In vivo DNA-binding and oligomerization properties of the Shigella flexneri AraC-like transcriptional regulator VirF as identified by random and site-specific mutagenesis.
In Shigella flexneri expression of the plasmid-encoded virulence genes is regulated via a complex mechanism involving both environmental signals and specific transactivators. The primary regulator protein, VirF, is a member of the AraC family of transcription factors and shares with other AraC-like proteins a conserved carboxy-terminal domain thought to be important for DNA binding. Random and site-directed mutagenesis of the virF gene encoding VirF yielded a number of mutations along the length of the protein which severely affected the ability of VirF to activate gene expression. The mutant proteins were shown to be affected in their ability to activate the virulence genes virB and icsA, both known to be regulated directly by VirF, as well as the virB-dependent virulence gene mxiC. Mutating key residues predicted to be important for DNA recognition had a significant negative effect, thereby suggesting that VirF interacts with its target sequence via two helix-turn-helix motifs. Two mutants that were dominant negative when coexpressed with the wild-type VirF protein were also isolated, indicating a role for protein-protein oligomerization in normal VirF function.
Porter
Megan E
ME
Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Republic of Ireland. meporter32@hotmail.com
Dorman
Charles J
CJ
D051777:AraC Transcription Factor; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D019077:Helix-Turn-Helix Motifs; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D012763:Shigella flexneri / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D037521:Virulence Factors
D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009842:Oligopeptides; D012097:Repressor Proteins; D014157:Transcription Factors; D037521:Virulence Factors; C058483:virG protein, Shigella flexneri; D004247:DNA