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

Abstract

Bacterial spores are among the most durable of cell forms, protected by robust envelopes of layered protein assemblies. Remarkably, these envelopes often share similar architectures across distantly related bacteria despite extensive divergence in their molecular components. How evolution has converged on such similar highly ordered and resilient cellular structures is relatively unexplored. Here, we combine targeted mutagenesis, cryo-electron microscopy, atomic force microscopy and structure prediction to determine the organisation and assembly of the outer spore envelope of Clostridium sporogenes, a genetically tractable surrogate for Group I Clostridium botulinum. We reveal a hierarchy of protein structures, including a semi-permeable two-dimensional crystalline exosporium; CsxA forms the exosporium scaffold with an outer “hairy nap” partially composed of BclA. We observe a previously uncharacterised multilayered three-dimensional crystalline parasporal assembly within the interspace between exosporium and coat (CsxC). These distinct structures are built from related cysteine-rich SPOCS (SpoVID-CotE-SipL)-domain proteins that self-assemble into highly ordered lattices. Such crystalline organisation, combined with high symmetry, can provide a template for local enhancement of cysteine concentration, favouring cooperative disulphide cross-linking and the formation of exceptionally stable supramolecular structures. Unexpectedly, the three-dimensional CsxC structure grows through screw dislocations, a mechanism familiar from inorganic and synthetic crystal growth but rarely demonstrated in native biological assemblies, with the exception of some biomineralisation processes. Our observations now suggest that this classical crystal-growth mechanism can be exploited in both mineralised and proteinaceous biological materials. Related SPOCS-domain proteins are implicated in spore-envelope assembly across diverse Clostridia, where they have diversified to act as both structural components and morphogenetic organisers. Remarkably, distantly related Bacilli construct similarly highly symmetric crystalline, cysteine-rich and disulphide-stabilised spore layers using proteins with very different protein folds. Thus, crystallisation and cooperative disulphide formation appear to represent a convergent physicochemical strategy for building diverse self-assembling proteins into exceptionally robust cellular assemblies. Together these findings provide the first molecular framework for understanding the organisation and assembly of the Clostridium spore envelope and reveal previously unrecognised principles governing the evolution of protective proteinaceous structures across the Bacillota (Firmicutes).