Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli
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This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min−1 mg protein−1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min−1 mg protein−1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (∆arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl2, ZnSO4, NiCl2, CoCl2, CuCl2, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H2O2), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.
KeywordsAnabaena sp. PCC7120 Arsenic alr1105 Overexpression Complementation Abiotic stress
L.C. Rai is thankful to the Department of Science and Technology New Delhi for J.C. Bose National Fellowship and the University Grant Commission for financial assistance through a project. Sarita Pandey, Rashmi Rai and Alok Kumar Shrivastava thank the Council of Scientific and Industrial Research for SRF. We thank Prof. B.P. Rosen, Department of Cellular Biology and Pharmacology, Florida International University, Herbert Wertheim College of Medicine, Miami, Florida-33199, USA for providing E. coli W3110 and WC3110 strain and to the Head, and the Programme Coordinator Centre of Advanced Study in Botany, Banaras Hindu University,Varanasi, India for facilities and DBT-BHU ISLS for MALDI-TOF/MS analysis.
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