Plant Molecular Biology

, Volume 11, Issue 3, pp 335–344

Expression of the Escherichia coli glutamate dehydrogenase gene in the cyanobacterium Synechococcus PCC6301 causes ammonium tolerance

  • D. A Lightfoot
  • A. J. Baron
  • J. C. Wootton
Article

Abstract

The unicellular cyanobacterium Synechococcus PCC6301 lacks a hybridisable homologue of the strongly conserved gdhA gene of E. coli that encodes NADP-specific glutamate dehydrogenase. This is consistent with the failure to find this enzyme in extracts of the cyanobacterium. The E. coli gdhA gene was transferred to Synechococcus PCC6301 by transformation with an integrative vector. High levels of glutamate dehydrogenase activity, similar to those found in ammonium grown E. coli cells, were found in these transformants. These transformed cyanobacteria displayed an ammonium tolerant phenotype, consistent with the action of their acquired glutamate dehydrogenase activity as an ammonium detoxification mechanism. Minor differences in colony size and in growth at low light intensity were also observed.

Key words

ammonia assimilation ammonia toxicity cyanobacteria glutamate dehydrogenase E. coli gdhA gene Synechococcus PCC6301 

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References

  1. 1.
    Beevers L, Hageman RH: Nitrate and nitrite reduction. In: Miflin BJ (ed.) The Biochemistry of Plants, Volume 5, pp. 115–168. Academic Press, New York (1980).Google Scholar
  2. 2.
    Bottomly PJ, vanBalen C: Characterization of heterotrophic growth in the blue-green alga Nostoc sp. strain MAC. J Gen Microbiol 107: 309–318 (1978).Google Scholar
  3. 3.
    Bravo A, Becerril B, Mora J: Introduction of the Escherichia coli gdhA gene into Rhizobium phaseoli: effect on nitrogen fixation. J Bact 170: 985–988 (1988).Google Scholar
  4. 4.
    Givan CV: Metabolic detoxification of ammonia in higher plants. Phytochem 18: 375–382 (1979).Google Scholar
  5. 5.
    Hanahan D: Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166: 557–580 (1983).Google Scholar
  6. 6.
    Haselkorn R: Organisation of the genes for nitrogen fixation in photosynthetic bacteria and cyanobacteria. Ann Rev Microbiol 40: 525–547 (1986).Google Scholar
  7. 7.
    Hoare DS, Hoare SL, Moore RB: The photoassimilation of organic compounds by autotrophic blue-green algae. J Gen Microbiol 49: 351–370 (1967).Google Scholar
  8. 8.
    Ish-Horowicz D, Burke JF: Rapid and efficient cosmid vector cloning. Nucl Acids Res 9: 2989–2998 (1981).Google Scholar
  9. 9.
    Janson CA, Kayne PS, Almassy RJ, Grunstein M, Eisenberg D: Sequence of glutamine synthetase from Salmonella typhimurium and implications for the protein structure. Gene 46: 297–300 (1986).Google Scholar
  10. 10.
    Krogmann DW, Jagendorf AT, Avron M: Uncouplers of spinach chloroplast photosynthetic phosphorylation. Plant Physiol 34: 272–277 (1959).Google Scholar
  11. 11.
    Lightfoot DA, Walters DE, Wootton JC: Transformation of the cyanobacterium Synechococcus PCC6301 using cloned DNA. J Gen Microbiol 134: 1509–1514 (1988).Google Scholar
  12. 12.
    Maniatis T, Fritsch E, Sambrook J: Molecular Cloning: A Laboratory Manual, 1st edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982).Google Scholar
  13. 13.
    Mattaj IW, McPherson MJ, Wootton JC: Localization of a strongly conserved section of coding sequence in glutamate dehydrogenase genes. FEBS Lett 147: 21–25 (1982).Google Scholar
  14. 14.
    Mazur BJ, Rice D, Haselkorn R: Identification of blue-green algal nitrogen fixation genes by using heterologous DNA hybridization probes. Proc Natl Acad Sci USA 77: 186–190 (1980).Google Scholar
  15. 15.
    McPherson MJ, Wootton JC: Complete nucleotide sequence of the Escherichia coli gdhA gene. Nucl Acids Res 11: 5257–5266 (1983).Google Scholar
  16. 16.
    Meeks JC, Wolk CP, Lockau W, Schilling N, Shaffer W, Chen WS: Pathways of assimilation of [13N]N2 and [13N]NH4 + by cyanobacteria with and without heterocysts. J Bact 134: 125–130 (1976).Google Scholar
  17. 17.
    Meister A: Catalytic mechanism of glutamine synthetase: overview of glutamine metabolism. In: Mora J, Palacios R (eds) Glutamine: Metabolism, Enzymes and Regulation, pp. 1–40. Academic Press, New York (1980).Google Scholar
  18. 18.
    Miflin BJ, Lea PJ: Ammonia assimilation. In: Miflin BJ (ed.) The Biochemistry of Plants, Volume 5, pp. 169–202. Academic Press, New York (1980).Google Scholar
  19. 19.
    Mountain A, McPherson MJ, Baron AJ, Wootton JC: The Klebsiella aerogenes glutamate dehydrogenase gene: cloning, high level expression and hybrid enzyme formation in Escherichia coli. Mol Gen Genet 199: 141–145 (1985).Google Scholar
  20. 20.
    Mulligan B, Schultes N, Chen L, Bogorad L: Nucleotide sequence of a multi-copy gene for the B-protein of photosystem II of a cyanobacterium. Proc Nat Acad Sci USA 81: 2693–2697 (1984).Google Scholar
  21. 21.
    Neilsonn AH, Doudoroff M: Ammonia assimilation in blue-green algae. Archiv Microbiol 89: 15–22 (1973).Google Scholar
  22. 22.
    Pearce J, Leach CK, Carr NG: The incomplete tricarboxylic acid cycle in Anabaena variabilis. J Gen Microbiol 55: 371–378 (1969).Google Scholar
  23. 23.
    Platt SG, Anthon GE: Ammonia accumulation and inhibition of photosynthesis in methionine sulphoxamine treated spinach. Plant Physiol 67: 509–513 (1981).Google Scholar
  24. 24.
    Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY: Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1–61 (1979).Google Scholar
  25. 25.
    Rocha M, Vazquez M, Garciarrubio A, Covarrubias AA. Nucletide sequences of the glnA-glnL intercistronic region of Escherichia coli. Gene 37: 91–99 (1985).Google Scholar
  26. 26.
    Sinden SL, Durbin RD: Glutamine synthetase inhibition: possible mode of action of wildfire toxin from Pseudomonas tabaci. Nature 219: 379–380 (1968).Google Scholar
  27. 27.
    Singh HN, Ladha JK, Kumar HD: Genetic control of heterocyst formation in the blue-green algae Nostoc muscorum and Nostoc linkea. Archiv Microbiol 144: 155–159 (1987).Google Scholar
  28. 28.
    Singh HN, Sonie KC, Singh HN: Nitrate regulation of heterocyst differentiation and nitrogen fixation in a chlorate resistant mutant of the blue-green alga Nostoc muscorum. Mutation Res 42: 447–452 (1977).Google Scholar
  29. 29.
    Smith FA, Raven JA: Intracellular pH and its regulation. Ann Rev Plant Physiol 30: 289–311 (1979).Google Scholar
  30. 30.
    Stanier RY, Adelberg EA, Ingraham JL: General Microbiology, 4th edn. McMillan Press, London (1977).Google Scholar
  31. 31.
    Stewart WDP: Some aspects of structure and function in N2-fixing cyanobacteria. Ann Rev Microbiol 34: 497–536 (1980).Google Scholar
  32. 32.
    Stewart GR, Mann AF, Fentem PA: Enzymes of glutamate formation: glutamate dehydrogenase, glutamine synthetase and glutamate synthase. In: Miflin BJ (ed.) The Biochemistry of Plants, Volume 5, pp. 169–202. Academic Press, New York (1980).Google Scholar
  33. 33.
    Tumer NE, Robinson SJ, Haselkorn R: Different promoters for the Anabaena glutamine synthetase gene during growth using molecular or fixed nitrogen. Nature 306: 337–343 (1983).Google Scholar
  34. 34.
    Windass JD, Worsey MJ, Pioli EM, Pioli D, Barth PT, Atherton KT, Byrom D, Powell K, Senior PJ: Inproved conversion of methanol to single-cell protein by Methylophilus methylotrophus. Nature 287: 396–401 (1981).Google Scholar
  35. 35.
    Wootton JC: Reassessment of ammonium ion affinities of NADP-specific glutamate dehydrogenase: activation of the Neurospora crassa enzyme by ammonium and rubidium ions. Biochem J 209: 527–531 (1983).Google Scholar
  36. 36.
    Wootton JC, McPherson MJ: Genes of nitrate and ammonium assimilation. Ann Proc Phytochem Soc Europe 23: 89–114 (1984).Google Scholar

Copyright information

© Kluwer Academic Publishers 1988

Authors and Affiliations

  • D. A Lightfoot
    • 1
  • A. J. Baron
    • 1
  • J. C. Wootton
    • 1
  1. 1.Department of GeneticsUniversity of LeedsLeedsUK
  2. 2.Department of HorticultureOregon State UniversityCorvallisUSA

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