Use of a Fluorescence-Based Assay To Measure Escherichia coli Membrane Potential Changes in High Throughput.
Antimicrob Agents Chemother
; 64(9)2020 08 20.
Article
en En
| MEDLINE
| ID: mdl-32631824
ABSTRACT
Bacterial membrane potential is difficult to measure using classical electrophysiology techniques due to the small cell size and the presence of the peptidoglycan cell wall. Instead, chemical probes are often used to study membrane potential changes under conditions of interest. Many of these probes are fluorescent molecules that accumulate in a charge-dependent manner, and the resulting fluorescence change can be analyzed via flow cytometry or using a fluorescence microplate reader. Although this technique works well in many Gram-positive bacteria, it generates fairly low signal-to-noise ratios in Gram-negative bacteria due to dye exclusion by the outer membrane. We detail an optimized workflow that uses the membrane potential probe, 3,3'-diethyloxacarbocyanine iodide [DiOC2(3)], to measure Escherichia coli membrane potential changes in high throughput and describe the assay conditions that generate significant signal-to-noise ratios to detect membrane potential changes using a fluorescence microplate reader. A valinomycin calibration curve demonstrates this approach can robustly report membrane potentials over at least an â¼144-mV range with an accuracy of â¼12 mV. As a proof of concept, we used this approach to characterize the effects of some commercially available small molecules known to elicit membrane potential changes in other systems, increasing the repertoire of compounds known to perturb E. coli membrane energetics. One compound, the eukaryotic Ca2+ channel blocker amlodipine, was found to alter E. coli membrane potential and decrease the MIC of kanamycin, further supporting the value of this screening approach. This detailed methodology permits studying E. coli membrane potential changes quickly and reliably at the population level.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Bioensayo
/
Escherichia coli
/
Potenciales de la Membrana
Idioma:
En
Revista:
Antimicrob Agents Chemother
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos