A single amino acid substitution (Glu134-->Ala) in NhaR1 increases the inducibility by Na+ of the product of nhaA, a Na+/H+ antiporter gene in Escherichia coli

EMBO J. 1994 Apr 15;13(8):1981-9. doi: 10.1002/j.1460-2075.1994.tb06467.x.

Abstract

The mutation nhaAup (antup) has now been identified as a Glu134 to Ala substitution in NhaR and designated nhaR1. This was demonstrated by sequence analysis showing that the mutant contains a wild-type nhaA but nhaR1 instead of nhaR and by the finding that nhaR1 cloned in a plasmid confers the NhaAup phenotype. Na+ (107 mM) increases by 5- to 10-fold the level of nhaA transcripts, similar to the effect on the NhaR-mediated expression of a nhaA'-'lacZ fusion. These results are in agreement with the notion that nhaR is a positive regulator which controls Na(+)-dependent transcription of nhaA. The promoter region of nhaR and nhaR1 was found to reside within the BglII-BamHI fragment of the C-terminal sequences of nhaA. The mutation nhaR1, while increasing dramatically the level of transcription, reduces the requirement for Na+ by 3- to 5-fold both for nhaA transcription and for the nhaR1-mediated expression of nhaA'-'lacZ fusion. NhaR1, like NhaR, binds specifically to the promoter region of nhaA. However, at equal protein concentration NhaR1 binds more DNA and the NhaR1-DNA complex shows higher mobility than that of NhaR-DNA, suggesting the existence of two different binding complexes. Yet in this assay the DNA binding pattern of neither NhaR nor NhaR1 was affected by the addition of Na+. The possible relevance of these two DNA-binding complexes to the Na(+)-induced NhaR-mediated expression is discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / analysis
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Base Sequence
  • DNA-Binding Proteins*
  • Dose-Response Relationship, Drug
  • Enzyme Induction
  • Escherichia coli / genetics*
  • Escherichia coli Proteins*
  • Gene Expression Regulation, Bacterial / drug effects*
  • Molecular Sequence Data
  • Phenotype
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • Recombinant Fusion Proteins / biosynthesis
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Sodium / analysis
  • Sodium / pharmacology*
  • Sodium-Hydrogen Exchangers / biosynthesis*
  • Transcription Factors / analysis
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Transcription, Genetic
  • beta-Galactosidase / biosynthesis

Substances

  • Bacterial Proteins
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Recombinant Fusion Proteins
  • Sodium-Hydrogen Exchangers
  • Transcription Factors
  • nhaR protein, E coli
  • Sodium
  • beta-Galactosidase

Associated data

  • GENBANK/J03879
  • GENBANK/L24072
  • GENBANK/S67239