Cloning And Functional Study Of Na~+/H~+ Transporter Gene From Moderately Halophilic Bacteria Halomonas Sp.19-A

Moderately halophilic bacteria can thrive under a broad range of 0.1%-32.5%NaCl which the reverse transporters play an important role in maintaining cell normal salt concentration and cytoplasmic pH homeostasis.The strain Halomonas sp.19-A studied in this paper was isolated from alkaline paper-making black liquor, It is abundant in the black liquor and shows superior ability in black liquor treatment. As a moderate halophilic and alkaliphilic bacterium, Halomonas sp.19-A has prominent resistance to salt and alkali resistant properties which can grow at the NaCl range of 3%-20%(optimum 3%) and pH 5-12 (optimum pH 10). By sequencing, we found that the strain has rich Na+ transport systems. In this study, four cation/proton reverse transport protein genes, designated nhaD1、nhaD2、 nhaP and mrp which gene mrp encodes a seven subunit protein, were cloned from chromosomal DNA of Halomonas sp.19-A by functional complementation. The deduced amino acid sequence of NhaD1 is 72% identical to NhaD2, and both have homology with NhaD from Halomonas elongata、Alkalimonas amylolytica、Vibrio parahaemolyticus and Vibrio cholera. Protein encoded by nhaP has the highest identity (46%) with the NhaP of Pseudomonas aeruginosa UCBPP-PA14 and Pseudomonas aeruginosa PAO1. Mrp has homology with Mrp encoded by gene from Bacillus pseudofirmus OF4 and the identity of both corresponding subunit is as described below:MrpA 37%, MrpB 32%, MrpC 46%, MrpD 48%, MrpE 35%, MrpF 30% and MrpG 40%. Similarly, the identity with Mrp of Bacillus subtilis168 is:MrpA 35%, MrpB 36%, MrpC 42%, MrpD 40%, MrpE 34%, MrpF 35%, MrpG 41%.The function of the nhaD1、nhaD2 nhaP and mrp was identified by heterologous expression of these genes in E. coli KNabc which Na+ extrusion genes have been destroyed. The mrp gene could successfully express in the E. coli KNabc and its production rendered the E. coli KNabc with both the resistance up to 500 mM Na+,10 mM Li+ and the ability to grow under pH 7.0-8.5 condition.NhaD2 could make E. coli KNabc grow in medium containing 0-500 mM Na+ or with 0-300 mM Li+ and strain E.coli KNabc/pEASYBlunt-nhaD2 can well survive under pH 8.0 alkaline conditions. E.coli KNabc/pEASYBlunt-nhaP can grow in LBK medium containing 0-400 mM Na+ while NhaD1 seems to make no obvious diffenence to E.coli KNabc on alkali resistance and salt resistance.Everted membrane vesicles assay indicated that Mrp had Na+(Li+, K+)/H+ antiporter activities, NhaD2 had Na+(Li+)/H+ antiporter activities, NhaP had Na+(Li+, K+)/H+ antiporter activities and NhaD1 had weak Li+/H+ antiporter activities. Na+/H+ antiporter activity of Mrp was detected under pH 7.0-9.0 condition while detection range of NhaD2 and NhaP reduced to pH 7.0-8.5 and pH 7.5-8.5 respectively, with both optimum pH 8.5. The Li+/H+antiporter activities of Mrp、NhaD2 and NhaP were pH dependent and for Mrp and NhaD2, the highest activity was at pH 8.0. Under pH 7.0-9.0, Mrp had K+/H+ antiporter activity with optimum pH 8.0. And NhaP was detected with K+/H+ antiporter activity in the scope of pH 7.5-8.5 with optimum pH 8.5. But NhaDl and NhaD2 were detected significantly K+/H+ antiporter activity only at pH7.Expression differences analysis showed that the highest expression amount of NhaD2 was under weak alkali (pH 8.0) condition, alkali (pH 10.5) condition comes second. Compared with neutral condition, in alkaline conditions expression amount of NhaD1 does not increase. With the pH increasing, the expression of MrpA is also increased. The results are consistent with the data of growth and everted membrane vesicles assay.