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

Abstract

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.

Bacillus subtilis NhaC, an Na + /H + Antiporter, Influences Expression of the phoPR Operon and Production of Alkaline Phosphatases

“…MrpA is encoded by the first gene of the mrp operon that is required for the maintenance of pH homeostasis and resistance to cholate and Na ϩ (16). While TetA(L) and MrpA antiporters play key roles in Na ϩ -dependent pH homeostasis and Na ϩ resistance, NhaC appeared to have only a modest role for maintaining pH homeostasis (17,38). This was the case even in tetA(L) deletion strains in which the expression of NhaC was increased (38).…”

“…NhaC appears to have only a modest role in Na ϩ -dependent pH homeostasis and no role in Na ϩ resistance (38). However, in Bacillus firmus the absence of NhaC led to a growth defect in medium containing low (25 mM) Na ϩ concentrations at pH 7.5 (17). In contrast, our data showed that growth rate and yield were unaffected by sodium in the absence of NhaC but were reduced progressively in both the wild type and the induced mutant as the concentration of Na ϩ was increased (Fig.…”

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation

“…Mrp antiporters are also broadly distributed among non-alkaliphilic Gram-positive and Gram-negative bacteria and play roles in diverse physiological processes that may depend upon pH and Na ϩ homeostasis (2). In addition to Na ϩ /H ϩ antiport, several of these Mrp antiporters support resistance to anions, such as arsenite and bile salts (13)(14)(15). We hypothesize that Mrp complexes are consortiums of more than one transporter type (2).…”

The ATP Synthase a-subunit of Extreme Alkaliphiles Is a Distinct Variant

“…All phenotypic measurements were done in the wild-type ␤-subunit background. Growth experiments were carried out as previously described in at least two independent experiments, each of which had duplicate cultures containing 2 ml of a semi-defined glucose or malate media that also contained 0.1% yeast extract (41). The pre-cultures were grown in glucose-containing medium at pH 7.5, and the cultures for the growth experiments were conducted at pH 7.5 and 10.5 and included controls with 0.1% yeast extract as the sole carbon source.…”