Bacterial and medium pH effects of urea addition to H. pylori-AGS coculture with either wild-type or ureI mutant strains. (a and b) The effect of urea addition on medium and bacterial pH in coculture with H. pylori expressing UreI. H. pylori and AGS cells in coculture were perfused with medium containing BCECF-free acid at the indicated pH and then 5 mM urea was added. A calibrated pH microelectrode was placed in the chamber to monitor medium pH and confocal microscopy was used to monitor the regions of change of pH after the addition of 5 mM urea using wild-type or ureI-deletion mutants as detailed in Methods. The numbering in the confocal images corresponds in time to the images above the pH tracings. (a) At a medium pH of 5.5, there was a rapid rise of medium pH after the addition of 5 mM urea. Within 2 minutes, the pH had risen to 6.0 and reached a steady-state pH of about 7.0 within 5 minutes. In the image sequence above the pH curve from the same experiment, the fluorescence increased markedly first on or at the bacteria, evident in the first two or three images. The increase in fluorescence then spread into the medium as medium pH increased and is close to maximum at about 4 minutes (see Table 1). (b) In contrast, when urea is added at 5 mM at a medium pH of 7.4, there was little change of medium pH or the pH around the organism. After 20 minutes, the pH had only increased by 0.2 U. (c and d) The effect of 5 mM urea addition on medium and bacterial pH in coculture with H. pylori ureI–negative mutants. (c) When urea was added at pH 5.5 to the ureI-ve strain, there was a slow rise of medium pH, reaching about 6.5 after 5 minutes and then rising slowly to 7.2 at the end of the experiments. There was no obvious change of fluorescence with the addition of urea in the vicinity of the organisms, although they have considerable surface urease activity (11). (d) Urea addition in the presence of 0.01% C₁₂E₈ to the ureI-deletion mutant resulted in a rapid change in medium pH, faster than that seen with the wild-type organisms, reaching pH 7.1 within 5 minutes and continuing to increase to more than 8.0, in contrast to the untreated wild-type organisms. In the fluorescence images, there was a similar increase in the fluorescence of the medium reflecting the pH changes monitored with the pH electrode. (e) When 5 mM urea was added in the presence of 10 μM flurofamide at pH 5.5, no change in either bacterial or medium pH could be observed, showing the dependence of these changes on urease activity.