FIG. 4.
Growth of mutants lacking multiple LytM factors. (A) TB28 (WT) (left) and TB156 (ΔenvC ΔnlpD) (right) cells were plated on LB agar and incubated overnight at 37°C. Notice the significant difference in the size and appearance of the TB156 colonies relative to those of TB28. (B) Overnight cultures of TB28(attλTD25) [WT(Para::envC)] and TD22(attλTD25) [Δlyt4(Para::envC)] were grown in M9 arabinose at 37°C. The cells were harvested by centrifugation, washed three times in LB, and resuspended to their original culture volume in LB. The suspensions were adjusted to the same OD600 and serially diluted, and 5 μl of each dilution was spotted onto LB agar with or without arabinose supplementation. Plates were incubated overnight at 37°C. The photos in panels A and B were taken with a Nikon D40 camera outfitted with a 60-mm f/2.8D AF Micro-Nikkor lens and mounted on a copy stand. (C and D) FM1-43FX-stained TD22(attλTD25) cells grown in LB at 37°C with or without arabinose, respectively. An overnight culture grown in LB supplemented with 0.2% arabinose at 37°C was diluted 1:200 into LB with or without 0.2% arabinose. Cultures were grown to an OD600 of 0.6 and processed as described in the legend for Fig. 2. Bar, 4 μm.