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

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.

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

mSphere ◽

10.1128/msphere.00007-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Armand Lablaine ◽

Mònica Serrano ◽

Christelle Bressuire-Isoard ◽

Stéphanie Chamot ◽

Isabelle Bornard ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Bacillus Cereus ◽

Basal Layer ◽

Super Resolution ◽

Host Cells ◽

Surface Layers ◽

Content Type ◽

Localization Pattern ◽

Morphogenetic Protein ◽

Environmental Interactions

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 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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Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores

Applied and Environmental Microbiology ◽

10.1128/aem.02626-15 ◽

2015 ◽

Vol 82 (1) ◽

pp. 232-243 ◽

Cited By ~ 13

Author(s):

Christelle Bressuire-Isoard ◽

Isabelle Bornard ◽

Adriano O. Henriques ◽

Frédéric Carlin ◽

Véronique Broussolle

Keyword(s):

Bacillus Cereus ◽

Optimal Growth ◽

Spore Surface ◽

Surface Layers ◽

Content Type ◽

Morphogenetic Protein ◽

Transmission Electron ◽

Wet Heat ◽

Uv C

ABSTRACTTheBacillus cereusspore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers ofB. cereusATCC 14579 spores and on the resulting spore properties. ThecotEdeletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotEspores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotEspores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotEspores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly ofB. cereusspore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.

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Intracellular localization of the mycobacterial stressosome complex

Scientific Reports ◽

10.1038/s41598-021-89069-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Malavika Ramesh ◽

Ram Gopal Nitharwal ◽

Phani Rama Krishna Behra ◽

B. M. Fredrik Pettersson ◽

Santanu Dasgupta ◽

...

Keyword(s):

Bacillus Subtilis ◽

Fluorescence Microscopy ◽

Bacillus Cereus ◽

Intracellular Localization ◽

Sigma Factors ◽

Alternative Sigma Factors ◽

Expression Of Genes ◽

Molecular Machinery ◽

Stress Signals ◽

Activation Pathways

AbstractMicroorganisms survive stresses by alternating the expression of genes suitable for surviving the immediate and present danger and eventually adapt to new conditions. Many bacteria have evolved a multiprotein "molecular machinery" designated the "Stressosome" that integrates different stress signals and activates alternative sigma factors for appropriate downstream responses. We and others have identified orthologs of some of the Bacillus subtilis stressosome components, RsbR, RsbS, RsbT and RsbUVW in several mycobacteria and we have previously reported mutual interactions among the stressosome components RsbR, RsbS, RsbT and RsbUVW from Mycobacterium marinum. Here we provide evidence that "STAS" domains of both RsbR and RsbS are important for establishing the interaction and thus critical for stressosome assembly. Fluorescence microscopy further suggested co-localization of RsbR and RsbS in multiprotein complexes visible as co-localized fluorescent foci distributed at scattered locations in the M. marinum cytoplasm; the number, intensity and distribution of such foci changed in cells under stressed conditions. Finally, we provide bioinformatics data that 17 (of 244) mycobacteria, which lack the RsbRST genes, carry homologs of Bacillus cereus genes rsbK and rsbM indicating the existence of alternative σF activation pathways among mycobacteria.

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The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00050-15 ◽

2015 ◽

Vol 79 (4) ◽

pp. 437-457 ◽

Cited By ~ 62

Author(s):

George C. Stewart

Keyword(s):

Bacillus Cereus ◽

Multilayered Structure ◽

Structure And Function ◽

Bacterial Spore ◽

Spore Surface ◽

Surface Layers ◽

Content Type ◽

Molecular Aspects ◽

And Function ◽

Structure Assembly

SUMMARYMuch of what we know regarding bacterial spore structure and function has been learned from studies of the genetically well-characterized bacteriumBacillus subtilis. Molecular aspects of spore structure, assembly, and function are well defined. However, certain bacteria produce spores with an outer spore layer, the exosporium, which is not present onB. subtilisspores. Our understanding of the composition and biological functions of the exosporium layer is much more limited than that of other aspects of the spore. Because the bacterial spore surface is important for the spore's interactions with the environment, as well as being the site of interaction of the spore with the host's innate immune system in the case of spore-forming bacterial pathogens, the exosporium is worthy of continued investigation. Recent exosporium studies have focused largely on members of theBacillus cereusfamily, principallyBacillus anthracisandBacillus cereus. Our understanding of the composition of the exosporium, the pathway of its assembly, and its role in spore biology is now coming into sharper focus. This review expands on a 2007 review of spore surface layers which provided an excellent conceptual framework of exosporium structure and function (A. O. Henriques and C. P. Moran, Jr., Annu Rev Microbiol61:555–588, 2007,http://dx.doi.org/10.1146/annurev.micro.61.080706.093224). That review began a process of considering outer spore layers as an integrated, multilayered structure rather than simply regarding the outer spore components as independent parts.

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Amino Acids in the Bacillus subtilis Morphogenetic Protein SpoIVA with Roles in Spore Coat and Cortex Formation

Journal of Bacteriology ◽

10.1128/jb.183.5.1645-1654.2001 ◽

2001 ◽

Vol 183 (5) ◽

pp. 1645-1654 ◽

Cited By ~ 27

Author(s):

Francesca A. Catalano ◽

Jennifer Meador-Parton ◽

David L. Popham ◽

Adam Driks

Keyword(s):

Amino Acids ◽

Bacillus Subtilis ◽

Immunofluorescence Microscopy ◽

Point Mutations ◽

Bacterial Spores ◽

Large Set ◽

Spore Coat ◽

Spore Surface ◽

Null Mutant ◽

Morphogenetic Protein

ABSTRACT Bacterial spores are protected from the environment by a proteinaceous coat and a layer of specialized peptidoglycan called the cortex. In Bacillus subtilis, the attachment of the coat to the spore surface and the synthesis of the cortex both depend on the spore protein SpoIVA. To identify functionally important amino acids of SpoIVA, we generated and characterized strains bearing random point mutations of spoIVA that result in defects in coat and cortex formation. One mutant resembles the null mutant, as sporulating cells of this strain lack the cortex and the coat forms a swirl in the surrounding cytoplasm instead of a shell around the spore. We identified a second class of six mutants with a partial defect in spore assembly. In sporulating cells of these strains, we frequently observed swirls of mislocalized coat in addition to a coat surrounding the spore, in the same cell. Using immunofluorescence microscopy, we found that in two of these mutants, SpoIVA fails to localize to the spore, whereas in the remaining strains, localization is largely normal. These mutations identify amino acids involved in targeting of SpoIVA to the spore and in attachment of the coat. We also isolated a large set of mutants producing spores that are unable to maintain the dehydrated state. Analysis of one mutant in this class suggests that spores of this strain accumulate reduced levels of peptidoglycan with an altered structure.

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Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽

10.3390/pharmaceutics13060861 ◽

2021 ◽

Vol 13 (6) ◽

pp. 861

Author(s):

Jacopo Cardellini ◽

Arianna Balestri ◽

Costanza Montis ◽

Debora Berti

Keyword(s):

Drug Delivery ◽

Fluorescence Microscopy ◽

Drug Delivery Systems ◽

Laser Scanning ◽

Super Resolution ◽

Laser Scanning Confocal Microscopy ◽

Delivery Systems ◽

Scanning Confocal Microscopy ◽

Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

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