a, Sacculi of S. aureus WT imaged in air, with different fragments containing single layers of PG (green dashed arrows) and double layers (black dotted arrow). DS = 75 nm. b, Sacculi from the S. aureus sagB mutant in an SH1000 background imaged in air. The fragments have two distinct regions with different thicknesses (pink arrows and orange arrowheads). These are distinct from the double-layer fragments (black dotted arrow). DS = 75 nm. c, Graph comparing the thickness of the cell wall in air and liquid between the sagB mutant and the WT. The box plot consists of boxes from the 25–75% percentile, the middle line represents the median, the black star represents the mean, and the error bars are the maximum and minimum values excluding any outliers (using the 1.5 interquartile range). The two different regions of the sagB cell wall are separately binned and labelled ‘sagB thin’ and ‘sagB thick’. Measurements were taken using the same approach as the values for the graph in Fig. 2k, Extended Data Fig. 3. The number of sacculi measured n = 25 for each bin. Mean ± s.d.: sagB thin air = 12 ± 3 nm, sagB thin liquid (liq) = 24 ± 7 nm, sagB thick air = 21 ± 1.5 nm, sagB thick liquid = 42 ± 5.7 nm, WT air and WT liquid corresponded to the same values as in Fig. 2k (left data, PG + WTA). All measurements in liquid were performed in buffer: 300 mM KCl + 10 mM Tris, pH 8. d, S. aureus sagB mutant sacculus in liquid with its external surface facing upwards. This sacculus is clearly divided into two sections: the region on the left is thinner than the region on the right. This irregularity in height was visualized in several sacculi from cells with this mutation. DS = 64 nm. e, Higher-resolution image from the thicker section in d corresponding to the finer mature cell wall architecture. This mesh is still in an early transition from rings, with some features reminiscent of rings that are visualized following the same orientation (blue arrows). These sorts of structures are uncommon on WT sacculi at the same stage in the cell cycle (exponential phase OD 600 = 0.5–0.7). However, in the sagB mutant, it was more common to find this early transition from rings to mesh. DS = 11 nm. f, Higher-resolution image from the thinner section in d showing the finer nascent cell wall architecture (that is, rings). The inset shows a lower-resolution image of the ring architecture in a different sacculus. The features from this image are not significantly different from the WT nascent PG architecture, such as in Fig. 2d. However, almost every sacculus containing nascent architecture in the sagB mutant had this part of the cell wall being thinner than the rest of the sacculus, something that is not seen in the WT. DS = 9 nm, inset DS = 29 nm. g, Sacculus fragment with the inner cell wall architecture facing upwards. DS = 14 nm. h, Higher-resolution image from g showing the fine inner structure of the sagB mutant, with no clear difference from the very tight and disordered structure seen in WT sacculi (for example, see Fig. 2f). DS = 4 nm. i, Sacculi from S. aureus pbp3 mutant cells in an SH1000 background. Here we show the fine external structure, with no apparent difference between this and the S. aureus WT mature external structure. It is important to highlight that PBP3 is not an essential PG synthesis enzyme, and the cells can survive without it and still maintain their spheroid shape. DS = 55 nm. j, The fine structure of an S. aureus pbp3 mutant sacculus with the internal PG surface exposed, which is similar to that seen in WT S. aureus. DS = 4 nm. k, Sacculus from S. aureus pbp4 mutant cells in an SH1000 background showing the nascent PG structure, with the concentric rings facing upwards. It is important to highlight that PBP4 is not an essential PG synthesis enzyme either, and the cells can survive without it and still maintain their spheroid shape. DS = 28 nm. l, Higher-resolution image taken within k showing the fine structure of the nascent PG surface: the concentric rings. There is no significant difference compared to the S. aureus WT nascent structure, except the fraction of sacculi with rings in a random area is higher in this sample than in the WT. DS = 8 nm. m, Sacculus showing a small area of the mature PG structure (green arrow), with its external surface facing upwards. The rest of the sacculus is covered by concentric rings that are already in the late phase of the transition to mesh (black arrow pointing to the centre of the concentric rings; the boundary with the mature structure is indicated by the red dashed line) and the newest generation fragment with rings (blue arrowhead; the boundary with the oldest rings is marked by the red dotted line). It is important to highlight that this is the first time that two perpendicular planes of rings have been visualized at exponential phase in our experience. OD 600 = 0.6. DS = 79 nm. n, Higher-resolution image from m showing the fine structure of the mature external cell wall of an S. aureus pbp4 mutant, with no apparent difference between this and the S. aureus WT mature external structure (see Fig. 2b, Extended Data Fig. 1). DS = 44 nm. o, Sacculus showing the fine internal structure of the S. aureus pbp4 mutant cell wall, with no apparent difference compared to the S. aureus WT internal PG architecture (see Fig. 2f). DS = 10 nm. These data show that removal of non-essential PBPs makes only relatively minor differences to the wall architecture.