Cloning and identification of a novel NhaD-type Na^sup +^/H^sup +^ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake

In this study, metagenomic DNA was screened for the Na^sup +^/H^sup +^ antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na^sup +^/H^sup +^ antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na^sup +^/H^sup...

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Bibliographic Details
Published in:Extremophiles : life under extreme conditions 2014-01, Vol.18 (1), p.89
Main Authors: Zhang, Hua, Wang, Zhenhui, Wang, Lei, Mu, Ren, Zou, Zhi, Yuan, Kun, Wang, Yuekun, Wu, Haiping, Jiang, Juquan, Yang, Lifu
Format: Article
Language:English
Subjects:
Bacteria
Cloning
Deoxyribonucleic acid
DNA
E coli
Genomics
Microbiology
Salt lakes
Sodium chloride
Soils
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Summary:In this study, metagenomic DNA was screened for the Na^sup +^/H^sup +^ antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na^sup +^/H^sup +^ antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na^sup +^/H^sup +^ 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^sup +^/H^sup +^ antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na^sup +^/H^sup +^ antiporters from Halomonas elongata (20.8 %), Alkalimonas amylolytica (19.0 %), Vibrio parahaemolyticus (18.9 %) and Vibrio cholerae (18.7 %). pH-dependent Na^sup +^(Li^sup +^)/H^sup +^ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na^sup +^(Li^sup +^)/H^sup +^ 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^sup +^/H^sup +^ antiporters. Also, the apparent K ^sub m^ values of Hb_NhaD for Na^sup +^ and Li^sup +^ 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^sup +^/H^sup +^ antiporter.[PUBLICATION ABSTRACT]
ISSN:1431-0651
1433-4909
DOI:10.1007/s00792-013-0600-2