1887

Abstract

Attempts to identify members of the antiporter complement of the alkali- and saline-adapted soda lake alkaliphile N10 have used screens of DNA libraries in antiporter-deficient KNabc. Earlier screens used Na or Li for selection but only identified one NhaD-type antiporter whose properties were inconsistent with a robust role in pH homeostasis. Here, new screens using elevated pH for selection identified three other putative antiporter genes that conferred resistance to pH ≥8.5 as well as Na resistance. The three predicted gene products were in the calcium/cation antiporter (CaCA), cation/proton antiporter-2 (CPA2) and cation/proton antiporter-1 (CPA1) families of membrane transporters, and were designated Aa-CaxA, Aa-KefB and Aa-NhaP respectively, reflecting homology within those families. Aa-CaxA conferred the poorest Na resistance and also conferred modest Ca resistance. Aa-KefB and Aa-NhaP inhibited growth of a K uptake-deficient mutant (TK2420), suggesting that they catalysed K efflux. For Aa-NhaP, the reversibility of the growth inhibition by high K concentrations depended upon an organic nitrogen source, e.g. glutamine, rather than ammonium. This suggests that as well as K efflux is catalysed by Aa-NhaP. Vesicles of KNabc expressing Aa-NhaP, which conferred the strongest alkali resistance, exhibited K/H antiport activity in a pH range from 7.5 to 9.5, and with an apparent for K of 0.5 mM at pH 8.0. The properties of this antiporter are consistent with the possibility that this soda lake alkaliphile uses K()/H antiport as part of its alkaline pH homeostasis mechanism and part of its capacity to reduce potentially toxic accumulation of cytoplasmic K or respectively, under conditions of high osmolarity or active amino acid catabolism.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/007450-0
2007-07-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/153/7/2168.html?itemId=/content/journal/micro/10.1099/mic.0.2007/007450-0&mimeType=html&fmt=ahah

References

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410 [CrossRef]
    [Google Scholar]
  2. Bakker E. P., Booth I. R., Dinnbier U., Epstein W., Gajewska A. 1987; Evidence for multiple K+ export systems in Escherichia coli. J Bacteriol 169:3743–3749
    [Google Scholar]
  3. Booth I. R., Edwards M. D., Murray E., Miller S. 2005; The role of bacterial channels in cell physiology. In Bacterial Ion Channels and Their Eukaryotic Homologs pp 291–312 Edited by Kubalski A., Marinac B. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  4. Brockman R. W., Heppel L. A. 1968; On the localization of alkaline phosphatase and cyclic phosphodiesterase in Escherichia coli. Biochemistry 7:2554–2562 [CrossRef]
    [Google Scholar]
  5. Buurman E. T., Neijssel O. M., Teixeira de Mattos M. J. 1991; Futile cycling of ammonium ions via the high affinity potassium uptake system (Kdp) of Escherichia coli. Arch Microbiol 155:391–395
    [Google Scholar]
  6. Cai X., Lytton J. 2004; The cation/Ca2+ exchanger superfamily: phylogenetic analysis and structural implications. Mol Biol Evol 21:1692–1703 [CrossRef]
    [Google Scholar]
  7. Epstein W. 2003; The roles and regulation of potassium in bacteria. Prog Nucleic Acid Res Mol Biol 75:293–320
    [Google Scholar]
  8. Epstein W., Buurman E., McLaggan D., Naprstek J. 1993; Multiple mechanisms, roles and controls of K+ transport in Escherichia coli. Biochem Soc Trans 21:1006–1010
    [Google Scholar]
  9. Falb M., Pfeiffer F., Palm P., Rodewald K., Hickmann V., Tittor J., Oesterhelt D. 2005; Living with two extremes: conclusions from the genome sequence of Natronomonas pharaonis. Genome Res 15:1336–1343 [CrossRef]
    [Google Scholar]
  10. Fujisawa M., Kusomoto A., Wada Y., Tsuchiya T., Ito M. 2005; NhaK, a novel monovalent cation/H+ antiporter of Bacillus subtilis. Arch Microbiol 183:411–420 [CrossRef]
    [Google Scholar]
  11. Goldberg E. B., Arbel T., Chen J., Karpel R., Mackie G. A., Schuldiner S., Padan E. 1987; Characterization of a Na+/H+ antiporter gene of Escherichia coli. Proc Natl Acad Sci U S A 84:2615–2619 [CrossRef]
    [Google Scholar]
  12. Grant W. D. 2004; Life at low water activity. Philos Trans R Soc Lond B Biol Sci 359:1249–1267 [CrossRef]
    [Google Scholar]
  13. Grant W. D., Tindall B. J. 1986; The alkaline saline environment. In Microbes in Extreme Environments Edited by Herbert R. A., Codd G. A. London: Academic Press;
    [Google Scholar]
  14. Guffanti A. A., Wei Y., Rood S. V., Krulwich T. A. 2002; An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+ . Mol Microbiol 45:145–153 [CrossRef]
    [Google Scholar]
  15. Harel-Bronstein M., Dibrov P., Olami Y., Pinner E., Schuldiner S., Padan E. 1995; MH1, a second-site revertant of an Escherichia coli mutant lacking Na+/H+ antiporters (Δ nhaA Δ nhaB ), regains Na+ resistance and a capacity to excrete Na+ in a ΔμH+-independent fashion. J Biol Chem 270:3816–3822 [CrossRef]
    [Google Scholar]
  16. Hellmer J., Patzold R., Zeilinger C. 2002; Identification of a pH regulated Na+/H+ antiporter of Methanococcus jannaschii. FEBS Lett 527:245–249 [CrossRef]
    [Google Scholar]
  17. Horikoshi K. 1991 Microorganisms in Alkaline Environments. New York: VCH Publishers;
    [Google Scholar]
  18. Ivey D. M., Guffanti A. A., Zemsky J., Pinner E., Karpel R., Padan E., Schuldiner S., Krulwich T. A. 1993; 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 268:11296–11303
    [Google Scholar]
  19. Jones B. E., Grant W. D., Duckworth A. W., Owenson G. G. 1998; Microbial diversity of soda lakes. Extremophiles 2:191–200 [CrossRef]
    [Google Scholar]
  20. Kakinuma Y., Igarashi K. 1995; Potassium/proton antiport system of growing Enterococcus hirae at high pH. J Bacteriol 177:2227–2229
    [Google Scholar]
  21. Krulwich T. A. 1995; Alkaliphiles: ‘basic’ molecular problems of pH tolerance and bioenergetics. Mol Microbiol 15:403–410 [CrossRef]
    [Google Scholar]
  22. Krulwich T. A., Hicks D. B., Swartz T. H., Ito M. 2007; Bioenergetic adaptations that support alkaliphily. In Physiology and Biochemistry of Extremophiles Edited by Gerday C., Glansdorff N. Washington, DC: American Society for Microbiology; in press
    [Google Scholar]
  23. Lewinson O., Padan E., Bibi E. 2004; Alkalitolerance: a biological function for a multidrug transporter in pH homeostasis. Proc Natl Acad Sci U S A 101:14073–14078 [CrossRef]
    [Google Scholar]
  24. Liu J., Xue Y., Wang Q., Wei Y., Swartz T. H., Hicks D. B., Ito M., Ma Y., Krulwich T. A. 2005; The activity profile of the NhaD-type Na+(Li+)/H+ antiporter from the soda lake haloalkaliphile Alkalimonas amylolytica is adaptive for the extreme environment. J Bacteriol 187:7589–7595 [CrossRef]
    [Google Scholar]
  25. Ma Y., Xue Y., Grant W. D., Collins N. C., Duckworth A. W., Van Steenbergen R. P., Jones B. E. 2004; Alkalimonas amylolytica gen. nov., sp. nov., and Alkalimonas delamerensis gen. nov., sp. nov., novel alkaliphilic bacteria from soda lakes in China and East Africa. Extremophiles 8:193–200 [CrossRef]
    [Google Scholar]
  26. MacLean M. J., Ness L. S., Ferguson G. P., Booth I. R. 1998; The role of glyoxalase I in the detoxification of methylglyoxal and in the activation of the KefB K+ efflux system in Escherichia coli. Mol Microbiol 27:563–571 [CrossRef]
    [Google Scholar]
  27. Miller S., Douglas R. M., Carter P., Booth I. R. 1997; Mutations in the glutathione-gated KefC K+ efflux system of Escherichia coli that cause constitutive activation. J Biol Chem 272:24942–24947 [CrossRef]
    [Google Scholar]
  28. Miller S., Ness L. S., Wood C. M., Fox B. C., Booth I. R. 2000; Identification of an ancillary protein, YabF, required for activity of the KefC glutathione-gated potassium efflux system in Escherichia coli. J Bacteriol 182:6536–6540 [CrossRef]
    [Google Scholar]
  29. Nakamura T., Tokuda H., Unemoto T. 1984; K+/H+ antiporter functions as a regulator of cytoplasmic pH in a marine bacterium, Vibrio alginolyticus. Biochim Biophys Acta 776:330–336 [CrossRef]
    [Google Scholar]
  30. Nakamura T., Kawasaki S., Unemoto T. 1992; Roles of K+ and Na+ in pH homeostasis and growth of the marine bacterium Vibrio alginolyticus. J Gen Microbiol 138:1271–1276 [CrossRef]
    [Google Scholar]
  31. Nakamura C., Burgess J. G., Sode K., Matsunaga T. 1995; An iron-regulated gene, magA , encoding an iron transport protein of Magnetospirillum sp. strain AMB-1. J Biol Chem 270:28392–28396 [CrossRef]
    [Google Scholar]
  32. Nozaki K., Kuroda T., Mizushima T., Tsuchiya T. 1998; A new Na+/H+ antiporter, NhaD, of Vibrio parahaemolyticus. Biochim Biophys Acta 1369213–220 [CrossRef]
    [Google Scholar]
  33. Ohyama T., Igarashi K., Kobayashi H. 1994; Physiological role of the chaA gene in sodium and calcium circulations at a high pH in Escherichia coli. J Bacteriol 176:4311–4315
    [Google Scholar]
  34. Padan E., Krulwich T. A. 2000; Sodium stress. In Bacterial Stress Responses pp 117–130 Edited by Storz G., Hengge-Aronis R. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  35. Padan E., Venturi M., Gerchman Y., Dover N. 2001; Na+/H+ antiporters. Biochim Biophys Acta 1505144–157 [CrossRef]
    [Google Scholar]
  36. Padan E., Bibi E., Ito M., Krulwich T. A. 2005; Alkaline pH homeostasis in bacteria: new insights. Biochim Biophys Acta 171767–88 [CrossRef]
    [Google Scholar]
  37. Plack R. H. Jr, Rosen B. P. 1980; Cation/proton antiport systems in Escherichia coli. Absence of potassium/proton antiporter activity in a pH-sensitive mutant. J Biol Chem 255:3824–3825
    [Google Scholar]
  38. Price G. D., Woodger F. J., Badger M. R., Howitt S. M., Tucker L. 2004; Identification of a SulP-type bicarbonate transporter in marine cyanobacteria. Proc Natl Acad Sci U S A 101:18228–18233 [CrossRef]
    [Google Scholar]
  39. Radchenko M. V., Tanaka K., Waditee R., Oshimi S., Matsuzaki Y., Fukuhara M., Kobayashi H., Takabe T., Nakamura T. 2006a; Potassium/proton antiport system of Escherichia coli. J Biol Chem 281:19822–19829 [CrossRef]
    [Google Scholar]
  40. Radchenko M. V., Waditee R., Oshimi S., Fukuhara M., Takabe T., Nakamura T. 2006b; Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli. Mol Microbiol 59:651–663 [CrossRef]
    [Google Scholar]
  41. Ren Q., Chen K., Paulsen I. T. 2007; TransportDB: a comprehensive database resource for cytoplasmic membrane transport systems and outer membrane channels. Nucleic Acids Res 35:D274–D279 [CrossRef]
    [Google Scholar]
  42. Rosen B. P. 1986; Ion extrusion systems in E. coli. Methods Enzymol 125:328–386
    [Google Scholar]
  43. Ruknudin A., Schulze D. H. 2002; Proteomics approach to Na+/Ca+ exchangers in prokaryotes. Ann N Y Acad Sci 976:103–108
    [Google Scholar]
  44. Saaf A., Baars L., von Heijne G. 2001; The internal repeats in the Na+/Ca2+ exchanger-related Escherichia coli protein YrbG have opposite membrane topologies. J Biol Chem 276:18905–18907 [CrossRef]
    [Google Scholar]
  45. Saier M. H. Jr, Eng B. H., Fard S., Garg J., Haggerty D. A., Hutchinson W. J., Jack D. L., Lai E. C., Liu H. J. other authors 1999; Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim Biophys Acta 1422:1–56 [CrossRef]
    [Google Scholar]
  46. Saier M. H. Jr, Tran C. V., Barabote R. D. 2006; TCDB: the Transporter Classification Database for membrane transport protein analyses and information. Nucleic Acids Res 34:D181–D186 [CrossRef]
    [Google Scholar]
  47. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  48. Southworth T. W., Guffanti A. A., Moir A., Krulwich T. A. 2001; GerN, an endospore germination protein of Bacillus cereus , is an Na+/H+-K+ antiporter. J Bacteriol 183:5896–5903 [CrossRef]
    [Google Scholar]
  49. Ventosa A., Nieto J. J., Oren A. 1998; Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62:504–544
    [Google Scholar]
  50. Verkhovskaya M. L., Barquera B., Wikström M. 2001; Deletion of one of two Escherichia coli genes encoding putative Na+/H+ exchangers ( ycgO ) perturbs cytoplasmic alkali cation balance at low osmolarity. Microbiology 147:3005–3013
    [Google Scholar]
  51. Waditee R., Hossain G. S., Tanaka Y., Nakamura T., Shikata M., Takano J., Takabe T. 2004; Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria. J Biol Chem 279:4330–4338
    [Google Scholar]
  52. Waser M., Hess-Bienz D., Davies K., Solioz M. 1992; Cloning and disruption of a putative NaH-antiporter gene of Enterococcus hirae. J Biol Chem 267:5396–5400
    [Google Scholar]
  53. Wei Y., Southworth T. W., Kloster H., Ito M., Guffanti A. A., Moir A., Krulwich T. A. 2003; Mutational loss of a K+ and NH+ 4 transporter affects the growth and endospore formation of alkaliphilic Bacillus pseudofirmus OF4. J Bacteriol 185:5133–5147 [CrossRef]
    [Google Scholar]
  54. Wutipraditkul N., Waditee R., Incharoensakdi A., Hibino T., Tanaka Y., Nakamura T., Shikata M., Takabe T. 2005; Halotolerant cyanobacterium Aphanothece halophytica contains NapA-type Na+/H+ antiporters with novel ion specificity that are involved in salt tolerance at alkaline pH. Appl Environ Microbiol 71:4176–4184 [CrossRef]
    [Google Scholar]
  55. Yumoto I. 2002; Bioenergetics of alkaliphilic Bacillus spp. J Biosci Bioeng 93:342–353 [CrossRef]
    [Google Scholar]
  56. Zheng X. 1992; Outline of salt lakes in Inner Mongolia: salt lakes of the Eerduosi Plateau of Inner Mongolia. In Salt Lakes of Inner Mongolia pp 219–287 Edited by Zheng X. Beijing: Academic Press;
    [Google Scholar]
  57. Zuker M. 2003; Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/007450-0
Loading
/content/journal/micro/10.1099/mic.0.2007/007450-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error