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Virulence and motility gene regulation in Vibrio cholerae.

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Call No.	QR201.C5 S76 2009
Author	Stonehouse, Emily Agnes.
Title	Virulence and motility gene regulation in Vibrio cholerae.
Date	2009
Abstract	The ctx and tcp genes that encode cholera toxin and the toxin co-regulated pilus, the major virulence determinants of Vibrio cholerae, are repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the ctxAB and tcp operons, H-NS binding at these promoters was characterized using gel mobility shift assays and DNase I footprinting. H-NS was found to have a higher relative binding affinity for the ctx promoter than for the tcpA promoter in vitro, a result that is consistent with the higher level of H-NS repression observed at the ctx promoter. In addition, the nucleoid-associated protein, IHF, was found to positively regulate virulence gene expression. Inactivation of ihfA or ihfB, the genes encoding the IHF subunits, decreased expression of the two main virulence factors tcpA and ctx and prevented TCP and CT production. While IHF did not bind to the ctx promoter, it is was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at -162 using gel mobility shift assays and DNase I footprinting experiments. In addition, disruption of ihfA in an hns or toxT mutant background had no effect on tcpA expression. These results suggest that IHF may function at the tcpA promoter to alleviate H-NS repression and demonstrates an additional difference in the regulation of the tcpA and ctx genes. Finally, ToxT and IHF were found to inversely affect motility and virulence. Deletion of toxT or ihfA led to increased motility and overexpression of these transcriptional regulators caused a repression of motility. Deletion of tcpA did not alter motility in any background examined. In addition, ToxT derivatives that were stable but non-functional as transcriptional activators were unable to repress motility upon overexpression. These results suggest that repression of motility by ToxT and IHF is independent of their role as activators of tcpA and that residues that are important for ToxT-dependent activation are important in the repression of motility.
Description	xvi, 337 leaves : ill. (some col.) ; 28 cm
Format	application/pdf
Advisor	Taylor, Ronald K., Guerinot, Mary Lou, O'toole, George
Subject	Vibrio cholerae -- Molecular genetics. DNA-binding proteins. Genetic transcription -- Regulation. Promoters (Genetics)
College	Dartmouth Medical School. Dept. of Microbiology and Immunology
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.301

Title	Page 1
Format	application/pdf
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Full Text	Virulence and motility gene regulation in Vibrio cholerae A Thesis Submitted to the Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology by Emily Agnes Stonehouse DARTMOUTH COLLEGE Hanover, New Hampshire October 2009 Examining Committee: ____________________________ Ronald K. Taylor (chair) ____________________________ Mary Lou Guerinot ____________________________ George A. O’Toole ____________________________ ____________________________ Brian W. Pogue, Ph.D. George P. Munson Dean of Graduate Studies

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Virulence and motility gene regulation in Vibrio cholerae.

Virulence and motility gene regulation in Vibrio cholerae.