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.

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.