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 nha A 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 nha A 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 nha A. 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 Δ nha R 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 nha A expression by undergoing a conformational change upon Na + binding which modifies the NhaR– nha A contact points.