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

Across the tree of life, cells rely on electrochemical gradients across membranes to fuel essential processes. In bacteria, this gradient, the proton-motive force (PMF), has been difficult to measure because of the small size of the cell. Although PMF is known to respond dynamically to internal and external cues, its real-time behavior under environmental stress remains poorly understood. Here, we use the bacterial flagellar motor as a sensitive, intrinsic reporter to investigate how PMF responds to hyperosmotic shock with high temporal resolution. We show that hyperosmotic stress causes a rapid, dose-dependent reduction in motor speed in Escherichia coli, reflecting a loss of PMF confirmed independently using the Nernstian fluorescent dye tetramethylrhodamine methyl ester (TMRM). The response is independent of the choice of nonionic osmolyte, the presence of potassium, and the direction of motor rotation, indicating that it originates upstream of stator-rotor interaction and is not specific to a particular osmotic agent or motor configuration. During sustained hyperosmotic shock, motor speed partially recovers over several minutes, consistent with cellular adaptation and restoration of PMF. Together, these results establish that hyperosmotic shock rapidly depolarizes E. coli and demonstrate the utility of the flagellar motor as a noninvasive, real-time reporter of bacterial electrophysiology in vivo.