UreR, the Transcriptional Activator of the Proteus mirabilis Urease Gene Cluster, Is Required for Urease Activity and Virulence in Experimental Urinary Tract Infections

P. mirabilis urease activity in wild-type HI4320, ureR::aphA mutant, and complemented strains. Urease activity (upper panel) was determined by using the phenol-hypochlorite reaction (31). Overnight cultures (5 ml) of the P. mirabilis wild-type strain HI4320, the ureR mutant (ureR::aphA), and the complemented strain (Comp.) were used to inoculate fresh medium (1:100) with or without 100 mM urea containing the appropriate antibiotics when necessary. Bacteria from mid-exponential-phase cultures (optical density at 600 nm, 0.4) (3 ml) were resuspended in 1 ml of 50 mM HEPES, pH 7.5, and lysed by passage through a French pressure cell at 20,000 lb/in². The protein concentration of crude extract was determined by the bicinchoninic acid assay (Pierce, Rockford, Ill.). Total protein (10 μg) was diluted into 1 ml of assay buffer (50 mM HEPES, pH 7.5, and 25 mM urea) and incubated for 20 min at 37°C. Ammonia production was measured by using the reaction of phenyl nitroprusside and alkali hypochlorite (31). Optical density was measured at 625 nm by using ammonium chloride as a standard. For Western blotting (lower panel), monoclonal anti-UreD serum was prepared from a murine hybridoma cell line as previously described (8). Crude extract prepared for urease assays was assessed for production of UreD. Protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred overnight onto Immobilon-P membrane (Millipore, Inc.). Anti-UreD serum (1:100) and anti-mouse IgG conjugated with horseradish peroxidase (1:2,000) were used as primary and secondary antibodies, respectively. Protein bands were visualized on X-ray film by using chemiluminescence reagents from Amersham Biosciences (Piscataway, N.J.).