Assembly of the outermost spore layer: pieces of the puzzle are coming together

Stewart, George C.

doi:10.1111/mmi.13651

Cited by 7 publications

(5 citation statements)

References 18 publications

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“…Taken together, these results show that CotY, ExsY and CotE share a common pattern of localization during sporulation, behaving as cap proteins at the MCP forespore pole during engulfment, then after engulfment completion forming a second cap at the MCD spore pole and finally completely encasing the forespore. Thus, CotY and ExsY do not seem to be restricted to the cap or the non-cap regions of the B. cereus exosporium, as previously proposed (5,12,33). We also found that CotY, ExsY and CotE already encased the forespore in most of the sporangia scored at hour 24 ( Fig.…”

Section: Coty Exsy and Cote Follow The Same Assembly Dynamic During supporting

confidence: 86%

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

Lablaine¹,

Serrano

Chamot³

et al. 2020

Preprint

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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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Section: Coty Exsy and Cote Follow The Same Assembly Dynamic During supporting

confidence: 86%

“…5Aiii-iv). Thus, the localization of CotY and ExsY is not confined to the cap or to the non-cap regions of the B. cereus exosporium, respectively, as previously thought (5,12,33).…”

Section: Cote Coty and Exsy Form A Complex In Vivo During Sporulatiosupporting

confidence: 61%

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

Lablaine¹,

Serrano

Chamot³

et al. 2020

Preprint

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show abstract

“…5Aiii and iv). Thus, the assembly of the B. cereus exosporium does not seem to be unidirectional, as had been deduced from B. anthracis studies (5,12,35).…”

Section: Discussionmentioning

confidence: 68%

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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“…Using MTG and a SNAP-fusion for the exosporium cap protein CotY, we showed the existence of a second small exosporium cap at the mother cell distal pole (MCD pole) of ∆ exsY spores not previously observed by TEM 27 , 28 . This indicates that exosporium formation shares more similarities with the B. subtilis model of coat formation than expected 1 , 2 , 42 , 43 . Here, we show that SNAP-cell TMR-star (TMR-star), a fluorescent substrate used to label SNAP-tag fusion proteins, and MTG are both able to specifically bind to the coat surface of B. cereus .…”

Section: Introductionmentioning

confidence: 66%

A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus

Lablaine,

Chamot,

Serrano

et al. 2023

Sci Rep

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The human pathogenic bacteria Bacillus cereus, Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of σK is not strictly linked to the state of forespore development in B. cereus.

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Assembly of the outermost spore layer: pieces of the puzzle are coming together

Cited by 7 publications

References 18 publications

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

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

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

A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus

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