Analysis of glucose-6-phosphate dehydrogenase of the cyanobacterium Synechococcus sp. PCC 7942 in the zwf mutant Escherichia coli DF214 cells
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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.
KeywordsSynechococcus sp. zwf gene Complementation E. coli DF214 Glucose-6-phosphate dehydrogenase Redox state
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.
- 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
- 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
- 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
- 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
- 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
- Marcus L, Hartnett J, Storts DR (1996) The pGEM-T and pGEM-T easy vector systems. Promega Notes Mag 58:36–38Google Scholar
- Pelroy RA, Bassham JA (1972) Photosynthetic and dark carbon metabolism in unicellular blue-green algae. Arch Microbiol 86:25–38Google Scholar
- 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
- Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- 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
- Vinapol RT, Hillmann JD, Schulman H, Reznikoff WS, Fraenkel DG (1975) New phosphoglucose isomerase mutants of Escherichia coli. J Bacteriol 122:1172–1174Google Scholar