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. 2021 Apr 21;6(2):e00007-21.
doi: 10.1128/mSphere.00007-21.

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

Armand Lablaine  1 , Mònica Serrano  2 , Christelle Bressuire-Isoard  1 , Stéphanie Chamot  1 , Isabelle Bornard  3 , Frédéric Carlin  1 , Adriano O Henriques  4 , Véronique Broussolle  5
Affiliations

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

Armand Lablaine et al. mSphere. .

Abstract

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 BacillaceaeIMPORTANCE 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.

Keywords: SR-SIM; endospores; exosporium; morphogenetic proteins; spore.

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Figures

FIG 1
FIG 1
Stages of CotY-SNAP localization during sporulation. Sporulating cells of B. cereus producing CotY-SNAP were labeled with the SNAP substrate TMR-Star and with the membrane dye MTG and were imaged by SR-SIM. Images of a representative cell for each pattern of localization identified at a particular stage of sporulation are shown, with schematic representations (patterns a to h) below. The white arrow points to the weak signal of CotY-SNAP detected at curved septa; pink arrows point to the exosporium at different stages of its formation; curved black arrows point to the mother cell-proximal (MCP) and -distal (MCD) forespore poles and to the outer forespore membranes (OFM). Bars, 0.5 μm. The stages of ExsY-SNAP, SNAP-CotE, and CotE-SNAP localization during sporulation are shown in Fig. S1A, Fig. S1B, and Fig. S2A, respectively.
FIG 2
FIG 2
CotE is required for cap formation, allowing CotY and ExsY to localize as caps. Samples were collected from cultures of a cotE mutant producing CotY-SNAP (A) or ExsY-SNAP (B) during sporulation, and the cells were imaged by SR-SIM following staining with TMR-Star and MTG. Cells representative of the different localization patterns observed are shown (panels a to f). Purple arrows point to the signal from CotY-SNAP or ExsY-SNAP (panels e and f). In the cotE mutant, CotY-SNAP and ExsY-SNAP never formed the cap or the subsequent patterns identified in the WT (Fig. 1 and Fig. S1A) but formed large patches or aggregates in the mother cell cytoplasm. Bars, 0.5 μm.
FIG 3
FIG 3
ExsY is required for encasement by CotY but not by CotE. (A and B) Samples were withdrawn at the indicated times from 37°C cultures of an exsY mutant producing CotY-SNAP (A) or CotE-SNAP (B). The cells were labeled with TMR-Star and MTG and were imaged by phase-contrast and fluorescence microscopy. Localization patterns a to g are identical to patterns a to g observed in WT cells in Fig. 1. Patterns c*, d*, and j* (white arrows) correspond to patterns c, d, and j observed in sporangia of phase-bright spores or in free spores. Numbers on the right show the percentage of cells with each of the indicated patterns relative to the total number of sporulating cells expressing the different SNAP fusions. ND; no signal detected. (C) Spores of the indicated strains were stained with MTG and TMR-STAR and were imaged by SR-SIM. The strains were ATCC 10876 (WT) and a derivative producing CotE-SNAP, as well as a congenic exsY mutant and derivatives producing CotE-SNAP or CotY-SNAP. Yellow arrows point to exosporia blocked at the two-cap stage. The pink arrow points to a complete exosporium. Cyan arrows point to nonspecific TMR signals. Bars, 1 μm in panels A and B and 0.5 μm in panel C.
FIG 4
FIG 4
CotE, CotY, and ExsY form complexes in vivo and interact in vitro. (A) Samples were collected at the indicated times from sporulating cultures of B. cereus strains producing various SNAP fusions. Whole-cell extracts were prepared and subjected to pulldown assays with a SNAP capture resin. Whole-cell extracts, flowthrough, and bound proteins were resolved by SDS-PAGE and subjected to immunoblot analyses with anti-CotE antibodies. A lane corresponding to a pulldown assay performed on proteins extracted from purified CotY-SNAP and ExsY-SNAP spores was added. One red asterisk indicates a monomer of CotE; two red asterisks, a potential dimer of CotE; three red asterisks, multimers; blue asterisks, possible proteolytic products of CotE. A nonspecific signal in the extracts from vegetative cells (hour 0) is indicated by a green asterisk. (B) Heterologous coexpression pulldown assays. E. coli BL21(DE3) cells either producing CotE alone or coproducing CotE with His6-CotY or His6-ExsY were lysed and subjected to pulldown assays. Proteins were subjected to immunoblot analysis with anti-CotE (top) or anti-His6 (bottom) antibodies. While CotE produced alone was not eluted from the Ni2+ beads (6th to 8th lanes, top), His6-CotY pulled down CotE in E1 (9th lane, top) and was detected in E1 and E2 (9th and 10th lanes, bottom). CotE was pulled down with His6-ExsY in E1 and E2 (12th and 13th lanes, top) and was detected in E1 and E2 (12th and 13th lanes, bottom). CE, cell extract; FT, flowthrough; w1 to w3, washes; E1 to E3, elutions. Red asterisks indicate the different species of CotE (upper panels), His6-tagged CotY (9th to 11th lanes, bottom), or His6-tagged ExsY (12th to 14th lanes, bottom). The positions of molecular weight (MW) markers (in kDa) are indicated on the left.
FIG 5
FIG 5
Successive localization, interactions, and interdependence among CotE, CotY, and ExsY during exosporium formation. (A) (Left) Assembly of the indicated spore structures as observed by TEM. Dotted lines show the new structures found in the present study. (Right) Diagrams showing the temporal sequence of localization of CotE, CotY, and ExsY inferred from our results. (i) CotE (red) forms a cap in the septal region at the onset of engulfment and recruits CotY (blue), which, in turn, recruits ExsY (gray). Once positioned, CotY and ExsY form the basal layer of the cap (mixed gray and blue). The cap remains unchanged until the completion of engulfment. (ii) After the completion of engulfment, the MCP cap is separated from the OFM by the formation of the interspace (IS), and CotE directs the localization of CotY, and therefore of ExsY, as a second cap on the MCD pole (dotted line). (iii) CotE progressively encases the spore starting from one longitudinal side of the forespore (red arrows), guiding the simultaneous encasement of ExsY (gray arrows), which, in turn, is required for the encasement of CotY (blue arrows). After the completion of encasement by CotE, CotY and ExsY cover the noncap region of the forespore as an immature morphogenetic scaffold (dotted line). (iv) After coat formation and insertion of the late-synthesized proteins, CotE, CotY, and ExsY are found in all the regions of the mature exosporium. (B) Formation of the morphogenetic scaffold and its maturation in B. cereus. Based on their roles in B. subtilis, SpoIVA and SpoVID homologs are good candidates for directing the recruitment and encasement, respectively, of CotE and CotE-controlled proteins. CotO, possibly through its interaction with CotE, may participate in the recruitment of CotY and/or ExsY. ExsA, a SafA homologue that controls B. cereus coat protein deposition, is also present in the morphogenetic scaffold. After sporulation completion, the proteins of the morphogenetic scaffold are positioned in the different layers of the mature spore.
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