Ti microscope

For the individual cell orientation experiments, cells were grown to mid-log (OD₆₀₀ = 0.6–0.8) and a 50 μl droplet was placed on a 60 × 22 mm glass cover slip. After 5 min of attachment, treatment was added to the cells and incubated for 5 min. Cells were imaged using a Nikon Ti microscope using a Plan Apo λ 40X PH2, Hamamatsu Orca Flash 4, and Nikon NIS Elements imaging software.

TagO depletions in Figure 2A were conducted using strain BEG300 in liquid culture. Cells were prepared as overnights, as described above, then grown at the specified xylose concentration at 37°C in LB with 20 mM magnesium chloride for 4 hr. The cells were then imaged as described above in the ‘Imaging – MreB particle tracking’ section.
Pbp2A depletions shown in Figure 2 were conducted in liquid culture using strain BRB785 and BRB786 with an IPTG‐inducible Pbp2A fusion at the native locus with the redundant transpeptidase PbpH deleted. This strain was grown overnight in the presence of 2 mM IPTG, and then inoculated into CH media containing 2 mM IPTG, 0.015% xylose, and 20 mM magnesium chloride to stabilize the cells against lysis. At an OD₆₀₀ of 0.6, cells were spun down in a tabletop centrifuge and washed three times in CH media lacking IPTG. Cells were placed under agar pads containing 20 mM magnesium chloride, and spinning disk confocal images were taken every 5 s on a Nikon Ti microscope with a 100 × 1.49 TIRF objective and a Hamamatsu ImagEM C9100-13 EM-CCD camera (effective pixel size of 160 nm).