Urea-dependent signal transduction by the virulence regulator UreR
Inessa Gendlina, Delia M. Gutman, Venetta Thomas, Carleen M. Collins
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.
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Signal Transduction Chemical Compounds 100%
Urea Medicine & Life Sciences 87%
Histidine Medicine & Life Sciences 25%
Alanine Chemical Compounds 19%
Urease Medicine & Life Sciences 19%
Substitution Reaction Chemical Compounds 12%
Binding Sites Medicine & Life Sciences 11%
Gendlina, I., Gutman, D. M., Thomas, V., & Collins, C. M. (2002). Urea-dependent signal transduction by the virulence regulator UreR. Journal of Biological Chemistry, 277(40), 37349-37358. https://doi.org/10.1074/jbc.M203462200
Urea-dependent signal transduction by the virulence regulator UreR. / Gendlina, Inessa; Gutman, Delia M.; Thomas, Venetta; Collins, Carleen M.
Gendlina, I, Gutman, DM, Thomas, V & Collins, CM 2002, 'Urea-dependent signal transduction by the virulence regulator UreR', Journal of Biological Chemistry, vol. 277, no. 40, pp. 37349-37358. https://doi.org/10.1074/jbc.M203462200
Gendlina I, Gutman DM, Thomas V, Collins CM. Urea-dependent signal transduction by the virulence regulator UreR. Journal of Biological Chemistry. 2002 Oct 4;277(40):37349-37358. https://doi.org/10.1074/jbc.M203462200
Gendlina, Inessa ; Gutman, Delia M. ; Thomas, Venetta ; Collins, Carleen M. / Urea-dependent signal transduction by the virulence regulator UreR. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 40. pp. 37349-37358.
@article{b2a4abb2766f491d8da4012f0a4f99fd,
title = "Urea-dependent signal transduction by the virulence regulator UreR",
abstract = "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.",
author = "Inessa Gendlina and Gutman, {Delia M.} and Venetta Thomas and Collins, {Carleen M.}",
T1 - Urea-dependent signal transduction by the virulence regulator UreR
AU - Gendlina, Inessa
AU - Gutman, Delia M.
AU - Thomas, Venetta
AU - Collins, Carleen M.
N2 - 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.
AB - 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.