The role of internal urease in acid resistance of Helicobacter pylori David R. Scott, David Weeks, Charlie Hong, Stefan Postius, Klaus Melchers, George Sachs  Gastroenterology  Volume 114, Issue 1, Pages 58-70 (January 1998) DOI: 10.1016/S0016-5085(98)70633-X Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 A comparison of the pH optimum of (A) urease external to H. pylori and (B) urease in intact organism showing the 10-fold activation in intact organisms observed between pH 6.5 and 5.5. External urease was obtained by washing a bacterial suspension in deionized water, and intact bacteria were measured in buffer as described in Materials and Methods. Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 A comparison of the pH optimum of (A) urease external to H. pylori and (B) urease in intact organism showing the 10-fold activation in intact organisms observed between pH 6.5 and 5.5. External urease was obtained by washing a bacterial suspension in deionized water, and intact bacteria were measured in buffer as described in Materials and Methods. Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Effect of the addition of 5 mmol/L urea on the transmembrane potential of H. pylori at various fixed pHout of 4.0, 5.0, and 6.0 showing the rapid increase of transmembrane potential without a measurable change in pHout. TCS was added to set the membrane potential to zero. (B) Transmembrane potential of H. pylori found at different fixed pHout values on the addition of 5 mmol/L urea (▨) compared with that in the absence of urea (■). Calibration of the potential difference was performed using the null point method (n = 3; mean ± SEM). Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Effect of the addition of 5 mmol/L urea on the transmembrane potential of H. pylori at various fixed pHout of 4.0, 5.0, and 6.0 showing the rapid increase of transmembrane potential without a measurable change in pHout. TCS was added to set the membrane potential to zero. (B) Transmembrane potential of H. pylori found at different fixed pHout values on the addition of 5 mmol/L urea (▨) compared with that in the absence of urea (■). Calibration of the potential difference was performed using the null point method (n = 3; mean ± SEM). Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of 1 (––––) and 10 μmol/L (——) flurofamide on the urea effect on transmembrane potential of H. pylori at pHout of 4.0. These concentrations did not inhibit the potential in the absence of urea during the time of observation. Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Typical effect on the fluorescence of BCECF loaded into H. pylori with the addition of urea at a fixed medium pH of 4.0 and 5.0 showing the biphasic increase in pH in the environment of the BCECF pH probe. (B) The average pH reached with urea addition on the assumption that BCECF is acting as a probe of periplasmic pH (n = 3; mean ± SEM). Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Typical effect on the fluorescence of BCECF loaded into H. pylori with the addition of urea at a fixed medium pH of 4.0 and 5.0 showing the biphasic increase in pH in the environment of the BCECF pH probe. (B) The average pH reached with urea addition on the assumption that BCECF is acting as a probe of periplasmic pH (n = 3; mean ± SEM). Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 A comparison of the protein synthesis performed by H. pylori at different fixed medium pH using 35S-methionine to label the protein. The image represents the phosphor image obtained from a 10% acrylamide SDS–polyacrylamide gel electrophoresis gel. This image reflects the typical pattern observed in at least three experiments. Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of the addition of 20 mmol/L urea at different fixed pHout on protein synthesis by H. pylori for 30 minutes as measured by 35S-methionine incorporation followed by SDS–polyacrylamide gel electrophoresis and phosphor imaging. Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Comparison of protein labeling as a function of time using 35S-methionine to label protein in intact H. pylori and in the medium in which these were suspended showing that significant quantities of labeled protein only appear in the medium after about 16 hours of incubation. The protein in the medium shows the same pattern as in the whole bacteria. This image is typical of at least three experiments. Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 A model showing the control of periplasmic pH and transmembrane potential by the stimulation of urease activity of H. pylori at a pHout of <6.5. The inner and outer membrane are separated by the periplasmic space. Cytoplasmic urease is able to compensate for medium acidity by the hydrolysis of urea when external pH decreases to <6.5. This enables buffering of the periplasmic space to about pH 6.2, which enables a transmembrane potential of −105 mV over a relatively wide range of gastric pH in the environment of the microorganism. Gastroenterology 1998 114, 58-70DOI: (10.1016/S0016-5085(98)70633-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions