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

O. Carmel, O. Rahav-Manor, N. Dover, B. Shaanan, E. Padan

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)5922-5929
Number of pages8
JournalEMBO Journal
Volume16
Issue number19
DOIs
StatePublished - 18 Oct 1997
Externally publishedYes

Keywords

  • Na-specific transcription regulation
  • Na/H antiporters
  • NhaR
  • nhaA-Na-specific footprint

ASJC Scopus subject areas

  • Neuroscience (all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology (all)
  • Immunology and Microbiology (all)

Fingerprint

Dive into the research topics of 'The Na+-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli'. Together they form a unique fingerprint.

Cite this