Protein architectures of the bacterial spore envelope - common principles of assembly

Bacterial spores are exceptionally resilient cellular forms, safeguarded by complex envelope layers composed of similar protein architectures. Although distantly related bacteria possess varying molecular constituents, their envelope structures exhibit striking similarities. The evolutionary convergence behind these robust cellular assemblies remains largely unexamined.

This study elucidates the inner workings of the outer spore envelope of Clostridium sporogenes, a convenient substitute for the more pathogenic Clostridium botulinum. By employing targeted mutagenesis, cryo-electron microscopy, atomic force microscopy, and structure prediction techniques, researchers determined the hierarchical organization and assembly of the spore envelope components.

They discovered a semi-permeable two-dimensional crystalline exosporium, with the exosporium scaffold primarily consisting of CsxA proteins. BclA proteins form a partially hairy nap on the outer layer of the exosporium. Additionally, a previously uncharacterized multilayered three-dimensional crystalline parasporal assembly exists within the interspace between the exosporium and coat (CsxC).

These distinct structures are composed of cysteine-rich SPOCS (SpoVID-CotE-SipL)-domain proteins that self-assemble into ordered lattices, showcasing a highly ordered, resilient, and stable protein architecture. The combination of crystalline organization and high symmetry may facilitate the concentration of cysteine, promoting cooperative disulfide cross-linking and the formation of exceptionally stable supramolecular structures.

Interestingly, the three-dimensional CsxC structure develops through screw dislocations, a mechanism typically associated with inorganic and synthetic crystal growth, although rarely observed in native biological assemblies save for specific biomineralisation processes. The findings suggest that the classical crystal-growth mechanism can be applied to both mineralized and proteinaceous biological materials.

Related SPOCS-domain proteins play crucial roles in spore-envelope assembly across various Clostridia, where they have diversified to function as structural components and morphogenetic organizers. Notably, distantly related Bacilli also construct highly symmetric crystalline, cysteine-rich, and disulfide-stabilized spore layers using proteins with distinct folds.

This research demonstrates that crystallization and cooperative disulfide formation constitute a convergent physicochemical strategy for constructing diverse self-assembling proteins into exceptionally robust cellular assemblies. Overall, these discoveries offer the first molecular framework for comprehending the organization and assembly of the Clostridium spore envelope and unveil previously unrecognized principles governing the evolution of protective proteinaceous structures within the Bacillota (Firmicutes).