a Time-lapse fluorescence microscopy of exponentially growing B. subtilis CM03 cells treated with 0.125 µg/ml ADEP2. Overlaid fluorescence and phase contrast images show the localization of mCherry-FtsZ or GFP-PBP2b, as indicated, during ADEP treatment over time. The micrographs show that the divisome succeeds to finalize cell division (closed triangles) in the presence of ADEP in situations when GFP-PBP2b is substantially detected at the septum area. In contrast, early-stage FtsZ rings, prior to the visible arrival of GFP-PBP2b at the septum, disintegrate during ADEP treatment (open triangles). For clarity, a phase contrast image of bacterial cells after 99 min is included at the end of the series to prove failure or success of septum formation. Scale bar, 5 µm. Images are representative of at least three biological replicate cultures of B. subtilis CM03 with >300 septa/FtsZ rings monitored over time. A time-lapse video is provided by Supplementary Movie 4. b Quantification analysis assessing divisome progression and success of cell division in cells with or without clear foci of PBP2b. In the absence of a substantial signal of GFP-PBP2b at the divisome, early-stage FtsZ rings disintegrate and cells fail to divide upon ADEP treatment (100%, N = 113), however, after substantial localization of GFP-PBP2b, the divisome progresses and cells finalize septum formation and division (100%, N = 174). In the transition phase of initial GFP-PBP2b localization, cells show a heterogeneous behavior with 18.5% of the divisomes abrogating division and 81.5% further constricting and finalizing division (N = 27). Bottom panels show representative fluorescence microscopy images of the different stages corresponding to the graphs in the upper panel. Images are representative of at least three biological replicate cultures of B. subtilis CM03. Source data underlying the graphs is presented in Supplementary Fig. 6.