Extended Data Fig. 2: S Protein is a membrane associated and forms a ring at midcell.
(a) Western immunoblot of whole-cell lysates of WT and mgfp-ess strains with specific anti-GFP antibody and anti-enolase antibody as a loading control. The expected mGFP-S Protein band is indicated by the black triangle. (b) Growth of WT and mgfp-ess strains in C + Y media at 37 °C. (c) Representative phase contrast microscopy images of WT and mgfp-ess strains. Scale bars, 2 µm. (d) Violin SuperPlots showing the distribution of the maximum cell width from three independent experiments, (n = 2000 cells for each strain). ns, not significant, p > 0.05 (e) Western immunoblot of whole-cell lysates of mgfp-ess, ∆ess and PcomX-mgfp-ess ∆ess cells, grown in the presence (3 µM) or absence (0 µM) of inducer (ComS), revealed with anti-GFP antibody and anti-enolase antibody as a loading control. The expected band for mGFP-S Protein is indicated with the black triangle. (f) Merge between phase contrast and GFP fluorescent signal of mgfp-ess, ∆ess and PcomX-mgfp-ess ∆ess cells, grown in the presence (3 µM) or absence (0 µM) of ComS inducer. Minicell-like compartments are highlighted by a white triangle. Scale bars, 2 µm. (g) Violin SuperPlots showing the distribution of the maximum cell width from three independent experiments, (n = 2000 cells for each strain). ***p < 0.001 and ns, not significant, p > 0.05. (h) Growth of mgfp-ess, ∆ess and PcomX-mgfp-ess ∆ess in C + Y medium at 37 °C. (i, j) Representative montage of images showing the dynamics of FtsZ-GFP (i) or mGFP-S Protein (j) in WT cells observed by conventional fluorescence microscopy with cells positioned vertically in agarose microholes for 60 s (9 s intervals). A summation of all the images and a kymograph are shown on the right and below the montage respectively. The kymographs (1 frame/s) were generated from a circular line around the circumference of the cell, highlighted in orange on the summation images. Scale bars, 0.5 µm. (k) Western immunoblot of whole-cell lysates of mgfp-ess fusion (Full length) and derivatives with specific anti-GFP antibody and anti-enolase antibody as a loading control.
