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Annals of Microbiology

, Volume 63, Issue 4, pp 1319–1325 | Cite as

Analysis of glucose-6-phosphate dehydrogenase of the cyanobacterium Synechococcus sp. PCC 7942 in the zwf mutant Escherichia coli DF214 cells

  • Haydar KarakayaEmail author
  • Funda Erdem
  • Kübra Özkul
  • Aylin Yilmaz
Original Article

Abstract

The aim of this study was to express the zwf gene of Synechococcus sp. PCC 7942 in zwf mutant Escherichia coli DF214 cells and to analyse glucose-6-phosphate dehydrogenase (G6PDH) activity. Initially, mutant cells were transformed with plasmid pNUT1 containing a Synechococcus sp. PCC 7942 zwf gene with a 1 kb upstream region that is expected to contain promoter elements. Transformant DF214 cells were not complemented by this fragment in a glucose minimal medium, nor did they exhibit statistically meaningful G6PDH activity. Therefore, the zwf gene was cloned in the lac operon to express the Zwf as a fusion protein; this yielded the construct pSG162. The pSG162 transformant E. coli DF214 cells were complemented in a glucose minimal medium, indicating that cyanobacterial Zwf protein fused with the part of LacZ′ polypeptide, enabling the cells to utilize glucose via the oxidative pentose phosphate pathway. Compared with wild-type E. coli cells, approximately ten times more G6PDH activity was measured in transformant cells. This indicated that the Synechococcus sp. PCC 7942 zwf gene was expressed under the control of the E. coli lac promoter as a fusion protein and the zwf product was converted into an active G6PDH form. Analyses was also carried out to determine whether dithiothreitol (DTT) was an in vitro reducing agent affected the enzyme activity, as was previously reported for this cyanobacterial strain. The results showed no variation in enzyme activity in the reduced assay conditions. Therefore, the zwf mutant E. coli strain DF214 was found to provide a rapid system for analysis of cyanobacterial G6PDH enzymes, but not for the redox state analysis of this enzyme.

Keywords

Synechococcus sp. zwf gene Complementation E. coli DF214 Glucose-6-phosphate dehydrogenase Redox state 

Notes

Acknowledgments

We would like to thank Prof. Dr. D.J. Scanlan of University of Warwick for kindly supplying us with pNUT1. This study was supported by the Research Fund of the University of Ondokuz Mayıs, Samsun, Turkey, through projects F-261 and PYO FEN 1904 09 21.

References

  1. Anderson LE, Nehrlich SC, Champigny M (1978) Light modulation of enzyme activity. Activation of the light effect mediators by reduction and modulation of enzyme activity by thiol-disulfide exchange? Plant Physiol 61:601–605PubMedCrossRefGoogle Scholar
  2. Austin PA, Ross IS, Mills JD (1992) Light/dark regulation of photosynthetic enzymes within intact cells of the cyanobacterium Nostoc sp. Mac. Biochim Biophys Acta 1099:226–232CrossRefGoogle Scholar
  3. 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 72:248–254PubMedCrossRefGoogle Scholar
  4. Copeland L, Turner JF (1987) The regulation of glycolysis and the pentose phosphate pathway. In: Davies DD (ed) The biochemistry of plants vol.11. Academic, San Diego, pp 107–128Google Scholar
  5. Cossar JD, Rowell P, Stewart WP (1984) Thioredoxin as a modulator of glucose -6- phosphate dehydrogenase in a N2-fixing cyanobacterium. J Gen Microbiol 130:991–998Google Scholar
  6. Gleason FK (1994) Thioredoxins in cyanobacteria. Structure and redox regulation of enzyme activity. In: Bryont DA (ed) The molecular biology of cyanobacteria. Kluwer Academic Publishers, Dordrecht, pp 714–729Google Scholar
  7. Gleason FK (1996) Glucose-6-phosphate dehydrogenase from the cyanobacterium, Anabaena sp. PCC 7120: purification and kinetics of redox modulation. Arch Biochem Biophys 384:277–283CrossRefGoogle Scholar
  8. Grant SGN, Jesseet J, Bloomt FR, Hanahan D (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci USA 87:4645–4649PubMedCrossRefGoogle Scholar
  9. Hylemon PB, Phibbs PV Jr (1972) Independent regulation of hexose catabolizing enzymes and glucose transport activity in Pseudomonas aeroginosa. Biochem Biophys Res Commun 48:1041–1048PubMedCrossRefGoogle Scholar
  10. Ihlenfeld MJA, Gibson A (1975) CO2 fixation and its regulation in Anacystis nidulans (Synechococcus). Arch Microbiol 102:13–21CrossRefGoogle Scholar
  11. Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Tabata S (1996) 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 3:109–136PubMedCrossRefGoogle Scholar
  12. Karakaya H, Mann NH (1998) zwf mutant Escherichia coli DF214 suşunun bir Anabaena sp. PCC7120 zwf fragmenti taşıyan plazmid ile genetik komplementasyonu üzerine araştırmalar. XIV. Ulusal Biyoloji Kongresi Cilt III: 100–113Google Scholar
  13. Karakaya H, Ay MT, Ozkul K, Mann NH (2008) A Δzwf (glucose-6-phosphate dehydrogenase) mutant of the cyanobacterium Synechocystis sp. PCC 6803 exhibits unimpaired dark viability. Annal Microbiol 58:281–286CrossRefGoogle Scholar
  14. Marcus L, Hartnett J, Storts DR (1996) The pGEM-T and pGEM-T easy vector systems. Promega Notes Mag 58:36–38Google Scholar
  15. Newman J, Karakaya H, Scanlan DJ, Mann NH (1995) A comparison of gene organisation in the zwf region of the genomes of cyanobacteria Synechococcus sp. PCC 7942 and Anabeana sp. PCC 7120. FEMS Lett 133:187–193CrossRefGoogle Scholar
  16. Pelroy RA, Bassham JA (1972) Photosynthetic and dark carbon metabolism in unicellular blue-green algae. Arch Microbiol 86:25–38Google Scholar
  17. Rowell P, Kerby NW (1992) Potential and commercial applications for photosynthetic prokaryots. In: Fay P, van Baalen C (eds) Photosynthetic prokaryotes. Biothecnology handbooks vol. 6. Plenium Press, New York, pp 233–266Google Scholar
  18. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  19. Scanlan DJ, Newman J, Sebaihia M, Mann NH, Carr NG (1992) Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus. sp PCC 7942. Plant Mol Biol 19:877–880PubMedCrossRefGoogle Scholar
  20. Scanlan DJ, Sundaram S, Newman J, Mann NH, Carr NG (1995) Characterisation of a zwf mutant of Synechococcus sp. strain PCC 7942. J Bacteriol 177:2550–2553PubMedGoogle Scholar
  21. Schaeffer F, Stanier RY (1978) Glucose-6-phosphate dehydrogenase of Anabaena sp. kinetic and molecular properties. Arch Microbiol 116:9–19PubMedCrossRefGoogle Scholar
  22. Smith AJ (1982) Modes of cyanobacterial carbon metabolism. In: Carr NG, Whitton BA (eds) The biology of cyanobacteria. Blackwell Scientific Publication, Oxford, pp 47–85Google Scholar
  23. Summers ML, Meeks JC, Chu S, Wolf RE Jr (1995a) Nucleotide sequence of an operon in Nostoc sp. strain ATCC 29133 encoding four genes of the oxidative pentose phosphate cycle. Plant Physiol 107:267–268PubMedCrossRefGoogle Scholar
  24. Summers ML, Wallis JG, Campbell EL, Meeks JC (1995b) 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 177:6184–6194PubMedGoogle Scholar
  25. Sundaram S, Karakaya H, Scanlan DJ, Mann NH (1998) Multiple oligomeric forms of glucose-6-phosphate dehydrogenase in cyanobacteria and the role of OpcA in the assembly process. Microbiol SGM 144:1549–1556CrossRefGoogle Scholar
  26. Tabita FR (1994) The biochemistry and molecular regulation of carbon dioxide metabolism in cyanobacteria. In: Bryont DA (ed) The molecular biology of cyanobacteria. Kluwer Academic Publishers, Dordrecht, pp 437–467CrossRefGoogle Scholar
  27. Tandeu de Marsac N, Houmard J (1993) Adaptation of cyanobacteria to environmental stimuli: new steps towards molecular mechanisms. FEMS Microbiol Rev 104:119–190CrossRefGoogle Scholar
  28. Vinapol RT, Hillmann JD, Schulman H, Reznikoff WS, Fraenkel DG (1975) New phosphoglucose isomerase mutants of Escherichia coli. J Bacteriol 122:1172–1174Google Scholar
  29. Wenderoth I, Scheibe R, Schaewen A (1997) Identification of the cystein residues involved in redox modification of plant plastitic glucose-6-phosphatedehydrogenase. J Biol Chem 272:26985–26990PubMedCrossRefGoogle Scholar
  30. Yanisch-Peron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mp18 and pUC19 vectors. Gene 33:103–119CrossRefGoogle Scholar
  31. Yee BC, de la Torre A, Crawford NA, Lara C, Charlson DE, Buchanan BB (1981) The ferredoxin/thioredoxin systhem of enzyme regulation on a cyanobacterium. Arch Microbiol 130:14–18CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg and the University of Milan 2012

Authors and Affiliations

  • Haydar Karakaya
    • 1
    Email author
  • Funda Erdem
    • 1
  • Kübra Özkul
    • 1
  • Aylin Yilmaz
    • 1
  1. 1.Faculty of Science and Arts, Department of BiologyUniversity of Ondokuz MayisSamsunTurkey

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