The exosporium is the outermost layer of spores of Bacillus cereus and its close relatives Bacillus anthracis and Bacillus thuringiensis. For these pathogens, it represents the surface layer that makes initial contact with the host. To date, only the BclA glycoprotein has been described as a component of the exosporium; this paper defines 10 more tightly associated proteins from the exosporium of B. cereus ATCC 10876, identified by N-terminal sequencing of proteins from purified, washed exosporium. Likely coding sequences were identified from the incomplete genome sequence of B. anthracis or B. cereus ATCC 14579, and the precise corresponding sequence from B. cereus ATCC 10876 was defined by PCR and sequencing. Eight genes encode likely structural components (exsB, exsC, exsD, exsE, exsF, exsG, exsJ, and cotE). Several proteins of the exosporium are related to morphogenetic and outer spore coat proteins of B. subtilis, but most do not have homologues in B. subtilis. ExsE is processed from a larger precursor, and the CotE homologue appears to have been C-terminally truncated. ExsJ contains a domain of GXX collagen-like repeats, like the BclA exosporium protein of B. anthracis. Although most of the exosporium genes are scattered on the genome, bclA and exsF are clustered in a region flanking the rhamnose biosynthesis operon; rhamnose is part of the sugar moiety of spore glycoproteins. Two enzymes, alanine racemase and nucleoside hydrolase, are tightly adsorbed to the exosporium layer; they could metabolize small molecule germinants and may reduce the sensitivity of spores to these, limiting premature germination.Spores of the Bacillus cereus family, which includes Bacillus anthracis and Bacillus thuringiensis, all possess a loose balloonlike exosporium (7). A similar layer is also found on spores of some other bacilli and clostridia. The particular adherence and hydrophobic properties conferred by the exosporium (4, 14) suggest that it may possibly be of significance to spore pathogenicity. Bacillus subtilis, the paradigm of sporeformers, has no such clearly defined exosporial layer, so the exosporium has not been studied in molecular detail. Scanning electron microscopy has revealed a paracrystalline basal layer, with hexagonal periodicity, and a hairlike outer layer (3, 11). There are also pilus-like structures on the surface (15). The exosporium contains protein, lipid, and carbohydrate (43 to 52, 15 to 18, and 23% of dry weight, respectively [3,19]). A spore glycoprotein of B. thuringiensis was purified and partially characterized (10); it was present as two forms according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-a 70-kDa monomer and a 205-kDa multimer. Another glycoprotein, BclA, important to the surface hairlike layer, has recently been identified in the B. anthracis exosporium (22). Crude exosporium extracts of B. cereus (5) contain at least 12 major and some minor protein components that can be readily solubilized, including GroEL and a zinc metalloprotease called immune inh...

The exosporium-defective phenotype of a transposon insertion mutant of Bacillus cereus implicated ExsY, a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZ, in assembly of an intact exosporium. Single and double mutants of B. cereus lacking ExsY and its paralogue, CotY, were constructed. The exsY mutant spores are not surrounded by an intact exosporium, though they often carry attached exosporium fragments. In contrast, the cotY mutant spores have an intact exosporium, although its overall shape is altered. The single mutants show altered, but different, spore coat properties. The exsY mutant spore coat is permeable to lysozyme, whereas the cotY mutant spores are less resistant to several organic solvents than is the case for the wild type. The exsY cotY double-mutant spores lack exosporium and have very thin coats that are permeable to lysozyme and are sensitive to chloroform, toluene, and phenol. These spore coat as well as exosporium defects suggest that ExsY and CotY are important to correct formation of both the exosporium and the spore coat in B. cereus. Both ExsY and CotY proteins were detected in Western blots of purified wild-type exosporium, in complexes of high molecular weight, and as monomers. Both exsY and cotY genes are expressed at late stages of sporulation. Endospores of the Bacillus cereus family, which includesBacillus anthracis and Bacillus thuringiensis, are enveloped by a large, balloon-like layer known as the exosporium (11,19). B. subtilis, the paradigm sporeformer, does not have this distinct layer, although the B. subtilis spore coat may have a tight-fitting outermost layer that can be visualized after extraction of coat material with urea or mercaptoethanol (23). Scanning electron microscopy has revealed that the exosporium is composed of two layers-a paracrystalline basal layer with hexagonal periodicity and a "hairy nap" outer layer (11). The exosporium is chemically complex and is composed of 53% protein, 20% amino and neutral polysaccharides, 18% lipids, and ϳ4% ash (17).Multiple proteins from the exosporium of both B. cereus and B. anthracis have been identified (22,24,31); the majority do not have homologues in B. subtilis. For example, the surface layers of B. cereus, B. anthracis, and B. thuringiensis spores all contain glycoproteins with characteristic collagen-like repeat regions (5,10,27). Of these, the B. anthracis glycoprotein BclA is an essential component of the hairy nap layer of the exosporium (27, 29).Analysis of genome sequence information from B. cereus ATCC 14579 and B. anthracis Ames identified a cluster of genes near bclA implicated in exosporium production, including the region whose genes were designated yjbX-exsYyjcA-yjcB-exsFA-cotY (31). The ExsY and CotY proteins are homologues (ca. 35% amino acid identity) of the B. subtilis coat proteins CotY and CotZ (37). ExsY and CotY proteins have both been detected in purified exosporium from B. anthracis endospores by N-terminal sequencing of separated peptides (22). ExsY and CotY are very sim...

SummaryWe report on the first step in mapping out the spatial location of structural proteins within the exosporium, namely a description of its three-dimensional architecture. Using electron microscopy and image analysis, we have characterized crystalline fragments from the exosporium of Bacillus cereus, B. thuringiensis and B. anthracis strains and identified up to three distinct crystal types. Type I and type II crystals were examined in three dimensions and shown to form arrays of interlinked crown-like structures each enclosing a cavity~26-34 Å deep with threefold symmetry. The arrays appear to be permeated by tunnels allowing access from one surface to the other, possibly indicating that the exosporium forms a semi-permeable barrier. The pore size of 23-34 Å would allow passage of the endospore germinants, alanine or inosine but not degradative enzymes or antibodies. Thus the structures appear compatible with a protective role for the exosporium. Furthermore the outermost crystalline layer must act as a scaffold for binding the BclA protein that contributes to the 'hairy nap' layer. The array of crowns may also act as a matrix for the binding or adsorption of other proteins that have been identified in the exosporium such as GroEL, immune inhibitor A and arginase.

Bacteria of the Bacillus cereus family form highly resistant spores, which in the case of the pathogen B. anthracis act as the agents of infection. The outermost layer, the exosporium, enveloping spores of the B. cereus family as well as a number of Clostridia, plays roles in spore adhesion, dissemination, targeting, and germination control. We have analyzed two naturally crystalline layers associated with the exosporium, one representing the "basal" layer to which the outermost spore layer ("hairy nap") is attached, and the other likely representing a subsurface ("parasporal") layer. We have used electron cryomicroscopy at a resolution of 0.8-0.6 nm and circular dichroism spectroscopic measurements to reveal a highly α-helical structure for both layers. The helices are assembled into 2D arrays of "cups" or "crowns." High-resolution atomic force microscopy of the outermost layer showed that the open ends of these cups face the external environment and the highly immunogenic collagen-like fibrils of the hairy nap (BclA) are attached to this surface. Based on our findings, we present a molecular model for the spore surface and propose how this surface can act as a semipermeable barrier and a matrix for binding of molecules involved in defense, germination control, and other interactions of the spore with the environment.electron crystallography | cryoelectron microscopy | two-dimensional crystal

The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn917-LTV1 transposon insertion mutants of B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortexbinding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coatassociated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants-two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region-showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus. Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensisare very closely related (16,22), and the possession of an exosporium is a major characteristic of this group. This outermost layer of the spore is the least understood of all the spore integuments. The paradigm of sporeformers, B. subtilis, lacks a distinct, separate exosporium, although there has been a report that a very outermost tightly fitting layer of the spore coat can be visualized after extracting some of the coat material from spores with urea-mercaptoethanol and might be considered an exosporium (26). The exosporium of Bacillus cereus is first observed as a small lamellar structure in the mother cell cytoplasm in proximity to, but not in contact with, the outer forespore membrane; it is synthesized concurrently with the spore coat, although the two structures are clearly separate within the mature spore (16). The exosporium contains a hexagonal crystal-like basal layer and a hairy-nap outer layer (9). It has been estimated as containing 53% protein, 20% amino and neutral polysaccharide, 18% lipids, and approximately 4% ash. The whole structure makes up approximately 2% of the dry weight of the spore (14). A number of the proteins from the exosporia of B. cereus (7, 37) and B. anthracis (25,28,30) have been identified, most notably the B. anthracis surface-exposed glycoprotein antigen BclA (30, 31).The hydrophobic properties of Bacillus me...