Sodium Ion Gradient Research Articles

Pompes d’efflux et résistance aux antibiotiques chez les bactéries

Bacteria can resist to antibiotics by active exportation mediated by membrane transporters called efflux pumps. These proteins can be specific of a class of antibiotics or responsible for multidrug resistance (MDR). Energy required by efflux pumps can be provided by transmembrane electrochemical gradient of protons (MFS, RND, SMR families) or sodium ions (MATE family) or by ATP hydrolysis (ABC family). Several physiological functions have been described in prokaryotes, such as protection from environmental toxics and regulation of cell homeostasis, which can indirectly contributes to bacterial virulence. In Gram-negative bacteria, efflux transporters usually are organized as multicomponent systems in wich the efflux pump located in the inner membrane works in conjunction with a periplasmic fusion protein and an outer membrane factor. The most frequently encountered pumps are of the RND-type such as AcrB in Escherichia coli or MexB in Pseudomonas aeruginosa. In Gram-positive bacteria, efflux is solely mediated by the pump protein, so described with MFS pumps such as NorA or QacA in Staphylococcus aureus and PmrA in Streptococcus pneumoniae. Efflux transporters have also been described in mycobacteria. Although numerous bacterial pumps have been characterized, the clinical consequences of efflux-mediated resistance are mostly unknown because of variable levels of expression and of the lack of specific markers in laboratory practice. Finally, associating pump-specific inhibitors to efflux-sensitive antibiotics might prove an interesting therapeutic perspective. However, inhibitors that are not toxic to eukaryotic cells remain to be identified.

Solubilization and purification of the MotA/MotB complex of Escherichia coli.

Bacterial flagella are driven at their base by a rotary motor fueled by the membrane gradient of protons or sodium ions. The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which function together to conduct ions across the membrane and couple ion flow to rotation. An invariant aspartate residue in MotB (Asp32 in the protein of E. coli) is essential for rotation and appears to have a direct role in proton conduction. A recent study showed that changes at Asp32 in MotB cause a conformational change in the complex, as evidenced by altered patterns of protease susceptibility of MotA [Kojima, S., and Blair, D. F. (2001) Biochemistry 40 (43), 13041-13050]. It was proposed that protonation/deprotonation of Asp32 might regulate a conformational change in the stator that acts as the powerstroke to drive rotation of the rotor. Biochemical studies of the MotA/MotB complex have been hampered by the absence of a suitable assay for its integrity in detergent solution. Here, we have studied the behavior of the MotA/MotB complex in a variety of detergents, making use of the protease-susceptibility assay to monitor its integrity. Among about 25 detergents tested, a few were found to solubilize the proteins effectively while preserving certain conformational properties characteristic of an intact complex. The detergent dodecylphosphocholine, or DPC, proved especially effective. MotA/MotB complexes purified in DPC migrate with an apparent size of approximately 300 kDa in gel-filtration columns, and retain the Asp32-modulated conformational differences seen in membranes. (35)S-radiolabeling showed that MotA and MotB are present in a 2:1 ratio in the complex. Purified MotA/MotB complexes should enable in vitro study of the proton-induced conformational change and other aspects of stator function.

Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1.

We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1). After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi). A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

Sodium ion cycling mediates energy coupling between complex I and ATP synthase.

We show here sodium ion cycling between complex I from Klebsiella pneumoniae and the F(1)F(0) ATP synthase from Ilyobacter tartaricus in a reconstituted proteoliposome system. In the course of NADH oxidation by complex I, an electrochemical sodium ion gradient was established and served as a driving force for the synthesis of ATP from ADP and phosphate. In the opposite direction, the deltamu(Na(+)) generated by ATP hydrolysis could be coupled to NADH formation by reversed electron transfer from ubiquinol to NAD. For reverse electron transfer, a transmembrane voltage larger than 30 mV was obligatory. No NADH-driven proton transport into the lumen of proteoliposomes was detected. We conclude that Na(+) is used as the exclusive coupling ion by the enterobacterial complex I.

Cell volume homeostasis: Ionic and nonionic mechanisms: The sodium pump in the emergence of animal cells

Plant cells and bacterial cells are surrounded by a massive polysaccharide wall, which constrains their high internal osmotic pressure (tens of atmospheres). Animal cells, in contrast, are in osmotic equilibrium with their environment, have no restraining surround, and can take on a variety of shapes and can change these from moment to moment. This osmotic balance is achieved, in the first place, by the action of the energy-consuming sodium pump, one of the P-type ATPase transport protein family, members of which are found also in bacteria. The pump's action brings about a transmembranal electrochemical gradient of sodium ions, harnessed in a range of transport systems which couple the dissipation of this gradient to establishing a gradient of the coupled substrate. These transport systems include many which are responsible for short-term regulation of the cell's volume in response to acute changes of their osmotic balance. Thus, the primary role of the sodium pump as a regulator of cell volume has been built upon to provide the basis for an enormous variety of physiological functions.

Targeted disulfide cross-linking of the MotB protein of Escherichia coli: evidence for two H(+) channels in the stator Complex.

Bacterial flagella are turned by rotary motors that obtain energy from the membrane gradient of protons or sodium ions. The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes that contain multiple copies of each protein. The complexes conduct ions across the membrane, and couple ion flow to motor rotation by a mechanism that appears to involve conformational changes [Kojima, S., and Blair, D. F. (2001) Biochemistry 40, 13041-13050]. Structural information on the MotA/MotB complex is very limited. MotA has four membrane-spanning segments, and MotB has one. We have begun a targeted disulfide-cross-linking study to probe the arrangement of membrane segments in the MotA/MotB complex, beginning with the single membrane segment of MotB. Cys residues were introduced in 21 consecutive positions in the segment, and disulfide cross-linking was studied in MotA/MotB complexes either in membranes or detergent solution. Most of the Cys-substituted MotB proteins formed disulfide-linked dimers in significant yield upon oxidation. The yield of dimer varied regularly with the position of the Cys substitution, following the pattern expected for a parallel, symmetric dimer of alpha-helices. In a structural model based on the cross-linking experiments, critical Asp32 residues that are believed to facilitate proton movement are positioned on separate surfaces of the MotB dimer and so probably function within two distinct proton channels. Regions accessible to solvent were mapped by measuring the reactivity of introduced Cys residues toward N-ethyl maleimide and a charged methanethiosulfonate reagent. Positions near the middle of the segment were inaccessible to sulhydryl reagents. Positions within 6-8 residues of either end, which includes residues around Asp32, were accessible.

Structure and function of mammalian sodium-dependent multivitamin transporter.

This review focuses on the advances made in recent years in the understanding of the multivitamin transporter, a unique and important transporter that transports not one but three different unrelated water-soluble vitamins namely, pantothenate, biotin and lipoate. The transport process mediated by the transporter is active and is energized by the transmembrane sodium ion gradient as well as the membrane potential. The transporter belongs to the sodium-coupled glucose transporter family. The ubiquitous expression of this transporter in mammalian tissues and the fact that it is highly conserved across the species indicate the nutritional relevance and importance of this transporter.

Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor.

Many motile species of bacteria are propelled by flagella, which are rigid helical filaments turned by rotary motors in the cell membrane. The motors are powered by the transmembrane gradient of protons or sodium ions. Although bacterial flagella contain many proteins, only three-MotA, MotB and FliG-participate closely in torque generation. MotA and MotB are ion-conducting membrane proteins that form the stator of the motor. FliG is a component of the rotor, present in about 25 copies per flagellum. It is composed of an amino-terminal domain that functions in flagellar assembly and a carboxy-terminal domain (FliG-C) that functions specifically in motor rotation. Here we report the crystal structure of FliG-C from the hyperthermophilic eubacterium Thermotoga maritima. Charged residues that are important for function, and which interact with the stator protein MotA, cluster along a prominent ridge on FliG-C. On the basis of the disposition of these residues, we present a hypothesis for the orientation of FliG-C domains in the flagellar motor, and propose a structural model for the part of the rotor that interacts with the stator.

Activation of the sodium/hydrogen exchanger via the fibronectin-integrin pathway results in hematopoietic stimulation.

The proliferative response of hematopoietic cells is regulated by many factors, including the presence and type of growth factors, the cellular microenvironment, and the physiochemical conditions prevailing in the tissue milieu. A process fundamental to all cells is the regulation of the intracellular acid-base conditions. One of the mechanisms by which intracellular pH (pHi) is regulated is through the sodium/hydrogen exchanger, a ubiquitous membrane protein which exploits the intra- and extracellular sodium ion gradient to drive hydrogen ions out of the cell. However, activation of the exchanger via mitogenic and nonmitogenic signals leads to an increase in pHi which, in turn, may directly or indirectly result in a proliferative response. It has been shown that interaction of fibronectin with its integrin receptor subunits alpha4 and alpha5 can result in activation of the Na+/H+ exchanger. In this report, we demonstrate that when mouse bone marrow cells are physically brought together in a preculture system we designate as high cell density culture (HCDC), in a small volume and at the same cellularity as that in the marrow, hematopoietic stem and progenitor cell populations are stimulated with no additional stimulation in the presence of growth factors. Neutralizing antibodies to the growth factors added to HCDC had little, if any, effect on the degree of stimulation. However, when antibodies to fibronectin or the alpha4 integrin subunit were added to HCDC, inhibition was observed, indicating that the observed hematopoietic stimulation occurred via the fibronectin-integrin pathway. Addition of 5 microM 5-(N,N-hexamethylene) amiloride (5-HMA), a specific inhibitor of the Na+/H+ exchanger, also resulted in inhibition of in vitro hematopoiesis. Since the exchanger was implicated, we then measured the pHi of normal and HCDC-treated bone marrow cells in the absence and presence of 5-HMA by flow cytometry using the fluorescent pH-sensitive indicator, carboxy SNARF-1 AM. It was found that cells subjected to HCDC exhibited a higher pH than normal fresh cells. In each case, the pH was lowered in the presence of 5-HMA. Furthermore, addition of antibodies to fibronectin or the alpha4 integrin subunit to HCDC also reduced the pH, to a similar level to that found for 5-HMA. Our results demonstrate, for the first time, that a hematopoietic stem and progenitor cell proliferative response can be initiated by activation of the Na+/H+ exchanger, leading to an increase in pHi, via cell-cell interaction through the fibronectin-integrin pathway. This pathway could, therefore, be significant not only in normal hematopoietic regulation, but also under pathophysiological conditions.

Energy conservation in the decarboxylation of dicarboxylic acids by fermenting bacteria.

Decarboxylation of dicarboxylic acids (oxalate, malonate, succinate, glutarate, and malate) can serve as the sole energy source for the growth of fermenting bacteria. Since the free energy change of a decarboxylation reaction is small (around -20 kJ per mol) and equivalent to only approximately one-third of the energy required for ATP synthesis from ADP and phosphate under physiological conditions, the decarboxylation energy cannot be conserved by substrate-level phosphorylation. It is either converted (in malonate, succinate, and glutarate fermentation) by membrane-bound primary decarboxylase sodium ion pumps into an electrochemical gradient of sodium ions across the membrane; or, alternatively, an electrochemical proton gradient can be established by the combined action of a soluble decarboxylase with a dicarboxylate/monocarboxylate antiporter (in oxalate and malate fermentation). The thus generated electrochemical Na+ or H+ gradients are then exploited for ATP synthesis by Na+- or H+-coupled F1F0 ATP synthases. This new type of energy conservation has been termed decarboxylation phosphorylation and is responsible entirely for ATP synthesis in several anaerobic bacteria.

High-affinity glutamate transporters in the rat retina: a major role of the glial glutamate transporter GLAST-1 in transmitter clearance.

Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. The reverse transcriptase-polymerase chain reaction (RT-PCR) has revealed the presence of three different high-affinity glutamate transporters in the rat retina, viz. GLAST-1, GLT-1 and EAAC-1. No message has been found in the retina for EAAT-4, a transporter recently cloned from human brain. By using membrane vesicle preparations of total rat retina, we show that glutamate uptake in the retina is a high-affinity electrogenic sodium-dependent transport process driven by the transmembrane sodium ion gradient. Autoradiography of intact and dissociated rat retinae indicates that glutamate uptake by Müller glial cells dominates total retinal glutamate transport and that this uptake is strongly influenced by the activity of glutamine synthetase. RT-PCR, immunoblotting and immunohistochemistry have revealed that Müller cells express only GLAST-1. The Km for glutamate of GLAST-1 is 2.1+/-0.4 microM. This study suggests a major role for the Müller cell glutamate transporter GLAST-1 in retinal transmitter clearance. By regulating the extracellular glutamate concentration, the action of GLAST-1 in Müller cells may extend beyond the protection of neurons from excitotoxicity; we suggest a mechanism by which Müller cell glutamate transport might play an active role in shaping the time course of excitatory transmission in the retina.

A study of the Na /H antiport in the archaeon Methanobacterium thermoautotrophicum strain ΔH

The ability of the cells of Mb. thermoautotrophicum strain delta H to generate a proton gradient (driven by a concentration gradient of sodium ions) at pH 6.8 as well as at pH 8 was demonstrated. The electrogenic Na+/H+ antiport responsible for this process was shown to be inhibited by EIPA and also by DCCD. Artificially increasing of intracellular concentration of Ca2+ in these cells enhanced the Na+/H+ antiport activity, while the lowering of external Ca2+ by EGTA significantly decreased this activity. The apparent K(m) values for Na+ about 14 and 3 mM at pH 6.8 and 8, respectively, and Vmax about 214 (pH 6.8) and 155 (pH 8) delta Q/min per mg of cell proteins, respectively, were calculated. It is concluded that the described processes are mediated by the Na+/H+ antiporter which might be a clue to the adaptive bioenergetic behaviour of the cells of Mb. thermoautotrophicum strain delta H under the different physiological conditions.

Generation of a proton motive force by the anaerobic oxalate-degrading bacterium Oxalobacter formigenes.

The generation of transmembrane ion gradients by Oxalobacter formigenes cells metabolizing oxalate was studied. The magnitudes of both the transmembrane electrical potential (delta psi) and the pH gradient (internal alkaline) decreased with increasing external pH; quantitatively, the delta psi was the most important component of the proton motive force. As the extracellular pH of metabolizing cells was increased, intracellular pH increased and remained alkaline relative to the external pH, indicating that O. formigenes possesses a limited capacity to regulate internal pH. The generation of a delta psi by concentrated suspensions of O. formigenes cells was inhibited by the K+ ionophore valinomycin and the protonophore carbonyl cyanide-m-chlorophenylhydrazone, but not by the Na+ ionophore monensin. The H+ ATPase inhibitor N,N'-dicyclohexyl-carbodiimide inhibited oxalate catabolism but did not dissipate the delta psi. The results support the concept that energy from oxalate metabolism by O. formigenes is conserved not as a sodium ion gradient but rather, at least partially, as a transmembrane hydrogen ion gradient produced during the electrogenic exchange of substrate (oxalate) and product (formate) and from internal proton consumption during oxalate decarboxylation.

Oral Absorption of Anti-Acquired Immune Deficiency Syndrome Nucleoside Analogues. 2. Carrier-Mediated Intestinal Transport of Stavudine in Rat and Rabbit Preparations

AbstractThe intestinal transport and metabolism of stavudine (d4T), a nucleoside analogue of thymidine used in the treatment of AIDS, was studied using single-pass intestinal perfusion (SPIP), intestinal brush-border membrane vesicles (BBMV), and mucosal homogenates in rats and rabbits. In the SPIP, d4T demonstrated concentration-dependent mean wall permeability (P *w) at perfusate concentrations ranging from 0.001 to 25mM. In coperfusion studies using 0.1mM thymidine, 1mM formycin B, or 5 μM NBTI as putative inhibitors of d4T transport, the P *w of 5 μM d4T was reduced to 48%, 62%, and 70% of the control value, respectively, suggesting the involvement of multiple nucleoside carriers in the intestinal uptake of d4T. d4T uptake in rat BBMV was significantly greater in the presence of a sodium ion gradient compared with a sodium-free (choline) gradient. The permeability of d4T, in the presence of a sodium gradient, was concentration-dependent and inhibited by 10mM thymidine but not significantly reduced by 10mM formycin B. In the presence of 10 μM NBTI, the permeability of d4T was not inhibited; however, the binding of d4T to rat and rabbit BBMV was significantly reduced. Formycin B did not significantly reduce the d4T uptake in rat or rabbit BBMV suggesting that d4T does not interact with the purine-selective N1 nucleoside carrier. However, because formycin B inhibited d4T uptake in the SPIP and since d4T inhibited formycin B uptake in rat but not rabbit BBMV, it appears to interact with the N3 carrier which has been demonstrated in rat but not rabbit intestine. Also, an interaction with the sodium-independent facilitative transporter at the basolateral membrane cannot be ruled out. The low hybrid Km and high passive permeability of d4T likely account for the lack of saturable absorption behavior observed in humans, whereas the brush-border and intracellular stability of d4T preserve the high bioavailability observed after oral dosing.

Pathways of energy conservation in methanogenic archaea

Methanogenic archaea are strictly anaerobic organisms that derive their metabolic energy from the conversion of a restricted number of substrates to methane. H2+CO2 and formate are converted to CH4 via the CO2-reducing pathway, while methanol and methylamines are metabolized by the methylotrophic pathway. A limited number of methanogenic organisms utilize acetate by the aceticlastic pathway. Redox reactions involved in these processes are partly catalyzed by membrane-bound enzyme systems that generate or, in the case of endergonic reactions, use electrochemical ion gradients. The H2:heterodisulfide oxidoreductase, the F420H2:heterodisulfide oxidoreductase and the CO:heterodisulfide oxidoreductase, are novel systems that generate a proton motive force by redox-potential-driven H+ translocation. The methyltetrahydromethanopterin:coenzyme M methyltransferase is a unique, reversible sodium ion pump that couples methyl transfer with the transport of Na+ across the cytoplasmic membrane. Formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation, of methanofuran. In summary, the pathways are coupled to the generation of an electrochemical sodium ion gradient and an electrochemical proton gradient. Both ion gradients are used directly for ATP synthesis via membrane integral ATP synthases. The function of the above-mentioned systems and their components in the metabolism of methanogens are described in detail.

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.

The sodium pump in the evolution of animal cells

Plant cells and bacterial cells are surrounded by a massive cellulose wall, which constrains their high internal osmotic pressure (tens of atmospheres). Animal cells, in contrast, are in osmotic equilibrium with their environment, have no restraining surround, can take on a variety of shapes and change these from moment to moment. This osmotic balance is achieved by the action of the energy-consuming sodium pump, one of the P-type ATPase transport protein family, members of which are indeed also found in bacteria. The pump's action brings about a transmembranal electrochemical gradient of sodium ions, harnessed in a range of transport systems that couple the dissipation of this gradient to establishing a gradient of the coupled substrate. The primary role of the sodium pump as a regulator of cell volume has evolved to provide the basis for an enormous variety of physiological functions.

Energetics of sulfate transport in Desulfomicrobium baculatum

The freshwater sulfate reducer Desulfomicrobium baculatum accumulated 35S-sulfate up to 120-fold by an energy-dependent transport system, as was concluded from inhibition of transport by tetrachlorosalicylanilide (TCS). Sulfate accumulation was completely reversible and depended on the presence of sodium ions. The sodium ion gradient ([Na+]out/[Na+]in) was eightfold and was built up by electrogenic Na+/H+ antiport. Together with a membrane potential of-145 m V, the sodium ion motive force was-199 m V, from which a symport stoichiometry of two sodium ions per sulfate was calculated. This is the first report of a freshwater sulfate reducer taking up sulfate electroneutrally in symport with sodium ions and not with protons.

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.