Extended Data Fig. 7: PBP2 suppressor mutants showed reduced peptidoglycan synthesis activity.
a, Spot dilutions showing that excess PBP2 is toxic in the absence of LytH. Wild-type pbp2 (pbp2WT) or a pbp2* suppressor allele (pbp2N220→KDLN) was expressed from an inducible promoter in strain HG003 ΔlytH. In these strains, the native pbp2WT allele was still present. b, Fluorescence images of Nile red-stained PBP2 overexpression strains. The controls in which the inducer was withheld to prevent PBP2 overexpression are shown. Quantitation of cellular phenotypes is summarized in the graph below: cells with a nascent or complete septum at midcell (Class A), cells showing only membrane fluorescence (Class B), and cells showing fluorescent punctate foci or multiple septa (Class C). Scale bars, 1 µm. c, A PBP2 suppressor variant was purified. Bocillin- labeling of purified proteins showed that PBP2F158L was properly folded. The Coomassie gel was a control for amounts of protein loaded. The experiment was performed once. d, Western blot comparing PBP2WT and PBP2F158L reactions with Lipid II substrate. PBP2 polymerizes Lipid II into glycan strands and crosslinks the glycan strands; PBP2 also incorporates BDL to enable visualization of crosslinked peptidoglycan. The PBP2 reactions were incubated for 5 or 15 min. When indicated, lysostaphin was added to cleave peptidoglycan crosslinks so that highly-crosslinked material could enter into the gel. Data are representative of two independent experiments (a-b, d).
