Cloning,Identification And Knock-out Of One Group 1 Mrp Operon Encoding A Multi-subuint Na~+/H~+ Antiporter From Halomonas Songenensis

Halomonas songnenensis is a strain of moderately halophile that could tolerate 15% of Na Cl and grow in the basic condition of pH 8.In order to obtain Na~+(Li~+)/H~+ antiporter genes from NEAU-ST10-39 T genomic DNA,a strategy used of combining gene library construction and gene function complementation method by selection in Escherichia coli KNabc lacking three major Na~+(Li~+)/H~+ antiporters(?nha A,?nha B,?cha A).One mrp operon encoding a multisubunit monovalent cation/proton antiporter was finally cloned.Here,we will use mrp to designate the homolog from H.songnenensis(Hs_mrp)and the denomination of its coding protein is Hs_Mrp.Hs_mrp operon(6,500 bp)consists of six complete open reading frames(ORFs)and a truncated fusion into the Lac Z ORF,all of which overlaping with or terminating very closely to the following one.Also,these seven ORFs are preceded by a predicted common promoter sequence and followed by a common terminator sequence Lac Z,each of which has one separate upstream Shine-Dalgarno(SD)sequence.Protein alignment showed that ORF7-13 has the highest identity(90.8%-99.3%)with the subunits A to G of Hs_Mrp system,a putative Group 1 Mrp system from H.zhanjiangensis.Phylogenetic analysis based on neighbour-joining algorithm showed that Hs_mrp system and its closest putative homolog including Hh_Mrp system,Hm_Mrp system Hzj_Mrp system and a identified Hz_Mrp system indeed consititute a separate clade with a bootstrap value higher than 50 %.Therefore,we propose that Hs_mrp system should represent a separate distinct sub-class of Group 1 Mrp systems.In order to further reveal the mechanism of salt tolerance of Hs_mrp,We carried out the relevant validation of functional experiments in the heterologous host of(E.coli)KNabc and homologous host of Hs_mrp gene deletion strain.The results are as follows:(1)In the heterology host,the growth test showed that the presence of Hs_mrp could confer tolerance of E.coli KNabc to 0.8 M Na Cl andalkaline p H 8.0,and a certain resistance to Li Cl(5mM Li Cl).Na~+(Li~+,K~+)/H~+ antiport assay based on everted membrane vesicles showed that Hs_mrp system exhibited Na~+(Li~+,K~+)/H~+ antiport activity at pH range from 6.5 to 9.5 with the highest activity at p H 9.5.Also,the apparent K0.5 values for Na~+,Li~+ and K~+ at pH 9.5 were 0.64?1.05 and 0.76,respectively.It indicates that Hs_Mrp operon encoding a p H-dependent Na~+(Li~+,K~+)/H~+ antiporter.(2)firstly,we successfully constructed a gene recombinant knock-out plasmids pK18-mrpNco I and p K18-mrp-Gm containing gentamicin resistance;Secondly,the knockout plasmids pK18-mrp-Nco I successfully entered the NEAU-ST10-39 T by electroporation;Then,two strains of the Hs_mrp gene deletion were successfully obtained by reverse screening,we designated them NEAU-ST10-39~T-?mrp-7 and NEAU-ST10-39~T-?mrp-23,respectively.Finally,we successfully transferred the shuttle recombinant plasmid p MCS5-mrp carrying the complete Hs_mrp operon into the deleted strain,and use NEAU-ST10-39~T-?mrp/pMCS5-mrp to designate the complementary mutant strain.(3)In the homology host,we conducted a salt tolerance and alkali resistance test for the deletion strains.It showed that: NEAU-ST10-39~T-?mrp/p MCS5-mrp(complementary mutants)exhibited similar salt tolerance to wild-type strains,both Hs_mrp operants can complement knockout strains;at p H 7 neutral conditions and present of 2% and 3% Na Cl concentration,the growth situation of knockout strains was not significantly different from that of wild-type and complementary mutants.However,with the increase of salt concentration,the growth of knockout strain was obviously inhibited.Under p H 8 or p H 9 alkaline conditions,starting from 2% or 3%Na Cl concentration,growth of knockout strain has been significantly inhibited.It is shown that under neutral conditions,Hs_mrp plays an important role in the growth of host bacteria under high salt.Under alkaline conditions,Hs_mrp has already played a role in the growth of host bacteria at lower salt concentration.Taken together,we proposed that Hs_mrp system should be categorized as a Group 1 Mrp system;and we successfully obtained the Hs_mrp gene deletion strain,NEAU-ST10-39~T-?mrp.