Cloning And Identification Of One Group 2 Mrp Operon Encoding A Multi-subunit Na~+/H~+ Antiporter And Construction Of Its Knock-out Mutant

The strain NEAU-ST10-39~T,isolated from saline-alkaline soils in the Songnen Plain,Heilongjiang Province,China,had been identified to represent a species of the genus Halomonas,Halomonas songnenensis.This strain is a moderate halophile which can grow at the range of NaCl concentrations of 0.2-15%(w/v)(optimum 4%,w/v)and at pH 5-10(optimum pH 7).it may have evolved the complex salt tolerance genes and their regulatory mechanisms to maintain their intracellular stable osmotic pressure and ion balance.Thus,it is likely that abundant Na~+(Li+)/H~+ transporters play an important role in salt tolerance in this strain.Therefore,the use of Halomonas songnenensis NEAU-ST10-39~T as a research object for bacterial salt-tolerant gene mining has a greater chance of obtaining important or new salt-tolerant genes.The methods of gene library construction and gene functional complementation were used in this study for Na~+(Li+)/H~+ antiporter genes from NEAU-ST10-39~T by selection in Escherichia coli KNabc lacking three major Na~+(Li+)/H~+ antiporters.One mrp operon was finally cloned and found to be able to confer tolerance of E.coli KNabc to 0.8 M NaCl,120 mM Li Cl and an alkaline pH.Sequence analysis and protein alignment showed that this operon containing six genes should belong to Group 2 mrp operons,and designated Hs_mrp2.The Hs_mrp2(6,709 bp)contained six complete open reading frames(ORF1-6),and ORF1-6 had a common promoter and terminator.Each reading frame had its own SD sequence.Sequence analysis results showed that ORF1-6 had the highest identities of 88.4% to 97.2% with the subunits A' to G of Hz_Mrp2 system,a putative Group 2 Mrp system from Halomonas zhanjiangensis.At present,only two Group 2 Mrps had been identified.ORF1-6 were also aligned with the subunits A' to G of two known Group 2 Mrp systems including Sm_Pha1 system from S.meliloti and Vc_Mrp2 system from V.cholerae.ORF1-6 had limited identity with the subunits A' to G of Sm_Pha1 system(28.7% to 50.9%),Vc_Mrp2 system(25.9% to 41.8%),respectively.The above results suggested that Hs_mrp2 might encode a novel Group 2 Mrp system,esspecially for the ones from the halophilic bacterium.Based on this,we constructed the phylogenetic tree of Hs_mrp2 based on neighbour-joining algorithm,the results showed that Hs_Mrp2 system,which was far from the identified Group 1 Mrp and Group 2 Mrp,together with the putative Hz_Mrp2 system indeed consitituted a separate clade.This indicated that Hs_mrp2 encode a novel Group 2 Mrp system.In order to demonstrat that Hs_Mrp2 system had Na~+/H~+ antiport activity.Fluorescence quenching measuerments of Na~+(Li+,K+)/H~+ activity with everted membrane vesicles prepared from E.coli KNabc cells carrying p UC-mrp2 or p UC18(as a negative control)revealed that not only Na~+/H~+ and Li+/H~+ but also K+/H~+ antiport activity was detected in everted membrane vesicles from KNabc/p UC-mrp2 especially at the pH 9.0 and pH 9.5,while no Na~+/H~+ and Li+/H~+ or K+/H~+antiport activity was detected in those from KNabc/p UC18.In order to further analyze the salt and halo-alkaline tolerance of Hs_mrp2 in NEAU-ST10-39~T,the recombinant plasmid p K18-mrp2-Nco I with Mrp2 homologous arm was constructed by gene knock-out plasmid p K18 mobsac B.The Group 2 Mrp was knocked by homologous recombination technique.Resistance of the host NEAU-ST10-39~T and knock-out strains to salts and alkaline pH explained that Group 2 Mrp found in this experiment was indeed essential effect to moderate halophilic bacteria NEAU-ST10-39~T in salt tolerance.Based on the above results,we considered that Hs_Mrp2 should be a new Group 2 Mrp system that played a major role in akaline response of the host NEAU-ST10-39~T and be the first case for the discovery of the Group 2 type Mrp system in moderate halophilic bacteria.