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Originally published In Press as doi:10.1074/jbc.M203462200 on July 29, 2002

J. Biol. Chem., Vol. 277, Issue 40, 37349-37358, October 4, 2002
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Urea-dependent Signal Transduction by the Virulence Regulator UreR*

Inessa Gendlina, Delia M. Gutman, Venetta ThomasDagger , and Carleen M. Collins§

From the Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33101

Identification of the environmental triggers involved in the expression of virulence genes is a fundamental objective in studies of bacterial pathogens. For uropathogens, urea, found in the urinary tract at concentrations of up to 500 mM, functions as an environmental signal. Urea freely diffuses into the bacterium Providencia stuartii and activates UreR, a member of the AraC family of transcriptional activators. Active UreR promotes transcription of virulence-associated urease genes and alerts the organisms of its immediate milieu. Thus, the UreR·urea complex has a dual role, acting as both a transcriptional activator as well as an environmental sensor. Here, we describe the molecular events associated with activation of gene expression by urea-bound UreR. The Kd of the urea·UreR binding reaction was measured as 0.2 mM by fluorescence quenching assays, and the shape of the binding curve indicated a single specific urea-binding site on UreR. Histidine residues are critical for urea binding in urease, and therefore to identify the urea-binding site in UreR, five mutant UreR forms were generated with histidine to alanine substitutions. Two of the mutants (UreRc) exhibited a constitutive phenotype by both activating transcription and binding to DNA with an increased affinity in the absence of urea. The UreRc bound urea with an affinity similar to that of wild-type UreR. We concluded, therefore, that the mutations resulting in constitutive activity were not involved in the UreR·urea interaction. UreR was activated, then, either by binding urea or by histidine to alanine substitutions at one of two positions. Circular dichroism indicated little change in the structure of UreR when activated, and size-exclusion chromatography demonstrated that both rUreR and rUreRc were dimers in both the presence and absence of urea. Thus, the structural changes associated with activation are subtle.

* This work was funded by Public Health Service Grants DK50495 and DK60163 (to C. M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: Dept. of Internal Medicine, Section of Rheumatology, P. O. Box 208031, New Haven, CT 06520.

§ To whom correspondence should be addressed (current address): Dept. of Microbiology, University of Washington School of Medicine, Box 357242, Seattle, WA 98195. Tel.: 206-616-0581; Fax: 206-543-8297; E-mail: carleen@u.washington.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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