The Na+-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli

We used partially purified NhaR and a highly purified His-tagged NhaR derivative to identify the cis-regulatory sequences of nhaA recognized by NhaR and to study the specific effect of Na+ on this interaction, Gel retardation assay,vith DNase I footprinting analysis showed that NhaR binds a region of nhaA which spans 92 bp and contains three copies of the conserved LysR-binding motif, Na+, up to 100 mM, had no effect on the binding of NhaR to nhaA. The dimethylsulfate methylation protection assay in vivo and in vitro, showed that bases G(-92), G(-60), G(-29) and A(-24) form direct contacts with NhaR; in the absence of added Na+ in vivo, these bases were protected but became exposed to methylation in a Delta nhaR strain; accordingly, these bases were protected in vitro by the purified His-tagged NhaR, 100 mM Na+, but not K+, removed the protection of G(-60) conferred by His-tagged NhaR in vitro, Exposure of intact cells to 100 mM Na+, but not K+, exposed G(-60), The maximal effect of Naf in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5, In contrast to G(-60), G(-92) was exposed to methylation by the ion only in vivo, suggesting a requirement for another factor existing only in vivo for this interaction, We suggest that NhaR is both sensor and transducer of the Na+ signal and that it regulates nhaA expression by undergoing a conformational change upon Na+ binding which modifies the NhaR-nhaA contact points.