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

EMBO J. 1997 Oct 1;16(19):5922-9. doi: 10.1093/emboj/16.19.5922.

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 DeltanhaR 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Binding Sites
  • DNA Footprinting
  • DNA Methylation
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / metabolism*
  • DNA-Binding Proteins*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins*
  • Gene Deletion
  • Histidine / metabolism
  • Molecular Sequence Data
  • Promoter Regions, Genetic
  • Sodium / metabolism*
  • Sodium-Hydrogen Exchangers / genetics*
  • Sodium-Hydrogen Exchangers / metabolism
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism*

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • NhaA protein, E coli
  • Sodium-Hydrogen Exchangers
  • Transcription Factors
  • nhaR protein, E coli
  • Histidine
  • Sodium