(A) Schematic illustrating the process of polar septation, chromosome translocation, engulfment and spore maturation. Membranes (red), PG (gray), chromosomes (blue), SpoIIIE (orange) and coat proteins (shades of brown) are highlighted. Outward arrows in the stage IIii forespore indicate increased turgor pressure due to chromosome translocation. (B) Revised engulfment model (Ojkic et al., 2016): new PG (purple) is synthesized ahead of the leading edge of the engulfing membrane by forespore-associated PG biosynthetic enzymes (blue) and is subsequently degraded by DMP (yellow pacman), making room for the engulfing membrane to advance. The coordinated synthesis and degradation of PG at the leading edge of the engulfing membrane can move the junction between the septum (pink) and the lateral cell wall (gray) around the forespore. (C) Schematic illustrating cryo-FIB-ET methodology for bacterial samples (see Materials and methods). (D–I) Slices through cryo-electron tomograms representing different stages of engulfment: (D) flat polar septum (Stage IIi), (F) curved septum (Stage IIii) and (H) engulfing septum (Stage IIiii). Scale bar for (D,F,I): 200 nm. The corresponding forespore membrane (green) and the mother cell membrane (yellow) are annotated in (E,G,I) respectively. n indicates the number of tomograms acquired for each cell type. Scale bars have been omitted for (E,G,I) as cells are shown in perspective views . (J–N) Zoomed-in slices through cryo-electron tomograms showing (J) a large ellipsoidal complex adjacent to the forespore membrane (black arrow), (K) a putative SpoIIIE channel (brown arrow) and (L) another putative channel (brown arrow), (L,M) coat filaments (green arrows), (M) basement coat layer (maroon arrow) and (N) amorphous coat proteins (purple arrow). Scale bar for (J-N): 50 nm.