A general workflow for characterization of nernstian dyes and their effects on bacterial physiology

The electrical membrane potential (Vm) is one of the components of the electrochemical potential of protons across the biological membrane (proton motive force), which powers many vital cellular processes. As Vm also plays a role in signal transduction, measuring it is of great interest. Over the years a variety of techniques has been developed for the purpose. In bacteria, given their small size, Nernstian membrane voltage probes are arguably the favourite strategy, and their cytoplasmic accumulation depends on Vm according to the Nernst equation. However, a careful calibration of Nernstian probes that takes into account the trade-offs between the ease with which the signal from the dye is observed, and the dyes’ interactions with cellular physiology, is rarely performed. Here we use a mathematical model to understand such trade-offs, and apply the results to asses the applicability of the Thioflavin T dye as Vm sensor in Escherichia coli. We identify the conditions in which the dye turns from a Vm probe into an actuator, and, based on the model and experimental results, propose a general work-flow for the characterization of Nernstian dye candidates.