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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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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.

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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.

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Authors and Affiliations

  1. Faculty of Science and Arts, Department of Biology, University of Ondokuz Mayis, 55139, Samsun, Turkey

    Haydar Karakaya, Funda Erdem, Kübra Özkul & Aylin Yilmaz

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  1. Haydar Karakaya
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  2. Funda Erdem
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  3. Kübra Özkul
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  4. Aylin Yilmaz
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Correspondence to Haydar Karakaya.

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Karakaya, H., Erdem, F., Özkul, K. et al. Analysis of glucose-6-phosphate dehydrogenase of the cyanobacterium Synechococcus sp. PCC 7942 in the zwf mutant Escherichia coli DF214 cells. Ann Microbiol 63, 1319–1325 (2013). https://doi.org/10.1007/s13213-012-0591-8

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  • DOI: https://doi.org/10.1007/s13213-012-0591-8

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