Rahav-Manor O, Carmel O, Karpel R, Taglicht D, Glaser G, Schuldiner S, Padan E
Division of Microbial and Molecular Ecology, Hadassah Medical School, Hebrew University of Jerusalem, Israel.
J Biol Chem. 1992 May 25;267(15):10433-8.
On the basis of protein homology, nhaR has previously been shown to belong to a large family of regulatory proteins, the LysR family (Henikoff, S., Haughn, G.W., Calvo, J.M., and Wallace, J.C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6602-6606). In this work we show that nhaR is a regulator of nhaA, a gene encoding a Na+/H+ antiporter in Escherichia coli. Multicopy plasmid bearing nhaR enhances the Na(+)-dependent induction of a chromosomal nhaA'-'lacZ fusion. Extracts derived from cells overexpressing nhaR exhibit specific DNA binding capacity to the upstream sequences of nhaA. Construction of an nhaR deletion mutant (OR100) shows that nhaR is required in addition to nhaA to tolerate the extreme conditions under which nhaA is indispensable. Whereas OR100 grows like the wild type at neutral pH even at high Na+ concentrations (700 mM), it becomes much more sensitive to Na+ (greater than 300 mM) at pH 8.5; furthermore, OR100 is more sensitive to Li+ (100 mM) than the wild type. Nevertheless, the phenotype of OR100, which is more resistant to Na+, Li+, and alkaline pH than a delta nhaA strain (NM81), implies that the regulation exerted by nhaR is not complete and that some expression of nhaA exists in OR100. Accordingly, the effect of nhaR in cells is dependent on the level of nhaA. OR200, a nhaA and nhaR deletion mutant, has the same phenotype as NM81. Multicopy plasmid bearing nhaR does not change the phenotype of either OR200 or NM81. On the other hand, multicopy nhaA renders the cells Li(+)- and and Na(+)-resistant even without nhaR.
基于蛋白质同源性，nhaR先前已被证明属于一大类调节蛋白，即LysR家族（亨尼科夫，S.，豪恩，G.W.，卡尔沃，J.M.，和华莱士，J.C.（1988年）《美国国家科学院院刊》85，6602 - 6606）。在这项工作中，我们表明nhaR是nhaA的调节因子，nhaA是大肠杆菌中编码Na⁺/H⁺反向转运蛋白的基因。携带nhaR的多拷贝质粒增强了染色体nhaA'-'lacZ融合基因的Na⁺依赖性诱导。过表达nhaR的细胞提取物对nhaA的上游序列表现出特异性DNA结合能力。构建nhaR缺失突变体（OR100）表明，除了nhaA之外，nhaR也是耐受nhaA不可或缺的极端条件所必需的。虽然OR100在中性pH下即使在高Na⁺浓度（700 mM）时也能像野生型一样生长，但在pH 8.5时它对Na⁺（大于300 mM）变得更加敏感；此外，OR100对Li⁺（100 mM）比野生型更敏感。然而，OR100比ΔnhaA菌株（NM81）对Na⁺、Li⁺和碱性pH更具抗性的表型意味着nhaR所施加的调节并不完全，并且在OR100中存在一些nhaA的表达。因此，nhaR在细胞中的作用取决于nhaA的水平。OR200，一种nhaA和nhaR缺失突变体，具有与NM81相同的表型。携带nhaR的多拷贝质粒不会改变OR200或NM81的表型。另一方面，多拷贝的nhaA即使没有nhaR也能使细胞对Li⁺和Na⁺具有抗性。
