title: Identification of the domains of UreR, an AraC-like transcriptional regulator of the urease gene cluster in Proteus mirabilis.
abstract: Proteus mirabilis urease catalyzes the hydrolysis of urea to CO(2) and NH(3), resulting in urinary stone formation in individuals with complicated urinary tract infections. UreR, a member of the AraC family, activates transcription of the genes encoding urease enzyme subunits and accessory proteins, ureDABCEFG, as well as its own transcription in the presence of urea. Based on sequence homology with AraC, we hypothesized that UreR contains both a dimerization domain and a DNA-binding domain. A translational fusion of the leucine zipper dimerization domain (amino acids 302 to 350) of C/EBP and the C-terminal half of UreR (amino acids 164 to 293) activated transcription from the ureD promoter (p(ureD)) and bound to a 60-bp fragment containing p(ureD), as analyzed by gel shift. These results were consistent with the DNA-binding specificity residing in the C-terminal half of UreR and dimerization being required for activity. To localize the dimerization domain of UreR, a translational fusion of the DNA-binding domain of the LexA repressor (amino acids 1 to 87) and the N-terminal half of UreR (amino acids 1 to 182) was constructed and found to repress transcription from p(sulA)-lacZ (sulA is repressed by LexA) and bind to the sulA operator site, as analyzed by gel shift. Since LexA binds this site only as a dimer, the UreR(1-182)-LexA(1-87) fusion also must dimerize to bind p(sulA). Indeed, purified UreR-Myc-His eluted from a gel filtration column as a dimer. Therefore, we conclude that the dimerization domain of UreR is located within the N-terminal half of UreR. UreR contains three leucines that mimic the leucines that contribute to dimerization of AraC. Mutagenesis of Leu147, Leu148, or L158 alone did not significantly affect UreR function. In contrast, mutagenesis of both Leu147 and Leu148 or all three Leu residues resulted in a 85 or 94% decrease, respectively, in UreR function in the presence of urea (P < 0.001). On the contrary, His102 and His175 mutations of UreR resulted in constitutive induction in the absence of urea. We conclude that a dimerization domain resides in the N-terminal half of the polypeptide, that Leu residues may contribute to this function, and that sequences within the C-terminal half of UreR are responsible for DNA binding to the urease promoter regions. Selected His residues also contribute significantly to UreR function.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland-Baltimore, School of Medicine, Baltimore, MD 21201, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Poore
authors.forename: C
authors.initials: C
authors.lastname: Coker
authors.forename: J D
authors.initials: JD
authors.lastname: Dattelbaum
authors.forename: H L
authors.initials: HL
authors.lastname: Mobley
mesh_terms: D051777:AraC Transcription Factor; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D011513:Proteus mirabilis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D014510:Urease / Q000235:genetics*; D000099160:LexA Repressor Protein
chemical_list: D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA; D006639:Histidine; D007930:Leucine; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D015534:Trans-Activators; D014157:Transcription Factors; D014510:Urease; D000099160:LexA Repressor Protein; C119767:UreR protein, bacteria