Ultrastructure of macromolecular assemblies contributing to bacterial spore resistance revealed by in situ cryo-electron tomography

Abstract

Bacterial spores owe their incredible resistance capacities to molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography on lamellae generated by cryo-focused ion beam micromachining provides insights into the ultrastructural organization of Bacillus subtilis sporangia. The reconstructed tomograms reveal that early during sporulation, the chromosome in the forespore adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a stack of amorphous or structured layers with distinct electron density, dimensions and organization. By analyzing mutant strains using cryo-electron tomography and transmission electron microscopy on resin sections, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of coat morphogenetic proteins.

Fig. 1. Cellular ultrastructures of B. subtilis sporangia.

a Slice through a tomogram (i) of a stage-III ΔspoIVB B. subtilis sporangium, used for the segmentation (ii) of various forespore and mother cell ultrastructures. The image is representative of 2 independent experiments, with 15 cells displaying similar features. Scale bar = 100 nm. b Zoom of a slice through a tomogram (i) showing the spore PG layer (small blue bracket) sandwiched between the IFM and OFM (lower magenta arrowheads), as well as the mother cell membrane (top magenta arrowhead), PG layer (large blue bracket) and surface glycans (light yellow arrowhead). Scale bar = 50 nm. Panel ii shows the corresponding segmentation. c Different views of the segmented mother cell envelope showing surface glycans. d Full view (i, scale bar = 100 nm) and zoom (ii, scale bar = 50 nm) of a slice through a cryo-electron tomogram in which the mother cell envelope bulges (violet arrow) toward the forespore. The images are representative of 2 independent experiments, with 27 cells displaying similar features. e Zooms of tomogram slices showing two thin dark lines (blue arrowheads) located close (i) to the mother cell cytoplasmic membrane (magenta arrowhead) or in the middle (ii) of the PG layer (blue bracket). Scale bars = 20 nm. f Large view (i) and zooms (blue insets) of a tomogram slice showing the region of the engulfing front, in which two thin dark lines (blue arrowheads) are observed in the middle of the IMS (ii) or close to the mother cell cytoplasmic membrane (iii). Scale bars = 20 nm.

Fig. 2. DNA organization in the B. subtilis forespore.

a, b Full views (i, scale bars = 100 nm) and zooms (ii, scale bars = 50 nm) of an engulfed forespore harboring organized crescent- (a) or toroid-shaped (b) DNA structures. DNA segmentation (iii) suggests that it forms short filamentous structures that organize around a DNA-free region. c Full view (i, scale bars = 100 nm) and zooms (ii and violet insets, scale bars = 50 nm) of an engulfed forespore harboring two discrete DNA-rich regions. The images are representative of 2 independent experiments, with 18 (a), 5 (b) and 10 (c) cells displaying similar features.

Fig. 3. Early coat assembly around the B. subtilis forespore.

a–d Slices through cryo-electron tomograms of ΔspoIVB forespores at different stages of development, shown in full view (scale bars = 100 nm) or as magnified views of specific regions (scale bars = 50 nm). The insets box the zoomed area. Five different regions can be distinguished above the OFM (magenta arrowhead): a crenelated layer (cyan beads), a light matrix (cyan bracket), a dark matrix (delineated in lime), two bead-like layers (red and orange beads) and a dark smooth layer (green line). e Two views of the segmented coat layers. The color code is identical to that described in Fig. 1. f Analysis of the 3D organization of early coat layers by observing their aspect in planes orthogonal to the section. The blue and red insets box the analyzed regions; the violet, magenta, cyan, lime, orange and green lines indicate the orientation of the plane used to generate panels i–vi. The symbol legend is the same as in a–d; the lime, orange and green arrowheads point to the patches formed by the dark matrix, the bead-like layers and the dark smooth layer, respectively. Scale bar = 100 nm. The images are representative of 2 independent experiments, with 6 (a), 5 (b), 15 (c), 7 (d) and 6 (f) cells displaying similar features.

Fig. 4. Impaired early coat assembly in the absence of SpoIIQ or CotE.

a–e Slices through cryo-electron tomograms of ΔspoIIQ or ΔcotE forespores, shown in full view (scale bars = 100 nm) or as magnified views of specific regions (scale bars = 50 nm). a The bead-like patterns (red and orange brackets) accumulate in ΔspoIIQ sporangia. b–c In the absence of CotE, the crenelated layer (cyan arrowhead), the light (cyan brackets) and dark (lime bracket) matrices and the dark smooth layer (green arrowhead) are visible but the bead-like patterns are absent. In addition, the dark smooth layer localizes right above the dark matrix (b), which abnormally extends toward the mother cell (c, lime arrowheads). d Analysis of the 3D organization of the coat layers by observing their aspect in planes orthogonal to the section. The blue inset boxes the analyzed region; the cyan lines indicate the orientation of the plane used to generate panels i–iii. e Slices through a cryo-electron tomogram of a ΔcotE forespore shown in full view and as magnified views with incremental depth (boxed in violet). A comb-like structure protrudes from the dark smooth layer in regions where it detaches from the dark matrix. The images are representative of 2 independent experiments, with 2 (a), 6 (b), 3 (c), 6 (d) and 1 (e) cells displaying similar features.

Fig. 5. Role of morphogenetic proteins in early coat assembly.

a–e Full views (i, scale bars = 100 nm) and zooms (ii, scale bars = 20 nm) of TEM micrographs collected on resin sections of B. subtilis sporangia. In the ΔspoIVB strain (a), different early coat layers can be distinguished above the OFM: a thin light matrix (cyan arrowhead), a thin dark matrix (cyan hatched arrowhead), a thick light matrix (cyan bracket), a thick dark matrix (lime bracket), two thin structured layers (red and orange arrowheads) and finally a thick structured layer (green arrowhead). In ΔspoVID (b), ΔcotE (c), ΔsafA (d) and ΔspoIVA (e) sporangia, the coat layer organization shows various architectural defects described in the text. The images are representative of 2 independent experiments, with 4 (a), 5 (b), 3 (c), 3 (d) and 10 (e) cells displaying similar features.

Fig. 6. Model of transient structures formed by the DNA and the coat, two cellular components involved in spore resistance.

In the cytoplasm of the forespore, the DNA (in violet) harbors a toroidal fibrillar conformation. A thin layer of PG is represented in the intermembrane space (IMS), delineated by the inner forespore membrane (IFM) and the outer forespore membrane (OFM). Morphogenetic proteins contributing to the seven coat regions evidenced by cryo-FIBM/ET and TEM are represented based on AlphaFold predictions. The crenelated layer (CL) observed by cryo-FIBM/ET likely corresponds to the thin light matrix (thin LM, in light blue) and thin dark matrix (thin DM, in blue) observed by TEM. Together with the thick light matrix (thick LM, in cyan), they would constitute the basement layer. In this model, the thick dark matrix (thick DM, in lime) would correspond to the inner coat, the CotE-dependent bead-like layers (BL, in red and orange) to the outer coat, and the dark smooth layer (DSL, in dark green) to the crust. The nascent coat layers are represented at the scale of the experimental measurements.