Extended Data Fig. 4: The spatiotemporal organization of cell wall biogenesis mirrors OMP biogenesis.

(A) Images of E. coli BW25113 cells after incubation with the PG stain HADA for 3 or 10 min. (B) Demographs of the normalised fluorescence intensity across the long axis of multiple cells after 3 or 10 min incubation with HADA. Cells are aligned so the more intense pole appears at the top. The charts highlight the cell cycle dependent patterns of PG biogenesis. (C) 3D-SIM images of single E. coli cells after incubation with HADA for 10 min. Left to right, cells representing different stages of the cell cycle. (D) The fluorescence intensity of FepA vs HADA (left) or BamA (right) following simultaneous co-labelling. The representative pixel by pixel cytofluorograms of single images show that OMP biogenesis correlates better with PG biogenesis than BAM localization. (E) Jitter plot showing the correlation between FepA biogenesis and either PG biogenesis or BamA distribution. Shown are Pearson coefficients of single images and the average value. Statistical significance was calculated using two-tailed Student’s unpaired t-test (P = 0.0001). (F) Co-labelling of BamA, PG and OMP biogenesis. HADA was added 3 min after FepA induction and the total induction time was 10 min. (G) Co-labelling of PG and OMP biogenesis incorporating a time delay. HADA was added 3 min after FepA induction (stained with ColB-GFP). The total induction time was 10 min. The white/red arrows indicate cells from groups 1 and 2, respectively (see Fig. 2b). (H) Demographs comparing the normalised fluorescence distribution of FepA and PG biogenesis in multiple cells. Cells were treated as in panel G. Scale bars, 1 μm.