ROLE OF THE NHAC-ENCODED NA+ H+ ANTIPORTER OF ALKALIPHILIC BACILLUS-FIRMUS OF4/

Application of protoplast transformation and single- and double-crosso ver 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+ a ntiporter, and the surrounding region, The nhaC gene is part of a like ly 2-gene operon encompassing nhaC and a small gene that was designate d 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 seg ments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influ enzae. 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 mu tants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deleti on strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ conce ntrations equal to or greater than 10 mM. Even at lower Na+ concentrat ions, N13 exhibited only a modest growth defect at pH 10.5, This was a ccompanied 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 af finity Na+/H+ antiport activity available to extrude the Na+ and to co nfer some initial protection in the face of a sudden upshift in extern al pH, i.e., before full induction of additional antiporters. Consiste nt with the inference that NhaC is a relatively high affinity, electro genic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential -energized efflux of Na-22(+) from right-side-out membrane resides fro m cells that were preloaded with 2 mM Na+ and energized at pH 7.5. Whe n the experiment was conducted with vesicles loaded with 25 mM Na+, co mparable efflux was observed in preparations from all the strains.