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. Because Vm also plays a role in signal transduction, measuring it is of great interest. Over the years, a variety of techniques have been developed for the purpose. In bacteria, given their small size, Nernstian membrane voltage probes are arguably the favorite 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 tradeoffs 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 tradeoffs and apply the results to assess the applicability of the Thioflavin T dye as a 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 workflow for the characterization of Nernstian dye candidates.

膜电位（electrical membrane potential, Vm）是跨生物膜的质子电化学势（即质子动力势，proton motive force）的组成部分之一，而质子动力势可为诸多关键细胞生理过程提供能量。由于Vm还参与信号转导过程，对其进行测量具有重要的研究价值。多年来，学界已开发出多种用于膜电位测量的技术手段。针对体积微小的细菌，能斯特型膜电位探针（Nernstian membrane voltage probes）通常被认为是首选的测量策略，这类探针在细胞质中的积累量遵循能斯特方程（Nernst equation），并与Vm呈直接依赖关系。然而，鲜有研究对能斯特型探针开展严谨校准——此类校准需要兼顾荧光染料信号观测的便捷性，以及染料与细胞生理过程的相互作用之间的权衡关系。本研究通过构建数学模型解析上述权衡关系，并将该模型应用于评估硫黄素T（Thioflavin T）染料作为Vm传感器在大肠杆菌（Escherichia coli）中的适用性。本研究明确了该染料从Vm探针转变为生理调控执行因子的临界条件，并基于数学模型与实验结果，提出了一套用于表征能斯特型染料候选物的通用工作流程。