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Vibrio cholerae H-NS: investigation of regulated genes and structure-function analysis.

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Call No.	QR201.C5 N94 2003
Author	Nye, Melinda Brooker
Title	Vibrio cholerae H-NS: investigation of regulated genes and structure-function analysis.
Date	2003
Abstract	H-NS, an abundant nucleoid associated protein, has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. A genetic approach was used to study H-NS regulation of genes in Vibrio cholerae. Transcriptional fusions were used to analyze expression of genes within the ToxR regulon. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin co-regulated pilus, and the ToxT regulatory protein. For the cholera toxin and ToxT encoding genes, elevated expression in a 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 regions upstream of toxT and ctx identified potential H-NS binding sites. Whole genome expression profiling using microarray analysis showed virulence genes were the most significantly derepressed in the absence of hns under the conditions tested and identified additional H-NS-regulated genes. Extensive mutagenesis of the structural and functional domains of the H-NS protein revealed the contribution of each to the regulation of gene expression. Insertions within, or truncations of, the C-terminal conserved DNA binding domain prevented repression of toxT and ctx, as expected. Dominant negative experiments demonstrated that V. cholerae H-NS represses gene expression as an oligomeric protein. Hydrophobic coiled-coil interactions, known to confer oligomerization capability in other H-NS orthologs, provide one mechanism for V. cholerae H-NS multimerization. Chimeric H-NS proteins, using sequences from both E. coli and V. cholerae H-NS orthologs, revealed that V. cholerae H-NS possesses a second oligomerization domain in the N-terminal 24 amino acids of the protein. Overall, 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. Our results demonstrate DNA binding and protein oligomerization, provided by either the central coiled-coil or N-terminal domain, are required for repression of H-NS regulated promoters within the ToxR regulon.
Description	1 online resource (xiv, 270 leaves) : illustrations (some color)
Format	application/pdf
Advisor	Taylor, Ronald K.
Subject	Vibrio cholerae -- Genetics. ; DNA-binding proteins. ; Genetic transcription -- Regulation. ; Bacterial proteins.
College	Dartmouth Medical School -- Department of Microbiology and Immunology.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D)--Dartmouth College.
Copyright	In Copyright. This material is protected by copyright and/or related rights. You are free to use this material in any way that is permitted by copyright law that applies to your use. For other uses, you need to obtain permission from the rights-holder(s). http://rightsstatements.org/page/InC/1.0/
DOI	https://doi.org/10.1349/ddlp.2850

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