Bacterial spore envelope

Leader : Cécile Morlot

Collaborations :

C. Rodrigues, Warwick University, UK
Benoit Gallet & Christine Moriscot (Team headed by G. Schoehn, IBS)

Bacterial spores are dormant cells that can withstand a wide range of stresses, including antibiotics, detergents, irradiation and high temperatures. Such resilience is an asset when spores are used for the benefit of humans (e.g. probiotics, spore-based delivery technologies) but it represents a major problem in terms of infectious diseases, food safety or biological warfare when it comes to spores of pathogenic bacteria (e.g. spores of Bacillus anthracis or Clostridium difficile).

Upon environmental stress, spore-formers enter a differentiation process, starting with an asymmetric division (stage I) that results in two morphologically different compartments : a large mother cell and a small forespore, into which a full copy of the chromosome is translocated (stage IIE). These two cells are genetically identical but they follow specific gene expression programs, governed by tightly regulated sporulation-specific sigma factors (σ).
Following asymmetric division, the mother cell and forespore are separated by the so-called intermembrane space (IMS), composed of two membranes and peptidoglycan (PG). The two cells then undergo dramatic morphological changes as the forespore is progressively internalized into the mother cell through a phagocytic process called engulfment (stage IIM), which requires coordinated PG synthesis and degradation. The engulfed forespore is eventually surrounded by its own cytoplasmic membrane and a second membrane derived from the mother cell (stage III). Tethering of these two membranes involves the assembly of a transenvelope nanomachine called the SpoIIIA-SpoIIQ complex (A-Q complex), which is also required to maintain the forespore physiology. During engulfment, protective protein layers (called the coat) self-assemble at the surface of the forespore. A modified PG, called the cortex, is then synthesized in the IMS. The mature spore is eventually released into the environment upon mother cell lysis (stage VI). The spore can remain dormant for thousands of years, while still being receptive to its environment, so that it can germinate and resume vegetative growth under appropriate conditions.

Illustration schématique des principaux changements ultrastructuraux au cours de la sporulation.

Despite their importance for the acquisition of resistance properties, the mechanisms involved in spore development are not yet fully elucidated, mainly because they involve macromolecular complexes of nanometric dimensions, whose assembly usually requires the cellular environment.
A first example is the transmembrane multi-protein A-Q complex (> 2 MDa). In its absence, the spore exhibits shape defects and fails to acquire the ability to withstand extreme environments. The structure and function of the A-Q complex remain enigmatic, but its structural similarities with specialized secretion systems and proton pumps suggest that it could be a new type of transport machinery, enabling the mother cell to "feed" the spore or transport specific molecules between the two cellular compartments (Morlot and Rodrigues, Trends Microbiol, 2018). We study this complex in Bacillus subtilis to unravel its structural and evolutionary relationship to other transport systems, and to determine the nature of the secreted molecule and its role in spore development (Collab. C. Rodrigues, Warwick Univ.). We have discovered that the A-Q component called SpoIIIAG (AG) forms large homo-oligomeric rings whose architecture and dimensions are reminiscent of the annular components of type-III secretion systems (Rodrigues et al., PNAS, 2016). In addition, our structural characterization of various proteins associated with the A-Q complex has revealed structural motifs required for ring formation and opened up avenues of research into alternative functions (Trouvé et al., J. Struct. Biol., 2018 ; Liu et al., J. Struct. Biol., 2022).
We are now developing cryo-tomography approaches to study the structure of the A-Q complex in cellulo.

A. Cellules de B. subtilis sporulantes exprimant une protéine localisée autour de la préspore et fusionnée à la GFP. Les membranes sont marquées avec un fluorophore (rouge). B. Modèle de l’assemblage du complexe A-Q à l’interface entre la cellule mère et la spore en développement. L’illustration montre l’anneau AG (cyan), des anneaux putatifs s’empilant dans l’espace intermembranaire et des pores membranaires hypothétiques (gris). C. Structure cristallographique d’une protéine de sporulation contenant un motif canonique de construction d’anneaux (vert) et des structures secondaires supplémentaires (orange).

Among the determinants of spore resistance is the extracellular shell made of proteinaceous layers called the coat. Its assembly is based on a complex network of interactions involving first a tens morphogenetic proteins, and eventually more than 80 different proteins. Despite their importance for the acquisition of resistance properties, the architecture of the different coat layers remains poorly understood, because their deposition and maturation is a long (> 7 h) and complex process. In collaboration with the group of Guy Schoehn at the IBS, we use cryo-electron tomography (cryo-ET) on spore lamellae generated by cryo-FIBM/SEM (cryo-focused ion beam milling coupled to scanning electron microscopy) to investigate coat formation in B. subtilis. We have recently shown that in early stages of sporulation, the nascent coat is made of a stack of distinct embryonic layers, whose architecture requires specific morphogenetic proteins (Bauda et al., Nature Comm., 2024).
This work lays the foundations for the dissection of molecular mechanisms involved in the development and the resistance of the bacterial spore. Our next challenging objective is to develop super-resolution cryo-CLEM (cryo-PALM coupled to cryo-FIB/SEM and cryo-tomography) to unravel the structure and composition of the coat layers all along the sporulation cycle.

Tranche de tomograme (panneau de gauche) montrant l’ultrastructure d’une cellule sporulante de B. subtilis. Le tomograme a permis de segmenter (panneaux du milieu et de droite) divers composants de la préspore et de la cellule mère.