Nisin is a cationic polycyclic bacteriocin secreted by some lactic acid bacteria. Nisin has previously been shown to permeabilize liposomes. The interaction of nisin was analyzed with liposomes prepared of the zwitterionic phosphatidylcholine (PC) and the anionic phosphatidylglycerol (PG). Nisin induces the release of 6-carboxyfluorescein and other small anionic fluorescent dyes from PC liposomes in a delta psi-stimulated manner, and not that of neutral and cationic fluorescent dyes. This activity is blocked in PG liposomes. Nisin, however, efficiently dissipates the delta psi in cytochrome c oxidase proteoliposomes reconstituted with PG, with a threshold delta psi requirement of about -100 mV. Nisin associates with the anionic surface of PG liposomes and disturbs the lipid dynamics near the phospholipid polar head group-water interface. Further studies with a novel cationic lantibiotic, epilancin K7, indicate that this molecule penetrates into the hydrophobic carbon region of the lipid bilayer upon the imposition of a delta psi. It is concluded that nisin acts as an anion-selective carrier in the absence of anionic phospholipids. In vivo, however, this activity is likely to be prevented by electrostatic interactions with anionic lipids of the target membrane. It is suggested that pore formation by cationic (type A) lantibiotics involves the local perturbation of the bilayer structure and a delta psi-dependent reorientation of these molecules from a surface-bound into a membrane-inserted configuration.

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...

Three mutants of Lactococcus lactis subsp. lactis MG1363, termed EthR, DauR, and RhoR, were selected for resistance to high concentrations of ethidium bromide, daunomycin, and rhodamine 6G, respectively. These mutants were found to be cross resistant to a number of structurally and functionally unrelated drugs, among which were typical substrates of the mammalian multidrug transporter (P-glycoprotein) such as daunomycin, quinine, actinomycin D, gramicidin D, and rhodamine 6G. The three multidrug-resistant strains showed an increased rate of energy-dependent ethidium and daunomycin efflux compared with that of the wild-type strain. This suggests that resistance to these toxic compounds is at least partly due to active efflux. Efflux of ethidium from the EthR strain could occur against a 37-fold inwardly directed concentration gradient. In all strains, ethidium efflux was inhibited by reserpine, a well-known inhibitor of P-glycoprotein. lonophores which selectively dissipate the membrane potential or the pH gradient across the membrane inhibited ethidium and daunomycin efflux in the wild-type strain, corresponding with a proton motive force-driven efflux system. The ethidium efflux system in the EthR strain, on the other hand, was inhibited by ortho-vanadate and not upon dissipation of the proton motive force, which suggests the involvement of ATP in the energization of transport.The partial inhibition of ethidium efflux by ortho-vanadate and nigericin in the DauR and RhoR strains suggest that a proton motive force-dependent and an ATP-dependent system are expressed simultaneously. This is the first report of an ATP-dependent transport system in prokaryotes which confers multidrug resistance to the organism.Multidrug resistance (MDR) is the intrinsic or acquired resistance of cells to various structurally and functionally unrelated toxic compounds. For various mammalian cells, it has been established that MDR is the result of active extrusion of drugs from the cells, a process that is catalyzed by an ATP-dependent transport protein termed P-glycoprotein or MDR1 (14). P-glycoprotein confers resistance to vinca alkaloids, anthracyclines, actinomycin D, valinomycin, gramicidin D, and phosphonium ions (4, 7, 9). P-glycoprotein is classified among members of the ATP-binding cassette (ABC) proteins (15) or traffic ATPases (26), to which belong prokaryotic and eukaryotic transport systems that facilitate either uptake or extrusion of substrates. Because of the importance of MDR in the failure of drug-based treatment of cancers and parasital infections, most attention has been focused on eukaryotic MDR systems. Recently, however, several MDR-like systems in both gram-positive (31, 42) and gram-negative (11, 20, 23) bacteria as well as in archaea (27) have been described. The occurrence of acquired resistance in bacteria, and especially in pathogenic bacteria like enterococci, staphylococci (35), and Mycobacterium tuberculosis (3), also poses serious problems to public health. MDR in these organisms is believed to be the...

Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics. It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential (⌬) and that involves local pertubation of the lipid bilayer structure. Here we show that nisin does not form exclusively voltage-dependent pores: even in the absence of a ⌬, nisin is able to dissipate the transmembrane pH gradient (⌬pH) in sensitive Lactococcus lactis cells and proteoliposomes. The rate of dissipation increases with the magnitude of the ⌬pH. Nisin forms pores only when the ⌬pH is inside alkaline. The efficiency of ⌬-induced pore formation is strongly affected by the external pH, whereas ⌬pH-induced pore formation is rather insensitive to the external pH. Lantibiotics are posttranslationally modified peptide antibiotics that owe their name to the presence of cyclic structures formed by lanthionines or 3-methyllanthionine residues. The lantibiotic nisin is produced by Lactococcus lactis subsp. lactis and has five such intramolecular rings, termed A, B, C, D, and E, in a total of 34 amino acids. Because of its bactericidal activity against a broad range of gram-positive bacteria, nisin is exploited as a food preservative. Its genetics (23) as well as the regulation of its synthesis (22) are known, and several mutants have been generated (24)(25)(26).Nisin forms nonselective, transient, multistate pores in membranes (38)(39)(40). In addition to having bactericidal activity, it inhibits the outgrowth of spores (11,18,29,33) and interferes with the activity of biosynthetic (36) and autolytic (2-5) enzymes. Nisin is able to form pores in cells, proteoliposomes, liposomes, and black lipid membranes. It has been reported that the pores are formed only when the transmembrane electrical potential (⌬), negative inside, is sufficiently high (15,38,39). In cells, pore formation induces the release of ions, amino acids, and ATP and causes the collapse of the proton motive force (⌬p) (7,16,21,35,37,39,40). Nisin requires anionic phospholipids for membrane binding and pore formation (12,15), while in the absence of anionic phospholipids, it acts as an anion carrier (15,17).The structure of nisin in solution (28,44) and in the bound state to micelles of dodecylphosphocholine and of sodium dodecyl sulfate (42, 43) has been studied by nuclear magnetic resonance (NMR) analysis. Nisin is a flexible molecule in solution but contains two relatively structured domains: an amino-terminal domain (residues 3 to 19) containing rings A, B, and C, in which rings A and B are particularly well structured; and a carboxyl-terminal domain (residues 22 to 28) containing rings D and E. Both domains are amphipathic, a property consistent with the ability of nisin to interact with phospholipid membranes. Based on structural and functional studies, we have proposed a wedge model for pore formation by nisin (15, 32a). A wedge-like pore composed of multiple nisin molecules may be formed once the ⌬ drives the membrane insertio...

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