Extended Data Fig. 7: B. subtilis live cells and sacculi.

a, High-pass-filtered image (filter size: 0.2 μm, horizontal) of the B. subtilis cell cylinder in exponential phase attached to a Cell-Tak-coated substrate. The inset (I) shows the unfiltered height data. DS = 1,417 nm. b–d, Higher-resolution images of the mature cell wall (mesh) within a in locations marked with white dashed squares. DS = 31 nm (b), 27 nm (c) and 21 nm (d). The external surface of B. subtilis is covered by disordered mesh all along the cylinder with the exception of the newly synthesized poles (which have a concentric ring architecture). e, A sacculus from a B. subtilis cell, showing the internal surface of a pole. We can differentiate between poles and completely formed septa because of the folded material on top (blue arrow). No septa were observed with this feature. DS = 130 nm. f, Higher-resolution image from within e showing the fine structure of the internal pole, similar to the internal structure of S. aureus, suggesting that this architecture is characteristic of the spherical part of Gram-positive cells. DS = 87 nm. g, External part of the pole facing upwards, with the interface between the pole and the rest of the cylinder indicated by blue arrows. DS = 118 nm. We presume that this image corresponds to a mature pole and that the concentric rings have become mesh, suggesting that the structural reorganization in time from rings to mesh also occurs in B. subtilis poles. h, Example of a newly synthesized pole facing upwards with a sharp interface between the pole and the rest of the cylinder (blue arrows). This pole displays very tight concentric rings in contrast to g and similar to Fig. 3e, f. DS = 800 nm. i, The same image as in h but processed with Gwyddion and JPK software (flattened to 0th order with the mask drawn manually over the sacculus and path level to remove lines), allowing the concentric rings (white arrow) to be visualized more clearly. The results from images shown in e–i support a common process of synthesis and maturation of the cell wall on the spherical parts of Gram-positive bacteria in both spherical and rod-shaped species. Images shown in e–i were taken with JPK NanoWizard 3 in QI mode (see Methods). Images shown in e–h were processed with Gwyddion³⁶, the image shown in i was processed with JPK data processing software, and the colour palettes are different. j, Two fragments from B. subtilis sacculi. The fragment on the left corresponds to the internal structure facing upwards (white arrowhead), and the fragment on the right has the external structure of the cylinder, mesh, facing upwards (blue arrow). The assumed cylindrical axis is marked (red dashed arrow). DS = 118 nm. k, Higher-resolution image from within j of the internal cell wall. Some larger pores are visualized here and there is some predominant orientation indicated by the angle distribution of the fibres shown in the inset, in agreement with the cylindrical axis (red dashed arrow). The method for obtaining these distributions is explained in Extended Data Fig. 8. DS = 46 nm. l, Example of B. subtilis sacculus fragments showing the two different structures: the internal cell wall in the two fragments (white arrowheads) and the top part of the cylinder corresponding to the external cell wall (blue arrow). DS = 146 nm. The distinction between this image and that shown in j is that these fragments are part of the cylinder that once belonged to the same cell. The blue dashed box marks the area corresponding to Fig. 3i, and the assumed cylindrical axis is marked (red dashed arrow). m, Higher-resolution image from l of the internal cell wall. This strongly aligned structure (see inset (I)) is the most commonly found in these sacculi in contrast with k. As the fragments in l are not totally broken, we assume that this will be more similar to the native structure of the cell wall. DS = 19 nm. n–p, The different structures from B. subtilis sacculi that have been HF treated so the WTAs have been removed: the mesh seen on the external surface (DS = 30 nm) (n); strands inside (DS = 30 nm), the circumferential axis direction is given by the red dashed arrow (strand orientation is shown in the inset (I)) (o); and the partially formed septum (DS = 130 nm) (p). This shows that none of the previous structures was due to teichoic acid organization, which is consistent with our findings for S. aureus cells.