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...

Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is the sole energy-coupling ion. To test whether sodium is the preferred coupling ion at high temperatures, the proton- and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature. The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures. Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature. In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature. The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.

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Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration. A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter. The longest-lived radioactive isotope of nitrogen, 13N-nitrate (half-life = 9.96 min) and the nitrite-sensitive fluorophore N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide have now been used to define the function of NarK. At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange. This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase.

Transport of acidic amino acids in Bacillus subtilis is an electrogenic process in which L-glutamate or L-aspartate is symported with at least two protons. This is shown by studies of transport in membrane vesicles in which a proton motive force is generated by oxidation of ascorbate-phenazine methosulfate or by artificial ion gradients. An inwards-directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake. The transporter is specific for L-glutamate and L-aspartate. L-Glutamate transport is inhibited by ␤-hydroxyaspartate and cysteic acid but not by ␣-methyl-glutamate. The gene encoding the L-glutamate transport protein of B. subtilis (gltP Bsu ) was cloned by complementation of Escherichia coli JC5412 for growth on glutamate as the sole source of carbon, energy, and nitrogen, and its nucleotide sequence was determined. Putative promoter, terminator, and ribosome binding site sequences were found in the flanking regions. UUG is most likely the start codon. gltP Bsu encodes a polypeptide of 414 amino acid residues and is homologous to several proteins that transport glutamate and/or structurally related compounds such as aspartate, fumarate, malate, and succinate. Both sodium-and proton-coupled transporters belong to this family of dicarboxylate transporters. Hydropathy profiling and multiple alignment of the family of carboxylate transporters suggest that each of the proteins spans the cytoplasmic membrane 12 times with both the amino and carboxy termini on the inside.The amino acid transporters in the thermophile Bacillus stearothermophilus studied to date facilitate an electrogenic symport reaction in which Na ϩ is used as the coupling ion. The apparent affinity constants for Na ϩ are in the range of 0.5 to 1 mM (14). The transport of glutamate and aspartate is driven by the proton motive force (⌬p) but also by an inwardly directed Na ϩ gradient (⌬pNa). The transport of glutamate occurs most likely in symport with one H ϩ and one Na ϩ (7); the apparent affinity constant for Na ϩ is Ͻ10 M. So far, sodium/proton/ glutamate transporters have been found in the thermophiles Bacillus sp. strain IS1 (gltT Bi ) (42), B. stearothermophilus (gltT Bs ), and Bacillus caldotenax (gltT Bc ). The genes encoding GltT Bs and GltT Bc have been cloned and functionally expressed in Escherichia coli (43).Studies on the transport of L-glutamate and L-aspartate in whole cells of B. subtilis W23, 60015, 6GM, and 8G5 suggested that ⌬pNa is not involved as a driving force in this mesophilic Bacillus species (41). The glutamate transporter of B. subtilis is likely to differ from those of thermophilic bacilli with respect to not only cation selectivity but also thermostability. In order to compare the sodium/proton/glutamate symport protein of the thermophile B. stearothermophilus with the glutamate transport protein of the closely related mesophile B. subtilis, the latter system was studied at the molecular level. This study confirms that glutamate uptake in B. subtilis is indeed coupled to th...