Figure 4. Distinct lytic phenotypes for cells defective for PBP1a and PBP1b upon pH shift.

(A–B) Representative phase contrast frames of time lapse imaging of ∆mrcB (PBP1b; EAM546) and ∆mrcA (PBP1a; EAM543) mutants upon acidic (A) or alkaline (B) pH shift, respectively, as compared to the parental strain (MG1655). White arrows indicate membrane bulges. Scale bar denotes 5 μm. Full videos can be viewed in Figure 4—videos 1 and 2. (C) Representative scanning electron microscopy micrographs for ∆mrcB (PBP1b; EAM546) mutant shifted to either pH 6.9 or pH 4.5 for two hours prior to fixation. Scale bar represents 1 μm. (D) Quantification of lysis phenotype between mutants. Lytic terminal phenotype was categorized into three groups: lysis via septal bulge, non-septal bulge, or no bulge. Determination of lytic phenotype was based on the frames preceding propidium iodide incorporation (time step = 3 min). Micrographs (top to bottom) depict representative images of no bulge, septal bulge, and non-septal bulge, respectively with arrows (scale bar = 2 μm). At least 50 cells across at least two independent biological replicates were assessed (ΔmrcA, n = 128; ΔmrcB, n = 278 cells). Bars are subdivided based on percent lytic phenotype in each mutant.

Figure 4—video 1. ∆mrcB cells undergo septal lysis upon exposure to acidic media.

Download video file ^{(2.4MB, mp4)}

DOI: 10.7554/eLife.40754.015

Representative video of ∆mrcB cell growth and lysis. Cells were cultured at pH 6.9 to early exponential phase (OD600 ~0.05–0.1) then spotted on agarose pads buffered to pH 4.5. Cells were allowed to dry for 10 min at room temperature prior to imaging at 37°C. Still images corresponding to this video are shown in Figure 4A.

Figure 4—video 2. Subpopulation of ∆mrcA cells lyse upon exposure to alkaline media.

Download video file ^{(599.8KB, mp4)}

DOI: 10.7554/eLife.40754.016

Representative video of ∆mrcA cell growth and lysis. Cells were cultured at pH 6.9 to early exponential phase (OD600 ~0.05–0.1) then spotted on agarose pads buffered to pH 8.0. Cells were allowed to dry for 10 min at room temperature prior to imaging at 37°C. Still images corresponding to this video are shown in Figure 4B.