Vibrio cholerae H-NS Silences Virulence Gene Expression at Multiple Steps in the ToxR Regulatory Cascade

Abstract

H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon of Vibrio cholerae. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin-and ToxT-encoding genes, elevated expression in an hns mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of toxT suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the V. cholerae virulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.

The H-NS protein represses transcription of the eltAB operon, which encodes heat-labile enterotoxin in enterotoxigenic Escherichia coli, by binding to regions downstream of the promoter

“…However, the promoter regions of the two operons have undergone considerable genetic changes during evolution with regard to regulation of transcriptional expression in their respective hosts. Although transcription of ctx is also negatively regulated by H-NS, the region responsible for H-NSmediated repression is situated upstream of the promoter core elements, between 269 and 2400 relative to the start site of transcription (Nye et al, 2000;Yu & DiRita, 2002). In the case of the eltAB operon, deleting the sequence between 265 and 2160 had no effect on H-NS-mediated repression.…”

“…It is a global regulator which controls the expression of a large number of genes whose products are involved in a wide range of cellular processes (Ussery et al, 1994;Rimsky, 2004;Dorman, 2004). In pathogenic enteric bacteria, such as enteropathogenic E. coli (EPEC) (Bustamante et al, 2001;Haack et al, 2003), ETEC (Murphree et al, 1997), enteroinvasive E. coli (Falconi et al, 2001), Shigella flexneri (Porter & Dorman, 1994) and Vibrio cholerae (Nye et al, 2000;Yu & DiRita, 2002), H-NS participates in the regulation of virulence gene expression. In these bacteria, H-NS functions mainly as a negative regulator which represses transcription in response to environmental changes, such as temperature and osmolarity (Atlung & Ingmer, 1997).…”

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

“…The result has been described as H-NSmediated transcriptional silencing (Bouffartigues et al, 2007;Fang & Rimsky, 2008;Göransson et al, 1990;Lang et al, 2007;Lucchini et al, 2006;Madhusudan et al, 2005;McGovern et al, 1994;Murphree et al, 1997;Navarre et al, 2006;Nye et al, 2000;Petersen et al, 2002;Westermark et al, 2000;Will et al, 2004). It has been estimated from single-molecule studies using optical tweezers that the force required to disrupt an H-NS-DNA bridge is 7 pN at an unzipping rate of 70 bp s 21 , which is the speed of RNA polymerase; RNA polymerase can exert a force of up to 25 pN (Dame et al, 2006).…”

New insights into transcriptional regulation by H-NS

“…ToxT, a transcriptional activator belonging to the AraC family, promotes the production of cholera toxin and other virulence factors of Vibrio cholerae by both activating transcription and countering H-NS silencing [58,59]. The VirB protein activates virulence gene expression in Shigella but resembles a plasmid partitioning protein rather than a classical transcriptional activator.…”