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Archives of Microbiology

, Volume 154, Issue 5, pp 471–474 | Cite as

A requirement for phosphoenolpyruvate carboxylase in the cyanobacterium Synechococcus PCC 7942

  • Irene Luinenburg
  • J. R. Coleman
Original Papers

Abstract

The gene encoding phosphoenolpyruvate carboxylase (PEPCase) in the cyanobacterium Synechococcus PCC 7942 has been isolated and characterized. As a first step in determining the role of this enzyme in cyanobacterial carbon metabolism we have attempted to generate PEPCase deficient mutants by insertional inactivation of the PEPCase gene (ppc) and recombination into the wild-type genome. Transformants generated by these constructs appear to be merodiploids in which some copies of ppc remain intact and PEPCase activity is present. Successful insertional inactivation of regions of the genome on either side of ppc suggest that the merodiploid state is a result of a requirement for PEPCase activity by the cyanobacteria. Attempts to select for ppc mutants by nutritional complementation during segregation are also described.

Key words

Cyanobacteria Synechocccus PEPCase gene inactivation merodiploid carbon metabolism 

Abbreviations

PEPCase

phosphenolpyruvate carboxylase

ppc

gene coding for PEPCase

amp

ampicillin

spec

spectinomycin

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References

  1. Allen MM (1968) Simple conditions for growth of uinicellular bluegreen algae on plates. J Phycol 4: 1–4CrossRefGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254CrossRefGoogle Scholar
  3. Coleman JR, Colman B (1980) Demonstration of C3 photosynthesis in the blue-green algae, Coccochloris peniocystis. Planta 149: 318–320CrossRefGoogle Scholar
  4. Coleman JR, Colman B (1981) Photosynthetic carbon assimilation in the blue-green algae, Coccochloris peniocystis. Plant Cell Environ 4: 285–290Google Scholar
  5. Colman B, Cheng KH, Ingle RK (1976) The relative activities of PEP carboxylase and RuDP carboxylase in blue-green algae. Plant Sci Lett 6: 123–127CrossRefGoogle Scholar
  6. Dohler G (1974) C-Weg der Photosynthese in der Blaualge Anacystis nidulans. Planta 118: 259–269CrossRefGoogle Scholar
  7. Golden SS, Brusslan J, Haselkorn R (1986) Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2. EMBOJ 5: 2789–2798CrossRefGoogle Scholar
  8. Golden SS, Brusslan J, Haselkorn R (1987) Genetic engineering of the cyanobacterial chromosome. Methods Enzymol 153: 215–231CrossRefGoogle Scholar
  9. Golden SS, Nalty MS, Cho DC (1989) Genetic relationship of two highly studied Synechococcus strains designated Anacystis nidulans. J Bacteriol 171: 24–29CrossRefGoogle Scholar
  10. Harrington TR, Glick BR, Lem NW (1986) Molecular cloning of the phosphoenolpyruvate gene of Anabaena variabilis. Gene 45: 113–116CrossRefGoogle Scholar
  11. Ishijima S, Katagiri F, Kodaki T, Izui K, Katsuki H, Nishikawa K, Nakashima H, Ooi T (1985) Comparison of amino acid sequences between phosphoenolpyruvate carboxylases from Escherichia coli and Anacystis nidulans: identification of conserved and variable regions. Biochem Biophys Res Commun 133: 436–441CrossRefGoogle Scholar
  12. Kodaki T, Katagiri F, Asano M, Izui K, Katsuki H (1985) Cloning of phosphoenolpyruvate carboxylase gene from a cyanobacterium, Anacystis nidulans, in Escherichia coli. J Biochem 97: 533–539CrossRefGoogle Scholar
  13. Lem NW, Penrose DM, Glick B (1986) Partial purification and characterization of phosphoeneolpyruvate carboxylase from the cyanobacterium Anabaena variabilis. Biochem Cell Biol 64: 427–433CrossRefGoogle Scholar
  14. Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  15. Maruyama H, Easterday RL, Chang HC, Lane MD (1986) The enzymatic carboxylation of phosphoenolpyruvate. I. Purification and properties of phosphoenolpyruvate carboxylase. J Biol Chem 241: 2405–2412Google Scholar
  16. Murphy RC, Gasparich GE, Bryant DA, Porter RD (1990) Nucleotide sequence and further characterization of the Synechococcus PCC 7002 recA gene: complementation of a cyanobacterial recA mutation by the E. coli recA gene. J Bacteriol 172: 967–976CrossRefGoogle Scholar
  17. Owttrim GW, Colman B (1986) Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium. J Bacteriol 168: 207–212CrossRefGoogle Scholar
  18. Owttrim GW, Coleman B (1987) Phosphoenolpyruvate carboxylase mediated carbon flow in a cyanobacterium. Biochem Cell Biol 66: 93–99CrossRefGoogle Scholar
  19. Owttrim GW, Coleman JR (1989) Regulation of expression and nucleotide sequence of the Anabaena variabilis recA gene. J Bacteriol 171: 5713–5719CrossRefGoogle Scholar
  20. Prentki P, Krisch HM (1984) In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29: 303–313CrossRefGoogle Scholar
  21. Smith AJ, London J, Stanier RY (1967) Biochemical basis of obligate autotrophy in blue-green algae and thiobacilli. J Bacteriol 94: 972–983PubMedPubMedCentralGoogle Scholar
  22. Vermaas WFJ, Williams JGK, Arntzen CJ (1987) Sequencing and modification of psbA, the gene encoding the CP-47 protein of photosystem II, in the cyanobacterium Synechocystis 6803. Plant Mol Biol 8: 317–326CrossRefGoogle Scholar
  23. Weathers PJ, Allen MM (1978) Variations in short-term products of inorganic carbon fixation in exponential in stationary phase cultures of Aphanocapsa 6308. Arch Microbiol 116: 231–234CrossRefGoogle Scholar
  24. Williams JGK (1988) Construction of specific mutations in photosystem II photosynthetic reaction centre by genetic engineering methods in Synechocystis 6803. Methods Enzymol 167: 766–778CrossRefGoogle Scholar
  25. Williams JGK, Szalay AA (1983) Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2. Gene 24: 37–51CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Irene Luinenburg
    • 1
  • J. R. Coleman
    • 1
  1. 1.Centre for Plant Biotechnology, Department of BotanyUniversity of TorontoTorontoCanada

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