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 (AGp) generally increased over the total pH range, except for a modest fall-off in the AGp 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 (Ap) and the AGp as the external pH is raised. Were a strictly bulk chemiosmotic coupling mechanism to account for oxidative phosphorylation over the entire range, the AGp/Ap ratio (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.Alkaliphilic Bacillus firmus OF4 grows at least as well at pH 10.5 as at pH 7.5 in batch and continuous culture on malatecontaining medium (5). At the more alkaline pH values, the cytoplasmic pH is maintained below pH 8.5, a remarkable, Na+-dependent capacity for pH homeostasis in which secondary Na+/H+ antiporters apparently play a central role (13,19). Having thus successfully confronted the primary biological challenge, the alkaliphile must further resolve a bioenergetic problem that results from this maintenance of a cytoplasmic pH (pHin) that is well below the external pH. Although a substantial transmembrane electrical potential (Az4, positive out) is generated by respiration-dependent proton extrusion, the magnitude of the electrochemical proton gradient (Ap, acid and positive out) is reduced by the chemiosmotically adverse pH gradient (ApH). Extreme alkaliphiles do not use the much larger electrochemical gradient of sodium via a sodium-coupled synthase, probably because outward sodium movements must be coupled to proton accumulation for purposes of pH homeostasis, and a primary sodium cycle would subvert t...
Summary Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cad‐mium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.
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 potentialenergized efflux of 22 Na ؉ 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.
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