The Na super(+)-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na super(+)/H super(+) 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 super(+) 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 super(+), 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 super(-92), G super(-60), G super(-29) and A super(-24) form direct contacts with NhaR; in the absence of added Na super(+) 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 Histagged NhaR. 100 mM Na super(+), but not K super(+), removed the protection of G super(-60) conferred by His-tagged NhaR in vitro. Exposure of intact cells to 100 mM Na super(+), but not K super(+), exposed G super(-60). The maximal effect of Na super(+) in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5. In contrast to G super(-60), G super(-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 super(+) signal and that it regulates nhaA expression by undergoing a conformational change upon Na super(+) binding which modifies the NhaR-nhaA contact points.