The Journal of Microbiology

, Volume 46, Issue 4, pp 415–421 | Cite as

Cloning and characterization of a Na+/H+ antiporter gene of the moderately halophilic bacterium Halobacillus aidingensis AD-6T

  • Ya Jie Zou
  • Li Fu Yang
  • Lei Wang
  • Su Sheng Yang
Articles
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Abstract

A gene encoding a Na+/H+ antiporter was obtained from the genome of Halobacillus aidingensis AD-6T, which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na+/H+ antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na+/H+ as well as Li+/H+ antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K+/H+ antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na+/H+ antiporter activity.

Keywords

Halobacillus aidingensis Na+/H+ antiporter nhaH Inverse PCR 
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References

  1. Bayer, A.S., P.M. Namara, M.R. Yeaman, N. Lucindo, T. Jones, A.L. Cheung, H.G. Sahl, and R.A. Proctor. 2006. Transposon disruption of the complex I NADH oxidoreductase gene (snoD) in Staphylococcus aureus is associated with reduced susceptibility to the microbicidal activity of thrombin-induced platelet microbicidal protein. J. Bacteriol. 188, 211–222.PubMedCrossRefGoogle Scholar
  2. Fujisawa, M., A. Kusomoto, Y. Wada, T. Tsuchiya, and M. Ito. 2005. NhaK, a novel monovalent cation/H+ antiporter of Bacillus subtilis. Arch. Microbiol. 183, 411–420.PubMedCrossRefGoogle Scholar
  3. Gerchman, Y., Y. Loamy, A. Rimon, D. Taglicht, S. Schuldiner, and E. Padan. 1993. Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli. Proc. Natl. Acad. Set USA 90, 1212–1216.CrossRefGoogle Scholar
  4. Goldberg, E.B., T. Arbel, J. Chen, R. Karpel, G.A. Mackie, S. Schuldiner, and E. Padan. 1987. Characterization of a Na+/H+ antiporter gene of Escherichia coli. Proc. Natl. Acad. Sci. USA 84, 2615–2619.PubMedCrossRefGoogle Scholar
  5. Gouda, T., M. Kuroda, T. Hiramatsu, K. Nozaki, T. Kuroda, T. Mizushima, and T. Tsuchiya. 2001. nhaG Na+/H+ antiporter gene of Bacillus subtilis ATCC9372, which is missing in the complete genome sequence of strain, and properties of the antiporter. J. Biochem. 130, 711–717.PubMedGoogle Scholar
  6. Hamada, A., T. Hibino, T. Nakamura, and T. Takabe. 2001. Na+/H+ antiporter from Synechocystis species PCC 6803, homologous to SOS1, contains an aspartic residue and long C-terminal tail important for the carrier activity. Plant Physiol. 125, 437–446.PubMedCrossRefGoogle Scholar
  7. Herz, K., S. Vimont, E. Padan, and P. Berche. 2003. Roles of NhaA, NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment. J. Bacteriol. 185, 1236–1244.PubMedCrossRefGoogle Scholar
  8. Hiramatsu, T., K. Kodama, X Kuroda, X Mizushima, and X Tsuchiya. 1998. A putative multisubunit Na+/H+ antiporter from Staphylococcus aureus. J. Bacteriol. 180, 6642–6648.PubMedGoogle Scholar
  9. Inoue, H., X Noumi, X Tsuchiya, and H. Kanazawa. 1995. Essential aspartic acid residues, Asp-133, Asp-163 and Asp-164, in the transmembrane helices of a Na+/H+ antiporter (NhaA) from Escherichia coli. FEBS Lett. 363, 264–268.PubMedCrossRefGoogle Scholar
  10. Inoue, H., Y. Tsuboi, and H. Kanazawa. 2001. Chimeric Na+/H+ antiporters constructed from NhaA of Helicobacter pylori and Escherichia coli: implications for domains of NhaA for pH sensing. J. Biochem. 129, 569–576.PubMedGoogle Scholar
  11. Ito, M., A.A. Guffanti, B. Oudega, and T.A. Krulwich. 1999. mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis. J. Bacteriol. 183, 411–420.Google Scholar
  12. Ito, M., A.A. Guffanti, J. Zemsky, D. Mackivey, and T.A. Krulwich. 1997. Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4. J. Bacteriol. 179, 3851–3857.PubMedGoogle Scholar
  13. Jiang, J.Q., W. Wei, B.H. Du, X.H. Li, L. Wang, and S.S. Yang. 2004. Salt tolerance genes involved in cation efflux and osmoregulation of Sinorhizobium fredii RT19 detected by isolation and characterization of Tn5 mutants. FEMS Microbiol. Lett. 239, 139–146.PubMedCrossRefGoogle Scholar
  14. Karpel, R., Y. Olami, D. Taglicht, S. Schuldiner, and E. Padan. 1988. Sequencing of the gene ant which affects the Na+/H+ antiporter activity in Escherichia coli. J. Biol. Chem. 263, 10408–10414.PubMedGoogle Scholar
  15. Kosono, S., S. Morotomi, M. Kitada, and X Kudo. 1999. Analyses of a Bacillus subtilis homologue of the Na+/H+ antiporter gene which is important for pH homeostasis of alkaliphilic Bacillus sp. C-125. Biochim. Biophys. Acta. 1409, 171–175.PubMedCrossRefGoogle Scholar
  16. Krulwich, T.A. 1983. Na+/H+ antiporters. Biochim. Biophys. Acta. 726, 245–264.PubMedGoogle Scholar
  17. Lewinson, O., E. Padan, and E. Bibi. 2004. Alkali tolerance: a biological function for a multidrug transporter in pH homeostasis. Proc. Natl. Acad. Sci. USA 101, 14073–14078.PubMedCrossRefGoogle Scholar
  18. Liu, W.Y., J. Zeng, L. Wang, Y.T. Dou, and S.S. Yang. 2005. Halobacillus dabanensis sp. nov. and Halobacillus aidingensis sp. nov., isolated from salt lakes in Xinjiang, China. Int. J. Syst. Evol. Microbiol. 55, 1991–1996.PubMedCrossRefGoogle Scholar
  19. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.PubMedGoogle Scholar
  20. Ochman, H., A.S. Gerber, and D.L. Hartl. 1988. Genetic application of an inverse polymerase chain reaction. Genetics 120, 621–623.PubMedGoogle Scholar
  21. Ohyama, T., K. Igarashi, and H. Kobayashi. 1994. Physiological role of the chaA gene in sodium and calcium circulations at a high pH in Escherichia coli. J. Bacteriol. 176, 4311–4315.PubMedGoogle Scholar
  22. Padan, E., N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner. 1989. Deletion of ant in Escherichia coli reveals its function in adaptation to high salinity and an alternative Na+/H+ antiporter system(s). J. Biol. Chem. 264, 20297–20302.PubMedGoogle Scholar
  23. Padan, E. and S. Schuldiner. 1994. Molecular physiology of Na+/H+ antiporter, key transporters in circulation of Na+ and H+ in cells. Biochim. Biophys. Acta. 1185, 129–151.PubMedCrossRefGoogle Scholar
  24. Pinner, E., E. Padan, and S. Schuldiner. 1992. Cloning, sequencing, and expression of the nhaB gene, encoding a Na+/H+ antiporter in Escherichia coli. J. Biol. Chem. 267, 11064–11068.PubMedGoogle Scholar
  25. Rosen, B.P. 1986. Ion extrusion systems in Escherichia coli. Methods Enzymol. 125, 328–336.PubMedCrossRefGoogle Scholar
  26. Rungaroon, W., H. Yakashi, X Yoshito, N. Tatsunosuke, I. Aran, and X Teruhiro. 2001. Halotolerant cyanobacterium Aphanothece halophytica contains an Na+/H+ antiporter, homologous to eukaryotic ones, with novel ion specificity affected by C-terminal tail. J. Biol. Chem. 276, 36931–36938.CrossRefGoogle Scholar
  27. Sambrook, J., E.F. Fritsch, and T.E. Maniatis. 1989. Molecular Cloning: Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, N.Y., USA.Google Scholar
  28. Tsuchiya, T., S.M. Hasan, and J. Raven. 1977. Glutamate transport driven by an electrochemical gradient of sodium ions in Escherichia coli. J. Bacteriol. 162, 794–798.Google Scholar
  29. Utsugi, J., K. Inaba, X Kuroda, M. Tsuda, and X Tsuchiya. 1998. Cloning and sequencing of a novel Na+/H+ antiporter gene from Pseudomonas aeruginosa. Biochim. Biophys. Acta. 1398, 330–334.PubMedGoogle Scholar
  30. Ventosa, A., J. Nieto, and A. Oren. 1998. Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 2, 504–544.Google Scholar
  31. Waser, M., B.D. Hess, K. Davies, and M. Solioz. 1992. Cloning and disruption of a putative Na+/H+ antiporter gene of Enterococcus hirae. J. Biol. Chem. 267, 5396–5400.PubMedGoogle Scholar
  32. Yang, L.F., J.Q. Jiang, B.S. Zhao, B. Zhang, D.Q. Feng, W.D. Lu, L. Wang, and S.S. Yang. 2006. A Na+/H+ antiporter gene of the moderately halophilic bacterium Halobacillus dabanensis D-8T: cloning and molecular characterization. FEMS Microbiol. Lett. 255, 89–95.PubMedCrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelber GmbH 2008

Authors and Affiliations

  • Ya Jie Zou
    • 1
  • Li Fu Yang
    • 1
    • 2
  • Lei Wang
    • 1
  • Su Sheng Yang
    • 1
  1. 1.Department of Microbiology and Immunology, College of Biological SciencesChina Agricultural University, and Key Laboratory of Agro-Microbial Resource and Application, Ministry of AgricultureBeijingP. R. China
  2. 2.Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, and Key Laboratory of Physiology for Tropical CropsMinistry of AgricultureDanzhou HainanP. R. China

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