Sensitive bacterial Vm sensors revealed the excitability of bacterial Vm and its role in antibiotic tolerance.

As an important free energy source, the membrane voltage (Vm) regulates many essential physiological processes in bacteria. However, in comparison with eukaryotic cells, knowledge of bacterial electrophysiology is very limited. Here, we developed a set of novel genetically encoded bacterial Vm sensors which allow single-cell recording of bacterial Vm dynamics in live cells with high temporal resolution. Using these new sensors, we reveal the electrically "excitable" and "resting" states of bacterial cells dependent on their metabolic status. In the electrically excitable state, frequent hyperpolarization spikes in bacterial Vm are observed, which are regulated by Na+/K+ ratio of the medium and facilitate increased antibiotic tolerance. In the electrically resting state, bacterial Vm displays significant cell-to-cell heterogeneity and is linked to the cell fate after antibiotic treatment. Our findings demonstrate the potential of our newly developed voltage sensors to reveal the underpinning connections between bacterial Vm and antibiotic tolerance.