PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
 
J Bacteriol. 1997 June; 179(12): 3851–3857.
PMCID: PMC179192

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 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.

Full Text

The Full Text of this article is available as a PDF (232K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed]
  • Ambudkar SV, Zlotnick GW, Rosen BP. Calcium efflux from Escherichia coli. Evidence for two systems. J Biol Chem. 1984 May 25;259(10):6142–6146. [PubMed]
  • Aono R, Ito M, Horikoshi K. Instability of the protoplast membrane of facultative alkaliphilic Bacillus sp. C-125 at alkaline pH values below the pH optimum for growth. Biochem J. 1992 Jul 1;285(Pt 1):99–103. [PubMed]
  • Biswas I, Gruss A, Ehrlich SD, Maguin E. High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol. 1993 Jun;175(11):3628–3635. [PMC free article] [PubMed]
  • Brey RN, Rosen BP. Properties of Escherichia coli mutants altered in calcium/proton antiport activity. J Bacteriol. 1979 Sep;139(3):824–834. [PMC free article] [PubMed]
  • Chang S, Cohen SN. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. [PubMed]
  • Cheng J, Guffanti AA, Wang W, Krulwich TA, Bechhofer DH. Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain. J Bacteriol. 1996 May;178(10):2853–2860. [PMC free article] [PubMed]
  • Cheng J, Hicks DB, Krulwich TA. The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline-cobalt/H+ and Na+(K+)/H+ exchange. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14446–14451. [PubMed]
  • Clejan S, Guffanti AA, Cohen MA, Krulwich TA. Mutation of Bacillus firmus OF4 to duramycin resistance results in substantial replacement of membrane lipid phosphatidylethanolamine by its plasmalogen form. J Bacteriol. 1989 Mar;171(3):1744–1746. [PMC free article] [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM, et al. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. [PubMed]
  • Fletcher CF, Lutz CM, O'Sullivan TN, Shaughnessy JD, Jr, Hawkes R, Frankel WN, Copeland NG, Jenkins NA. Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell. 1996 Nov 15;87(4):607–617. [PubMed]
  • Goldberg EB, Arbel T, Chen J, Karpel R, Mackie GA, Schuldiner S, Padan E. Characterization of a Na+/H+ antiporter gene of Escherichia coli. Proc Natl Acad Sci U S A. 1987 May;84(9):2615–2619. [PubMed]
  • Guffanti AA, Krulwich TA. Oxidative phosphorylation by ADP + P(i)-loaded membrane vesicles of alkaliphilic Bacillus firmus OF4. J Biol Chem. 1994 Aug 26;269(34):21576–21582. [PubMed]
  • Guffanti AA, Krulwich TA. Tetracycline/H+ antiport and Na+/H+ antiport catalyzed by the Bacillus subtilis TetA(L) transporter expressed in Escherichia coli. J Bacteriol. 1995 Aug;177(15):4557–4561. [PMC free article] [PubMed]
  • Hamamoto T, Hashimoto M, Hino M, Kitada M, Seto Y, Kudo T, Horikoshi K. Characterization of a gene responsible for the Na+/H+ antiporter system of alkalophilic Bacillus species strain C-125. Mol Microbiol. 1994 Dec;14(5):939–946. [PubMed]
  • Ivey DM, Guffanti AA, Bossewitch JS, Padan E, Krulwich TA. Molecular cloning and sequencing of a gene from alkaliphilic Bacillus firmus OF4 that functionally complements an Escherichia coli strain carrying a deletion in the nhaA Na+/H+ antiporter gene. J Biol Chem. 1991 Dec 5;266(34):23483–23489. [PubMed]
  • Ivey DM, Guffanti AA, Zemsky J, Pinner E, Karpel R, Padan E, Schuldiner S, Krulwich TA. Cloning and characterization of a putative Ca2+/H+ antiporter gene from Escherichia coli upon functional complementation of Na+/H+ antiporter-deficient strains by the overexpressed gene. J Biol Chem. 1993 May 25;268(15):11296–11303. [PubMed]
  • Karpel R, Alon T, Glaser G, Schuldiner S, Padan E. Expression of a sodium proton antiporter (NhaA) in Escherichia coli is induced by Na+ and Li+ ions. J Biol Chem. 1991 Nov 15;266(32):21753–21759. [PubMed]
  • Krulwich TA. Alkaliphiles: 'basic' molecular problems of pH tolerance and bioenergetics. Mol Microbiol. 1995 Feb;15(3):403–410. [PubMed]
  • Krulwich TA, Cheng J, Guffanti AA. The role of monovalent cation/proton antiporters in Na(+)-resistance and pH homeostasis in Bacillus: an alkaliphile versus a neutralophile. J Exp Biol. 1994 Nov;196:457–470. [PubMed]
  • Krulwich TA, Federbush JG, Guffanti AA. Presence of a nonmetabolizable solute that is translocated with Na+ enhances Na+-dependent pH homeostasis in an alkalophilic Bacillus. J Biol Chem. 1985 Apr 10;260(7):4055–4058. [PubMed]
  • Krulwich TA, Ito M, Gilmour R, Sturr MG, Guffanti AA, Hicks DB. Energetic problems of extremely alkaliphilic aerobes. Biochim Biophys Acta. 1996 Jul 18;1275(1-2):21–26. [PubMed]
  • Lebovitz RM, Takeyasu K, Fambrough DM. Molecular characterization and expression of the (Na+ + K+)-ATPase alpha-subunit in Drosophila melanogaster. EMBO J. 1989 Jan;8(1):193–202. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Nakamura T, Komano Y, Itaya E, Tsukamoto K, Tsuchiya T, Unemoto T. Cloning and sequencing of an Na+/H+ antiporter gene from the marine bacterium Vibrio alginolyticus. Biochim Biophys Acta. 1994 Mar 23;1190(2):465–468. [PubMed]
  • Nakamura T, Enomoto H, Unemoto T. Cloning and sequencing of nhaB gene encoding an Na+/H+ antiporter from Vibrio alginolyticus. Biochim Biophys Acta. 1996 Jul 31;1275(3):157–160. [PubMed]
  • Ohyama T, Igarashi K, Kobayashi H. Physiological role of the chaA gene in sodium and calcium circulations at a high pH in Escherichia coli. J Bacteriol. 1994 Jul;176(14):4311–4315. [PMC free article] [PubMed]
  • Padan E, Schuldiner S. Na+/H+ antiporters, molecular devices that couple the Na+ and H+ circulation in cells. J Bioenerg Biomembr. 1993 Dec;25(6):647–669. [PubMed]
  • Pinner E, Carmel O, Bercovier H, Sela S, Padan E, Schuldiner S. Cloning, sequencing and expression of the nhaA and nhaR genes from Salmonella enteritidis. Arch Microbiol. 1992;157(4):323–328. [PubMed]
  • Pinner E, Kotler Y, Padan E, Schuldiner S. Physiological role of nhaB, a specific Na+/H+ antiporter in Escherichia coli. J Biol Chem. 1993 Jan 25;268(3):1729–1734. [PubMed]
  • Pinner E, Padan E, Schuldiner S. Kinetic properties of NhaB, a Na+/H+ antiporter from Escherichia coli. J Biol Chem. 1994 Oct 21;269(42):26274–26279. [PubMed]
  • Plack RH, Jr, Rosen BP. Cation/proton antiport systems in Escherichia coli. Absence of potassium/proton antiporter activity in a pH-sensitive mutant. J Biol Chem. 1980 May 10;255(9):3824–3825. [PubMed]
  • Sturr MG, Guffanti AA, Krulwich TA. Growth and bioenergetics of alkaliphilic Bacillus firmus OF4 in continuous culture at high pH. J Bacteriol. 1994 Jun;176(11):3111–3116. [PMC free article] [PubMed]
  • Taglicht D, Padan E, Schuldiner S. Overproduction and purification of a functional Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli. J Biol Chem. 1991 Jun 15;266(17):11289–11294. [PubMed]
  • Tani K, Watanabe T, Matsuda H, Nasu M, Kondo M. Cloning and sequencing of the spore germination gene of Bacillus megaterium ATCC 12872: similarities to the NaH-antiporter gene of Enterococcus hirae. Microbiol Immunol. 1996;40(2):99–105. [PubMed]
  • Thelen P, Tsuchiya T, Goldberg EB. Characterization and mapping of a major Na+/H+ antiporter gene of Escherichia coli. J Bacteriol. 1991 Oct;173(20):6553–6557. [PMC free article] [PubMed]
  • Waser M, Hess-Bienz D, Davies K, Solioz M. Cloning and disruption of a putative NaH-antiporter gene of Enterococcus hirae. J Biol Chem. 1992 Mar 15;267(8):5396–5400. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)