The bactericidal activity of several antibiotics partially relies on production of reactive oxygen species (ROS), which requires the Fenton reaction. Here, the authors show that membrane depolarizatio...

Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic ...

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 [DiSC3(5)]. We also discuss the confounding effects caused by the presence of the outer membrane, or by measurements performed in buffers rather than growth medium. We hope that the discussed methods and protocols provide an easily accessible basis for the use of voltage-sensitive dyes in Gram-negative organisms, and raise awareness of potential experimental pitfalls associated with their use.

PhD Project - BBSRC NLD Doctoral Training Partnership: Interplay between bacterial multidrug efflux and outer membrane permeability creates an unexpected vulnerability towards antibiotics at Newcastle...

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