Identification of the domains of UreR, an AraC-like transcriptional regulator of the urease gene cluster in Proteus mirabilis.

Proteus mirabilis infects the urinary tract of humans and is most commonly responsible for causing disease in individuals with structural abnormalities of the urinary tract or in patients who undergo long-term catheterization (16). Cystitis, acute pyelonephritis, and urinary stone formation are all possible consequences of P. mirabilis infection (17). P. mirabilis produces a urea-inducible urease, a high-molecular-weight, multimeric, cytoplasmic nickel metalloenzyme. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide (18). During the course of infection, the production of ammonia by urea hydrolysis raises the pH in the local environment, subsequently precipitating polyvalent ions that are normally soluble in urine. The result is the formation of urinary stones. The elevated pH also creates an environment that is more favorable for growth of this species (4). Increased ammonia production can also lead to acute inflammation with possible tissue necrosis (18). The P. mirabilis urease gene cluster is found in single copy on the chromosome and consists of eight contiguous genes, ureRDABCEFG (12, 19, 24). The ureA (UreA, 11 kDa), ureB (UreB, 12 kDa), and ureC (UreC, 61 kDa) genes encode the structural polypeptides required for the assembly of a catalytically inactive urease apoenzyme (18). The accessory genes, ureD (UreD, 31 kDa), ureE (UreE, 18 kDa), ureF (UreF, 23 kDa), and ureG (UreG, 22 kDa), encode proteins required for insertion of nickel ions into the metalloenzyme resulting in catalytically active urease (18). The urease gene cluster is regulated by the gene product of ureR (UreR, 33 kDa). P. mirabilis UreR and the plasmid-encoded UreR found in Escherichia coli are positive transcriptional activators of the urease genes. The two proteins share 70% amino acid identity (6) and are functionally interchangeable in the activation of transcription from the ureR (pureR) and ureD (pureD) promoters in both the P. mirabilis and plasmid-encoded urease gene clusters (6). The UreR binding sites of both promoters have the consensus sequence T(A/G)(T/C)(A/T)(T/G)(C/T)T(A/T)(T/A)ATTG (25). Both UreR proteins have been shown to activate transcription from pureD in the presence of urea (11, 6). In addition, UreR regulates its own transcription in the presence of urea from pureR in the direction opposite the rest of the gene cluster (6). In the absence of urea induction, H-NS represses ureR expression (3). Because UreR activates transcription in a urea-inducible manner, it is hypothesized that UreR binds urea; however, this has not been directly demonstrated. UreR is a member of the AraC family of transcriptional regulators and contains a putative helix-turn-helix in addition to an AraC signature sequence (5, 19). The AraC signature sequence, found within all AraC family members, is a second helix-turn-helix that is hypothesized to also bind DNA (7). Moreover, UreR also contains three conserved leucine residues (Leu147, Leu148, and Leu158) in the same relative location with the same spatial distance relative to each other as in AraC (Leu150, Leu151, and Leu161). These leucine residues are critical for AraC dimerization (23), and we therefore also hypothesize that UreR dimerizes via this mechanism. In the presence of arabinose, AraC uses these three critical leucines for dimerization via an antiparallel coiled-coil in a “knobs-into-holes” manner, as elucidated by X-ray crystallographic studies (23). This coiled-coil is also the primary dimerization face in the absence of arabinose, shown by both size exclusion chromatography and sedimentation velocity analytical ultracentrifugation of an AraC mutant with mutations in Leu150, Leu151, Asn154, and Leu161 (15). A secondary dimerization face in the β barrel of AraC is evident; however, it does not appear to represent the primary means of dimer interaction (15). AraC contains two separate and independent domains, each with a distinct function, namely, dimerization and DNA binding; UreR is predicted to have similar domains with similar functions. Previously, chimeric proteins containing the two domains of AraC to characterize each of the domain's functions were synthesized (2). The predicted AraC DNA-binding domain was fused to C/EBP, a known eukaryotic transcriptional activator that dimerizes via a leucine zipper. The C/EBP-AraC fusion was found to bind to pBAD and activate transcription (2). The hypothesized AraC dimerization domain was fused to the LexA DNA-binding domain. This fusion was predicted to mimic full-length LexA and demonstrated the need for dimerization in order to repress transcription of genes normally turned off by LexA. A chromosomal transcriptional fusion of psulA to lacZ was repressed in the presence of the AraC-LexA fusion protein. This strategy was used to identify both domains of AraC (2). In this study, we constructed fusion proteins to identify putative domains of UreR and assign dimerization and DNA-binding functions to each of the domains as well as identifying key amino acid residues involved in dimerization and urea induction.