A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes.

Transmembrane potential is one of the main bioenergetic parameters of bacterial cells, and is directly involved in energizing key cellular processes such as transport, ATP synthesis and motility. The most common approach to measure membrane potential levels is through use of voltage-sensitive fluorescent dyes. Such dyes either accumulate or are excluded from the cell in a voltage-dependent manner, which can be followed by means of fluorescence microscopy, flow cytometry, or fluorometry. Since the cell's ability to maintain transmembrane potential relies upon low and selective membrane ion conductivity, voltage-sensitive dyes are also highly sensitive reporters for the activity of membrane-targeting antibacterials. However, the presence of an additional membrane layer in Gram-negative (diderm) bacteria complicates their use significantly. In this paper, we provide guidance on how membrane potential and its changes can be monitored reliably in Gram-negatives using the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide [DiSC