The role of H‐NS in silencing F transfer gene expression during entry into stationary phase

Abstract

SummaryThe conjugative ability of the F plasmid of Escherichia coli is highly growth phase dependent, with plasmid transfer efficiency dropping rapidly as donor cells progress through the growth cycle towards stationary phase. Transfer is dependent on the expression of the plasmid transfer ( tra ) genes, which are controlled by three plasmid-encoded regulatory proteins: TraJ, TraY and TraM. Here, we show that the nucleoid-associated host protein, H-NS, acts to repress the expression of traM and traJ as cells enter stationary phase, thereby decreasing mating ability to barely detectable levels. Sequence analysis identified regions of predicted intrinsic curvature, to which H-NS preferentially binds, at the promoters of both traM and traJ . H-NS binding at these regions was then confirmed by electrophoretic mobility shift and DNase I protection footprinting assays. Immunoblot assays displayed a significant increase in TraJ and TraM levels in an hns mutant strain. These findings were further supported by Northern and primer extension analyses which showed that whereas both genes were only expressed in early exponential phase in wild-type cells, hns mutant cells exhibited drastic derepression throughout the growth cycle. Transcriptional fusion studies of the individual promoters demonstrated that H-NS-mediated repression was observed when the promoters of both traM and traJ were present in cis to each other. This suggests that H-NS may bind to an extended region of the F plasmid, acting as a regional silencer of promoters for traJ and traM .

Regulation of Expression of Uropathogenic Escherichia coli Nonfimbrial Adhesin TosA by PapB Homolog TosR in Conjunction with H-NS and Lrp

“…An H-NS and Lrp regulatory switch drives tos operon transcriptional regulation. It has been previously proposed and noted that Lrp might act to anti-silence H-NS repression (35,38,59,68). We hypothesize, therefore, that an H-NS and Lrp regulation switch (i.e., predominance of either H-NS or Lrp) explains the observed regulation of the tos operon.…”

“…In agreement with this, others have noted the possibility that Lrp and H-NS antagonize the activity of each other or could interact together to potentiate gene regulation (35,38,59,68). This type of regulation resembles a regulatory switch, in which one nucleoid-structuring protein switches in predominance at key regulatory elements to perturb gene regulation.…”

Silencing and activating type IV secretion genes of the F-like conjugative resistance plasmid R1

“…1). We therefore set up experiments to elucidate whether regulation of tra gene expression at the P Y promoter in the case of the naturally repressed plasmid R1 displays similar characteristics to those found for the F plasmid (Will & Frost, 2006;Will et al, 2004).…”

“…For the F plasmid it has been recently proposed that TraJ does not act as a classical transcriptional activator but rather counteracts H-NS mediated repression and thereby activates transcription (Will & Frost, 2006;Will et al, 2004). In R1, H-NS is involved in silencing the P Y promoter and there are additional binding sites for H-NS in the P J and P M promoter regions, the latter overlapping oriT; in addition we could demonstrate direct binding of H-NS to P Y DNA by EMSA and DNase I footprinting (unpublished observations).…”

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).…”