Urease activities were determined for each of the ten hypA Zn-site variants, the wild type H. pylori strain, the hypA::kan-sacB mutant, and the hypA restorant strain. For each strain, 8mL liquid cultures of H. pylori were inoculated to an optical density (600nm) of 0.05 from overnight liquid grown bacterial cells and then allowed to grow for approximately 22 hours. At that point, 1mL aliquots were removed from the culture and pelleted by centrifugation (~108 cells). The supernatants were removed and the bacterial pellets were stored at −20°C until ready for urease assays. The frozen cells were thawed and then re-suspended in 750µL of ice-cold HEPES buffer (pH 7.0), 1mM phenylmethanesulfonyl fluoride (PMSF) (MP Biomedicals, LLC), and 1× protease inhibitor cocktail (Sigma-Aldrich) and then lysed by sonication at 70% power for 6 pulses (2-second each) on ice. Lysate was centrifuged at 15,000-g for 10 minutes to remove insoluble fractions from soluble whole cell extracts. Soluble whole cell extracts were kept at 4°C for up to one month and insoluble fractions were stored at −20°C. Total protein concentration in soluble whole cell extract was assessed by Bradford Assay using the Coomassie Protein Assay Kit (Thermo Scientific).
Urease activities for each strain were determined using a modified phenol-hypochlorite method to assay the amount of ammonia released in the soluble whole cell extract of H. pylori lysate in the presence of urea16 (link). For each strain, 5µL of whole cell extract was added to 245µL of urease reaction buffer (50mM HEPES, 25mM Urea, pH 7.0), and incubated at 37°C for 20 minutes to allow for ammonia production. The reaction was quenched with the sequential addition of 375µL of phenol-hypochlorite buffer A (100mM phenol, 167.8µM sodium nitroprusside) and then the addition of 375µL of phenol-hypochlorite buffer B (125mM NaOH, 0.044% NaClO); samples were mixed with quick vortexing after the addition of each buffer. The assay mixture was incubated at 37°C for 30 minutes to allow for color development (the conversion of ammonia to indophenol) and the absorbance was evaluated at 625nm. Assays were performed alongside a standard curve created using known amounts of ammonium chloride (0.24 – 500nmol) in place of whole cell extract. The urease activity of ΔureB strain was set as background and subtracted from the activity of all other strains. Urease activity for the various mutants was normalized to the hypA-restorant (hypA-R; DSM1295) H. pylori as 100%. All experiments were performed in triplicate with two independently grown cultures.
Urease activities for each strain were determined using a modified phenol-hypochlorite method to assay the amount of ammonia released in the soluble whole cell extract of H. pylori lysate in the presence of urea16 (link). For each strain, 5µL of whole cell extract was added to 245µL of urease reaction buffer (50mM HEPES, 25mM Urea, pH 7.0), and incubated at 37°C for 20 minutes to allow for ammonia production. The reaction was quenched with the sequential addition of 375µL of phenol-hypochlorite buffer A (100mM phenol, 167.8µM sodium nitroprusside) and then the addition of 375µL of phenol-hypochlorite buffer B (125mM NaOH, 0.044% NaClO); samples were mixed with quick vortexing after the addition of each buffer. The assay mixture was incubated at 37°C for 30 minutes to allow for color development (the conversion of ammonia to indophenol) and the absorbance was evaluated at 625nm. Assays were performed alongside a standard curve created using known amounts of ammonium chloride (0.24 – 500nmol) in place of whole cell extract. The urease activity of ΔureB strain was set as background and subtracted from the activity of all other strains. Urease activity for the various mutants was normalized to the hypA-restorant (hypA-R; DSM1295) H. pylori as 100%. All experiments were performed in triplicate with two independently grown cultures.
