| The moderate halophiles are a group of microorganisms that can grow optimally in the presence of3-15%NaCl. Compared with the common non-halophiles, the halophiles possess more salt-alkali tolerant genes and the corresponding molecular mechanism of salt-alkali tolerance. Halophiles are regarded as ideal objects used for screening halo-alkaline tolerant genes, which have increasingly attracted wide spread attentions in the academic community. Therefore, it’s of more prossiblity that novel and key salt-alkali tolerant genes wil be cloned from the halophiles. Identification of these genes is very helpful for further understanding bacterial molecular mechanism of salt-alkali tolerance. Moreover, key salt-alkali tolerant genes may be used for the construction of bacterial gene-engineered strains with high-efficiency capacity of salt-alkali tolerance.In order to explore its halo-alkaline tolerant mechanism, genomic DNA was screened in this study for Na+(Li+)/H+antiporter genes from NEAU-ST10-251by selection in Escherichia coli KNabc lacking three major Na+(Li+)/H+antiporters (AnhaA, ΔnhaB, ΔchaA). One mrp operon encoding a multisubunit monovalent cation/proton antiporter and one oadGAB gene cluster encoding a Na+-transporting oxaloactate decarboxylase were finally cloned. The former was previously mainly designated mrp (also mnh, pha or sha) due to its multiple resistance and pH-related activity. Here, we will also use mrp to designate the homolog from H. zhaodongensis (Hz_mrp). The latter were designated Hz_oadGAB. The growth test showed that the presence of Hz_mrp could confer tolerance of E. coli KNabc to0.8M NaCl and100mM LiCl, and an alkaline pH8.0while the presence of Hz_oadGAB could confer tolerance of E. coli KNabc to0.4M NaCl and10mM LiCl, and an alkaline pH8.0.Hz_mrp operon (6,400bp) consists of seven open reading frames (ORFs), all of which overlap with or terminate very closely to the following one. Also, these seven ORFs are preceded by a predicted common promoter sequence and followed by a possible common terminator sequence, each of which has one separate upstream Shine-Dalgarno (SD) sequence. Protein alignment showed that ORF1-7has the highest identity (90.8%-99.3%) with the subunits A to G of Hs_Mrp system, a putative Group1Mrp system from Halomonas sp. GFAJ-1. In contrast, ORF1-7have limited identity (33.6%-42.7%,30.9%-38.7%,35.1%-45.0%,34.0%-48.9%,28.2%-37.4%,25.8%-38.2%and32.8%-38.3%) with the subunits A to G of Sf_Pha2system from Sinorhizobium fredii, Bh_Mrp system from Bacillus halodurans, Bf_Mrp system from B. firmus, Bs_Mrp system from B. subtilis and Sa_Mnh system from Staphylococcus aureus, respectively. Phylogenetic analysis based on neighbour-joining algorithm showed that Hz_Mrp system and its closest putative homologs including Hs_Mrp system, Hzh_Mrp system and Is_Mrp system indeed constitute a separate clade with a bootstrap value higher than50%. Therefore, we propose that Hz_Mrp system should represent a separate distinct sub-class of Group1Mrp systems.Hz_oadGAB gene cluster (4,800bp) contains three ORFs preceded by their respective promoter-like and SD sequences. Protein alignment showed that ORF1-3has the highest identity with γ (54.3%), α (79.5%), β (78.8%) subunits of a putative Na+-transporting oxaloactate decarboxylase from Halomonas elongata. We also analyzed the identity of Hz_OadGAB with their characterized homologs from Vibiro cholerae, Salmonella typhimurium, Klebsiella pneumoniae and Enterobacter aerogenes. Hz_OadGAB showed higher identity (62.5-65.2%for Hz_OadA,68.1-77.1%for Hz_OadB and34.7-45.3%Hz_OadG) for with the above homologs, which suggests that oadGAB gene cluster should encode α, β,γ subunits of Na+-transporting oxaloactate decarboxylase. The oxaloacetate decarboxylase assay showed that Hz_OadGAB exihit the optimum enzymatic activity at37℃, pH7.0and in the presence of100mM NaCl. Everted membrane vesicles prepared from E. coli KNabc cells carrying Hz_OadGAB exhibited Na+/H+antiport activity. These results reveal that Hz_OadGAB should function as a complex protein with the dual properties of both oxaloacetate decarboxylase and Na+/H+antiporter..Na+(Li+, K+)/H+antiport assay based on everted membrane vesicles showed that Hz_Mrp system exhibited pH-dependent Na+(Li+, K+)/H+antiport activity at pH range from6.5to9.5with the highest activity at pH8.5. Also, the apparent Km values for Na+, Li+and K+at pH8.5were0.70,0.93and0.83, respectively. Therefore, Hz_Mrp system exhibits also a significant difference in the specificity for the transported monovalent cations or pH-dependent activity profile compared with all the known Mrp systems. Taken together, we proposed that Hz_Mrp system should be categorized as a novel Group1Mrp system. |
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