Extended Data Fig. 3. FtsW-RFP molecules in stationary state display confined diffusion.

a. Example of an FtsW-RFP molecule (arrowhead) remaining within a small region of the septum. As indicated by the kymograph, this molecule showed little observable directional movement for at least 80 seconds. b. Unwrapped and decomposed 1d-trajectory of the FtsW-RFP molecule along the long (gray) and short (orange) axis. c. 1d-MSD curves (short axis) of 204 ‘stationary’ molecules in live BW25113 WT cells growing in M9-glucose medium without (blue) or with 1μg/mL Aztreonam (dark green), and in cells grown without Aztreonam upon para-formaldehyde fixation (gray). The BW25113 curve (blue) is fit by Kusumi equation⁵¹ with D = 0.0007 ± 0.0002 μm²/s, L = 95 ± 6 nm. Note that Aztreonam treatment causes confinement of ‘stationary’ FtsW-RFP molecules to smaller areas, similar to what is observed in fixed cells. d. Percentage of FtsW-RFP molecules in stationary state in the presence (blue bars) and absence (orange bars) of drug to inhibit sPG synthesis (MTSES for FtsW^I302C and PBP1B^S247C, Aztreonam for FtsI, and Fosfomycin for precursor synthesis). e. Mean dwell time of FtsW-RFP molecules staying in stationary state in the presence (blue dots) and absence (orange dots) of drug to inhibit sPG synthesis. c and d. Data are presented as mean ± SEM where the SEM is estimated by bootstrapping (For details regarding statistics and box plot definitions see the “Statistics” subheading in “Methods”). e. Data are presented as mean ± SEM. For the sample size of each point see Supplementary Table 6.