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

Area of Science:

• Microbiology
• Cellular Bioenergetics
• Bacterial Physiology

Background:

• Transmembrane potential is a critical bioenergetic parameter in bacteria, influencing essential processes like transport, ATP synthesis, and motility.
• Voltage-sensitive fluorescent dyes are commonly used to measure membrane potential, but their application in Gram-negative bacteria is complicated by the presence of an outer membrane.

Purpose of the Study:

• To provide reliable methods for monitoring transmembrane potential in Gram-negative bacteria using the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide [DiSC3(5)].
• To address and discuss confounding factors that affect membrane potential measurements in Gram-negative organisms.

Main Methods:

• Utilizing the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide [DiSC3(5)] for fluorescence-based measurements.
• Applying fluorescence microscopy, flow cytometry, or fluorometry to detect dye accumulation or exclusion.
• Investigating the impact of the outer membrane and buffer conditions on measurement accuracy.

Main Results:

• Established protocols for reliable monitoring of transmembrane potential in Gram-negative bacteria.
• Identified and discussed specific experimental pitfalls related to the outer membrane and buffer composition.
• Demonstrated the utility of DiSC3(5) as a reporter for membrane potential in diderm bacteria.

Conclusions:

• The developed methods offer a basis for the accurate use of voltage-sensitive dyes in Gram-negative bacteria.
• Increased awareness of potential experimental challenges is crucial for obtaining valid results.
• This work facilitates the study of membrane potential and the activity of membrane-targeting antibacterials in Gram-negative organisms.