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bioRxiv · 10.1101/2020.12.11.422311

The morphogenetic protein CotE drives exosporium formation by positioning CotY and ExsY during sporulation of Bacillus cereus

Lablaine, A.·Serrano, M.·Chamot, S.·Bornard, I.·Carlin, F.·Henriques, A. O.·Broussolle, V.
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Abstract

The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates interactions of spores with their environment, modulates spore adhesion and germination and could be implicated in pathogenesis. The exosporium is composed of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, and surrounded by a glycoprotein hairy nap. The morphogenetic protein CotE is necessary for Bacillus cereus exosporium integrity, but how CotE directs exosporium assembly remains unknown. Here, we followed the localization of SNAP-tagged CotE, -CotY and -ExsY during B. cereus sporulation, using super-resolution fluorescence microscopy and evidenced interactions among these proteins. CotE, CotY and ExsY are present as complexes at all sporulation stages and follow a similar localization pattern during endospore formation that is reminiscent of the localization of Bacillus subtilis CotE. We show that B. cereus CotE drives 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 it is performed through the formation of two caps, as observed during B. subtilis coat morphogenesis. It appears that a general principle governs the assembly of the spore surface layers of Bacillaceae. IMPORTANCESpores of Bacillaceae are enveloped in a glycoprotein outermost layer. In the B. cereus group, encompassing the B. anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance separated 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 formation of the exosporium during sporulation of B. cereus and we studied the localization and interactions of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaf-fold, before a complete exosporium or coat are 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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Lablaine, A., Serrano, M., Chamot, S., Bornard, I., Carlin, F., Henriques, A. O., Broussolle, V.. 2020-12-14. The morphogenetic protein CotE drives exosporium formation by positioning CotY and ExsY during sporulation of Bacillus cereus. https://doi.org/10.1101/2020.12.11.422311

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