Urea-dependent signal transduction by the virulence regulator UreR.
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 K(d) 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 (UreR(c)) exhibited a constitutive phenotype by both activating transcription and binding to DNA with an increased affinity in the absence of urea. The UreR(c) 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 rUreR(c) were dimers in both the presence and absence of urea. Thus, the structural changes associated with activation are subtle.
Gendlina
Inessa
I
Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Gutman
Delia M
DM
Thomas
Venetta
V
Collins
Carleen M
CM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019077:Helix-Turn-Helix Motifs; D008969:Molecular Sequence Data; D010957:Plasmids; D011532:Providencia / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D015534:Trans-Activators / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014508:Urea / Q000494:pharmacology*
D001426:Bacterial Proteins; D003062:Codon; D015534:Trans-Activators; C119767:UreR protein, bacteria; D014508:Urea