FIGURE 4.

Response of the wild type and the rapidly lysing strains to cephalexin. (A) Shown are the micrographs of the wild type and the rapidly lysing strains after seeding on agar pads containing 50 μg/ml of cephalexin. The wild type elongates and starts to lyse only after 30 min whereas all the mutants start lysing immediately. After 1 h, a small fraction of ΔtolQ, ΔtolR, and Δslt cells stay intact but their morphology is grossly altered. These cells display stable large bulges (shown by red arrowheads). The yellow and pink square insets are enlarged in (B). (B) The bulge morphology of Δslt is different from ΔtolQ and ΔtolR. Bulges in Δslt protrude on both sides whereas ΔtolQ and ΔtolR cells have protrusion only on one side. (C) Time-kill curves of cephalexin against all rapidly lysing strains and the wild type is shown. Cephalexin 50 μg/ml was added at time 0 h. The wild type is shown by a black curve in all plots. Due to cell lysis, the number of surviving cells declines very quickly for all mutants. mrcB and lpoB deletion strains exhibit the fastest decline in viable cells. All data points correspond to 3 replicates. Phase contrast and fluorescence images of bulging wild-type (D), Δslt and ΔtolR (E) cells. Fluorescent images show cell walls stained with WGA-Tetramethylrhodamine. The wild type was imaged 25 min after adding cephalexin, while Δslt and ΔtolR cells were imaged after 10 min. In the image of the wild type we can see debris from lysed cells and one intact cell with a bulge. In the wild type and ΔtolR cells, the bulge protrudes on one side. Their cell wall is also fractured only on one side, whereas Δslt cell has protrusion and cell wall damage on both sides (albeit unequal in severity). Scale bar corresponds to 30 μm in (D) and 20 μm in (E).