1998;Moll et al, 1996Moll et al, , 1998Nissen-Meyer et al, 1992;Oppegård et al, 2007Oppegård et al, , 2008Rogne et al, 2008). The bacteriocin consists of two peptides, LcnG-a (39 amino acid residues) and LcnG-b (35 amino acid residues) ( Fig.…”

“…The bacteriocin consists of two peptides, LcnG-a (39 amino acid residues) and LcnG-b (35 amino acid residues) ( Fig. 1), and both peptides must be present in about equal molar amounts in order to obtain optimal antimicrobial activity (Moll et al, 1996;Nissen-Meyer et al, 1992). The genes encoding the two peptides are located next to each other on the same operon, along with the gene encoding the lactococcin G immunity protein (Håvarstein et al, 1995;Nes et al, 1995), which protects the producer cell from being killed by its own bacteriocin.…”

“…The genes encoding the two peptides are located next to each other on the same operon, along with the gene encoding the lactococcin G immunity protein (Håvarstein et al, 1995;Nes et al, 1995), which protects the producer cell from being killed by its own bacteriocin. The bacteriocin causes cell death by inducing membrane leakage of small monovalent cations, such as Na + , K + , Li + , Cs + and Rb + , but not H + (Moll et al, 1996(Moll et al, , 1998. Circular dichroism (CD) and NMR spectroscopy show that the two peptides, LcnG-a and LcnG-b, are unstructured in aqueous environments, but adopt mainly a-helical structures when they are individually exposed to membranes or membranemimicking environments (Hauge et al, 1998;Rogne et al, 2008).…”