Abstract
In this study, metagenomic DNA was screened for the Na+/H+ antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na+/H+ antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6 %) with a putative NhaD-type Na+/H+ antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na+/H+ antiporters from Halomonas elongata (20.8 %), Alkalimonas amylolytica (19.0 %), Vibrio parahaemolyticus (18.9 %) and Vibrio cholerae (18.7 %). pH-dependent Na+(Li+)/H+ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na+(Li+)/H+ antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na+/H+ antiporters. Also, the apparent K m values of Hb_NhaD for Na+ and Li+ at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na+/H+ antiporter.
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Acknowledgments
We are so grateful to Dr Terry A. Krulwich (Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine of the City University, New York) for the kind gift of E. coli strain KNabc. We are also so thankful to Editor Dr. Sonja-Verena Albers and two anonymous reviewers for such valuable suggestions about the improvement of our manuscript. This work was supported by National Natural Science Foundation of China (Grant No. 30960009, 31000055 and 31370087), Fund for Fostering Talents in Basic Science of the National Natural Science Foundation of China (Grant No. J1210069), Natural Science Foundation of Heilongjiang Province of China (Grant No. LC201025), Key Project of Returned Overseas Chinese Scholars of Heilongjiang Province of China (Grant No. 1251HZ001), China Postdoctoral Science Foundation (Grant No. 20100471244 and 201104408), Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (Grant No. 2012940), Heilongjiang Postdoctoral Funds (Grant No. LBH-TZ1107 and LBH-Z10239), Science and Technology Innovation Fund of Harbin City of China (Grant No. 2012RFLXN004), Doctor Start-up Fund of Northeast Agricultural University (Grant No. 2009RC23), Key Laboratory Open Fund of Soybean Biology of Ministry of Education (Grant No. SB11A05) and Key Laboratory Open Fund of Agricultural Biological Functional Genes of Heilongjiang Province (Grant No. NSGJ2012-09).
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Communicated by S. Albers.
H. Zhang and Z. Wang contributed equally to this work.
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Zhang, H., Wang, Z., Wang, L. et al. Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake. Extremophiles 18, 89–98 (2014). https://doi.org/10.1007/s00792-013-0600-2
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DOI: https://doi.org/10.1007/s00792-013-0600-2