Coordinated Assembly of the Bacillus anthracis Coat and Exosporium during Bacterial Spore Outer Layer Formation

Boone, Tyler J.; Mallozzi, Michael; Nelson, Alex; Thompson, Brian M.; Khemmani, Mark; Lehmann, Dörte; Dunkle, Alexis; Hoeprich, Paul D.; Rasley, Amy; Stewart, George C.; Driks, Adam

doi:10.1128/mbio.01166-18

Cited by 25 publications

(58 citation statements)

References 50 publications

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“…Moreover, the absence of ExsY or CotY possibly affects the association of CotE with the released spores. Hence, since cotY spores possess a seemingly normal exosporium (7,9), our results suggest that CotE is needed during sporulation, only to guide cap localization and encasement by ExsY and CotY, but seems dispensable later for maintaining the integrity of the structure once assembled. Finally, while CotE completely encased exsY forespores, CotY assembly was blocked at the two-cap stage.…”

Section: Resultsmentioning

confidence: 83%

The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

Lablaine

Serrano

Bressuire-Isoard

et al. 2021

mSphere

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The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates the interactions of spores with their environment, modulates spore adhesion and germination, and has been implicated in pathogenesis. In Bacillus cereus, the exosporium consists of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, surrounded by a hairy nap composed of glycoproteins. The morphogenetic protein CotE is necessary for the integrity of the B. cereus exosporium, but how CotE directs exosporium assembly remains unknown. Here, we used super-resolution fluorescence microscopy to follow the localization of SNAP-tagged CotE, CotY, and ExsY during B. cereus sporulation and evidenced the interdependencies among these proteins. Complexes of CotE, CotY, and ExsY are present at all sporulation stages, and the three proteins follow similar localization patterns during endospore formation that are reminiscent of the localization pattern of Bacillus subtilis CotE. We show that B. cereus CotE guides the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process, as previously proposed, but occurs through the formation of two caps, as observed during B. subtilis coat morphogenesis, suggesting that a general principle governs the assembly of the spore surface layers of Bacillaceae. IMPORTANCE Spores of Bacillaceae are enveloped in an outermost glycoprotein layer. In the B. cereus group, encompassing the Bacillus anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance and separation from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize the formation of the exosporium during the sporulation of B. cereus, and we studied the localization and interdependencies of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaffold before a complete exosporium or coat is detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore, and we present a model for the assembly of the exosporium.

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Section: Resultsmentioning

confidence: 83%

The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

Lablaine

Serrano

Bressuire-Isoard

et al. 2021

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“…Sporulation is blocked at the late stage of engulfment in spoIIQ mutants of B. subtilis [11]. CotE plays an essential role in assembly of both the coat and exosporium in B. anthracis and B. cereus spores [13,21,41,42]. Thus, SpoIIB Bs may be able to stimulate sporulation of the B. anthracis pBspoIIB Bs /∆spoVG strain to the engulfment stage (Figure 7b).…”

Section: Discussionmentioning

confidence: 99%

SpoVG Is Necessary for Sporulation in Bacillus anthracis

Chen¹,

Lyu²,

Feng³

et al. 2020

Microorganisms

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The Bacillus anthracis spore constitutes the infectious form of the bacterium, and sporulation is an important process in the organism’s life cycle. Herein, we show that disruption of SpoVG resulted in defective B. anthracis sporulation. Confocal microscopy demonstrated that a ΔspoVG mutant could not form an asymmetric septum, the first morphological change observed during sporulation. Moreover, levels of spoIIE mRNA were reduced in the spoVG mutant, as demonstrated using β-galactosidase activity assays. The effects on sporulation of the ΔspoVG mutation differed in B. anthracis from those in B. subtilis because of the redundant functions of SpoVG and SpoIIB in B. subtilis. SpoVG is highly conserved between B. anthracis and B. subtilis. Conversely, BA4688 (the protein tentatively assigned as SpoIIB in B. anthracis) and B. subtilis SpoIIB (SpoIIBBs) share only 27.9% sequence identity. On complementation of the B. anthracis ΔspoVG strain with spoIIBBs, the resulting strain pBspoIIBBs/ΔspoVG could not form resistant spores, but partially completed the prespore engulfment stage. In agreement with this finding, mRNA levels of the prespore engulfment gene spoIIM were significantly increased in strain pBspoIIBBs/ΔspoVG compared with the ΔspoVG strain. Transcription of the coat development gene cotE was similar in the pBspoIIBBs/ΔspoVG and ΔspoVG strains. Thus, unlike in B. subtilis, SpoVG appears to be required for sporulation in B. anthracis, which provides further insight into the sporulation mechanisms of this pathogen.

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“…CotO is conserved in Bacillus species that also have orthologs of CotY and CotZ. It was recently shown that cotO plays a related role in B. anthracis , where it is involved in the coordination of coat and exosporium assembly (Boone et al , ).…”

Section: Discussionmentioning

confidence: 99%

Contributions of crust proteins to spore surface properties in Bacillus subtilis

Shuster

Khemmani

Abe

et al. 2019

Molecular Microbiology

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Summary Surface properties, such as adhesion and hydrophobicity, constrain dispersal of bacterial spores in the environment. In Bacillus subtilis, these properties are influenced by the outermost layer of the spore, the crust. Previous work has shown that two clusters, cotVWXYZ and cgeAB, encode the protein components of the crust. Here, we characterize the respective roles of these genes in surface properties using Bacterial Adherence to Hydrocarbons assays, negative staining of polysaccharides by India ink and Transmission Electron Microscopy. We showed that inactivation of crust genes caused increases in spore relative hydrophobicity, disrupted the spore polysaccharide layer, and impaired crust structure and attachment to the rest of the coat. We also found that cotO, previously identified for its role in outer coat formation, is necessary for proper encasement of the spore by the crust. In parallel, we conducted fluorescence microscopy experiments to determine the full network of genetic dependencies for subcellular localization of crust proteins. We determined that CotZ is required for the localization of most crust proteins, while CgeA is at the bottom of the genetic interaction hierarchy.

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Coordinated Assembly of the Bacillus anthracis Coat and Exosporium during Bacterial Spore Outer Layer Formation

Cited by 25 publications

References 50 publications

The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

SpoVG Is Necessary for Sporulation in Bacillus anthracis

Contributions of crust proteins to spore surface properties in Bacillus subtilis

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