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. 1997 Jul;179(14):4513–4522. doi: 10.1128/jb.179.14.4513-4522.1997

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803.

F Valverde 1, M Losada 1, A Serrano 1
PMCID: PMC179286  PMID: 9226260

Abstract

The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD- to NADP-dependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis cells grown in different metabolic regimens, even under chemoheterotrophic conditions. A possible amphibolic role of the cyanobacterial GAPDH2, namely, anabolic for photosynthetic carbon assimilation and catabolic for carbohydrate degradative pathways, is discussed.

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Selected References

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  1. Anderson S. L., McIntosh L. Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process. J Bacteriol. 1991 May;173(9):2761–2767. doi: 10.1128/jb.173.9.2761-2767.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Cai Y. P., Wolk C. P. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J Bacteriol. 1990 Jun;172(6):3138–3145. doi: 10.1128/jb.172.6.3138-3145.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen J. H., Gibson J. L., McCue L. A., Tabita F. R. Identification, expression, and deduced primary structure of transketolase and other enzymes encoded within the form II CO2 fixation operon of Rhodobacter sphaeroides. J Biol Chem. 1991 Oct 25;266(30):20447–20452. [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Conway T., Sewell G. W., Ingram L. O. Glyceraldehyde-3-phosphate dehydrogenase gene from Zymomonas mobilis: cloning, sequencing, and identification of promoter region. J Bacteriol. 1987 Dec;169(12):5653–5662. doi: 10.1128/jb.169.12.5653-5662.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Corbier C., Clermont S., Billard P., Skarzynski T., Branlant C., Wonacott A., Branlant G. Probing the coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenases by site-directed mutagenesis. Biochemistry. 1990 Jul 31;29(30):7101–7106. doi: 10.1021/bi00482a022. [DOI] [PubMed] [Google Scholar]
  8. Elhai J. Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120. FEMS Microbiol Lett. 1993 Dec 1;114(2):179–184. doi: 10.1111/j.1574-6968.1993.tb06570.x. [DOI] [PubMed] [Google Scholar]
  9. Flores E., Schmetterer G. Interaction of fructose with the glucose permease of the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol. 1986 May;166(2):693–696. doi: 10.1128/jb.166.2.693-696.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fothergill-Gilmore L. A., Michels P. A. Evolution of glycolysis. Prog Biophys Mol Biol. 1993;59(2):105–235. doi: 10.1016/0079-6107(93)90001-z. [DOI] [PubMed] [Google Scholar]
  11. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ganter C., Plückthun A. Glycine to alanine substitutions in helices of glyceraldehyde-3-phosphate dehydrogenase: effects on stability. Biochemistry. 1990 Oct 9;29(40):9395–9402. doi: 10.1021/bi00492a013. [DOI] [PubMed] [Google Scholar]
  13. Grillo J. F., Gibson J. Regulation of phosphate accumulation in the unicellular cyanobacterium Synechococcus. J Bacteriol. 1979 Nov;140(2):508–517. doi: 10.1128/jb.140.2.508-517.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henze K., Badr A., Wettern M., Cerff R., Martin W. A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9122–9126. doi: 10.1073/pnas.92.20.9122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hillman J. D., Fraenkel D. G. Glyceraldehyde 3-phosphate dehydrogenase mutants of Escherichia coli. J Bacteriol. 1975 Jun;122(3):1175–1179. doi: 10.1128/jb.122.3.1175-1179.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kaneko T., Sato S., Kotani H., Tanaka A., Asamizu E., Nakamura Y., Miyajima N., Hirosawa M., Sugiura M., Sasamoto S. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 1996 Jun 30;3(3):109–136. doi: 10.1093/dnares/3.3.109. [DOI] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Liaud M. F., Valentin C., Martin W., Bouget F. Y., Kloareg B., Cerff R. The evolutionary origin of red algae as deduced from the nuclear genes encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases from Chondrus crispus. J Mol Evol. 1994 Apr;38(4):319–327. doi: 10.1007/BF00163149. [DOI] [PubMed] [Google Scholar]
  19. Martin W., Brinkmann H., Savonna C., Cerff R. Evidence for a chimeric nature of nuclear genomes: eubacterial origin of eukaryotic glyceraldehyde-3-phosphate dehydrogenase genes. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8692–8696. doi: 10.1073/pnas.90.18.8692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mougin A., Corbier C., Soukri A., Wonacott A., Branlant C., Branlant G. Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase. Protein Eng. 1988 Apr;2(1):45–48. doi: 10.1093/protein/2.1.45. [DOI] [PubMed] [Google Scholar]
  21. Pearce J., Carr N. G. The incorporation and metabolism of glucose by Anabaena variabilis. J Gen Microbiol. 1968 Dec;54(3):451–462. doi: 10.1099/00221287-54-3-451. [DOI] [PubMed] [Google Scholar]
  22. Pelroy R. A., Rippka R., Stanier R. Y. Metabolism of glucose by unicellular blue-green algae. Arch Mikrobiol. 1972;87(4):303–322. doi: 10.1007/BF00409131. [DOI] [PubMed] [Google Scholar]
  23. Robertson E. F., Dannelly H. K., Malloy P. J., Reeves H. C. Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Anal Biochem. 1987 Dec;167(2):290–294. doi: 10.1016/0003-2697(87)90166-7. [DOI] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Serrano A., Mateos M. I., Losada M. ATP-driven transhydrogenation and ionization of water in a reconstituted glyceraldehyde-3-phosphate dehydrogenases (phosphorylating and non-phosphorylating) model system. Biochem Biophys Res Commun. 1993 Dec 30;197(3):1348–1356. doi: 10.1006/bbrc.1993.2625. [DOI] [PubMed] [Google Scholar]
  26. Serrano A., Mateos M. I., Losada M. Differential regulation by trophic conditions of phosphorylating and non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenases in Chlorella fusca. Biochem Biophys Res Commun. 1991 Dec 31;181(3):1077–1083. doi: 10.1016/0006-291x(91)92047-n. [DOI] [PubMed] [Google Scholar]
  27. Shih M. C., Lazar G., Goodman H. M. Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases. Cell. 1986 Oct 10;47(1):73–80. doi: 10.1016/0092-8674(86)90367-3. [DOI] [PubMed] [Google Scholar]
  28. Soukri A., Mougin A., Corbier C., Wonacott A., Branlant C., Branlant G. Role of the histidine 176 residue in glyceraldehyde-3-phosphate dehydrogenase as probed by site-directed mutagenesis. Biochemistry. 1989 Mar 21;28(6):2586–2592. doi: 10.1021/bi00432a036. [DOI] [PubMed] [Google Scholar]
  29. Summers M. L., Wallis J. G., Campbell E. L., Meeks J. C. Genetic evidence of a major role for glucose-6-phosphate dehydrogenase in nitrogen fixation and dark growth of the cyanobacterium Nostoc sp. strain ATCC 29133. J Bacteriol. 1995 Nov;177(21):6184–6194. doi: 10.1128/jb.177.21.6184-6194.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tamoi M., Ishikawa T., Takeda T., Shigeoka S. Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide. Biochem J. 1996 Jun 1;316(Pt 2):685–690. doi: 10.1042/bj3160685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vioque A. Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria. Nucleic Acids Res. 1992 Dec 11;20(23):6331–6337. doi: 10.1093/nar/20.23.6331. [DOI] [PMC free article] [PubMed] [Google Scholar]

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