Use of a Fluorescence-Based Assay To Measure Escherichia coli Membrane Potential Changes in High Throughput

Area of Science:

• Microbiology
• Biophysics
• Cell Biology

Background:

• Classical electrophysiology is unsuitable for bacterial membrane potential measurement due to cell size and cell wall.
• Fluorescent probes are commonly used but show low signal-to-noise in Gram-negative bacteria like E. coli due to outer membrane dye exclusion.

Purpose of the Study:

• To detail an optimized workflow for measuring E. coli membrane potential using the fluorescent probe DiOC2(3).
• To achieve high throughput and significant signal-to-noise ratios for reliable detection of membrane potential changes.
• To demonstrate the utility of this method for screening compounds that affect E. coli membrane energetics.

Main Methods:

• Utilized the membrane potential probe 3,3'-diethyloxacarbocyanine iodide [DiOC2(3)].
• Optimized assay conditions for high-throughput analysis using a fluorescence microplate reader.
• Performed valinomycin calibration to assess accuracy and range of membrane potential reporting.

Main Results:

• Established an optimized workflow for measuring E. coli membrane potential with high signal-to-noise ratios.
• Demonstrated robust reporting of membrane potentials over a ~144-mV range with ~12 mV accuracy via valinomycin calibration.
• Identified amlodipine as a compound altering E. coli membrane potential and decreasing kanamycin's Minimum Inhibitory Concentration (MIC).

Conclusions:

• The optimized DiOC2(3) probe workflow enables rapid and reliable high-throughput measurement of E. coli membrane potential changes.
• This method expands the screening capabilities for compounds affecting bacterial membrane energetics.
• The findings highlight the potential of targeting bacterial membrane potential for antimicrobial strategies.