A novel NhaD-type Na+/H+ antiporter from the moderate halophile and alkaliphile Halomonas alkaliphila

Abstract

In this study, a NhaD-type Na⁺/H⁺ antiporter gene designated Ha-nhaD was obtained by selection of genomic DNA from the moderate halophile and alkaliphile Halomonas alkaliphila in Escherichia coli KNabc lacking 3 major Na⁺/H⁺ antiporters. The presence of Ha-NhaD conferred tolerance of E. coli KNabc to NaCl up to 0.6 mol·L^-1 and to LiCl up to 0.2 mol·L^-1 and to an alkaline pH. pH-dependent Na⁺(Li⁺)/H⁺ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc/pUC-nhaD but not those of KNabc/pUC18. Ha-NhaD exhibited Na⁺(Li⁺)/H⁺ antiport activity over a wide pH range from 7.0 to 9.5, with the highest activity at pH 9.0. Protein sequence alignment and phylogenetic analysis revealed that Ha-NhaD is significantly different from the 7 known NhaD-type Na⁺/H⁺ antiporters, including Dw-NhaD, Dl-NhaD, Vp-NhaD, Vc-NhaD, Aa-NhaD, He-NhaD, and Ha-NhaD1. Although Ha-NhaD showed a closer phylogenetic relationship with Ha-NhaD2, a significant difference in pH-dependent activity profile exists between Ha-NhaD and Ha-NhaD2. Taken together, Ha-nhaD encodes a novel pH-dependent NhaD-type Na⁺/H⁺ antiporter.

Keywords: Halomonas alkaliphila; Na+/H+ antiporter; NhaD; antiport Na+/H+; halophile et alcaliphile modéré; moderate halophile and alkaliphile; pH-dependent activity profile; profil d’activités dépendantes du pH.