Cflow

A BD Accuri C6 flow cytometer (Becton Dickinson Biosciences, Oxford, United Kingdom) was used for FC analysis. For analysis of membrane permeability, samples were stained directly with 4 μg/mL Propidium iodide (PI) (Sigma, United Kingdom) and incubated at room temperature in the dark for 10 min. Untreated bacteria and bacteria treated with 3 M H₂O₂ for 30 min, served as “healthy” and “dead or membrane compromised” controls, respectively. For all assays, samples were excited using a 488 nm solid-state laser. PI fluorescence was detected using 670 LP filters. 25,000 data points were collected at a maximum rate of 2,500 events/sec and the data were analyzed using CFlow (BD) software.

Membrane potential was assessed using a flow cytometry assay based on the BacLight bacterial membrane potential kit (Life Technologies). Cells from overnight cultures were inoculated in 10 ml TSB in 100-ml Erlenmeyer flasks and grown to an optical density at 600 nm (OD₆₀₀) of 0.2. Fifteen microliters of culture was transferred to 1 ml filtered PBS. To each cell solution, 10 μl of the fluorescent membrane potential indicator dye DiOC₂ (3 (link)) was added and cells were stained for 5 min at room temperature. Data were recorded on a BD Biosciences Accuri C₆ flow cytometer (Becton, Dickinson and Company), with emission filters suitable for detecting red and green fluorescence. Settings on the flow cytometer were as follows: 50,000 recorded events at a forward scatter (FSC) threshold of 15,000 and medium flow rate. Gating of the stained cell population and analysis of flow cytometry data were performed in CFlow (BD Accuri). As an indicator of membrane potential, the ratio of red to green fluorescence intensity was calculated. The assay was verified with the NTML mutant containing a transposon insertion in menD (NE1345), which displays depolarization of the membrane (45 (link)).

At each time point, samples were diluted in PBS, stained with PI (Sigma) and Bis‐(1,3‐Dibutylbarbituric Acid) Trimethine Oxonol (DiBAC₄(3) or BOX; Life Technologies), and analyzed with a BD Accuri C6 flow cytometer (BD, Oxford, UK). The stock solution of PI was prepared by dissolving PI in dH₂O at concentration of 300.12 μM. The stock solution of BOX was prepared by first dissolving the BOX in dimethyl sulfoxide (19.36 mM) followed by dilution in PBS in the ratio of 1:1,000 (19.36 μM). BOX was added to the samples at final concentration of 191.64 nM. EDTA was also added at a final concentration of 400 μM in order to facilitate staining with BOX. In total, 20,000 events were recorded per sample. Fluorescence was detected using 533/30 bandpass and 670 longpass filters corresponding to GFP/BOX and PI fluorescence, respectively. Data were analyzed using CFlow (BD). In addition, samples were serially decimally diluted in maximum recovery diluent (MRD; 8.5 g/L NaCl, 1 g/L peptone) and plated on nutrient agar plates (Oxoid), which were incubated at 37°C for 48 hr in order to determine the aerobic plate count. Throughout, t test analysis using Microsoft Excel was used, with a significant p value of 0.05. Error bars shown on figures are the ±SD of the mean value obtained at each data point.