1887

Abstract

Transposon mutagenesis of sp. PCC7120 led to the isolation of a mutant strain, PHB11, which grew poorly at pH values above 10. The mutant strain exhibited pronounced Na sensitivity; this sensitivity was higher under basic conditions. Mutant PHB11 also showed an inhibition of photosynthesis that was much more pronounced at alkaline pH. Reconstruction of the transposon mutation of PHB11 in the wild-type strain reproduced the phenotype of the original mutant. The wild-type version of the mutated gene was cloned and the mutation complemented. In mutant strain PHB11, the transposon had inserted within an ORF that is part of a seven-ORF operon with significant sequence similarity to a family of bacterial operons that are believed to code for a novel multiprotein cation/proton antiporter primarily involved in resistance to salt stress and adaptation to alkaline pH. The operon was denoted (multiple resistance and pH adaptation) following the nomenclature of the operon; the ORF mutated in PHB11 corresponded to . Computer analysis suggested that all seven predicted Mrp proteins were highly hydrophobic with several transmembrane domains; in fact, the predicted protein sequences encoded by , and showed significant similarity to hydrophobic subunits of the proton pumping NADH : ubiquinone oxidoreductase. expression studies indicated that is induced with increasing external Na concentrations and alkaline pH; is also upregulated under inorganic carbon (Ci) limitation. The biological significance of a putative cyanobacterial Mrp complex is discussed.

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2005-05-01
2024-03-28
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References

  1. Allen M. B., Arnon D. I. 1955; Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol 30:366–372 [CrossRef]
    [Google Scholar]
  2. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
    [Google Scholar]
  3. Avendaño M. C., Fernández-Valiente E. 1994; Effect of sodium on phosphate uptake in unicellular and filamentous cyanobacteria. Plant Cell Physiol 35:1097–1101
    [Google Scholar]
  4. Belkin S., Mehlhorn R., Packer L. 1987; Proton gradients in intact cyanobacteria. Plant Physiol 84:25–30 [CrossRef]
    [Google Scholar]
  5. Black T. A., Cai Y., Wolk C. P. 1993; Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena. Mol Microbiol 9:77–84 [CrossRef]
    [Google Scholar]
  6. Booth I. R. 1985; Regulation of cytoplasmatic pH in bacteria. Microbiol Rev 49:359–378
    [Google Scholar]
  7. Brock T. D. 1973; Lower pH limit for the existence of blue-green algae: evolutionary and ecological implications. Science 197:480–483
    [Google Scholar]
  8. Cai Y., Wolk C. P. 1990; Use of a conditionally lethal gene in Anabaena sp. strain PCC7120 to select for double recombinants and to entrap insertion sequences. J Bacteriol 181:3981–3993
    [Google Scholar]
  9. Cohen M. F., Meeks J. C., Cai Y., Wolk C. P. 1998; Transposon mutagenesis of heterocyst-forming filamentous cyanobacteria. Methods Enzymol 297:3–17
    [Google Scholar]
  10. Devereux J. P., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analyses for the VAX. Nucleic Acids Res 12:387–395 [CrossRef]
    [Google Scholar]
  11. Elanskaya I. V., Karandashova I. V., Bogachev A. V., Hagemann M. 2002; Functional analysis of the Na+/H+ antiporter encoding genes of the cyanobacterium Synechocystis PCC 6803. Biochemistry (Mosc) 67:432–440 [CrossRef]
    [Google Scholar]
  12. Elhai J. A., Wolk C. P. 1988; Conjugal transfer of DNA to cyanobacteria. Methods Enzymol 167:747–754
    [Google Scholar]
  13. Elhai J. A., Vepritsky A., Muro-Pastor A. M., Flores E., Wolk C. P. 1997; Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC7120. . J Bacteriol 179:1998–2005
    [Google Scholar]
  14. Espie G. S., Miller A. G., Canvin D. T. 1988; Characterization of the Na+ requirement in cyanobacterial photosynthesis. Plant Physiol 88:757–763 [CrossRef]
    [Google Scholar]
  15. Figge R. M., Cassier-Chauvat C., Chauvat F., Cerff R. 2001; Characterization and analysis of an NAD(P)H dehydrogenase transcriptional regulator critical for the survival of cyanobacteria facing inorganic carbon starvation and osmotic stress. Mol Microbiol 39:455–468 [CrossRef]
    [Google Scholar]
  16. Finnegan F., Sherratt D. 1982; Plasmid ColE1 conjugal mobility: the nature of bom, a region required in cis for transfer. Mol Gen Genet 185:344–351 [CrossRef]
    [Google Scholar]
  17. Friedrich T., Steinmuller K., Weiss H. 1995; The proton-pumping respiratory complex I of bacteria and mitochondria and its homologue in chloroplasts. FEBS Lett 367:107–111 [CrossRef]
    [Google Scholar]
  18. Hamamoto T., Hashimoto M., Hino M., Kitada M., Seto Y., Kudo T., Horikoshi K. 1994; Characterization of a gene responsible for the Na+/H+ antiporter system of alkalophilic Bacillus species strain C-125. Mol Microbiol 14:939–946 [CrossRef]
    [Google Scholar]
  19. Hastings J. W., Weber G. 1963; Total quantum flux of isotropic sources. J Opt Soc Am 53:1410–1415 [CrossRef]
    [Google Scholar]
  20. Hiramatsu T., Kodama K., Kuroda T., Mizushima T., Tsuchiya T. 1998; A putative multisubunit Na+/H+ antiporter from Staphylococcus aureus. J Bacteriol 180:6642–6648
    [Google Scholar]
  21. Ito M., Guffanti A. A., Oudega B., Krulwich T. A. 1999; mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis. J Bacteriol 181:2394–2402
    [Google Scholar]
  22. Ito M., Guffanti A. A., Wang W., Krulwich T. A. 2000; Effects of nonpolar mutations in each of the seven Bacillus subtilis mrp genes suggest complex interactions among the gene products in support of Na+ and alkali but not cholate resistance. J Bacteriol 182:5663–5670 [CrossRef]
    [Google Scholar]
  23. Ito M., Guffanti A. A., Krulwich T. A. 2001; Mrp-dependent Na+/H+ antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters. FEBS Lett 496:117–120 [CrossRef]
    [Google Scholar]
  24. Kaneko T., Sato S., Kaneko T. 23 other authors 1996; Sequence analysis of the genome of the unicellular cyanobacterium Synechocystissp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3:109–136 [CrossRef]
    [Google Scholar]
  25. Kaneko T., Nakamura Y., Wolk C. P. 19 other authors 2001; Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. Strain PCC 7120. DNA Res8205–213 [CrossRef]
    [Google Scholar]
  26. Klughammer B., Sultemeyer D., Badger M. R., Price G. D. 1999; The involvement of NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in Synechococcus sp. PCC7002 gives evidence for multiple NDH-1 complexes with specific roles in cyanobacteria. Mol Microbiol 32:1305–1315 [CrossRef]
    [Google Scholar]
  27. Kosono S., Morotomi S., Kitada M., Kudo T. 1999; Analyses of a Bacillus subtilis homologue of the Na+/H+ antiporter gene which is important for pH homeostasis of alkaliphilicBacillus sp. C-125. BBA Bioenergetics 1409171–175 [CrossRef]
    [Google Scholar]
  28. Kosono S., Ohashi Y., Kawamura F., Kitada M., Kudo T. 2000; Function of a principal Na+/H+ antiporter, ShaA, is required for initiation of sporulation in Bacillus subtilis. J Bacteriol 182:898–904 [CrossRef]
    [Google Scholar]
  29. Krulwich T. A. 1995; Alkaliphiles: ‘basic’ molecular problems of pH tolerance and bioenergetics. Mol Microbiol 15:403–410 [CrossRef]
    [Google Scholar]
  30. Krulwich T. A., Gufanti A. A., Ito M. 1990; pH tolerance in Bacillus: alkaliphiles vs. non-alkaliphiles. In Bacterial Responses to pH pp 167–182 Edited by Chadwick D. J., Cardew G. Chichester: Wiley;
    [Google Scholar]
  31. Krulwich T. A. J., Cheng J., Guffanti A. A. 1994; The role of monovalent cation/proton antiporters in Na+-resistance and pH homeostasis in Bacillus: an alkaliphile versus a neutrophile. J Exp Biol 196:457–470
    [Google Scholar]
  32. Lara C., Guerrero M. G, Rodríguez R. 1993; Sodium-dependent nitrate transport and energetics of cyanobacteria. J Phycol 29:389–395 [CrossRef]
    [Google Scholar]
  33. Lien S. 1978; Hill reaction and photophosphorylation with chloroplast preparations from Chlamydomonas reinhardii. In Handbook of Phycological Methods: Physiological & Biochemical Methods pp 305–315 Edited by Hellebust J. A., Cragie J. S. Cambridge: University Press;
    [Google Scholar]
  34. Marker A. F. H. 1972; The use of acetone and methanol in the estimation of chorophyll in the presence of phaeophytin. Freshwater Biol 2:361–385 [CrossRef]
    [Google Scholar]
  35. Mathiesen C., Hägerhäll C. 2003; The ‘antiporter module’ of respiratory chain complex I includes the MrpC/NuoK subunit – a revision of the modular evolution scheme. FEBS Lett 549:7–13 [CrossRef]
    [Google Scholar]
  36. Mi H., Endo U., Ogawa T., Asada K. 1995; Thylakoid membrane-bound pyridine nucleotide dehydrogenase complex mediated cyclic electron transport in the cyanobacterium Synechocystis PCC 6803. Plant Cell Physiol 36:661–668
    [Google Scholar]
  37. Ohkawa H., Pakrasi H. B., Ogawa T. 2000; Two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis sp. strain PCC6803. J Biol Chem 275:31630–31634 [CrossRef]
    [Google Scholar]
  38. Padan E., Schuldiner S. 1993; Na+/H+ antiporters, molecular devices that couple the Na+ and H+ circulation in cells. J Bioenerg Biomembr 25:647–669
    [Google Scholar]
  39. Padan E., Schuldiner S. 1994; Molecular physiology of the Na+/H+ antiporter inEscherichia coli . J Exp Biol 196:443–456
    [Google Scholar]
  40. Padan E., Schuldiner S. 1996; Bacterial Na+/H+ antiporters – molecular biology, biochemistry and physiology. In Handbook of Biological Physics pp 501–531 Edited by Konings W. N., Kaback H. R., Loikema J. S. Amsterdam: Elsevier Science;
    [Google Scholar]
  41. Prommeenate P., Lennon A. M., Markert C., Hippler M., Nixon P. J. 2004; Subunit composition of NDH-1 complexes of Synechocystis sp. PCC 6803: identification of two new ndh gene products with nuclear-encoded homologues in the chloroplast Ndh complex. J Biol Chem 279:28165–28173 [CrossRef]
    [Google Scholar]
  42. Putnoky P., Kereszt A., Nakamura T., Endre G. E., Grosskopf E., Kiss P., Kondorosi A. 1998; The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system. Mol Microbiol 28:1091–1101 [CrossRef]
    [Google Scholar]
  43. Reed R. H., Stewart W. D. P. 1985; Osmotic adjustment and organic solute accumulation in unicellular cyanobacteria from freshwater and marine habitats. Mar Biol 88:1–9
    [Google Scholar]
  44. Ritchie R. J. 1992; Sodium transport and the origin of the membrane potential in the cyanobacterium Synechococcus R-2 (Anacystis nidulans) PCC 7942. J Plant Physiol 139:320–330 [CrossRef]
    [Google Scholar]
  45. Ritchie R. J. 1998; Bioenergetics of membrane transport in SynechococcusR-2 (Anacystis nidulans, S. leopoliensis) PCC 7942. Can J Bot 76:1127–1145
    [Google Scholar]
  46. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. 1997; The clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 [CrossRef]
    [Google Scholar]
  47. Wang H. L., Postier B. L., Burnap R. L. 2002; Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis ofSynechocystis sp. PCC 6803. Mol Microbiol 44:1493–1506 [CrossRef]
    [Google Scholar]
  48. Wang H. L., Postier B. L., Burnap R. L. 2004; Alterations in global patterns of gene expression in Synechocystis sp. PCC 6803 in response to inorganic carbon limitation and the inactivation ofndhR, a LysR family regulator. J Biol Chem 279:5739–5751 [CrossRef]
    [Google Scholar]
  49. Weidner U., Geier S., Ptock A., Friedrich T., Leif H., Weiss H. 1993; The gene locus of the proton-translocating NADH : ubiquinone oxidoreductase in Escherichia coli. Organization of the 14 genes and relationship between the derived proteins and subunits of mitochondrial complex I. J Mol Biol 233:109–122 [CrossRef]
    [Google Scholar]
  50. Wolk C. P., Vonshak A., Kehoe P., Elhai J. 1984; Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc Natl Acad Sci U S A 81:1561–1565 [CrossRef]
    [Google Scholar]
  51. Wolk C. P., Cai Y., Panoff J.-M. 1991; Use of a transposon with luciferase as a reporter to identify environmentally responsive genes in a cyanobacterium. Proc Natl Acad Sci U S A 88:5355–5359 [CrossRef]
    [Google Scholar]
  52. Zhang P., Battchikova N., Jansen T., Appel J., Ogawa T., Aro E. 2004; Expression and functional roles of the two distinct NDH-1 complexes and the carbon acquisition complex NdhD3/NdhF3/CupA/Sll1735 in Synechocystissp. PCC6803. Plant Cell 16:3326–3340 [CrossRef]
    [Google Scholar]
  53. Zhao J. D., Brand J. J. 1988; Sequential effects of sodium depletion on photosystem II in Synechocystis. Arch Biochem Biophys 264:657–664 [CrossRef]
    [Google Scholar]
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