Proteus mirabilis urease: operon fusion and linker insertion analysis of ure gene organization, regulation, and function

Abstract

Urease is an inducible virulence factor of uropathogenic Proteus mirabilis. Although eight contiguous genes necessary for urease activity have been cloned and sequenced, the transcriptional organization and regulation of specific genes within the Proteus gene cluster has not been investigated in detail. The first gene, ureR, is located 400 bp upstream and is oriented in the direction opposite the other seven genes, ureDABCEFG. The structural subunits of urease are encoded by ureABC. Previously, UreR was shown to contain a putative helix-turn-helix DNA-binding motif 30 residues upstream of a consensus sequence which is a signature for the AraC family of positive regulators; this polypeptide is homologous to other DNA-binding regulatory proteins. Nested deletions of ureR linked to either ureD-lacZ or ureA-lacZ operon fusions demonstrated that an intact ureR is required for urea-induced synthesis of LacZ from either ureA or ureD and identified a urea-regulated promoter in the ureR-ureD intergenic region. However, lacZ operon fusions to fragments encompassing putative promoter regions upstream of ureA and ureF demonstrated that no urea-regulated promoters occur upstream of these open reading frames; regions upstream of ureR, ureE, and ureG were not tested. These data suggest that UreR acts as a positive regulator in the presence of urea, activating transcription of urease structural and accessory genes via sequences upstream of ureD. To address the role of the nonstructural regulatory and accessory genes, we constructed deletion, cassette, and linker insertion mutations throughout the ure gene cluster and determined the effect of these mutations on production and regulation of urease activity in Escherichia coli. Mutations were obtained, with locations determined by DNA sequencing, in all genes except ureA and ureE. In each case, the mutation resulted in a urease-negative phenotype.Proteus mirabilis is a common etiologic agent of urinary tract infections, especially in the elderly catheterized population, and is second only to Escherichia coli as a cause of urinary tract infection (49). A number of putative virulence factors have been identified in this organism, including (i) the MR/P (4), PMF (31), ATF (32), and UCA (58) fimbriae, implicated in adhesion to epithelial cells of the urinary tract; (ii) flagella generated during swarming differentiation (3, 5); (iii) the poreforming HpmA hemolysin (35,53,54); (iv) an immunoglobulin A (IgA) protease (48); (v) an intracellular deaminase whose keto-acid products chelate extracellular iron (12, 33); and (vi) urea-inducible urease which hydrolyzes urea to ammonia and carbon dioxide (34). The ammonia generated by urease is responsible for the increase in urine pH and subsequent formation of struvite and apatite stones which are a hallmark of P. mirabilis infection. Urease has been shown to be a critical virulence factor in vivo in the CBA mouse model of ascending urinary tract infection (19, 23).The P. mirabilis urease gene cluster has been cloned and sequenced and comprise...

Nickel enzyme maturation in Helicobacter hepaticus: roles of accessory proteins in hydrogenase and urease activities

“…Our study also showed that H. hepaticus ureE and ureG mutants are totally devoid of urease activity. These results were expected, because UreE and UreG have been shown to be required for urease activity in several micro-organisms, including H. pylori (Mehta et al, 2003a;Voland et al, 2003), Proteus mirabilis (Island & Mobley, 1995) and K. aerogenes (Mulrooney & Hausinger, 1990). As shown previously for H. pylori , nickel supplementation of the growth medium did not restore urease activity in either H. hepaticus mutant.…”

Detection of Urer and Urec Among Proteus Mirabilis

“…Several studies referred to using of 16SrRNA for identification of P. mirabilis isolated from ocular infections [14], cerebrospinal fluid [15,16] who used specific primers for 16SrRNA gene to identify P. mirabilis and Proteus vulgaris that isolated from UTIs. 16SrRNA was described as a high discriminatory power for identification of bacteria and to differentiate between closely related genera because it exists in almost all bacteria, often existing as a gene cluster or operon, and also the function of this gene has not changed over time [17].…”

Differential regulation of the Proteus mirabilis urease gene cluster by UreR and H-NS

“…The P. mirabilis urease gene cluster, ureDABCEFG, is induced in the presence of urea and UreR, the transcriptional regulator of the gene cluster (D'Orazio & Collins, 1993D'Orazio et al, 1996;Island & Mobley, 1995;Nicholson et al, 1993). P. mirabilis UreR, a dimer of 33?4 kDa polypeptides is an AraC-like transcriptional regulator that activates transcription from p ureD and p ureR in a urea-inducible manner (D'Orazio et al, 1996;Island & Mobley, 1995).…”

“…P. mirabilis UreR, a dimer of 33?4 kDa polypeptides is an AraC-like transcriptional regulator that activates transcription from p ureD and p ureR in a urea-inducible manner (D'Orazio et al, 1996;Island & Mobley, 1995). Thomas & Collins (1999) have shown, using electrophoretic mobility shift assay, that there are two binding sites for UreR in the ureR-ureD intergenic region, resulting in a major band shift at low concentrations of UreR (occupation of a single binding site) and an additional minor band shift at higher concentrations of UreR (occupation of two sites).…”