Cloning And Identification Of A Novel NhaD-type Na~+/H~+ Antiporter From Halomonas Alkaliphila

The moderately halophile Halomonas alkaliphila DSM 16354 T can tolerate 10% of Na Cl and grow in the basic condition of pH 9.0.Moderate halophilic bacteria refers to microbial groups that are normally grown and metabolized in the 0.1%-32.5% Na Cl environment.Moderate halophiles not only have a wide variety of genes,but also the salt tolerance mechanism is very complex.Therefore,the research on salt tolerance mechanism of moderately halophile has been current research focus.compared with general of non-halophilic bacteria,it has a relatively unique physiological and biochemical structure,metabolic pathway and the characteristics of genetics.In this study,genomic DNA was screened for salt-alkali tolerant genes p UC-SN11 from Halomonas alkaliphila DSM 16354 T by selecting in Escherichia coli Na~+(Li~+)/H~+ antiporters lacking strains KNabc(?nha A,?nha B,?cha A).It coded Na~+/H~+ aniporter.The growth test showed that the presence of Ha_nhaD could confer tolerance of E.coli KNabc to 0.2 M Na Cl.preliminary forecast the plasmid containing sequences may coding Na~+/H~+ antiporter.Sequence analysis found that the pUC-SN11 carries DNA fragments of 5662 bp long,there are three complete ORF.The result of sequence analysis showed that ORF1(1-1324 bp)had the highest identity(99%)with TRAP dicarboxylate transporter subunit Dct M from Halomonas campaniensis.ORF2(1414-2389bp)had the highest identity(99%)with TRAP ABC transporter substrate-binding proteinthe from Halomonas campaniensis.ORF3(2833-4309 bp)had the highest identity(99%)with sodium:proton antiporter(NhaD)from Halomonas campaniensis.Because of the three complete reading frame including a single subunit Na~+/H~+ antiporter,reading frame is ORF3 so that works.So this study directly to the clone ORF3.Screening the gene designated Ha_nhaD(nhaD from the Halomonas alkaliphila DSM 16354T).Then,ORF3 was also aligned with all the known NhaD-type Na~+/H~+ antiporters including Ha_NhaD2(from Halomonas sp.Y2),Aa_NhaD(from Alkalimonas amylolytica),Ha_NhaD1(from Halomonas sp.Y2),He_NhaD(from Halomonas elongata),Vc_NhaD(from Vibrio cholera),Vp_NhaD(from Vibrio parahaemolyticus),Dl_NhaD(from the metagenome of halophilic bacteria in Daban Salt Lake)and Dw_NhaD(from the metagenome of halophilic bacteria in Dagong Ancient Brine Well)that have been identified experimentally.It showed a certain identity with fore-mentioned NhaD-type Na~+/H~+ antiporters(91% for Ha_NhaD2,72% for Aa_NhaD,70% for Ha_NhaD1,64% for He_NhaD,60% for Vc_NhaD,56% for Vp_NhaD,17% for Dl_NhaD and 11% for Dw_NhaD).To judge whether this novel protein indeed represents a separate distinct sub-class,phylogenetic analysis based on neighbour-joining algorithm was carried out.For the construction of phylogenetic tree,seven closest homologs with 76%-99% identities,seven closer homologs with50%-75% identities,and seven distant homologs with 20%-49% identities and including eight identified NhaD-type Na~+/H~+ antiporters and were selected based on the alignment result.The results show that it gets together with homologue and formed its own independent branch.Therefore,we propose that Ha-NhaD should represent a novel NhaD-type Na~+/H~+ antiporter.Salt-tolerant experiments showed that the recombinant plasmid containing nhaD gene pUC-nhaD in(E.coli)KNabc(? nha A,? nha B,? cha A),can make the host bacterium growth under the concentration of 0.6 M Na Cl and 0.2 M LiCl and alkaline p H 8.0.The analysis of topological structure contained 12 TMS.This research tested the cation/H~+ antiporter activity of E.coli KNabc.The results showed that KNabc/p UC-nhaD showed Na~+(Li~+)/H~+ antiporter activity.In order to verify the p H effect on the cation/H~+ antiporter activity of NhaD,we determined the Na~+(Li~+)/H~+ reverse transporter activity in the p H range of 6.5-9.5.The result shows that Ha_NhaD exhibited Na~+(Li~+)/H~+ antiporter activity p H dependent.In order to determine the affinity of Ha_NhaD for Na~+ or Li~+,the apparent K0.5 values of Na~+ or Li~+ in everted membrane vesicles from KNabc/p UC-nhaD at the optimum pH were determined.The results showed that the affinity of protein Ha_NhaD for cations is Na~+ > Li~+.In summary,Ha_nhaD gene should encode a novel NhaD-type Na~+/H~+ antiporter.