In Vivo DNA-Binding and Oligomerization Properties of the Shigella flexneri AraC-Like Transcriptional Regulator VirF as Identified by Random and Site-Specific Mutagenesis

Abstract

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-turnhelix 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.

Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri

“…37 uC and virF : virB). Expression of virB was markedly decreased in the virF mutant compared with the wild-type strain and expression of all genes that were less expressed in the virB mutant was also decreased in the virF mutant, consistent with previous results (Adler et al, 1989;Porter & Dorman, 2002;Sakai et al, 1988;Watanabe et al, 1990). In addition to virB (and genes that were less expressed in the virB mutant), genes that were less expressed in the virF mutant compared with the wild-type strain included ipgB2, ipaH4, orf82, orf94, orf136, phoN1, orf163, repA and ospE1/2.…”

Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

“…Therefore, Rns may use both HTHs to bind DNA as has been proposed or demonstrated for the AraC family members ToxT (Childers et al, 2007), VirF (Porter & Dorman, 2002) and PerA (Porter et al, 2004). The other Cterminal a-helices of Rns may not be directly involved in DNA binding.…”

Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria

“…Temperature sensitivity is a common theme among those H-NS-antagonizing AraC-like proteins that are involved in virulence gene activation, yet the molecular basis of temperature sensitivity remains obscure in most cases (Porter & Dorman, 2002). Perhaps the effect of temperature on the topology of the DNA to which the AraC-like protein binds influences its interaction with RNA polymerase, allowing it to act as a transcription activator as well as an anti-repressor that disrupts H-NS-DNA complexes.…”

“…In those cases where the matter has been investigated, the AraC-like protein has been found both to antagonize H-NS repression and to exert a positive influence (albeit a modest one) on The virB regulatory virulence gene in Shigella flexneri and enteroinvasive E. coli Falconi et al (1998); Porter & Dorman (2002) promoter function in the absence of the repressor (Jordi et al, 1992;Murphree et al, 1997). Those AraC-like proteins that derepress and activate thermally responsive virulence genes do not appear to bind chemical ligands; they respond instead to a physical signal (temperature).…”