Fig. 2 |. Sporulation-specific PG synthases, SpoVD and SpoVE, are important for asymmetric but not vegetative division.
a, Growth profiles of C. difficile wildtype (WT), ΔspoVD, and ΔspoVE strains in BHIS. Data are from a single experiment; mean and standard deviation curves are plotted from three biological replicates. b, Violin plots showing cell length distributions and representative micrographs of WT, ΔspoVD, and ΔspoVE cells sampled from BHIS cultures during exponential growth (OD600 ~0.5). White circles indicate means from each replicate, black lines indicate average means, and the dotted line indicates the WT average mean for comparison across strains. Data from three biological replicates; >1,500 cells per sample. Scale bar, 5 μm. c, d, Cytological profiling of WT, ΔspoVD, and ΔspoVE cells sampled from sporulation-inducing 70:30 plates after 18–20 hours of growth. Cells were assigned to five distinct stages based on their membrane (FM4–64) and DNA (Hoechst) staining and their phase-contrast morphological phenotypes. For representative micrographs and stage assignment information, see Supplementary Fig. 3. Bars indicate means; error bars indicate standard deviation. ***p<0.001; statistical significance was determined using ordinary one-way ANOVA with Dunnett’s test. Data from three independent experiments; >1,000 total cells and >100 visibly sporulating cells per sample. e, Representative merged phase-contrast and fluorescence micrographs visualizing PspoIIE::mScarlet transcriptional reporters in sporulating WT, ΔspoVD, and ΔspoVE cells sampled from 70:30 plates after 14–16 hours of growth. PspoIIE is induced immediately upon sporulation initiation. The Δspo0A strain was used as a negative control because it does not initiate sporulation. Scale bar, 10 μm. f, Violin plots showing quantified mean fluorescence intensities. Black dots represent median values from each replicate. Data from three independent experiments; >3,000 cells per sample. g, Transmission electron micrographs of ΔspoVD and ΔspoVE sporulating cells that fail to complete septum formation 24 hrs after sporulation induction (white arrows). Scale bars, 500 nm.