<p><b>(A)</b><i>Salmonella</i> cultures of I<sub>A488</sub>/I<sub>A647-</sub>incorporated WT, <i>ompR</i> null mutant, and <i>ompR</i> null complemented with pWSK29-<i>ompR</i> (encoding <i>ompR</i>) were incubated at either acidic pH<sub>e</sub> (5.6) or neutral pH<sub>e</sub> (7.2) at indicated time points. Representative epifluorescence of the D/A ratio images are shown for WT <i>Salmonella</i>, the <i>ΔompR</i> mutant, and the <i>ΔompR</i> mutant with <i>ompR</i> provided in <i>trans</i>. Scale bar, 3 μm. <b>(B)</b> A plot of the intracellular pH of <i>Salmonella</i> WT at pH<sub>e</sub> 5.6 and pH<sub>e</sub> 7.2, an <i>ompR</i> null mutant at pH<sub>e</sub> 5.6 and pH<sub>e</sub> 7.2, and the <i>ompR</i> null mutant complemented with <i>ompR</i> supplied in <i>trans</i> at pH<sub>e</sub> 5.6 and pH<sub>e</sub> 7.2 over time. The D/A ratios of 50 cells were analyzed at each time point and the pH values were determined from the intracellular standard curve. Error bars represent mean ± standard error of the mean (SEM) (<i>n</i> = 3). (<b>C</b>) To demonstrate the reversibility of the I-switch and mimic vacuolar pH conditions in vitro, WT <i>Salmonella</i> cultures were incubated at pH<sub>e</sub> (4.5) for 1 h, and samples were collected at 30 and 60 min for imaging. The cultures were then washed and incubated for an additional 1 h at pH<sub>e</sub> 7.2. The D/A ratios of 50 cells were analyzed at the indicated time points. Representative epifluorescence of the D/A ratio images are shown. Scale bar, 3 μm. The average pH<sub>i</sub> from 50 cells is listed under the image.</p
