a Time-lapse fluorescence microscopy of exponentially growing B. subtilis 2020 cells treated with 0.25 µg/ml ADEP2. Overlaid fluorescence and phase contrast images show the localization of GFP-tagged FtsZ (in green) and the progression of FtsZ rings over time. The micrographs indicate that early FtsZ rings (open triangles) disintegrate during ADEP treatment while more progressed FtsZ rings (closed triangles) constrict and finish septum formation to yield two separated daughter cells. Numbers indicate previously finished septa. For clarity, numbers remain positioned to the corresponding cell pole of the daughter cell on the right. A phase contrast image of bacterial cells after 65 min is included at the end of the series to prove failure or success of septum formation. Overlay (0–35 min) or single channel (65 min) images are provided. Scale bar, 5 µm. Images are representative of at least three biological replicate cultures of B. subtilis 2020 with >600 FtsZ rings analyzed over time. A time-lapse video is provided by Supplementary Movie 1. b Super-resolution fluorescence microscopy of different stages of FtsZ ring formation in B. subtilis 2020. While all of the early-stage FtsZ rings disintegrate upon ADEP treatment (100%, N = 323), all late-stage FtsZ rings further constrict and finalize septum formation (100%, N = 140). Intermediate-stage FtsZ rings, which were in transition from early to late stages, show a heterogeneous behavior with 71% of FtsZ rings abrogating division and 29% further constricting and finalizing septum formation (N = 178). Of note, we deliberately analyzed immediate daughter cells comprising both early and further progressed FtsZ rings, of which all late-stage FtsZ rings finalized division while all early FtsZ rings did not, thus making it unlikely that the cells with late-stage FtsZ rings may by chance happened to have considerably higher FtsZ levels compared to cells with early FtsZ rings. This is further supported by the fact that ADEP leads to a rapid depletion of the FtsZ pool within 15–20 min, where potential minor differences of the FtsZ level may be negligible in relation to the doubling time of the bacteria of ~30–45 min on microscopy slides. Images are representative of at least three biological replicate cultures. Source data underlying the graphs is presented in Supplementary Fig. 5.