Fig 1.

SpoVD catalytic activity is partially dispensable for its function during asymmetric division. (a) Schematic of a SEDS-bPBP peptidoglycan synthase complex. The SEDS glycosyltransferase polymerizes nascent glycan strands from lipid-linked PG precursors in the cytoplasmic membrane, while the class B penicillin-binding protein (bPBP) cross-links the stem peptides between the growing strands. (b) Cytological profile of individual cells representing each of the five morphological stages of sporulation, as indicated. Representative phase-contrast and fluorescence micrographs show wild-type (WT) cells sampled from sporulation-inducing 70:30 plates after 18 h of growth. The nucleoid was stained using Hoechst, and the cell membrane was stained using FM4-64. Cells undergoing asymmetric division (AD) have a flat polar septum; cells undergoing engulfment (EI) have a curved polar septum; cells that have completed engulfment (EC) are indicated by bright-membrane staining around a fully engulfed forespore; phase-visible forespores (PFs) indicate forespores completing maturation visible as phase-dark or phase-bright forespores (yellow arrowheads) associated with the mother cell; mature free spores (FSs) are observable as independent phase-bright particles. (c and d) Quantification of the cytological profiling of cells sampled from sporulation-inducing plates after 20–22 h of growth. White circles indicate data from each replicate; bars indicate the average means; and error bars indicate standard deviation. More than 1,000 total cells and over 100 visibly sporulating cells were analyzed per sample from a minimum of three biological replicates, except for ∆spoVD/spoVD^S311A, for which two biological replicates were conducted. For representative micrographs, see Fig. S2. (c) Distribution of visibly sporulating cells among the indicated stages of sporulation. (d) Proportion of cells that complete and progress beyond asymmetric division, i.e., all visibly sporulating cells, as a percentage of the total cells profiled. Note that the data are normalized to WT (dotted line). ****P < 0.0001. Statistical significance was determined using one-way ANOVA and Tukey’s test. (e) Western blot analyses of SpoVD levels in the indicated strains 14 h after growth on sporulation-inducing plates. The anti-Spo0A antibody was used as a proxy for measuring sporulation induction.