An extended role for the nucleoid structuring protein H-NS in the virulence gene regulatory cascade of Shigella flexneri

Abstract

The H-NS nucleoid structuring protein has been shown previously to play a negative role in controlling virulence gene expression in Shigella flexneri by repressing transcription of the virF and virB regulatory genes and the VirF-dependent icsA structural gene under non-permissive growth conditions. Here, we show that H-NS also acts at the promoters of the VirB-dependent structural genes in the regulatory cascade. H-NS protein binds to the promoter regions in vivo and in vitro. The promoters were shown physically and by in silico analysis to contain regions of DNA curvature, a feature of H-NS binding sites. H-NS binding sites were determined by DNase I footprinting at the icsB and the virA promoters. The locations of these sites were consistent with a role for H-NS as a transcription repressor. The VirB-dependent structural gene promoters were found to respond directly to the H-NS repressor, revealing a level of control that is additional to that exerted by the H-NS-dependent virB activator gene. Moreover, the promoters were sensitive to the level of VirB protein in the cell, requiring a threshold level of VirB to be reached before becoming active. A model is discussed in which the levels of expression of the structural genes reflect the outcome of competition between the countervailing regulatory activities of the H-NS and VirB proteins.