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The NADP-glutamate dehydrogenase of the cyanobacterium Synechocystis 6803: cloning, transcriptional analysis and disruption of the gdhA gene

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Abstract

The gdhA gene of Synechocystis PCC 6803, which encodes an NADP-dependent glutamate dehydrogenase (NADP-GDH), has been cloned by complementation of an Escherichia coli glutamate auxotroph. This gene was found to code for a polypeptide of 428 amino acid residues, whose sequence shows high identity with those of archaebacteria (42–47%), some Gram-positive bacteria (40–44%) and mammals (37%). The minimal fragment of Synechocystis DNA required for complementation (2 kb) carries the gdhA gene preceded by an open reading frame (ORF2) encoding a polypeptide of 130 amino acids. ORF2 and gdhA are co-transcribed as a 1.9 kb mRNA, but shorter transcripts including only gdhA were also detected. Two promoter regions were identified upon transcriptional fusion to the cat reporter gene of a promoter probe plasmid. Transcription from the promoter upstream of ORF2 was found to be regulated depending on the growth phase of Synechocystis, in parallel to NADP-GDH activity. This promoter is expressed in Escherichia coli too, in contrast to the second promoter, located between ORF2 and gdhA, which was silent in E. coli and did not respond to the stage of growth in Synechocystis. Disruption of the cyanobacterial gdhA gene with a chloramphenicol resistance cassette yielded a mutant strain totally lacking NADP-GDH activity, demonstrating that this gene is not essential to Synechocystis 6803 under our laboratory conditions.

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References

  1. Amuro N, Mayumi Y, Goto Y, Okazaki T: Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor. Biochem Biophys Res Commun 152: 1395–1400 (1988).

    Google Scholar 

  2. Baker PJ, Britton KL, Engel PC, Farrants GW, Lilley KS, Rice DW, Stillman TJ: Subunit assembly and active site location in the structure of glutamate dehydrogenase. Proteins 12: 75–86 (1992).

    Google Scholar 

  3. Bansal A, Dayton MA, Zalkin H, Colman RF: Affinity labeling of a glutamyl peptide in the coenzyme binding site of NADP+-specific glutamate dehydrogenase of Salmonelia typhimurium by 2-[(4-bromo-2,3-dioxobutyl)thio]-1, N 6-ethenoadenosine 2′, 5′-bisphosphate. J Biol Chem 264: 9827–9835 (1989).

    Google Scholar 

  4. Bascomb NF, Schmidt RR: Purification and partial kinetic and physical characterization of two chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes and their preferential accumulation in Chlorella sorokiniana cells cultured at low or high ammonium levels. Plant Physiol 83: 75–84 (1987).

    Google Scholar 

  5. Benachenhou N, Baldacci G: The gene for a halophilic glutamate dehydrogenase: sequence, transcription analysis and phylogenetic implications. Mol Gen Genet 230: 345–352 (1991).

    Google Scholar 

  6. Benachenhou-Lahfa N, Forterre P, Labedau B: Evolution of glutamate dehydrogenase genes: evidence for two paralogous protein families and unusual branching patterns of the Archaebacteria in the universal tree of life. J Mol Evol 36: 335–346 (1993).

    Google Scholar 

  7. Bonete MJ, Camacho ML, Cadenas E: A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity. Int J Biochem 19: 1149–1155 (1987).

    Google Scholar 

  8. Börmann ER, Eikmanns BJ, Sahm H: Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. Mol Microbiol 6: 317–326 (1992).

    Google Scholar 

  9. Börmann-EI Kholy ER, Eikmans BJ, Gutmann M, Sahm H: Glutamate dehydrogenase is not essential for glutamate formation by Corynebacterium glutamicum. Appl Environ Microbiol 59: 2329–2331 (1993).

    Google Scholar 

  10. Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).

    Google Scholar 

  11. Brett M, Chambers GK, Holder AA, Fincham JRS, Wootton JC: Mutational amino acid replacements in Neurospora crassa NADP-specific glutamate dehydrogenase. J Mol Biol 106: 1–22 (1976).

    Google Scholar 

  12. BroedelJr SE, WolfJr RE: Growth-phase-dependent induction of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase in the cyanobacterium Synechococcus sp. PCC 7942. Gene 109: 71–79 (1991).

    Google Scholar 

  13. Chauvat F, de Vries L, van der Ende A, van Arkel GA: A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803. Mol Gen Genet 204: 185–191 (1986).

    Google Scholar 

  14. Chauvat F, Labarre J, Ferino F: Development of genes transfer system for the cyanobacterium Synechocystis PCC 6803. Plant Physiol Biochem 26: 629–637 (1988).

    Google Scholar 

  15. Chávez S, Candau P: An NAD-specific glutamate dehydrogenase from cyanobacteria. Identification and properties. FEBS Lett 285: 35–38 (1991).

    Google Scholar 

  16. Cock JM, Kim KD, Miller PW, Hutson RG, Schmidt RR: A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) in Chlorella sorokiniana. Plant Mol Biol 17: 1023–1044 (1991).

    Google Scholar 

  17. Colman RF: Glutamate dehydrogenase (bovine liver). In: Kuby SA (ed) A Study of Enzymes, vol II. Mechanism of Enzyme Action, pp. 173–192. CRC Press, Boca Raton, FL (1991).

    Google Scholar 

  18. Consalvi V, Chiaraluce R, Politi L, Gambacorta A, De Rosa M, Scandurra R: Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Eur J Biochem 196: 459–467 (1991).

    Google Scholar 

  19. Curry KA, Tomich CSC: Effect of ribosome binding site on gene expression in Escherichia coli. DNA 7: 173–179 (1988).

    Google Scholar 

  20. Das AT, Moerer P, Charles R, Moorman AFM, Lamers WH: Nucleotide sequence of rat liver glutamate dehydrogenase cDNA. Nucl Acids Res 17: 2355 (1989).

    Google Scholar 

  21. Dayhoff MO, Barker WC, Hunt LT: Establishing homologies in protein sequences. Meth Enzymol 91: 524–545 (1983).

    Google Scholar 

  22. De Boer HA, Kastein RA: Biased codon usage: an exploration of its role in optimization of translation. In: Reznikoff W, Gold L (eds) Maximizing Gene Expression, pp. 225–285. Butterworths, Boston (1986).

    Google Scholar 

  23. Devereux J, Haeberli P, Smithies O: A comprehensive set of sequences analysis programs for the VAX. Nucl Acids Res 12: 387–395 (1984).

    Google Scholar 

  24. De Zoysa PA, Connerton IF, Watson DC, Johnston JR: Cloning, sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene. Curr Genet 20: 219–224 (1991).

    Google Scholar 

  25. Di Ruggiero J, Robb FT, Jagust R, Klump HH, Borges KM, Kessel M, Mai X, Adams MWW: Characterization, cloning, and in vitro expression of the extremely thermostable glutamate dehydrogenase from the hyperthermophilic Archaeon, ES4. J Biol Chem 268: 17767–17774 (1993).

    Google Scholar 

  26. Douglas SE, Turner S: Molecular evidence for the origin of plastids from a cyanobacterium-like ancestor. J Mol Evol 33: 267–273 (1991).

    Google Scholar 

  27. Ferino F, Chauvat F: A promoter-probe vector-host system for the cyanobacterium Synechocystis PCC 6803. Gene 84: 257–266 (1989).

    Google Scholar 

  28. Fickett JW: Recognition of protein coding regions in DNA sequences. Nucl Acids Res 10: 5302–5319 (1982).

    Google Scholar 

  29. Florencio FJ, Marqués S, Candau P: Identification and characterization of a glutamate dehydrogenase in the unicellular cyanobacterium Synechocystis PCC 6803. FEBS Lett 223: 37–41 (1987).

    Google Scholar 

  30. Gribskov M, Devereux J, Burguess RR: The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression. Nucl Acids Res 12: 539–549 (1984).

    Google Scholar 

  31. Haeffner-Gormley L, Chen Z, Zalkin H, Colman RF: Evaluation of cysteine 238 and glutamic acid 284 in the coenzyme binding site of Salmonella typhimurium glutamate dehydrogenase by site-directed mutagenesis and reaction with the nucleotide analogue 2-((4-bromo-2,3-dioxobutyl)thio)1,N 6-ethenoadenosine2′,5′-bisphosphate. J Biol Chem 266: 5388–5394 (1991).

    Google Scholar 

  32. Hajnsdorf E, Steier O, Coscoy L, Teysset L, Régnier P: Roles of RNase E, RNase II and PNPase in the degradation of the rpsO transcripts of Escherichia coli: stabilizing function of RNase II and evidence for efficient degradation in an ams pnp rnb mutant. EMBO J 13: 3368–3377 (1994).

    Google Scholar 

  33. Hanahan D: Techniques for transformation of E. coli. In: Glover DM (ed) DNA Cloning, vol. 1, pp. 109–135. IRL Press Oxford (1985).

    Google Scholar 

  34. Hawkins AR, Gurr SJ, Montague P, Kinghorn JR: Nucleotide sequence and regulation of expression of the Aspergillus nidulans gdhA gene encoding NADP dependent glutamate dehydrogenase. Mol Gen Genet 218: 105–111 (1989).

    Google Scholar 

  35. Henikoff S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359 (1984).

    Google Scholar 

  36. Higgins DG, Bleasby AJ, Fuchs R: CLUSTAL V: improved software for multiple sequence alignment. CABIOS 8: 189–191 (1992).

    Google Scholar 

  37. Jones KM, McPherson MJ, Baron AJ, Mattaj IW, Riordan CL, Wootton JC: The gdhA1 point mutation in Escherichia coli K12 CLR207 alters a key lysine residue of glutamate dehydrogenase. Mol Gen Genet 240: 286–289 (1993).

    Google Scholar 

  38. Kinnaird JH, Fincham RS: The complete nucleotide sequence of the Neurospora crassa am (NADP-specific glutamate dehydrogenase) gene. Gene 26: 253–260 (1983).

    Google Scholar 

  39. Liaud M-F, Zhang DX, Cerff R: Differential intron loss and endosymbiotic transfer of chloroplast glyceraldehyde-3-phosphate dehydrogenase gene to the nucleus. Proc Natl Acad Sci USA 87: 8918–8922 (1990).

    Google Scholar 

  40. Lightfoot DA, Baron AJ, Wootton JC: Expression of the Escherichia coli glutamate dehydrogenase gene in the cyanobacterium Synechococcus PCC 6301 causes ammonium tolerance. Plant Mol Biol 11: 335–344 (1988).

    Google Scholar 

  41. Lyerly DM, Barroso LA, Wilkins TD: Identification of the latex test-reactive protein of Clostridium difficile as glutamate dehydrogenase. J Clin Microbiol 29: 2639–2642 (1991).

    Google Scholar 

  42. Mackinney G: Absorption of light by chlorophyll solutions. J Biol Chem 140: 315–322 (1941).

    Google Scholar 

  43. Maras B, Consalvi V, Chiaraluce R, Politi M, De Rosa L, Bossa F, Scandurra R, Barra D: The protein sequence of glutamate dehydrogenase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium. Is the presence of N-e-methyllysine related to thermostability? Eur J Biochem 203: 81–87 (1992).

    Google Scholar 

  44. Marraccini P, Cassier-Chauvat C, Bulteau S, Chávez S, Chauvat F: Light-regulated promoters from Synechocystis PCC6803 share a consensus motif involved in photoregulation. Mol Microbiol 12: 1005–1012 (1994).

    Google Scholar 

  45. Magalhães JR, Ju GC, Rich PJ, Rhodes D: Kinetics of 15NH4 + assimilation in Zea mays: preliminary studies with a glutamate dehydrogenase (GDH1) null mutant. Plant Physiol 94: 647–656 (1990).

    Google Scholar 

  46. Markwell MA, Haas SM, Bieber LL, Tolber NE: A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87: 206–210 (1978).

    Google Scholar 

  47. Mattaj IW, McPherson MJ, Wootton JC: Localisation of a strongly conserved section of coding sequence in glutamate dehydrogenase genes. FEBS Lett 147: 21–25 (1982).

    Google Scholar 

  48. Mayes SR, Cook KM, Self SJ, Zhang Z, Barber J: Deletion of the gene encoding the Photosystem II 33 kDa protein from Synechocystis sp. PCC 6803 does not inactivate water-splitting but increase vulnerability to photoinhibition. Biochim Biophys Acta 1060: 1–12 (1991).

    Google Scholar 

  49. McPherson MJ, Wootton JC: Complete nucleotide sequence of the Escherichia coli gdhA gene. Nucl Acids Res 15: 5257–5266 (1983).

    Google Scholar 

  50. Meeks JC, Wolk CP, Lockau W, Schilling N, Shaffer PW, Chien W-S: Pathways of assimilation of [13N]N2 and 13NH4 by cyanobacteria with and without heterocysts. J Bact 134: 125–130 (1978).

    Google Scholar 

  51. Meredith MJ, Gronotajski RM, Schmidt RR: Physical and kinetic properties of the nicotinamide adenine dinucleotide-specific glutamate dehydrogenase purified from Chlorella sorokiniana. Plant Physiol 61: 967–974 (1978).

    Google Scholar 

  52. Mérida A, Candau P, Florencio FJ: Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium. J Bact 173: 4095–4100 (1991).

    Google Scholar 

  53. Metz J, Nixon PJ, Diner B: Nucleotide sequence of the psbA3 gene from the cyanobacterium Synechocystis PCC 6803. Nucl Acids Res 18: 6715–6715 (1990).

    Google Scholar 

  54. Mohamed A, Jansson C: Influence of light on accumulation of photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803. Plant Mol Biol 13: 693–700 (1989).

    Google Scholar 

  55. Mountain A, McPherson MJ, Baron AJ, Wootton JC: The Klebsiella aerogenes (gdhA) gene: cloning, high level expression and hybrid enzyme formation in Escherichia coli. Mol Gen Genet 199: 141–145 (1985).

    Google Scholar 

  56. Moye WS, Amuro N, Rao JKM, Zalkin H: Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase. J Biol Chem 260: 8502–8508 (1985).

    Google Scholar 

  57. Neilson AH, Doudoroff M: Ammonia assimilation in blue-green algae. Arch Mikrobiol 89: 15–22 (1973).

    Google Scholar 

  58. Pearce J, Leach CK, Carr NG: The incomplete tricarboxilic acid cycle in the blue-green alga Anabaena variabilis. J Gen Microbiol 55: 371–378 (1969).

    Google Scholar 

  59. Pearson WR, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85: 2444–2448 (1988).

    Google Scholar 

  60. Petersen C: Control of functional mRNA stability in bacteria: multiple mechanisms of nucleolytic and non-nucleolytic inactivation. Mol Microbiol 6: 277–282 (1992).

    Google Scholar 

  61. Philbrick JB, Zilinskas BA: Cloning, nucleotide sequence and mutational analysis of the gene encoding the photosystem II manganese-stabilizing polypeptide of Synechocystis 6803. Mol Gen Genet 212: 418–425 (1988).

    Google Scholar 

  62. Reitzer LJ, Magasanik B: Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate, asparagine, l-alanine and d-alanine. In: Neidhart FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology, vol I, pp. 302–320. American Society for Microbiology, Washington, DC (1987).

    Google Scholar 

  63. Robinson SA, Slade AP, Fox GG, Philips R, Ratcliffe RG, Stewart GR: The role of glutamate dehydrogenase in plant nitrogen metabolism. Plant Physiol 95: 509–516 (1991).

    Google Scholar 

  64. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  65. Sanger F, Nicklen F, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).

    Google Scholar 

  66. Shaw WV: Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Meth Enzymol 43: 737–755 (1975).

    Google Scholar 

  67. Snedecor B, Chu H, Chen E: Selection, expression, and nucleotide sequencing of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus. J Bact 173: 6162–6167 (1991).

    Google Scholar 

  68. Sogin ML, Gunderson JH, Elwood HE, Alonso RA, Peattie DA: Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science 243: 75–77 (1991).

    Google Scholar 

  69. Stewart GR, Mann AF, Fentem PA: Enzymes of glutamate formation: glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. In: Stumpf K, Conn EE (eds) The Biochemistry of Plants, vol. 5, pp. 271–327. Academic Press, New York (1980).

    Google Scholar 

  70. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of T7 RNA polymerase to direct expression of cloned genes. Meth Enzymol 185: 60–89 (1990).

    Google Scholar 

  71. Tandeau de Marsac N, Borrias WE, Kuhlemeier CJ, Castets AM, van Arkel GA, van den Hondel CAMJJ: A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant. Gene 20: 111–119 (1982).

    Google Scholar 

  72. Teller JK, Smith RJ, McPherson MJ, Engel PC, Guest JR: The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli. Eur J Biochem 206: 151–159 (1992).

    Google Scholar 

  73. Tzimagiorgis G, Moschonas NK: Molecular cloning, structure and expression analysis of a full-length mouse brain glutamate dehydrogenase cDNA. Biochim Biophys Acta 1089: 250–253 (1991).

    Google Scholar 

  74. Vicente M, Kushner SR, Garrido T, Aldea M: The role of the ‘gearbox’ in the transcription of essential genes. Mol Microbiol 5: 2085–2091 (1991).

    Google Scholar 

  75. Wierenga RK, De Maeyer MCH, Hol WGJ: Interaction of pyrophosphate moieties with α-helixes in dinucleotide binding proteins. Biochemistry 24: 1346–1357 (1985).

    Google Scholar 

  76. Williams JGK: Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Meth Enzymol 167: 766–778 (1988).

    Google Scholar 

  77. Yee J, Dennis PP: Isolation and characterization of a NADP-dependent glutamate dehydrogenase gene from the primitive eucaryote Giardia lamblia. J Biol Chem 267: 7539–7544 (1992).

    Google Scholar 

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  1. Sebastián Chávez

    Present address: Institut für Molekularbiologie und Tumorforschung, Phillips-Universität, 35037, Marburg, Germany

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  1. Departamento de Bioquímica Vegetal y Biologéa Molecular and Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC, Apartado 1113, 41080, Sevilla, Spain

    Sebastián Chávez, José Carlos Reyes, Francisco J. Florencio & Pedro Candau

  2. Service de Biochimie et Génétique Moléculaire, Département de Biologie Cellulaire et Moléculaire, C.E. Saclay, 91191, Gif sur Yvette cedex, France

    Franck Chauvat

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Chávez, S., Reyes, J.C., Chauvat, F. et al. The NADP-glutamate dehydrogenase of the cyanobacterium Synechocystis 6803: cloning, transcriptional analysis and disruption of the gdhA gene. Plant Mol Biol 28, 173–188 (1995). https://doi.org/10.1007/BF00042048

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