Permeabilization of the bacterial membrane by RumC1 was measured using the cell-impermeable DNA/RNA probe PI as previously explained (53, 54). A bacterial culture of Cp was grown until it reached 109 bacteria/ml and then centrifuged for 5 min at 300s. Bacteria pellet was then resuspended in sterile PBS in the original volume. PI (Sigma-Aldrich) was then added to the suspension at a final concentration of 60 μM. One hundred microliters of this suspension was then transferred into 96-well black plate (Greiner Bio-One) and treated with RumC1 or nisin at 5× their MIC values. Water and CTAB diluted at 300 μM final concentration were used as negative and positive controls, respectively. After 15- and 120-min incubations at 37°C under anaerobic conditions, fluorescence was measured (excitation at 530 nm and emission at 590 nm) using a microplate reader (Synergy Mx, BioTek). Results were expressed as the percentage of total permeabilization obtained by treatment with CTAB. All experiments were done in triplicate. For the permeabilization assay with SYTOX Green (Thermo Fisher Scientific), an overnight culture of Cp was diluted at 1:100 in BHI-YH and grown at 37°C under anaerobic conditions until OD600 = 0.2. Cells were then treated with RumC1 or nisin at 5× their MIC values for 15 min before being stained with SYTOX Green at 0.5 μM. Then, cells were rinsed with Hanks’ balanced salt solution +/+ (Gibco) and resuspended in VECTASHIELD (Vector Laboratories, CliniSciences H-1000). Observations were lastly performed with a Leitz DMRB microscope (Leïca), equipped with a Leïca DFC 450C camera.

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