The bacterial cytoplasmic membrane regulates ion concentrations inside the cell, in response to the cell’s requirements. This generates an electric potential difference, known as the membrane potential (MP). This MP powers the essential bacterial functions of energy generation, nutrient uptake and transport, metabolism, osmoregulation, etc. Recent studies have shown that bacteria communicate with one another via rudimentary electrical signaling networks, enabling collective decision-making and adaptation to environmental challenges. The MP of a bacterium is therefore not static but instead undergoes modulation in response to its own physiological activities and the surrounding environment. Quantifying MP changes could thus provide valuable insights into bacterial functions and interactions. However, measuring MP is challenging. Traditional electrode-based methods are unsuitable for bacteria, and Nernstian indicators lack the precision needed for accurate MP quantification. In this work, we introduced a novel fluorescence lifetime–based method using a voltage-sensitive dye, VoltageFluor (VF2.1.Cl), and phasor-FLIM analysis to measure MP in Bacillus subtilis cells, with subcellular resolution. The phasor-FLIM method makesit easy to quantify and visualize MP changes in a high-throughput manner, enabling the study of bacterial behavior and communication.
To calibrate the response of VF2.1.Cl, we controlled the potassium ion (K+) concentration outside the bacteria by using ionophores, which act as molecular shuttles across the membrane and bypass all bacterial native K+ concentration regulation mechanisms. In this case, the MP can be computed from the Nernst equation. Correlating observed VF2.1.Cl lifetimes and computed MPs at different external K+ concentrations, we obtained a calibration curve relating VF2.1.Cl lifetime and MP.
The cover image is an artistic representation of bacterial cells labeled with VF2.1.Cl in the presence of ionophores and K+ in solution. VF2.1.Cl’s fluorescence lifetime increases and the dye becomes brighter as the membrane depolarizes, which occurs due to efflux of K+ from the cytoplasm. Those lifetime changes are color-coded for easy visualization.
Beyond measuring MP for unperturbed and increasingly depolarized B. subtilis, we also observed significant MP heterogeneity within bacterial populations, reflecting diverse physiological and metabolic states. This variability may explain why some bacteria are more resilient to antibiotics or environmental stresses than others.
Our method opens up exciting perspectives beyond simply understanding bacterial behavior. If bacteria indeed communicate through electrical signals, being able to monitor bacterial electrical communication may lead to new ways to combat infections by observing the way external interventions disrupt these signals, prevent bacterial coordination, and make them more susceptible to treatments. Conversely, manipulating bacterial electrical signaling in a controlled manner could lead to new ways to program bacteria to perform specific tasks, such as small molecule production or degradation, more efficiently.
— Debjit Roy, Xavier Michalet, Evan W. Miller, Kiran Bharadwaj, and Shimon Weiss