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

As an important free energy source, the membrane voltage (V_m) 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 V_m sensors which allow single-cell recording of bacterial V_m 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 V_m are observed, which are regulated by Na⁺/K⁺ ratio of the medium and facilitate increased antibiotic tolerance. In the electrically resting state, bacterial V_m 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 V_m and antibiotic tolerance.