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. 2019 Apr 9;8:e40754. doi: 10.7554/eLife.40754

Figure 3. Cells defective for class A PBPs lyse upon exposure to non-permissive pH conditions.

(A–B) Single cell elongation rates for wild-type (MG1655), ∆mrcA (EAM543), and ∆mrcB (EAM546) cells during growth on agarose pads at pH 4.5 (A; n = 134, 81, and 155 cells) and pH 8.0 (B; n = 386, 257, and 246 cells). Rates were determined in the MATLAB-based program SuperSegger as described in the methods. Error bars represent SD. (C–D) Micrographs depicting representative images of propidium iodide incorporation in ∆mrcA (D, left) and ∆mrcB (C, left) mutants at t = 0 or 60 min post indicated pH shift. Scale bar represents 5 μm. Cell viability curves for wild-type, ∆mrcA (PBP1a), and ∆mrcB (PBP1b) strains after acidic (D, right) or alkaline pH (C, right) shift as indicated. Cell death was determined by uniform cytoplasmic staining with propidium iodide. Markers indicate mean percent viability ± SD of three biological replicates. Greater than 100 cells were analyzed for each strain at each time point per replicate.

Figure 3.

Figure 3—figure supplement 1. Cytoplasmic GFP loss correlates with propidium iodide staining.

Figure 3—figure supplement 1.

Representative micrographs for ∆mrcB (EAM546) (A) and time course of fluorescence signal (B–C) for ∆mrcA (EAM543) and ∆mrcB mutants expressing gfp from an IPTG-inducible promoter (pBH234) when exposed to indicated non-permissive pH conditions. EAM543 and EAM546 were induced with 10 and 20 μM IPTG, respectively. Markers represent percent of cells straining positive for GFP (triangles) or PI (squares) ± SD for three independent biological replicates. (D) Propidium iodide staining for ∆mrcA (EAM543) cells grown in liquid culture at pH 8.2 at indicated time points. Markers indicate mean percent viability ± SD of three biological replicates. For each time point, at least 100 cells were analyzed per replicate (panels B-D).