Abstract
Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro, we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity.IMPORTANCE This work dramatically improves our understanding of the assembly of the outermost layer of the B. anthracis spore, the exosporium, a layer that encases spores from many bacterial species and likely plays important roles in the spore's interactions with the environment, including host tissues. Nonetheless, the mechanisms directing exosporium assembly into a shell surrounding the spore are still very poorly understood. In this study, we clarify these mechanisms by the identification of a novel protein interaction network that directs assembly to initiate at a specific subcellular location in the developing cell. Our results further suggest that the presence or absence of an exosporium has a major impact on the assembly of other more interior spore layers, thereby potentially explaining long-noted differences in spore assembly between B. anthracis and the model organism B. subtilis.
Highlights
Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function
In this study, we address a fundamental question in spore formation: how is exosporium formation directed to initiate at one pole of the forespore surface? Our findings argue that, to a large degree, this is achieved by a protein network, consisting at a minimum of the proteins CotE, CotO, and CotY, which assemble at the forespore prior to engulfment (Fig. 9A, left image, and B)
We infer, based on previous work, that the coat proteins SpoIVA and ExsA likely reside between CotE and the forespore membrane and anchor the network to the forespore [13, 39,40,41,42]. This model of assembly resembles the coat protein network in B. subtilis, suggesting that there may be a core outer layer assembly program that is conserved among all spore-forming Bacillaceae
Summary
Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. The bacterial endospore, an abundant cell type found in many environments (including the soil and human gastrointestinal [GI] tract), is highly resistant to diverse stresses and survives under extreme conditions for extended time periods These properties are important for persistence in the environment, colonization of the human. Ongoing investigations in B. anthracis and the model organism Bacillus subtilis (which lacks the interspace and exosporium) seek to identify the functions of these layers and elucidate the mechanisms directing their assembly. B. anthracis spores lacking the exosporium are largely functional [12] and cause significant disease in animal models [13, 14] These results argue that the exosporium is not required for infection per se but may play an important role in natural infection or in other environmental adaptations. The exosporium likely has additional unknown functions in B. anthracis, as well as in the large number of other phylogenetically diverse species possessing this structure
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