Bacterial Membrane Potential Assay

Differences in membrane potential between the strains were estimated using a flow cytometry assay based on the BacLight Bacterial Membrane Potential Kit according to the manufacturer (Life Technologies^®, cat. no. B34950). Briefly, bacterial strains were grown overnight shaking at 37°C in TSB. Fifty μL of cells from each overnight culture was inoculated into 10 mL of TSB in 100 mL Erlenmeyer flasks and grown with shaking at 37°C to an OD₆₀₀ of 0.2. Then 15 μL of culture was transferred to 1 mL of PBS and 10 μL of fluorescent membrane potential indicator dye, DiOC₂(3) was added. Cells were stained for 30 min at room temperature followed by analysis on a BD Biosciences Accuri C6 flow cytometer (Becton, Dickinson and Company), with emission filters suitable for detecting red and green fluorescence. Fifty-thousand events were recorded at a forward scatter threshold of 15 000 and medium flow rate. Gating of stained cell population and analysis of flow cytometry data were performed in CFlow^® (Becton, Dickinson and Company). As an indicator of membrane potential, the ratio of red to green fluorescence intensity was calculated. The assay was verified using the protonophore CCCP and two strains derived from the Nebraska Transposon Mutant Library^{25 (link)} that display reduced membrane potential (ΔmenD, NE1345 and ΔhemB, NE1845^{32 (link)}).