Characterization And Molecular Mechanism Analysis Of NhaD Type Na~+/H~+ Antiporters From Halomonas Sp.Y2

Halomonas hydrothermalis Y2 is a halotolerant and alkaliphilic strain isolated from the Na+-rich pulp mill wastewater.The strain is dominant in the bacterial community of pulp mill wastewater and closely related with Halomonas elongata DSM 2581T.Here we present the genome sequence of this strain,which comprises a circular chromosome 3,933,432 bp in size with GC content of 60.2%.Diverse genes encoding Na+(Li+,K+)/H+antiporters were identified from the genome.The abundant Na+(Li+,K+)/H+systems play important roles in controlling intracellular pH,cellular Na+concentrations,germination of spores and cell volume.For the great potential of Y2 used in industrial fermentation production and developed into the chassis,the study on the ion transport system of Halomonas sp.Y2 will provide theoretical basis for the practical application of Y2 strain.This study only analysis of the NahDl and NhaD2 protein,both of them exhibited 72%identity in amino acid sequence while exhibits similar,high in vitro activity,but remarkably different in vivo functions.The chimera of N39D2,N39D1,NhaD2?C4,NhaDl-C4,resulted in the complete loss of antiport activity.Further chimera construction by combining 39 N-terminal residues and seven C-terminal residues of NhaD2 partially recovered the activity for Na+ and Li+expulsion and the complementary growth.The results of the alkaline phosphatase experiment and software prediction showed that the C-terminal and N-terminal of NhaD1,NhaD2 are very conservative,and different with Nout-Cin of V.Cholera,the transmembrane region is distributed as Nin-Cout.Fluorescence resonance energy transfer analysis further showed that the fusion fluorescence donor and receptor in 39 site and C end of the NhaD2,could occur fluorescence resonance energy transfer,the distance is relatively close,within the 10 nm,indicated that the N and C termini are structurally dependent on each other and function synergistically.In the NhaD2 and N39D2-C7,the replacement of Glu38 with Pro38 of NhaD1,completely abolished the recovered complementation ability and transport activity.In addition,this amino acid substitution in NhaDl also resulted in a similar result,suggested that acidic amino acids Glu38 and Pro38 played an important role and could not be replaced by each other.To search for critical domains or residues involved in the differences of physiological functions,various chimeras composed of NhaDl and NhaD2 segments were generated.In particular,the completely abolished activity of KNabc/N463r and the partially recovered ion resistance of the KNabc/N463r-C7 chimera,suggested that transmembrane helix(TM)XIII is crucial for the robust ion resistance of NhaD2 Using site-directed mutagenesis,seven hydrophobic residues in TM XIII,especially 468 and 482,the transcript levels of A468V and M482W showed a significant downregulation than those of other targeted genes,indicating that poor transport activities of these two mutants should be attributed to their decreased transcript levels in the KNabc strain.Compared with the wild-type NhaD2,the reduced FRET efficiency of N463r chimeras provided solid evidence for conformational changes in the N463r fusion protein and consequently verified the important roles of TM XIII in the ion transport activity and the ion passage and physiological functions of NhaD2.Through evolutionary conservative analysis,we found two conservative regions in NhaD2,TM ?-? and TM ?-?,20 conservative amino acids with the potential of sensing pH changes,and a total of 30 mutants were successfully constructed.After transport activity,growth complementary analysis and the experiment covering to Y2/AnhaD2 strain,we further determine the critical areas and sites of NhaD2 associated with ion combination,combining the three dimensional structure of Vc-INDY,E.coli NhaA transporters,and other study about the ion binding sites of similar protein,we analyzed the D166,N167,D405,N406 may be ion binding sites of NhaD2 and the T170 is the important amino acid affect the substrate binds to the protein,the apparent Km values of mutation T170S increased 10-15 times compared with wild strains.We suspect that most of these key sites are located at the bottom of similar funnel channels formed by TM ?-? and TM X-XII in the core area,directly or indirectly participate in the transfer of substrate.