FIG. 3.

Mutant SpoIID proteins show engulfment defects. Sporulation was induced by resuspension at 37°C and samples were collected at indicated times of sporulation (in hours). Scale bar, 1 μm. (A to E) Membranes were stained with FM 4-64 in strains expressing a single copy of either wild-type or mutant SpoIID at the amyE locus. A, wild type (KP718); B, spoIID null (KP7); C, SpoIID^E88A (KP1072); D, SpoIID^R106A (KP1079); E, SpoIID^K203A (KP1099); F, SpoIID^D210A (KP1102). Membranes in the wild type (A) show membrane migration and no septal bulges, while membranes in spoIID (B), SpoIID^E88A (C), and SpoIID^K203A (E) show flat or slightly curved septa with bulges. SpoIID^D210A (F) shows some open bulges but also membrane migration. Migration is often strikingly asymmetric in this strain. SpoIID^R106A (D) shows an intermediate phenotype with slow migration and persistent bulges. (G to L) Fusion assays. Membranes were stained with FM 4-64 and Mitotracker Green (MTG) to examine the completion of engulfment. Forespores that stain green but not red have completed engulfment membrane fission. Wild-type (G) cells have completed engulfment. The spoIID null (H), SpoIID^E88A (I), and SpoIID^K203A (K) show no migration and no fusion but retain septal bulges. SpoIID^R106A (J) shows some migration but very little fusion; an arrow illustrates septal bulge persistence even when some migration is present. SpoIID^D210A (L) shows significant membrane migration but little fusion (no fusion is shown in this panel, although 3% of cells have fused at t₃; see Table SA1 in the supplemental material).