Proton-coupled bioenergetic processes in extremely alkaliphilic bacteria

Oxidative phosphorylation, which involves an exclusively proton-coupled ATP synthase, and pH homeostasis, which depends upon electrogenic antiport of cytoplasmic Na⁺ in exchange for H⁺, are the two known bioenergetic processes that require inward proton translocation in extremely alkaliphilic bacteria. Energy coupling to oxidative phosphorylation is particularly difficult to fit to a strictly chemiosmotic model because of the low bulk electrochemical proton gradient that follows from the maintenance of a cytoplasmic pH just above 8 during growth at pH 10.5 and higher. A large quantitative and variable discrepancy between the putative chemiosmotic driving force and the phosphorylation potential results. This is compounded by a nonequivalence between respiration-dependent bulk gradients and artificially imposed ones in energizing ATP synthesis, and by an apparent requirement for specific respiratory chain complexes that do not relate solely to their role in generation of bulk gradients. Special features of the synthase may contribute to the mode of energization, just as novel features of the Na⁺ cycle may relate to the extraordinary capacity of the extreme alkaliphiles to achieve pH homeostasis during growth at, or sudden shifts to, an external pH of 10.5 and above.     

Replacement of amino acid sequence features of a- and c-subunits of ATP synthases of Alkaliphilic Bacillus with the Bacillus consensus sequence results in defective oxidative phosphorylation and non-fermentative growth at pH 10.5

Mitchell's (Mitchell, P. (1961) Nature 191, 144-148) chemiosmotic model of energy coupling posits a bulk electrochemical proton gradient (Deltap) as the sole driving force for proton-coupled ATP synthesis via oxidative phosphorylation (OXPHOS) and for other bioenergetic work. Two properties of proton-coupled OXPHOS by alkaliphilic Bacillus species pose a challenge to this tenet: robust ATP synthesis at pH 10.5 that does not correlate with the magnitude of the Deltap and the failure of artificially imposed potentials to substitute for respiration-generated potentials in energizing ATP synthesis at high pH (Krulwich, T. (1995) Mol. Microbiol. 15, 403-410). Here we show that these properties, in alkaliphilic Bacillus pseudofirmus OF4, depend upon alkaliphile-specific features in the proton pathway through the a- and c-subunits of ATP synthase. Site-directed changes were made in six such features to the corresponding sequence in Bacillus megaterium, which reflects the consensus sequence for non-alkaliphilic Bacillus. Five of the six single mutants assembled an active ATPase/ATP synthase, and four of these mutants exhibited a specific defect in non-fermentative growth at high pH. Most of these mutants lost the ability to generate the high phosphorylation potentials at low bulk Deltap that are characteristic of alkaliphiles. The aLys(180) and aGly(212) residues that are predicted to be in the proton uptake pathway of the a-subunit were specifically implicated in pH-dependent restriction of proton flux through the ATP synthase to and from the bulk phase. The evidence included greatly enhanced ATP synthesis in response to an artificially imposed potential at high pH. The findings demonstrate that the ATP synthase of extreme alkaliphiles has special features that are required for non-fermentative growth and OXPHOS at high pH.     

Two-dimensional gel electrophoresis analyses of pH-dependent protein expression in facultatively alkaliphilic Bacillus pseudofirmus OF4 lead to characterization of an S-layer protein with a role in alkaliphily

The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well. slpA RNA abundance was, moreover, unchanged by growth pH. SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. An slpA mutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na(+)-replete or suboptimal Na(+) concentrations. The extent of the adaptation deficit increased with pH elevation and suboptimal Na(+). By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7. 5 under Na(+)-replete and suboptimal Na(+) conditions, respectively. Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na(+)-dependent transport of alpha-aminoisobutyric acid, or H(+)-dependent synthesis of ATP. However, the capacity for Na(+)-dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8. 5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.     

ATP synthesis is driven by an imposed delta pH or delta mu H+ but not by an imposed delta pNa+ or delta mu Na+ in alkalophilic Bacillus firmus OF4 at high pH

Starved whole cells of alkalophilic Bacillus firmus OF4 that are equilibrated at either pH 10.2, 9.5, or 8.5 synthesize ATP in response to a pH gradient that is imposed by rapid dilution of the cyanide-treated cells into buffer at pH 7.5. If a valinomycin-mediated potassium diffusion potential (positive out) is generated simultaneously with the pH gradient, then the rate of ATP synthesis and the level of synthesis achieved is much higher than upon imposition of a pH gradient alone. By contrast, imposition of a large chemical gradient of Na+, either in the presence or absence of a concomitant diffusion potential, fails to result in ATP synthesis. We conclude that this organism does not possess a sodium-motive ATPase that can be made to synthesize detectable levels of ATP by imposition of a suitably large chemical or electrochemical gradient of Na+. On the other hand, a proton-translocating ATPase is in evidence when protons are provided at very high pH, corroborating our earlier work on extremely alkalophilic bacilli. Oxidative phosphorylation must, then, be catalyzed in these organisms by a proton-translocating ATPase even though the putative bulk driving forces for such a catalyst are low under optimal growth conditions. Stable, imposed pH gradients of 1 unit, comparable to the magnitude of the total electrochemical proton gradient of growing cells, result in much lower ATP concentrations than observed in such cells. We hypothesize that ATP synthesis in growing cells utilizes protons that are made available by some localized pathway between proton pumps and the ATP synthase.     

Growth and bioenergetics of alkaliphilic Bacillus firmus OF4 in continuous culture at high pH.

The effect of external pH on growth of alkaliphilic Bacillus firmus OF4 was studied in steady-state, pH-controlled cultures at various pH values. Generation times of 54 and 38 min were observed at external pH values of 7.5 and 10.6, respectively. At more alkaline pH values, generation times increased, reaching 690 min at pH 11.4; this was approximately the upper limit of pH for growth with doubling times below 12 h. Decreasing growth rates above pH 11 correlated with an apparent decrease in the ability to tightly regulate cytoplasmic pH and with the appearance of chains of cells. Whereas the cytoplasmic pH was maintained at pH 8.3 or below up to external pH values of 10.8, there was an increase up to pH 8.9 and 9.6 as the growth pH was increased to 11.2 and 11.4, respectively. Both the transmembrane electrical potential and the phosphorylation potential (delta Gp) generally increased over the total pH range, except for a modest fall-off in the delta Gp at pH 11.4. The capacity for pH homeostasis rather than that for oxidative phosphorylation first appeared to become limiting for growth at the high edge of the pH range. No cytoplasmic or membrane-associated organelles were observed at any growth pH, confirming earlier conclusions that structural sequestration of oxidative phosphorylation was not used to resolve the discordance between the total electrochemical proton gradient (delta p) and the delta Gp as the external pH is raised. Were a strictly bulk chemiosmotic coupling mechanism to account for oxidative phosphorylation over the entire range, the deltaGp/deltap ration (which would equal the H+/ATP ratio) would rise from about 3 at pH 7.5 to 13 at pH 11.2, dropping to 7 at pH 11.4 only because of the rise in cytoplasmic pH relative to other parameters. Moreover, the molar growth yields on malate were higher at pH 10.5 than at pH 7.5, indicating greater rather than lesser efficiency in the use of substrate at the more alkaline pH.     

A tridecameric c ring of the adenosine triphosphate (ATP) synthase from the thermoalkaliphilic Bacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemical proton potential

Despite the thermodynamic problem imposed on alkaliphilic bacteria of synthesizing adenosine triphosphate (ATP) against a large inverted pH gradient and consequently a low electrochemical proton potential, these bacteria still utilize a proton-coupled F(1)F(o)-ATP synthase to synthesize ATP. One potential solution to this apparent thermodynamic problem would be the operation of a larger oligomeric c ring, which would raise the ion to ATP ratio, thus facilitating the conversion of a low electrochemical potential into a significant phosphorylation potential. To address this hypothesis, we have purified the oligomeric c ring from the thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1 and determined the number of c-subunits using a novel mass spectrometry method, termed 'laser-induced liquid bead ion desorption' (LILBID). This technique allows the mass determination of non-covalently assembled, detergent-solubilized membrane protein complexes, and hence enables an accurate determination of c ring stoichiometries. We show that the Bacillus sp. strain TA2.A1 ATP synthase harbours a tridecameric c ring. The operation of a c ring with 13 subunits renders the thermodynamic problem of ATP synthesis at alkaline pH less severe and may represent a strategy for ATP synthesis at low electrochemical potential.     

Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4.

Application of protoplast transformation and single- and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na+/H+ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na+-sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na+/H+-deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ concentrations equal to or greater than 10 mM. Even at lower Na+ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na+ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na+/H+ antiport activity available to extrude the Na+ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential-energized efflux of 22Na+ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na+ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na+, comparable efflux was observed in preparations from all the strains.     

A specific adaptation in the a subunit of thermoalkaliphilic F1FO-ATP synthase enables ATP synthesis at high pH but not at neutral pH values

Analysis of the atp operon from the thermoalkaliphilic Bacillus sp. TA2.A1 and comparison with other atp operons from alkaliphilic bacteria reveals the presence of a conserved lysine residue at position 180 (Bacillus sp. TA2.A1 numbering) within the a subunit of these F(1)F(o)-ATP synthases. We hypothesize that the basic nature of this residue is ideally suited to capture protons from the bulk phase at high pH. To test this hypothesis, a heterologous expression system for the ATP synthase from Bacillus sp. TA2.A1 (TA2F(1)F(o)) was developed in Escherichia coli DK8 (Deltaatp). Amino acid substitutions were made in the a subunit of TA2F(1)F(o) at position 180. Lysine (aK180) was substituted for the basic residues histidine (aK180H) or arginine (aK180R), and the uncharged residue glycine (aK180G). ATP synthesis experiments were performed in ADP plus P(i)-loaded right-side-out membrane vesicles energized by ascorbate-phenazine methosulfate. When these enzyme complexes were examined for their ability to perform ATP synthesis over the pH range from 7.0 to 10.0, TA2F(1)F(o) and aK180R showed a similar pH profile having optimum ATP synthesis rates at pH 9.0-9.5 with no measurable ATP synthesis at pH 7.5. Conversely, aK180H and aK180G showed maximal ATP synthesis at pH values 8.0 and 7.5, respectively. ATP synthesis under these conditions for all enzyme forms was sensitive to DCCD. These data strongly imply that amino acid residue Lys(180) is a specific adaptation within the a subunit of TA2F(1)F(o) to facilitate proton capture at high pH. At pH values near the pK(a) of Lys(180), the trapped protons readily dissociate to reach the subunit c binding sites, but this dissociation is impeded at neutral pH values causing either a blocking of the proposed H(+) channel and/or mechanism of proton translocation, and hence ATP synthesis is inhibited.     

Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis

ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (ΔpH) across the membrane and transmembrane electrical potential (Δψ). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted, and previous experimental results vary. Here, we show that with the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the ε subunit, ΔpH imposed by acid-base transition and Δψ produced by valinomycin-mediated K(+) diffusion potential contribute equally to the rate of ATP synthesis within the experimental range examined (ΔpH -0.3 to 2.2, Δψ -30 to 140 mV, pH around the catalytic domain 8.0). Either ΔpH or Δψ alone can drive synthesis, even when the other slightly opposes. Δψ was estimated from the Nernst equation, which appeared valid down to 1 mm K(+) inside the proteoliposomes, due to careful removal of K(+) from the lipid.     

The Na(+)-dependence of alkaliphily in Bacillus

A Na(+) cycle plays a central role in the remarkable capacity of aerobic, extremely alkaliphilic Bacillus species for pH homeostasis. The capacity for pH homeostasis, in turn, appears to set the upper pH limit for growth. One limb of the alkaliphile Na(+) cycle consists of Na(+)/H(+) antiporters that achieve net H(+) accumulation that is coupled to Na(+) efflux. The major antiporter on which pH homeostasis depends is thought to be the Mrp(Sha)-encoded antiporter, first identified from a partial clone in Bacillus halodurans C-125. Mrp(Sha) may function as a complex. While this antiporter is capable of secondary antiport energized by an imposed or respiration-generated protonmotive force, the possibility of a primary mode has not been excluded. In Bacillus pseudofirmus OF4, at least two additional antiporters, including NhaC, have supporting roles in pH homeostasis. Some of these additional antiporters may be especially important for antiport at low [Na(+)] or at near-neutral pH. The second limb of the Na(+) cycle facilitates Na(+) re-entry via Na(+)/solute symporters and, perhaps, the ion channel associated with the Na(+)-dependent flagellar motor. The process of pH homeostasis is also enhanced, perhaps especially during transitions to high pH, by different arrays of secondary cell wall polymers in the two alkaliphilic Bacillus species studied most intensively. The mechanisms whereby alkaliphiles handle the challenge of Na(+) stress at very elevated [Na(+)] are just beginning to be identified, and a hypothesis has been advanced to explain the finding that B. pseudofirmus OF4 requires a higher [Na(+)] for growth at near-neutral pH than at very alkaline pH values.     

Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles

The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of new facultatively alkalophilic strains of Bacillus species.

Four facultatively alkalophilic isolates were purified from enrichment cultures initiated with lime-treated garden soil. Four isolates, OF1, OF3, OF4, and OF6, were obligately aerobic, spore-forming, gram-positive, motile rods which were capable of growth at both pH 7.5 and pH 10.5. Strains OF1 and OF6 grew best at the lower pH value; and whereas growth of these strains at pH 10.5 was completely dependent on added Na+, growth at pH 7.5 was only partially dependent on added Na+. Strains OF3 and OF4 grew better at pH 10.5 than at pH 7.5, with strain OF3 growing modestly over its entire pH range, while OF4 grew well. Growth of OF3 and OF4 was completely dependent on added Na+ at both pH 7.5 and pH 10.5. DNA-DNA hybridization studies indicated that OF1 and OF6 are closely related strains but are not related to the other isolates, Bacillus subtilis, or two previously studied obligately alkalophilic bacilli. OF3 was unrelated to any of the other organisms examined in the study, whereas OF4 showed complete homology with obligately alkalophilic Bacillus firmus RAB. All four isolates maintained a cytoplasmic pH that was considerably lower than the external pH when the latter was 10.5. Although substantial transmembrane electrical potentials were observed, the total electrochemical proton gradient (delta mu H+) was low at pH 10.5 in all the strains. By contrast, delta mu H+ was substantial at pH 7.5 and at that pH was composed entirely of an electrical potential. These results are in contrast to previous findings that obligately alkalophilic bacilli generate only small electrical potentials at near neutral pH. All the isolates exhibited substantial rates of respiration as measured by oxygen consumption. Neither respiration nor NADH oxidation by everted membrane vesicles was significantly stimulated by Na+. Analyses of reduced versus oxidized difference spectra of membranes from OF4 showed that the total membrane cytochrome content was considerably higher in cells grown at pH 10.5 than at pH 7.5, with the levels of c- and a-type cytochromes exhibiting the largest pH-dependent differences. Initial examination of membrane protein profiles on gel electrophoresis also indicated a number of changes in pattern in each isolate, depending on the growth pH.     

Tetracycline/H+ antiport and Na+/H+ antiport catalyzed by the Bacillus subtilis TetA(L) transporter expressed in Escherichia coli.

The properties of TetA(L)-dependent tetracycline/proton and Na+/proton antiport were studied in energized everted vesicles of Escherichia coli transformed with a cloned tetA(L) gene (pJTA1) from Bacillus subtilis. Inhibition patterns by valinomycin and nigericin indicated that both antiports were electrogenic, in contrast to the tetracycline/proton antiport encoded by gram-negative plasmid tet genes. Tetracycline uptake in the everted system was dependent upon a divalent cation, with cobalt being the preferred one. The apparent Km for tetracycline was markedly increased at pH 8.5 versus pH 7.5, whereas the Vmax was unchanged. The much higher apparent Km for Na+ decreased at pH 8.5 relative to that at pH 7.5, as did the Vmax. Na+ did not affect tetracycline uptake, nor did Co2+ and/or tetracycline affect Na+ uptake; complex patterns of inhibition by amiloride and analogs thereof were observed.     

Difference in ionic specificity of ATP synthesis in extremely alkalophilic sulfate-reducing and acetogenic bacteria

Ionic specificity of oxidative phosphorylation was studied in Natroniella acetigena and Desulfonatronum lacustre, which are new alkaliphilic anaerobes that were isolated from soda lakes and have a pH growth optimum of 9.5-9.7. The ability of their cells to synthesize ATP in response to the imposition of artificial delta pH+ and delta pNa+ gradients was studied. As distinct from other marine and freshwater sulfate reducers and extremely alkaliphilic anaerobes, D. lacustre uses a Na(+)-translocating ATPase for ATP synthesis. The alkaliphilic acetogen N. acetigena, which develops at a much higher Na+ concentration in the medium, generated primary delta pH+ for ATP synthesis. Thus, the high Na+ concentrations and alkaline pH values typical of soda lakes do not predetermine the type of bioenergetics of their inhabitants.     

Properties of two different Na+/H+ antiport systems in alkaliphilic Bacillus sp. strain C-125.

Na+/H+ antiport was studied in alkaliphilic Bacillus sp. strain C-125, its alkali-sensitive mutant 38154, and a transformant (pALK2) with recovered alkaliphily. The transformed was able to maintain an intracellular pH (pHin) that was lower than that of external milieu and contained an electrogenic Na+/H+ antiporter driven only by delta psi (membrane potential, interior negative). The activity of this delta psi-dependent Na+/H+ antiporter was highly dependent on pHin, increasing with increasing pHin, and was found only in cells grown at alkaline pH. On the other hand, the alkali-sensitive mutant, which had lost the ability to grow above pH 9.5, lacked the delta psi-dependent Na+/H+ antiporter and showed defective regulation of pHin at the alkaline pH range. However, this mutant, like the parent strain, still required sodium ions for growth and for an amino acid transport system. Moreover, another Na+/H+ antiporter, driven by the imposed delta pH (pHin > extracellular pHout), was active in this mutant strain, showing that the previously reported delta pH-dependent antiport activity is probably separate from delta psi-dependent antiporter activity. The delta pH-dependent Na+/H+ antiporter was found in cells grown at either pH 7 or pH 9. This latter antiporter was reconstituted into liposomes by using a dilution method. When a transmembrane pH gradient was applied, downhill sodium efflux was accelerated, showing that the antiporter can be reconstituted into liposomes and still retain its activity.     

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.     

Failure of an alkalophilic bacterium to synthesize ATP in response to a valinomycin-induced potassium diffusion potential at high pH

Starved whole cells of the obligately alkalophilic Bacillus firmus RAB synthesize ATP upon addition of L-malate at pH 9.0 as expected of an aerobic organism that grows oxidatively on nonfermentable carbon sources at pH values as high as 11.0. The current study was a detailed examination of the perplexing inability of such cells to exhibit ATP synthesis in response to a valinomycin-mediated potassium diffusion potential at pH 9.0. While there were minor differences in the patterns of generation of the potential and the proton influx that accompanies its generation in the three different buffering systems employed, the magnitude of the transmembrane electro-chemical potential of protons was at least as high as pH 9.0 as at pH 7.0. Nevertheless, a diffusion potential consistently energized ATP synthesis at pH 7.0 but not at 9.0; these findings were independent of the presence or absence of Tris or of Na+. By contrast, the artificial electron donor ascorbate, in the presence of phenazine methosulfate, energized ATP synthesis by the starved whole cells at both pH values. The same phenomenon, i.e., efficacy of a respiration-derived potential but not of a diffusion potential at pH 9.0, was demonstrated in ADP + Pi-loaded membrane vesicles. On the other hand, electrogenic Na+-coupled solute transport could be energized by both ascorbate/phenazine and methosulfate and a diffusion potential in the vesicles at pH 9.0. The results are discussed in connection with models of a localized path of proton flow between proton pumps and the ATP synthase.     

Molar growth yields and bioenergetic parameters of extremely alkaliphilic Bacillus species in batch cultures, and growth in a chemostat at pH 10.5.

Alkaliphilic Bacillus species that grow at pH 10.5 must cope with a low protonmotive force (-50 mV) due to a reversed transmembrane pH gradient at least 2 pH units more acid inside. Here we demonstrate that strictly alkaliphilic B. firmus RAB and two strains of B. alcalophilus (ATCC 27467 and DSM 485) grow exponentially in batch cultures with a doubling time of less than 1 h in 100 mM buffered medium, while the actual medium pH remains above 10.2. The ATCC strain continued to grow rapidly for at least 7 h, but the growth rate of the DSM strain declined dramatically after 3 h. However, both the B. alcalophilus strains, B. firmus RAB and facultatively alkaliphilic B. firmus OF4 were readily maintained for at least 24 h between pH 10.4 and 10.6 in a chemostat where nutrients were constantly replenished. A critical nutrient may be limiting in batch cultures of the DSM strain of B. alcalophilus. The facultative alkaliphile grew equally well in batch cultures at an initial pH of 7.5 or 10.5. Its molar growth yield (23 mg dry wt mmol-1) on malate (Ymal) was the same at the two pH values and was comparable to Ymal for B. subtilis grown at neutral pH. B. firmus RAB and B. alcalophilus ATCC 27467 grown at pH 10.5 also showed Ymal values at least as high as the neutralphile, indicating efficient use of the energy source even at low protonmotive force. Moreover, the phosphorylation potential of B. firmus OF4 grown at pH 7.5 (45.2 kJ mol-1) or pH 10.5 (46 kJ mol-1) was in a conventional range for bacteria.     

Millisecond kinetics of ATP synthesis driven by externally imposed electrochemical potentials in chloroplasts

We have used rapid mixing and quenching techniques to measure the initial ATP synthesis rates and the duration of the ATP synthetic capacity derived from artificially imposed proton gradients and valinomycin-mediated K+ diffusion potentials in chloroplasts. The initial rate of ATP synthesis driven by a K+ diffusion potential was 10-fold slower than that driven by an acid-base transition of equivalent electrochemical potential. Total yields of ATP resulting from a K+ concentration shift were only slightly affected by the absence of Cl-, indicating that Cl- permeability does not significantly reduce the K+ diffusion potential. The ATP synthetic capacity decayed with a half-life of 0.2 s in the case of a K+ diffusion potential and a half-life of 1.0 s in the case of an acid-base shift. In both cases, ATP, added at the time of the pH or [KCl] shift, slowed the decay of the ATP synthesis rates, indicating that the coupling factor controls a channel for proton efflux, as proposed earlier (Portis, A.R., and McCarty, R.E. (1974) J. Biol. Chem. 249, 6250-6254). Because the proton gradient has a longer half-life than the K+ diffusion potential, when combinations of the two are employed to drive ATP synthesis, the proton gradient will make a larger contribution to the initial rate and total yield than that predicted from a strictly linear proportionality of the initial magnitudes of the two gradients.     

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na+/H+ antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na+-resistance on an antiporter-deficient E. coli strain, nor did they confer Na+/H+ antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.