Fig. 3

The activity of the PBSX endolysin creates holes in the bacterial cell wall. a Staining of the PG with FDL in cells expressing the holin–endolysin genes and in uninduced control cells. Upper panel shows strain 168 (P _xylA -xhlAB-xlyA), in which the holin–endolysin genes were expressed; the lower panel shows the control strain 168 (P _xylA ). Membranes were stained with FM4-64 (red, left) and the PG was stained with FDL (green, central). Corresponding bright field images are shown in the right panel. Scale bar, 5 μm. b Thin section of induced B. subtilis 168 (P _xylA -xhlAB-xlyA) cells (upper panel) and of the control strain 168 (P _xylA ) (lower panel). Arrows indicate holes in the bacterial cell wall. Scale bar, 1 μm. c–k ECT images of different Bacillus ΔponA strains. c Uninduced B. subtilis ΔponA (P _xylA -xhlAB-xlyA) cells have an intact peptidoglycan (PG) layer and cytoplasmic membrane (CM) and a dense cytoplasm. d–h Induced cells extrude MVs through holes in the PG layer. We also observed cells with lowered cytoplasmic densities that harbored intracellular vesicles (h). i–k Likewise, cells of a MMC-induced B. subtilis ΔponA culture showed MV formation (i) in addition to phage particles (PBSX) inside cells (j) and sometimes within extracellular MVs (k). Shown are 1.38 nm (c, f, h, k), 6.9 nm (g), 11.86 nm (i), 29.64 nm (j) thick slices through cryotomograms and a model (e) of the cell shown in d. White, peptidoglycan; orange, cytoplasmic membrane. Scale bars, 100 nm