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.
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.
Carmel
O
O
Division of Microbial and Molecular Ecology, Hebrew University of Jerusalem, Israel.
Dover
N
N
Rahav-Manor
O
O
Dibrov
P
P
Kirsch
D
D
Karpel
R
R
Schuldiner
S
S
Padan
E
E
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000032:analysis / Q000494:pharmacology*; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis*; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D014157:Transcription Factors; C074687:nhaR protein, E coli; D012964:Sodium; D001616:beta-Galactosidase