Proton motive force during growth of Streptococcus lactis cells

Experiments with the aerotolerant anaerobe Streptococcus lactis provide the opportunity for determining the proton motive force (Δp) in dividing cells. The two components of Δp, ΔΨ (the transmembrane potential) and ΔpH (the chemical gradient of H⁺), were determined by the accumulation of radiolabeled tetraphenylphosphonium (TPP⁺) and benzoate ions. The ΔΨ was calibrated with the K⁺ diffusion potential in starved, valinomycin-treated cells. With resting, glycolyzing cells, the Δp was measured also by the accumulation of the non-metabolizable sugar thiomethyl-β-galactoside (TMG). In resting cells the Δp, calculated either by adding ΔΨ and ZΔpH or from the levels of TMG, was relatively constant between pH 5 to 7, decreasing from 160 to 150 mV and decreasing further to 100 mV at pH 8.0. With the TPP⁺ probe for ΔΨ, we confirmed our previous finding that the K⁺ ions dissipate ΔΨ and increase ΔpH, whereas Na⁺ ions have little effect on ΔΨ and no effect on ΔpH. [³H]TPP⁺ and [¹⁴C]benzoate were added during exponential phase to S. lactis cells growing at pH 5 to 7 at 28°C in a defined medium with glucose as energy source. As with resting cells, the ΔpH and ΔΨ were dependent on the pH of the medium. At pH 5.1, the ΔpH was equivalent to 60 mV (alkaline inside) and decreased to 25 mV at pH 6.8. The ΔΨ increased from 83 mV (negative inside) at pH 5.1 to 108 mV at pH 6.8. The Δp, therefore, was fairly constant between pH 5 and 7, decreasing from 143 to 133 mV. The values for Δp in growing cells, just as in resting cells, are consistent with a system in which the net efflux of H⁺ ions is effected by a membrane-bound adenosine triphosphatase and glycolytically generated adenosine triphosphate. The data suggest that in both growing and resting cells the pH of the medium and its K⁺ concentration are the two principal factors that determine the relative contribution of ΔpH and ΔΨ to the proton motive force.     

The antibacterial activity of vancomycin towards Staphylococcus aureus under aerobic and anaerobic conditions

AIMS: To investigate the antibacterial efficacy of vancomycin towards Staphylococcus aureus under aerobic and anaerobic conditions, and to assess the influence of oxygen on the duration of the post-antibiotic effect (PAE) after exposure to vancomycin. METHODS AND RESULTS: Culture-based techniques and flow cytometric measurements of 5-cyano-2,3-ditolyl tetrazolium chloride (an indicator of redox activity) and the membrane potential-sensitive fluorophore Sytox Green, were used to test four staphylococcal strains. The MICs for all strains, and the duration of PAE, were similar whether tested with or without oxygen. However, a fivefold logarithmic reduction in cell counts was observed in 10-15 h aerobically, depending on strain, compared with longer than 60 h in an anaerobic environment. Flow cytometric data correlated well with counts of colony-forming units under both aerobic and anaerobic conditions. CONCLUSIONS: The death rate of Staph. aureus exposed to vancomycin was greater in the presence of oxygen, although MIC values and PAE durations were similar whether tested aerobically or anaerobically. Also, flow cytometry provided a rapid and sensitive alternative to plate counts for the assessment of antibiotics in oxygen-free conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study underlines the need for further anaerobic testing using different strain/antibiotic combinations, the results of which will have clinical significance due to the anaerobic nature of some sites of infection.     

Proton motive force generation from stored polymers for the uptake of acetate under anaerobic conditions

The bacteria facilitating enhanced biological phosphorus removal gain a selective advantage from intracellularly stored polymer-driven substrate uptake under anaerobic conditions during sequential anaerobic : aerobic cycling. Mechanisms for these unusual membrane transport processes were proposed and experimentally validated using selective inhibitors and highly-enriched cultures of a polyphosphate-accumulating organism, Accumulibacter, and a glycogen-accumulating organism, Competibacter. Acetate uptake by both Accumulibacter and Competibacter was driven by a proton motive force (PMF). Stored polymers were used to generate the PMF -Accumulibacter used phosphate efflux through the Pit transporter, while Competibacter generated a PMF by proton efflux through the ATPase and fumarate reductase in the reductive TCA cycle.     

Metabolic profiling of Staphylococcus aureus cultivated under aerobic and anaerobic conditions with (1)H NMR-based nontargeted analysis

Staphylococcus aureus is a major pathogen in the medical area and food-producing sector. Detailed analyses of its basic cell physiology will help comprehensively understand this pathogen, which will be useful for developing novel diagnostic and treatment tools. Oxygen is one of the most crucial growth-limiting factors for S. aureus. In this study, to characterize and distinguish metabolic profiles of S. aureus cultivated under aerobic and anaerobic conditions, nontargeted analyses of both types of cultures were carried out using (1)H nuclear magnetic resonance spectroscopy. Fifty compounds were identified by Chenomx software. Characteristics of metabolic profiles were achieved by using principal components analysis. During aerobic growth, S. aureus mainly consumed glucose, alanine, arginine, glycine, isoleucine, leucine, phenylalanine, and acetate. Meanwhile, it accumulated 17 metabolites, mainly 2-oxoglutarate, isobutyrate, isovalerate, succinate, and ethanol. Under anaerobic condition, S. aureus mainly consumed glucose, arginine, and threonine. Meanwhile, it accumulated 13 metabolites, mainly ethanol, lactate, and ornithine. The representative metabolites that could most significantly differentiate metabolic profiles of S. aureus were isobutyrate, isovalerate, and succinate in aerobic cultivation; and lactate, ethanol, and ornithine in anaerobic cultivation. Among these metabolites, isobutyrate and ornithine were present only in aerobic and anaerobic culture, respectively.     

Interaction of H+ and K+ transport systems in E. coli growing under anaerobic and aerobic conditions

The interaction of H+-ATPase complex F1 X F0 with the Trk system of K+ accumulation in E. coli grown quasi-anaerobically in pepton media with glucose (anaerobia) and aerobically in the salt medium with succinate (aerobia) treated with cyanide was studied. The ratio of H+ fluxes via F1 X F0 and K+ fluxes via the Trk system is stable and equals 2 in anaerobia and is changed from 0.5 to 5.0 in aerobia treated with cyanide in response to pH variation, K+ activity and temperature variations. Q10 is about 2.8 both for F1 X F0 and the Trk system in anaerobia, but 2.4 and 1.0 respectively in aerobia. K+ distribution in anaerobia reaches high values, K+ equilibrium potential is much higher than the measured membrane potential. K+ distribution in aerobia is smaller, which is in conformity with the measured membrane potential. Structural association of F1 X F0 and the Trk system with the formation of H+--K+-pump is assumed to take place in anaerobia, and separate operation of these systems occurs in aerobia, transfer of K+ via Trk system being energized by the electric field on the membrane.     

Proton motive force in washed cells of Rhizobium japonicum and bacteroids from Glycine max.

The components of the proton motive force (delta p), namely the membrane potential and the transmembrane pH gradient, were measured in washed cells of Rhizobium japonicum CC705 grown in cultures (5% O2-95% N2) in the presence of 10 mM KNO3 and in bacteroids from Glycine max. The delta p and its components remained reasonably constant in cells as well as in bacteroids at various stages of growth. The effects of uncouplers and ATPase inhibitors on the delta p and its components were determined in both cultured cells and bacteroids. The data indicated that a respiration-driven H+ translocation is the source of the delta p in both cultured cells and bacteroids.     

Proton motive force-driven and ATP-dependent drug extrusion systems in multidrug-resistant Lactococcus lactis.

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. Ionophores 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.     

Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides.

In Leuconostoc mesenteroides subsp. mesenteroides 19D, citrate is transported by a secondary citrate carrier (CitP). Previous studies of the kinetics and mechanism of CitP performed in membrane vesicles of L. mesenteroides showed that CitP catalyzes divalent citrate HCit2-/H+ symport, indicative of metabolic energy generation by citrate metabolism via a secondary mechanism (C. Marty-Teysset, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Biol. Chem. 270:25370-25376, 1995). This study also revealed an efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate cometabolism, suggesting that under physiological conditions, CitP may function as a precursor/product exchanger rather than a symporter. In this paper, the energetic consequences of citrate metabolism were investigated in resting cells of L. mesenteroides. The generation of metabolic energy in the form of a pH gradient (delta pH) and a membrane potential (delta psi) by citrate metabolism was found to be largely dependent on cometabolism with glucose. Furthermore, in the presence of glucose, the rates of citrate utilization and of pyruvate and lactate production were strongly increased, indicating an enhancement of citrate metabolism by glucose metabolism. The rate of citrate metabolism under these conditions was slowed down by the presence of a membrane potential across the cytoplasmic membrane. The production of D-lactate inside the cell during cometabolism was shown to be responsible for the enhancement of the electrogenic uptake of citrate. Cells loaded with D-lactate generated a delta psi upon dilution in buffer containing citrate, and cells incubated with citrate built up a pH gradient upon addition of D-lactate. The results are consistent with an electrogenic citrate/D-lactate exchange generating in vivo metabolic energy in the form of a proton electrochemical gradient across the membrane. The generation of metabolic energy from citrate metabolism in L. mesenteroides may contribute significantly to the growth advantage observed during cometabolism of citrate and glucose.     

Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions.

When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.     

Measurements of bovine sperm velocities under true anaerobic and aerobic conditions.

Velocities of bovine spermatozoa in a medium containing glucose were similar under true anaerobic and aerobic conditions. Spermatozoa were not able to sustain motility under anaerobic conditions when glycolysis was inhibited, but regained motility when re-aerated. This demonstrates that immobilisation was due to lack of oxygen and that conditions under which motility was analysed were truly anaerobic. Sperm motility parameters were not significantly different in the presence and absence of 4 microM antimycin A and 4 microM rotenone when glucose was present in the medium. After each incubation, functionality of sperm mitochondria was assayed by washing sperm into the medium which supported respiration but not glycolysis, and motility was visually assessed. All sperm samples were highly motile in this medium indicating that their mitochondria were functional. When glycolysis was inhibited, antimycin and rotenone abolished sperm motility immediately after addition. Bovine sperm can maintain similar levels of motility aerobically and anaerobically if a glycolysable substrate is available. Available data on bovine sperm energetics support this view.     

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.