Doctor of Philosophy in Biological Sciences
First Committee Member
Patricia Cruz, Chair
Second Committee Member
Third Committee Member
Fourth Committee Member
Number of Pages
Shigella species are gram-negative intracellular pathogens that cause bacillary dysentery in humans. Many genes required for virulence of Shigella are carried on a large 230 kb plasmid and many of these are under the transcriptional control of the histone-like nucleoid structuring protein (H-NS) and by the major virulence regulator VirB. At the non-permissive temperature of 30⁰C, H-NS represses transcription, while at 37⁰C VirB alleviates this repression. This mechanism of gene regulation has been coined "silencing/anti-silencing" and is commonly found in many important bacterial pathogens including Salmonella spp. and Yersinia spp. The icsP gene, encoded by the Shigella virulence plasmid, is positively controlled by VirB and negatively by H-NS. The icsP gene encodes an outermembrane protease respsonsible for maintaining the tight polar cap of the actin polymerization protein IcsA, which is involved in the inter-, and intracellular spread of Shigella.
Our work has revealed that sequences located over 1 kb upstream of the icsP annotated transcription start site (+1) are needed for the VirB-dependent regulation of the icsP promoter. Using site directed mutagenesis we identified two DNA sequences that are required for the VirB dependent regulation of the icsP promoter, both of which display good matches to the reported consensus VirB binding site. We demonstrate that sequences located upstream of position -665 relative to the annotated transcription start site are needed for complete H-NS-mediated silencing of the icsP promoter.
Using electrophoretic mobility shift assays (EMSAs), and DNase I footprint analysis we show that VirB and H-NS bind directly to DNA sequences located both upstream of -665 and downstream of -213 by and that these regions are predicted to display high levels of intrinsic curvature. While we demonstrate that VirB functions to solely de-repress the icsP promoter, our EMSA data indicate that VirB and H-NS are capable of binding to the full upstream intergenic region (1232 bp) of the icsP gene simultaneously. These in vitro data suggest that VirB may function to relocate H-NS bound to the region immediately upstream of the icsP gene, rather than displacing H-NS from the DNA.
Our data also suggest that disruption of single regions of predicted curvature, contained within the upstream intergenic region of icsP, does not have an effect on either the ability of H-NS to silence the promoter or the ability of VirB to alleviate the H-NS induced repression.
Taken together our data suggest that remote regulation of the icsP promoter requires promoter proximal sequences that act in concert with upstream sequences. Our findings raise the possibility that other bacterial promoters may be regulated by DNA-binding proteins binding to remote DNA sequence elements. My work improves our understanding of transcriptional silencing and anti-silencing, a regulatory event that controls the expression of many virulence genes in many important enteric pathogens.
Anti-silencing; H-NS; Nucleotide sequence; Shigella; Silencing; VirB; Virulence (Microbiology) — Genetic aspects
Bacteriology | Genetics | Microbiology
University of Nevada, Las Vegas
Harrison, Dustin, "Transcriptional regulation of the Shigella flexneri icsp Promoter: Silencing and anti-silencing by H-NS and VirB" (2010). UNLV Theses, Dissertations, Professional Papers, and Capstones. 847.
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