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 with 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 ΔnhaR 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 Na⁺ 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.