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. 1997 Jun;179(12):3851–3857. doi: 10.1128/jb.179.12.3851-3857.1997

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

M Ito 1, A A Guffanti 1, J Zemsky 1, D M Ivey 1, T A Krulwich 1
PMCID: PMC179192  PMID: 9190799

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.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Ambudkar S. V., Zlotnick G. W., Rosen B. P. Calcium efflux from Escherichia coli. Evidence for two systems. J Biol Chem. 1984 May 25;259(10):6142–6146. [PubMed] [Google Scholar]
  3. 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. doi: 10.1042/bj2850099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biswas I., Gruss A., Ehrlich S. D., Maguin E. High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol. 1993 Jun;175(11):3628–3635. doi: 10.1128/jb.175.11.3628-3635.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brey R. N., Rosen B. P. Properties of Escherichia coli mutants altered in calcium/proton antiport activity. J Bacteriol. 1979 Sep;139(3):824–834. doi: 10.1128/jb.139.3.824-834.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
  7. Cheng J., Guffanti A. A., Wang W., Krulwich T. A., Bechhofer D. H. 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. doi: 10.1128/jb.178.10.2853-2860.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng J., Hicks D. B., Krulwich T. A. 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. doi: 10.1073/pnas.93.25.14446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clejan S., Guffanti A. A., Cohen M. A., Krulwich T. A. 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. doi: 10.1128/jb.171.3.1744-1746.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  12. Fletcher C. F., Lutz C. M., O'Sullivan T. N., Shaughnessy J. D., Jr, Hawkes R., Frankel W. N., Copeland N. G., Jenkins N. A. Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell. 1996 Nov 15;87(4):607–617. doi: 10.1016/s0092-8674(00)81381-1. [DOI] [PubMed] [Google Scholar]
  13. Goldberg E. B., Arbel T., Chen J., Karpel R., Mackie G. A., 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. doi: 10.1073/pnas.84.9.2615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guffanti A. A., Krulwich T. A. 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] [Google Scholar]
  15. Guffanti A. A., Krulwich T. A. 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. doi: 10.1128/jb.177.15.4557-4561.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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. doi: 10.1111/j.1365-2958.1994.tb01329.x. [DOI] [PubMed] [Google Scholar]
  17. Ivey D. M., Guffanti A. A., Bossewitch J. S., Padan E., Krulwich T. A. 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] [Google Scholar]
  18. Ivey D. M., Guffanti A. A., Zemsky J., Pinner E., Karpel R., Padan E., Schuldiner S., Krulwich T. A. 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] [Google Scholar]
  19. 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] [Google Scholar]
  20. Krulwich T. A. Alkaliphiles: 'basic' molecular problems of pH tolerance and bioenergetics. Mol Microbiol. 1995 Feb;15(3):403–410. doi: 10.1111/j.1365-2958.1995.tb02253.x. [DOI] [PubMed] [Google Scholar]
  21. Krulwich T. A., Cheng J., Guffanti A. A. 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. doi: 10.1242/jeb.196.1.457. [DOI] [PubMed] [Google Scholar]
  22. Krulwich T. A., Federbush J. G., Guffanti A. A. 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] [Google Scholar]
  23. Krulwich T. A., Ito M., Gilmour R., Sturr M. G., Guffanti A. A., Hicks D. B. Energetic problems of extremely alkaliphilic aerobes. Biochim Biophys Acta. 1996 Jul 18;1275(1-2):21–26. doi: 10.1016/0005-2728(96)00044-8. [DOI] [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. Lebovitz R. M., Takeyasu K., Fambrough D. M. Molecular characterization and expression of the (Na+ + K+)-ATPase alpha-subunit in Drosophila melanogaster. EMBO J. 1989 Jan;8(1):193–202. doi: 10.1002/j.1460-2075.1989.tb03364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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. doi: 10.1016/0005-2728(96)00034-5. [DOI] [PubMed] [Google Scholar]
  27. 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. doi: 10.1016/0005-2736(94)90109-0. [DOI] [PubMed] [Google Scholar]
  28. 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. doi: 10.1128/jb.176.14.4311-4315.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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. doi: 10.1007/BF00770252. [DOI] [PubMed] [Google Scholar]
  30. 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. doi: 10.1007/BF00248676. [DOI] [PubMed] [Google Scholar]
  31. 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] [Google Scholar]
  32. 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] [Google Scholar]
  33. Plack R. H., Jr, Rosen B. P. 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] [Google Scholar]
  34. Sturr M. G., Guffanti A. A., Krulwich T. A. Growth and bioenergetics of alkaliphilic Bacillus firmus OF4 in continuous culture at high pH. J Bacteriol. 1994 Jun;176(11):3111–3116. doi: 10.1128/jb.176.11.3111-3116.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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] [Google Scholar]
  36. 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. doi: 10.1111/j.1348-0421.1996.tb03323.x. [DOI] [PubMed] [Google Scholar]
  37. Thelen P., Tsuchiya T., Goldberg E. B. Characterization and mapping of a major Na+/H+ antiporter gene of Escherichia coli. J Bacteriol. 1991 Oct;173(20):6553–6557. doi: 10.1128/jb.173.20.6553-6557.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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] [Google Scholar]

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