Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions.

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Map location of the pcbA mutation and physiology of the mutant.

The obligate aerobe Cowpea Rhizobium sp. strain 32H1 in axenic culture is able to fix N2 when grown under 0.2% O2 but not when grown under 21% O2. It was, therefore, of interest to investigate ATP synthesis in these cells grown under the two conditions. When respiring in buffers having pHs ranging from 6 to 8.5, cells grown under either O2 tension maintained an intracellular pH more alkaline than the exterior. The transmembrane chemical gradient of H+ (delta pH) was essentially the same under both conditions of growth, decreasing from ca. 90 mV at medium pH 6 to ca. 30 mV at pH 8.5. However, the transmembrane electrical gradient (delta psi) was significantly higher in cells grown under 21% O2 (150 to 166 mV) than in cells grown under 0.2% O2, the latter being 16 mV at pH 6 and increasing to 88 mV at pH 8.5. Therefore, the proton motive force of 21% O2-grown cells ranged from 237 mV at external pH 6 to 185 mV at pH 8.5, compared with a proton motive force of 114 to 121 mV in the 0.2% O2-grown cells. The cells grown in 0.2% O2 had the same proton motive force whether tested at 21 or at 0.2% O2. The phosphorylation potential, calculated from the intracellular ATP, ADP, and Pi concentrations, was 424 mV in the 21% O2-grown cells and 436 mV in the 0.2% O2-grown cells. Thus, the 21% O2-grown cells translocated 1.8 to 2.3 H+/ATP synthesized by the H+-ATPase, whereas the H+/ATP ratio for 0.2% O2-grown cells was 3.7 to 3.8.     

Lactose transport system of Streptococcus thermophilus. Functional reconstitution of the protein and characterization of the kinetic mechanism of transport.

The kinetic mechanism of the lactose transport system of Streptococcus thermophilus was studied in membrane vesicles fused with cytochrome c oxidase containing liposomes and in proteoliposomes in which cytochrome c oxidase was coreconstituted with the lactose transport protein. Selective manipulation of the components of the proton (and sodium) motive force indicated that both a membrane potential and a pH gradient could drive transport. The galactoside/proton stoichiometry was close to unity. Experiments which discriminate between the effects of internal pH and delta pH as driving force on galactoside/proton symport showed that the carrier is highly activated at alkaline internal pH values, which biases the transport system kinetically toward the pH component of the proton motive force. Galactoside efflux increased with increasing pH with a pKa of about 8, whereas galactoside exchange (and counterflow) exhibited a pH optimum around 7 with pKa values of 6 and 8, respectively. Imposition of delta pH (interior alkaline) retarded the rate of efflux at any pH value tested, whereas the rate of exchange was stimulated by an imposed delta pH at pH 5.8, not affected at pH 7.0, and inhibited at pH 8.0 and 9.0. The results have been evaluated in terms of random and ordered association/dissociation of galactoside and proton on the inner surface of the membrane. Imposition of delta psi (interior negative) decreased the rate of efflux but had no effect on the rate of exchange, indicating that the unloaded transport protein carries a net negative charge and that during exchange and counterflow the carrier recycles in the protonated form.     

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.     

Depletion of proton motive force by nisin in Listeria monocytogenes cells.

The basal proton motive force (PMF) levels and the influence of the bacteriocin nisin on the PMF were determined in Listeria monocytogenes Scott A. In the absence of nisin, the interconversion of the pH gradient (Z delta pH) and the membrane potential (delta psi) led to the maintenance of a fairly constant PMF at -160 mV over the external pH range 5.5 to 7.0. The addition of nisin at concentrations of greater than or equal to 5 micrograms/ml completely dissipated PMF in cells at external pH values of 5.5 and 7.0. With 1 microgram of nisin per ml, delta pH was completely dissipated but delta psi decreased only slightly. The action of nisin on PMF in L. monocytogenes Scott A was both time and concentration dependent. Valinomycin depleted only delta pH, whereas nigericin and carbonyl cyanide m-chlorophenylhydrazone depleted only delta psi, under conditions in which nisin depleted both. Four other L. monocytogenes strains had basal PMF parameters similar to those of strain Scott A. Nisin (2.5 micrograms/ml) also completely dissipated PMF in these strains.     

Proton motive force and Na+/H+ antiport in a moderate halophile.

The influence of pH on the proton motive force of Vibrio costicola was determined by measuring the distributions of triphenylmethylphosphonium cation (membrane potential, delta psi) and either dimethyloxazolidinedione or methylamine (osmotic component, delta pH). As the pH of the medium was adjusted from 5.7 to 9.0, the proton motive force steadily decreased from about 170 to 100 mV. This decline occurred, despite a large increase in the membrane potential to its maximum value at pH 9.0, because of the loss of the pH gradient (inside alkaline). The cytoplasm and medium were of equal pH at 7.5; membrane permeability properties were lost at the pH extremes of 5.0 and 9.5. Protonophores and monensin prevented the net efflux of protons normally found when an oxygen pulse was given to an anaerobic cell suspension. A Na+/H+ antiport activity was measured for both Na+ influx and efflux and was shown to be dissipated by protonophores and monensin. These results strongly favor the concept that respiratory energy is used for proton efflux and that the resulting proton motive force may be converted to a sodium motive force through Na+/H+ antiport (driven by delta psi). A role for antiport activity in pH regulation of the cytosol can also explain the broad pH range for optimal growth, extending to the alkaline extreme of pH 9.0.     

The proton motive force generated in Leuconostoc oenos by L-malate fermentation.

In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi. Synthesis of ATP was observed in cells performing malolactic fermentation.     

Effects of potassium ions on proton motive force in Rhodobacter sphaeroides.

The proton motive force (PMF) was determined in Rhodobacter sphaeroides under anaerobic conditions in the dark and under aerobic-dark and anaerobic-light conditions. Anaerobically in the dark in potassium phosphate buffer, the PMF at pH 6 was -20 mV and was composed of an electrical potential (delta psi) only. At pH 7.9 the PMF was composed of a high delta psi of -98 mV and was partially compensated by a reversed pH gradient (delta pH) of +37 mV. ATPase inhibitors did not affect the delta psi, which was most likely the result of a K+ diffusion potential. Under energized conditions in the presence of K+ the delta psi depolarized due to electrogenic K+ uptake. This led to the generation of a delta pH (inside alkaline) in the external pH range of 6 to 8. This delta pH was dependent on the K+ concentration and was maximal at external K+ concentrations larger than 1.2 mM. In energized cells in 50 mM KPi buffer containing 5 mM MgSO4, a delta pH (inside alkaline) was present at external pHs from pH 6 to 8. As a result the overall magnitude of the PMF at various external pHs remained constant at -130 mV, which was significantly higher than the PMF under anaerobic-dark conditions. In the absence of K+, in 50 mM NaPi buffer containing 5 mM MgSO4, no depolarization of the delta psi was found and the PMF was composed of a large delta psi and a small delta pH. The delta pH became even reversed (inside acidic) at alkaline pHs (pH>7.3), resulting in a lowering of the PMF. These results demonstrate that in R. sphaeroides K+ uptake is essential for the generation of a delta pH and plays a central role in the regulation of the internal pH.     

Effects of aerobiosis and nitrogen source on the proton motive force in growing Escherichia coli and Klebsiella pneumoniae cells.

The electrochemical gradient of hydrogen ions, or proton motive force (PMF), was measured in growing Escherichia coli and Klebsiella pneumoniae in batch culture. The electrical component of the PMF (delta psi) and the chemical component (delta pH) were calculated from the cellular accumulation of radiolabeled tetraphenylphosphonium, thiocyanate, and benzoate ions. In both species, the PMF was constant during exponential phase and decreased as the cells entered stationary phase. Altering the growth rate with different energy substrates had no effect on the PMF. The delta pH (alkaline inside) varied with the pH of the culture medium, resulting in a constant internal pH. During aerobic growth in media at pH 6 to 7, the delta psi was constant at 160 mV (negative inside). The PMF, therefore, was 255 mV in cells growing at pH 6.3, and decreased progressively to 210 mV in pH 7.1 cultures. K. pneumoniae cells and two E. coli strains (K-12 and ML), including a mutant deficient in the H+-translocating ATPase and a pleiotropically energy-uncoupled mutant with a normal ATPase, had the same PMF during aerobic exponential phase. During anaerobic growth, however, both species had delta psi values equal to 0. Therefore, the PMF in anaerobic cells consisted only of the delta pH component, which was 75 mV or less in cells growing at pH 6.2 or greater. These data thus met the expectation that cells deriving metabolic energy from respiration have a PMF above a threshold value of about 200 mV when the ATPase functions in the direction of H+ influx and ATP synthesis; in fermenting cells, a PMF below a threshold value was expected since the enzyme functions in the direction of H+ extrusion and ATP hydrolysis. K. pneumoniae cells growing anaerobically had no delta psi whether the N source added was N2, NH+4 or one of several amino acids; the delta pH was unaffected. Therefore, any energy cost incurred by the process of nitrogen fixation could not be detected as an alteration of the proton gradient.     

Quantitative measurements of proton motive force and motility in Bacillus subtilis.

The protein motive force of metabolizing Bacillus subtilis cells was only slightly affected by changes in the external pH between 5 and 8, although the electrical component and the chemical component of the proton motive force contributed differently at different external pH. The electrical component of the proton motive force was very small at pH 5, and the chemical component was almost negligible at pH 7.5. At external pH values between 6 and 7.7, swimming speed of the cells stayed constant. Thus, either the electrical component or the chemical component of the proton motive force could drive the flagellar motor. When the proton motive force of valinomycin-treated cells was quantitatively decreased by increasing the external K+ concentration, the swimming speed of the cells changed in a unique way: the swimming speed was not affected until about--100 mV, then decreased linearly with further decrease in the proton motive force, and was almost zero at about--30 mV. The rotation rate of a flagellum, measured by a tethered cell, showed essentially the same characteristics. Thus, there are a threshold proton motive force and a saturating proton motive force for the rotation of the B. subtilis flagellar motor.     

Effects of K+ and Na+ on the proton motive force of respiring Escherichia coli at alkaline pH.

The role of K+ and Na+ in the maintenance of the proton motive force (delta p) was studied in Escherichia coli incubated in alkaline media. Cells respiring in Tris buffer (pH 7.8) that contained less than 100 microEq of K+ and Na+ per liter had a normal delta p of about -165 mV. At pH 8.2, however, the delta p was reduced significantly. The decrease in delta p at pH 8.2 was due to a marked decrease in the transmembrane potential (delta psi), while the internal pH remained at 7.5 to 7.7. When KCl or NaCl, but not LiCl or choline chloride, was added to the cells, the delta psi rose to the values seen at an external pH of 7.8. In addition, choline chloride inhibited the enhancement of delta psi by K+. None of the salts had a significant effect on the internal pH. The effects can be attributed to alterations of K+ or Na+ cycling in and out of the cells via the known K+ and Na+ transport systems.     

Proton motive force in growing Streptococcus lactis and Staphylococcus aureus cells under aerobic and anaerobic conditions.

Measurements of the electrochemical gradient of hydrogen ions, which gives rise to the proton motive force (PMF), were carried out with growing Streptococcus lactis and Staphylococcus aureus cells. The facultative anaerobe was chosen in order to compare the PMF of cells growing aerobically and anaerobically. It was expected that during aerobic growth the cells would have a higher PMF than during anaerobic growth, because the H+-translocating ATPase (BF0F1) operates in the direction of H+ influx and ATP synthesis during respiration, whereas under anaerobic conditions the BF0F1 hydrolyzes glycolytically generated ATP and establishes the proton gradient by extruding H+. The electrical component of the PMF, delta psi, and the chemical gradient of H+, delta pH, were measured with radiolabeled tetraphenylphosphonium and benzoate ions. In both S. lactis and S. aureus cells, the PMF was constant during the exponential phase of batch growth and decreased in the stationary phase. In both species of bacteria, the exponential-phase PMF was not affected by varying the growth rate by adding different sugars to the medium. The relative contributions of delta psi and delta pH to the PMF, however, depended on the pH of the medium. The internal pH of S. aureus was constant at pH 7.4 to 7.6 under all conditions of growth tested. Under aerobic conditions, the delta psi of exponential phase S. aureus remained fairly constant at 160 to 170 mV. Thus, the PMF was 250 to 270 mV in cells growing aerobically in media at pH 6 and progressively lower in media of higher pH, reaching 195 to 205 mV at pH 7. Under anaerobic conditions, the delta psi ranged from 100 to 120 mV in cells at pH 6.3 to 7, resulting in a PMF of 150 to 140 mV. Thus, the mode of energy metabolism (i.e., respiration versus fermentation) and the pH of the medium are the two important factors influencing the PMF of these gram-positive cells during growth.     

Effects of K+ on the proton motive force of Bradyrhizobium sp. strain 32H1.

In previous studies, respiring Bradyrhizobium sp. strain 32H1 cells grown under 0.2% O2, conditions that derepress N2 fixation, were found to have a low proton motive force of less than -121 mV, because of a low membrane potential (delta psi). In contrast, cells grown under 21% O2, which do not fix N2, had high proton motive force values of -175 mV or more, which are typical of respiring bacteria, because of high delta psi values. In the present study, we found that a delta psi of 0 mV in respiring cells requires growth in relatively high-[K+] media (8 mM), low O2 tension, and high internal [K+]. When low-[O2], high-[K+]-grown cells were partially depleted of K+, the delta psi was high. When cells were grown under 21% O2 or in media low in K+ (50 microM K+), the delta psi was again high. The transmembrane pH gradient was affected only slightly by varying the growth or assay conditions. In addition, low-[O2], high-[K+]-grown cells had a greater proton permeability than did high-[O2]-grown cells. To explain these findings, we postulate that cells grown under conditions that derepress N2 fixation contain an electrogenic K+/H+ antiporter that is responsible for the dissipation of the delta psi. The consequence of this alteration in K+ cycling is rerouting of proton circuits so that the putative antiporter becomes the major pathway for H+ influx, rather than the H+-ATP synthase.     

Interconversion of components of the bacterial proton motive force by electrogenic potassium transport.

The influence of K+ ions on the components of the transmembrane proton motive force (delta mu H+) in intact bacteria was investigated. In K+-depleted cells of the glycolytic bacterium STreptococcus faecalis the addition of K+ ions caused a depolarization of the membrane by about 60 mV. However, since the depolarization was compensated for by an increase in the transmembrane pH gradient (delta pH), the total proton motive force remained almost constant at about 120 mV. Half-maximal changes in the potential were observed at K+ concentrations at which the cells accumulated K+ ions extensively. In EDTA-treated, K+-depleted cells of Escherichia coli K-12, the addition of K+ ions to the medium caused similar, although smaller changes in the components of delta mu H+. Experiments with various E. coli K-12 K+ transport mutants showed that for the observed potential changes the cells required either a functional TrkA or Kdp K+ transport system. These data are interpreted to mean that the inward movement of K+ ions via each of these bacterial transport systems is electrogenic. Consequently, it leads to a depolarization of the membrane, which in its turn allows the cell to pump more protons into the medium.     

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.     

Electrochemical proton gradient and lactate concentration gradient in Streptococcus cremoris cells grown in batch culture.

The lactate concentration gradient and the components of the electrochemical proton gradient (delta micro H+) were determined in cells of Streptococcus cremoris growing in batch culture. The membrane potential (delta psi) and the pH gradient (delta pH) were determined from the accumulation of the lipophilic cation tetraphenylphosphonium and the weak acid benzoate, respectively. During growth the external pH decreased from 6.8 to 5.3 due to the production of lactate. Delta pH increased from 0 to -35 mV, inside alkaline (at an external pH of 5.7), and fell to zero directly after growth stopped. Delta psi was nearly constant at -90 mV during growth and also dissipated within 40 min after termination of growth. The internal lactate concentration decreased from 200 mM at the beginning of growth (at pH 6.8) to 30 mM at the end of growth (at pH 5.3); the external lactate concentration increased from 8 to 30 mM due to the fermentation of lactose. Thus, the lactate gradient decreased from 80 mV to zero as growth proceeded and the external pH decreased. From the data obtained on delta psi, delta pH, and the lactate concentration gradient, the H+/lactate stoichiometry (n) was calculated. The value of n varied with the external pH from 1.9 (at pH 6.8) to 0.9 (at pH values below 6). This implies that especially at high pH values the carrier-mediated efflux of lactate supplies a significant quantity of metabolic energy to S. cremoris cells. At pH 6.8 this energy gain was almost two ATP equivalents per molecule of lactose consumed if the H+/ATP stoichiometry equals 2. These results supply strong experimental evidence for the energy recycling model postulated by Michels et al.     

Proton motive force across the membrane of Mycoplasma gallisepticum and its possible role in cell volume regulation.

A proton motive force (delta (-) microH+) of 70 to 130 mV was measured across the membrane of Mycoplasma gallisepticum cells. The membrane potential was measured utilizing the lipid-soluble cation tetraphenylphosphonium. The method was validated by showing that in the presence of valinomycin the ratio of the concentrations (in/out) of tetraphenylphosphonium agreed well with those for K+ and Rb+. The pH gradient was calculated from the measured distribution ratio of benzoic acid. The proton motive force was approximately the same in cells harvested at early exponential, midexponential, and stationary phases of growth. The proportion of pH gradient to membrane potential varied with external pH. In the absence of glucose, cells incubated in an isosmotic NaCl solution showed low adenosine triphosphate and delta (-) microH+ levels and a tendency to swell and lyse compared with cells incubated with added glucose. It is concluded that energy is required for normal cell volume regulation.     

Characterization of the Na+/H+ antiporter of alkalophilic bacilli in vivo: delta psi-dependent 22Na+ efflux from whole cells.

The Na+/H+ antiporter of Bacillus alcalophilus was studied by measuring 22Na+ efflux from starved, cyanide-inhibited cells which were energized by means of a valinomycin-induced potassium diffusion potential, positive out (delta psi). In the absence of a delta psi, 22Na+ efflux at pH 9.0 was slow and appreciably inhibited by N-ethylmaleimide. Upon imposition of a delta psi, a very rapid rate of 22Na+ efflux occurred. This rapid rate of 22Na+ efflux was competitively inhibited by Li+ and varied directly with the magnitude of the delta psi. Kinetic experiments with B. alcalophilus and alkalophilic Bacillus firmus RAB indicated that the delta psi caused a pronounced increase in the Vmax for 22Na+ efflux. The Km values for Na+ were unaffected by the delta psi. Upon imposition of a delta psi at pH 7.0, a retardation of the slow 22Na+ efflux rate at pH 7.0 was caused by the delta psi. This showed that inactivity of the Na+/H+ antiporter at pH 7.0 was not secondary to a low delta psi generated by respiration at this pH. Indeed, 22Na+ efflux activity appeared to be inhibited by a relatively high internal proton concentration. By contrast, at a constant internal pH, there was little variation in the activity at external pH values from 7.0 to 9.0; at an external pH of 10.0, the rate of 22Na+ efflux declined. This decline at typical pH values for growth may be due to an insufficiency of protons when a diffusion potential rather than respiration is the driving force. Non-alkalophilic mutant strains of B. alcalophilus and B. firmus RAB exhibited a slow rate of 22Na+ efflux which was not enhanced by a delta psi at either pH 7.0 or 9.0.     

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.