Antibiotics Mode of Action in Bacillus

Details on antibiotics, bacterial strains, and experimental procedures can be found in Text S2 in the supplemental material. A list of B. subtilis strains used in this study is displayed in Table S1. All strains were grown at 30°C under steady agitation in Luria-Bertani broth (LB). MICs were determined in a standard serial dilution assay. Growth experiments were carried out in 96-well format in a temperature-controlled BioTek Synergy MX plate reader under continuous shaking. All mode-of-action assays were performed with log-phase B. subtilis cells at an OD at 600 nm (OD₆₀₀) of 0.3 at 30°C under steady agitation. Unless otherwise noted, cells were treated with 1× MIC of the respective antibiotics for 10 min. Fluorescence microscopy and staining of cells with fluorescent dyes were carried out as described previously (44 (link), 60 (link), 98 (link)). Electron microscopy was performed using a recently described flat embedding technique (60 (link)). The membrane potential was measured with DiSC(3)5 as described by Te Winkel et al. (98 (link)). Propidium iodide influx and laurdan spectroscopic assays were essentially performed as described by Müller et al. (44 (link)). Electrophysiological and NMR measurements were performed in 3:1 POPG-POPE or pure POPE, respectively, as described in Text S2. Time-lapse microscopy and SIM were essentially performed as described by Saeloh et al. (60 (link)). Activity against stationary-phase cells was determined using overnight cultures of B. subtilis and S. aureus. Antibiotic concentrations were adjusted to the higher cell count, and cells were incubated with antibiotics for 9 h prior to CFU determination.