Osmotic stress triggers fast and reversible PMF collapse in Escherichia coli.

Environmental stressors routinely impact bacteria and affect their physiology. Among the most important physiological parameters is the proton motive force (PMF), that powers vital cellular processes and molecular machines. Measuring how PMF responds to environmental stress in real time requires tools that capture rapid physiological changes with high temporal resolution. Here, we use the bacterial flagella motor as an in vivo voltmeter to probe PMF dynamics during hyperosmotic shock. Because motor rotation frequency scales linearly with PMF, this approach enables single-cell electrophysiology with high temporal resolution. We find that hyperosmotic stress causes a rapid and reversible loss of PMF in Escherichia coli, independent of the osmolyte used or the presence of potassium ions. We corroborate these findings by using the Nernstian dye, tetramethyl rhodamine methyl ester (TMRM), showing that hyperosmotic shock leads to membrane depolarization. Together, our results highlight the efficacy of the flagellar motor as powerful tool for probing bacterial electrophysiology and reveal that hyperosmotic stress directly disrupts cellular energetics in addition to its mechanical effects.