Extended Data Fig. 9: B. subtilis septa.

A comparison of B. subtilis cell wall fragments that were acquired in liquid with the same fragments acquired in air, allowing our current findings to be understood in the context of our previous study²¹. a, b, B. subtilis septa at different stages of formation, imaged in liquid. DS = 70 nm (a) and 158 nm (b). In both cases, a disordered mesh structure is apparent with pores penetrating deep into the cell wall. c, d, Corresponding images from the same septa as a and b in the same orientation in air. DS = 31 nm (c) and 60 nm (d). Some features that were observed in our previous work, such as the concentric rings around the circumference of the septa (green arrowheads) and the ridges around the outer diameter of the septa (blue arrow), were identified. We attribute these features to the superposition of the mesh structures on both sides of the septa, with the concentric rings formed in the middle between them, coupled with the influence of differential drying. The experiment of correlating S. aureus sacculi in ambient and liquid environments (Extended Data Fig. 3) to measure the difference in thickness, was repeated for B. subtilis sacculus fragments and also showed a significant difference. e, f, Examples of single-layer fragments, marked 1 and 2, imaged in water (e) and air (f). The thickness of each individual fragment was measured using the 1D statistical analysis from the Gwyddion software (see Extended Data Fig. 3e for details). g, Graph showing the thickness for 19 sacculi measured in both air and liquid. There is a significant increase in thickness in liquid after a two-tailed, paired t-test was performed (P = 1.2 × 10⁻⁹; see Methods). The box plot consists of boxes from the 25–75% percentile, the middle line represents the median, the black star indicates the mean, and the error bars are the maximum and minimum values excluding any outliers (using the 1.5 interquartile range). Mean ± s.d.: 9 ± 1 nm (air) and 34 ± 5 nm (liquid). These results are in agreement with the sacculus thickness of B. subtilis measured by transmission electron microscopy (figure 4 of ref. ⁵). h, Higher-resolution image from Fig. 3j. The internal surface of this partially formed septum is composed of randomly orientated mesh. Large pores are present that go partially through the cell wall (blue arrows). DS = 137 nm. i, In contrast to h, in this partially formed septum, most of the material has been synthesized already, and the pores are not as deep or as wide as in h (yellow arrows). DS = 160 nm. These two septa differ in the size of their aperture by 100 nm, meaning that they are probably representing consecutive stages of septal formation. The structural difference between them suggests that there is more than one type of synthesis happening simultaneously: one type that occurs at the leading edge of the aperture making it smaller, and the other type that is responsible for in-filling the pores that go partially through the cell wall as seen in h, finally resulting in the tighter mesh seen in i.