ToxR proteins with substitutions in residues conserved with OmpR fail to activate transcription from the cholera toxin promoter.
The ToxR protein of Vibrio cholerae is an integral membrane protein that coordinately regulates the expression of virulence genes required for successful infection. ToxR has been shown to bind directly to and activate transcription of the cholera toxin (ctx) promoter. Within the amino-terminal cytoplasmic region of ToxR, several amino acids are strictly conserved among ToxR, OmpR, and the other members of a family of bacterial regulatory proteins. To better understand the function of this region, two approaches were taken: conserved residues were changed by site-directed mutagenesis, and random mutations that eliminated ToxR-mediated transcriptional activation were isolated. Several classes of mutations were identified: those that abolish promoter DNA binding and transcriptional activation (toxR R96K, toxR R68K, and toxR R68L), those that abolish transcriptional activation but retain the ability to bind promoter DNA (toxR R96L), and those that have an intermediate phenotype (toxR R77L, toxR E51K, and toxR E51D). The toxR E51K allele had reduced activity in both Escherichia coli and V. cholerae but also exerted a dominant-negative effect over wild-type ToxR when assayed in V. cholerae. This result provides additional evidence that ToxR acts as an oligomer in the transcriptional activation process. From this mutational analysis of conserved amino acid residues within the OmpR-homologous region of ToxR, we conclude that this region is essential for transcriptional activation at the level of DNA binding and other steps that lead to activation of the ctx promoter.
Ottemann
K M
KM
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
DiRita
V J
VJ
Mekalanos
J J
JJ
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000494:pharmacology; D001426:Bacterial Proteins; D001483:Base Sequence; D002772:Cholera Toxin / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005799:Genes, Dominant / Q000235:genetics; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic; D014157:Transcription Factors / Q000235:genetics* / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014734:Vibrio cholerae / Q000235:genetics* / Q000378:metabolism; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C499324:toxR protein, Vibrio cholerae; C066719:toxR protein, bacteria; D002772:Cholera Toxin; D001618:beta-Lactamases