Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale

Abstract

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.

Bacterial endospores are uniquely resistant survival structures that result from a complex differentiation process. The dormancy, resistance (1), and widespread dispersal of bacterial endospores mean that they are ubiquitous in the environment. Spores of a few species can be very effective infectious agents, remaining dormant in the environment until the opportunity for infection arises, in addition to being resistant to host defenses after infection. Formation of spores involves a tightly controlled sequence of steps of gene expression and cellular morphological change. Although these steps have been well-studied (2), the structure of the fully assembled spore remains poorly understood at the molecular scale. Coupled with previous genetic and morphological studies, our recent work on electron crystallography of spore proteins suggests that spores may present an attractive model system for exploring the molecular mechanisms driving the formation of complex supramolecular structures (3).

Spores contain a relatively dehydrated cellular core that is membrane-bound and surrounded by a peptidoglycan cortex and an outer protective coat containing multiple protein layers (4–6). Some species, including, but by no means limited to, pathogens such as Bacillus cereus and Clostridium botulinum (food-borne pathogens), B. thuringiensis (an insect pathogen), and B. anthracis (animal and human pathogen), possess a further outermost layer called the “exosporium” (7), a loosely fitting shell surrounding the coat. The volume between the spore coat and exosporium, the “interspace,” contains proteins but has not been well-studied. In B. cereus and the closely related B. anthracis and B. thuringiensis, the exosporium is made up of a “basal” layer supporting a filamentous “hairy nap” (8) and is composed of proteins, lipid, and carbohydrate (9). Proteins of the exosporium are likely to have intimate interactions with the environment and are also potential candidates for vaccines and ligands for spore detection (10, 11).

Two exosporium glycoproteins, BclA and BclB, have been identified in B. anthracis (8, 12, 13). These contain a central trimeric collagen-like domain, a processed N-terminal domain anchoring the protein to the basal layer, and a C-terminal head domain whose structure has been elucidated to atomic resolution in the case of BclA (14). BclA filaments are the major components of the hairy nap (8, 13). ExsFA/BxpB and its homolog ExsFB are required for attachment of BclA to the basal layer (15, 16). BclA, ExsFA, and ExsY form high-molecular weight complexes, ExsY being required for the complete assembly of the exosporium (17–19). The exosporium is likely to contain a number of other structural proteins (many reviewed in ref. 4) as well as loosely bound proteins such as immune inhibitor A (20) and arginase (21).

The exosporium is likely to play multiple roles in the interaction of the spore with its environment. For example, in B. anthracis, BclA–integrin interaction promotes spore uptake by macrophages in vitro (22). The exosporium contributes protection against macrophage-mediated damage (21), whereas enzymes in the exosporium (23) moderate responses to germinants. However, although the exosporium may have a role in natural anthrax infections, a BclA-containing exosporium is not required for anthrax infection in animal models (24). The adherent properties exhibited by the exosporium (25) point to a role in attachment to surfaces; this has important implications for development of decontamination protocols.

A substantial part of the basal layer of the exosporium from members of the B. cereus family takes the form of a natural 2D crystal (7). Ball et al. have isolated fragments of this layer (type II crystals) from B. cereus, B. anthracis, and B. thuringiensis, describing the 3D structure to ∼30 Å resolution (3). The basic architecture of the crystalline basal layer, including unit cell dimensions, is identical in all three species and the following description applies to all of them. The layer forms a network of “crown”-like structures, which were cautiously proposed to be trimeric although the density showed strong indications of higher symmetry. The open ends of the crowns are linked together to form tunnels connecting one surface of the basal layer with the other (figure 5 in ref. 3). In some spores, there is evidence of another crystalline layer, a “parasporal” layer (type I crystals), located within the interspace of the spore. This layer does not appear to envelop the spore completely; it follows the same architectural theme as the basal layer, but the crowns appear smaller and more closely packed (3). The parasporal layer may represent a different conformation of the same complex found in the basal layer, and/or it may be an intermediate assembly state on the way to forming a mature exosporium basal layer.

Our aim is to move on to the next stage in visualizing the detailed molecular architecture of the exosporium, to map identified proteins onto the structure, and to understand how these various proteins determine function and architecture. A few key questions are: (i) Which surface revealed in the B. cereus family basal layer structure of Ball et al. (3) corresponds to the spore surface exposed to the environment? (ii) Where and how is the hairy nap attached to this outer surface? (iii) Where are the “core” structural proteins located in the exosporium? These are likely to be the proteins resistant to all washing regimes and making up the crystalline lattice. (iv) Where are the more easily removed “accessory” proteins such as enzymes located? (v) What is the relation between the crystalline basal and parasporal layers? To address these questions, we need higher resolution and to exploit noncrystallographic techniques to visualize the less regular components of the spore surface. In this study, we have used electron cryomicroscopy (cryo-EM) to reveal the internal structural details of the crystalline layers and circular dichroism (CD) spectroscopy to confirm the high helical content; atomic force microscopy (AFM) has revealed the fine details of the surfaces facing both the environment and the spore interior.

Results

Parasporal Layer Crystals: Projection Structure at 6 Å Resolution.

Electron micrographs of exosporium fragments from B. thuringiensis kurstaki HD1 embedded in glucose were recorded and six of them were processed. A representative Fourier transform is displayed in Fig. S1A. Phases were consistent with p3 symmetry (Table S1). After phase origin alignment and averaging, the mean phase error was significantly less than that expected for random phases (90°) to a resolution of 6 Å, and we have truncated all subsequent calculations to this resolution (Table S2). Individual images were corrected for fall-off in the envelope of the contrast transfer function (26); temperature factors for individual films ranged from 158 to 521 Ų. The projection map calculated from the averaged Fourier terms is shown in Fig. 1A. The unit cell has dimensions of a = b = ∼67 Å. By comparing this map with the 3D map in negative stain (3) and also with maps calculated from crystals embedded in a glucose/uranyl formate mixture, we have been able to superimpose the 6-Å map onto a projection of the negatively stained protein assembly, whose approximate envelope is indicated by the filled gray circles (Fig. 1A). This allowed us to identify which threefold symmetry axis corresponds to the central symmetry axis of the trimeric crown. The envelope of the trimeric crown encloses a number of very dense circular features, which have the appearance of projected α-helices; we have circled the six densest features in an arbitrary cluster in magenta with their trimeric partners in blue.

Fig. 1.

Projection maps for the two crystal types. (A) Projection map with p3 symmetry at 6 Å resolution for parasporal layer crystals. Contours are at intervals ∼0.3× root-mean-square density. Contours below average density are not shown. The approximate projection of the envelope of an individual trimer as determined by Ball et al. (3) is indicated by the filled gray circle. The six densest features are circled in magenta with their symmetry mates in blue. a = b = ∼67 Å. (B) Projection map with p6 symmetry at 8 Å resolution for basal layer crystals. Features are as described above. a = b = ∼80 Å.

Basal Layer Crystals: Projection Structure at 8 Å Resolution.

Electron micrographs of exosporium fragments from B. cereus 10876 embedded in ice were recorded and 16 of them were processed. A representative Fourier transform computed after unbending is shown in Fig. S1B. Fourier phases were consistent with p6 plane group symmetry (Table S3). Table S4 shows that the mean phase error is significantly less than that expected for random phases (45°) to a resolution of 8 Å, and we have truncated all subsequent calculations to this resolution. Temperature factors applied to individual films ranged from 546 to 1,000 Ų. The calculated projection map is shown in Fig. 1B. The unit cell has dimensions of a = b = ∼80 Å. There is only one unique sixfold symmetry axis, so we can match this to the previously determined structure in negative stain (3), which has its sixfold axis centered on the crown (although only p3 symmetry had been imposed in this previous study). The approximate maximum extent of the projected density of a negatively stained crown is indicated by filled gray circles in Fig. 1B. The six highest unique density features have been circled in magenta with their symmetry partners in blue; some or all of these could correspond to projections of α-helices.

CD Spectroscopy of Exosporium.

The CD spectrum recorded from an exosporium preparation of B. cereus (Fig. 2) had the appearance of a protein with a high α-helical content, with a positive peak at 193 nm and approximately equal magnitude negative peaks at 210 and 224 nm. Secondary structural analyses indicated a helical content of 75% with no β-sheet, 20% “other” (random coil), and ∼5% collagen. The low normalized root-mean-square deviation (NRMSD) value (0.07) indicated a strong correspondence between the data and the calculated secondary structure.

Fig. 2.

CD spectrum of exosporium fragments from B. cereus.

AFM of Basal Layer Crystals.

The height and phase images from a folded fragment of B. cereus 10876 exosporium (Fig. 3 A and B) show the two surfaces. Fig. 3B shows patches of ordered crystalline lattice on one side (indicated by the dashed white arrows) and disordered surface (indicated by the solid white arrows) on the other side of the folded exosporium. The crystalline surface appears to be made up of an array of “dome”-shaped structures. Additional representative AFM images can be seen in Fig. S2. The ordered surface is more clearly illustrated in an ultra-high-resolution image of B. cereus exosporium obtained using torsional tapping that reveals a studded surface of hexagonally close-packed units of ∼8-nm diameter (Fig. 3 C and D). The exosporium in B. thuringiensis 4D11 strain retains the same basal layer architecture as B. cereus, but has little or no nap; therefore, the outer surface of the basal layer can be visualized by AFM without obstruction from the hairy nap (Fig. 4). The height images from the B. thuringiensis 4D11 (Fig. 4C) exosporium fragments show that the crystalline lattice on one side of the B. thuringiensis exosporium appears to be made up of hexagonally close-packed concave “cups” forming a honeycomb-like pattern. Fig. 4B shows that the B. thuringiensis exosporium has two different crystalline surfaces on opposite sides of the basal layer with no apparent disorder on either side. An additional image with a wider field of view showing the honeycomb pattern can be seen in Fig. S3.

Fig. 3.

AFM height (A and C) and phase (B and D) images showing a folded fragment of B. cereus exosporium. [Scale bars, 200 nm (A and B) and 50 nm (C and D).] Dark to bright variation in color represents a change in height of 100 nm (A) and 10 nm (C) and a change in phase of 40° (B) and 8° (D). Images A and B were taken in normal tapping mode using high-resolution diamond-like carbon tips, and C and D were taken in torsional tapping mode using T-shaped cantilevers. The ordered surface is indicated by the dashed white arrows, and the disordered surface is indicated by the solid white arrows in B.

Fig. 4.

AFM height images of exosporium fragments from B. thuringiensis 4D11. [Scale bars, 500 nm (A), 50 nm (B), and 10 nm (C and D).] Dark to bright variation in color represents a change in height of 80 nm (A) and 15 nm (B). B is a magnified image of the square area depicted in A. C denotes the area indicated by the white arrow, and D represents the area indicated by the black arrow in B. Images were taken in torsional tapping mode using T-shaped cantilevers.

Discussion

One of the main advantages of the B. cereus family exosporium for structural studies is its in vivo crystalline nature, which allows analysis by electron crystallography. Moreover, the basic architecture of the exosporium in B. cereus, B. anthracis, and B. thuringiensis is identical (3), meaning that we can directly compare different strains for variations in structural detail and draw general conclusions for the whole B. cereus family. In this study, we report exceptionally high resolution (6–8 Å) electron crystallographic projection maps and have thus taken the first step in building up a high-resolution 3D map of the exosporium. Both parasporal and basal layer crystals show density features (circled in Fig. 1 A and B) that are entirely consistent with end-on projection views of α-helices and appear remarkably similar to helices seen in projection maps calculated from integral membrane proteins (27); these features have diameters of ∼10 Å and are packed with minimum center-to-center spacings on the order of 10 Å, as expected for close-packed helical bundles. The CD spectrum of exosporium from B. cereus (Fig. 2) indicated a very high α-helical content of ∼75%, which is qualitatively consistent with the observations by EM. Notably, the CD spectrum also indicated ∼5% polyproline (collagen-like) helix content; this is likely to arise from BclA and possibly other proteins of the hairy nap. We are unsure of the precise molecular composition of the two crystal types, but a very approximate molecular mass of 100 kDa was estimated for the hexameric crown found in basal layer crystals (3). If we assume a 75% helical content, then each crown would contain ∼650 amino acids in a helical conformation equivalent to an ∼100-nm length of helix, which could span the thickness of the crystal (as determined by AFM measurements; see below) about 20–25 times, equivalent to roughly three to four crystal-spanning helices per subunit. If we examine Fig. 1B, we see one exceptionally dense feature (marked with an asterisk) with other less dense features (circled) clustered nearby at a center–center distance of ∼10 Å. Other, less dense (circled) features nearby could also correspond to shorter, tilted, or more mobile helices. Similar clusters of dense helix-like features can be seen in the projection of parasporal layer crystals, but we have not been able to find any broad match of density features in the two maps.

Ball et al. (3) had proposed that the type I structure originated from a parasporal layer located in the interspace between the exosporium and the spore coat. It was suggested that this layer might represent an immature or nascent form of the type II crystal, which makes up the true exosporium basal layer. The unambiguous threefold and sixfold symmetries now determined for the parasporal and basal layer crowns, respectively, show that at the very least, the two forms must represent different assembly states. Although we can show that the two forms have features in common such as the overall crown-like architecture and probable clusters of α-helices traversing the layer, there are clearly differences in detail. These differences may arise from both conformational and compositional differences within the subunits. A further notable feature is that the density in parasporal layer crystals appears much more tightly packed than in the basal layer crystals, consistent with its smaller unit cell. Of more certain origin are the type II crystals, which make up a substantial part of the exosporium basal layer to which the hairy nap is attached. Because one surface of the basal layer must be involved in the interaction with the hairy nap and the other surface is likely to interact with proteins deeper within the spore, the assignment of the relative orientation of the crystalline basal lattice is essential if we are to model a 3D structure of the spore and to understand how other spore proteins and factors from the external environment interact with the crystalline layers. We found from negative-stain electron micrographs that B. cereus 10876 exosporium has a very clear hairy nap whereas B. thuringiensis 4D11 has little or no nap. It should be noted, however, that the basic basal layer architecture is identical for all B. cereus, B. anthracis, and B. thuringienis strains examined, and in general B. thuringiensis strains do have a hairy nap (3). The AFM images from the B. cereus samples (Fig. 3 and Fig. S2) suggest that one side of the basal layer is made up of a well-ordered array of dome-shaped structures and the other side is highly disordered. This disordered side is most likely to represent the hairy nap collapsed onto the external surface of the spore. Therefore, the ordered array of domes must correspond to the interior-facing side of the basal layer. The domes appear to have a diameter ranging between ∼7.5 and 9 nm that fits the unit cell dimensions of the basal layer crystals determined by EM. In contrast to B. cereus, the AFM images from the B. thuringiensis 4D11 samples show that both surfaces of the basal layer are well-ordered (Fig. 4), with one surface showing the same dome structures as seen in B. cereus. However, the other surface shows a honeycomb-like pattern with concave cups. This cup view must correspond to the views into the crowns described by Ball et al. (3) for both B. cereus and B. thuringiensis strains and also inferred for B. anthracis. Because this B. thuringiensis strain is found to have little or no nap and the AFM images from the B. cereus strain show the inner surface to be made of domes, the side having the concave cups must correspond to the external face of the basal layer. From this, we can infer that the crowns described in ref. 3 open out toward the external face of the spore.

The use of high-resolution AFM to complement the EM work has provided insights into the detailed architecture of the exosporium. AFM studies had previously been done on spore surfaces (28–30), but the exosporium appeared to be featureless (28) or to have a granular texture (30), as the hairy nap present on the exosporium would not have revealed the underlying ordered structure of the basal layer. Because we had fragmented the exosporium as well as looked at B. cereus family strains with and without the hairy nap (but with identical basal layer architecture), we were able to image both the outer and inner side of the crystalline basal layer and this has revealed the well-ordered crystalline lattice. In our studies with fragmented exosporium, we have calculated the thickness of the exosporium in B. cereus to be ∼10 nm, whereas that in B. thuringiensis 4D11 is ∼4 nm. As this B. thuringiensis sample was apparently devoid of the hairy nap, we can conclude that the crystalline basal lattice is ∼4-nm thick and the collapsed hairy nap is contributing to ∼6 nm in height on the B. cereus basal layer.

Fig. 5 shows a proposed model of the exosporium of the Bacillus cereus family, bringing together data from negative-stain electron microscopy, cryo-EM, and AFM. One primary structural role for the basal layer is to act as a platform to which the hairy nap is attached. Our comparative AFM analysis of a B. cereus strain with nap and a very closely related B. thuringiensis strain without nap has established that the nap is attached to the surface corresponding to the crown openings. The positions of the nap anchor points on the surface are unknown, but are most likely to be on the outer perimeter of the crowns. In Fig. 5, we have placed BclA fibrils attached near the threefold connectors, but they could be elsewhere on this outer surface. This could be an attractive hypothesis, because these connections have threefold symmetry and could then dock specifically onto the three-stranded collagen-like fibers found in the proteins of the nap such as BclA (14) in a specific fashion. However, there is evidence that one BclA trimer is linked to only one EsxFA/BxpB monomer (16), which would break such symmetry. At the base of the hairy nap fibrils there is likely to be a complex of ExsF/BxpB and ExsY proteins, which would make up part of the observed basal layer crystal structure. There are likely to be other less abundant glycoprotein fibers, such as BclB, attached to the same face; these have not been shown.

Fig. 5.

A schematic diagram illustrating a possible model for the exosporium of the B. cereus family. This outermost layer of the spore contains a 2D lattice of cups opening out to the environment on the convex outer face of the layer. On the concave inner face, the cups may or may not be completely sealed, but there are pores or channels between them. The collagen-like fibrils of the nap are shown schematically attached to the convex face, terminating in the globular tumor necrosis factor-like C-terminal domain. The lengths of the fibrils vary between strains, and those shown here are not necessarily to scale. (Inset) A higher-magnification schematic view of a small segment of the exosporium. The crystalline basal layer is represented by the 3D surface determined for B. cereus by Ball et al. (3). The crowns are indicated schematically to have a high α-helical content, and we have shown the fibers of BclA to be docked in the vicinity of the threefold symmetric linkers between the crowns. This may be via ExsFA/ExsFB, shown as small pillars anchoring BclA to the crystalline matrix. However, the docking sites may be elsewhere on the crown perimeter.

What are the protein–protein interactions that hold the crowns together in the lattice? This is an intriguing question, because the exosporium is very flexible. ExsFA and BclA are clearly not essential to hold the lattice together, because exosporium from ΔexsFA and ΔbclA, mutants maintains the crystal lattice (8, 15). We speculate that ExsY and CotY could be good candidates for major components of the lattice because a ΔexsY strain of B. cereus makes only a small terminal cap of exosporium and a ΔexsY ΔcotY strain is completely devoid of exosporium (17, 18, 31). It is possible that the main bulk of the exosporium contains ExsY whereas the cap contains CotY, which is 85% identical to ExsY (17). CotY could confer subtly different structural properties on the cap compared with the rest of the exosporium. The projection structure of the basal layer crystals shows that the crystalline part of the basal layer is made up of hexameric assemblies linked together. The putative helices clustered around the sixfold symmetry axis (Fig. 1B) could have roles in lining the wall of the crown cavity, whereas the putative peripheral helices could have roles in defining pore size and also linking individual crowns together through helix–helix and other interactions.

The exosporium clearly forms a semipermeable barrier, because it fully encloses the spore. The projection map shown in Fig. 1B shows relatively low density at the threefold crystallographic symmetry positions, which coincide with the “linkers” revealed in the 3D negative-stain map. With the caveat that there is always additional uncertainty in interpretation of features on symmetry axes due to the enhanced noise level, this is consistent with the large stain-filled volume that is seen to be capped by the threefold linker (3). Centered on these threefold positions in the unstained projection (Fig. 1B), we have drawn a circle (dotted in red) whose circumference just grazes the three prominent symmetry-related putative helices. This circle is less than 20 Å across, suggesting a minimum diameter for the cavity capped by the linker; this cavity is connected to the three pores distributed around the threefold axis (Fig. 5). A 20-Å constriction is entirely consistent with pore sizes estimated from permeation experiments (32). A passage of this size is clearly large enough to allow entry of the endospore germinants alanine or inosine (33) but not degradative enzymes or antibodies; equally, it would act as a barrier to the loss of proteins from the interspace between coat and exosporium.

Fig. 5 summarizes the main conclusions of this paper, although it should be noted that in different strains the basal layer thickness may vary depending on whether the layer has a single tier or is made up of multiple layers stuck together: (i) The exosporium forms a semipermeable barrier enclosing the spore and breached by passages as narrow as ∼20 Å in diameter and small enough to exclude typical proteolytic enzymes but large enough to allow entry of spore germinants. The barrier may not be identically structured around the whole spore surface because a specialized “cap” structure directs the escape of the germinating cell enclosed within the exosporium (31). (ii) The exosporium is made up of interlinked cups opening to the external environment of the spore. (iii) These cups are constructed in large part from α-helices traversing some or all of the thickness of the crystalline part of the basal layer; the bottom of the cups could be closed off or might have a small depression or opening, which would be revealed only with higher-resolution data. (iv) The threefold symmetric linkers between the cups form open caps over the relatively empty volume between crowns, and there could be docking sites for the hairy nap fibers in the vicinity of these or elsewhere on the crown perimeter. The docking sites may be formed by a complex between BclA (and/or potentially other glycoprotein homologs), ExsFA/BxpB, and ExsY and/or their cognate homologs ExsFB and CotY. (v) The cups could act as a matrix to sequester various additional proteins associated with the exosporium such as arginase, alanine racemase, and so forth. (vi) The tapering of the cups toward the interior of the spore would facilitate the expected concave curvature of the inner surface of the exosporium as it wraps around the spore. (vii) The exosporium is highly deformable, which may allow a larger surface-to-surface contact area than would be achieved with the more tightly wrapped spore coat. This could be an advantage for attachment and spore dispersal in the environment. This deformability may come partly through flexible cross-links between cups and/or partly through weak boundaries or defects between crystalline domains that would allow bending and folding. To test the structural aspects of this model, we require higher-resolution structural information in three dimensions.

Materials and Methods

Exosporium Preparation.

Crystalline fragments of exosporium were prepared as described in ref. 23, which included a French press step and salt and detergent washes. A detailed description of the spore preparation and exosporium isolation is given in SI Materials and Methods. Basal layer crystals were from B. cereus American Type Culture Collection (ATCC) 10876 and B. thuringiensis 4D11. Parasporal layer crystals were from B. thuringiensis kurstaki HD1.

Electron Microscopy.

Crystals from B. cereus ATCC 10876 were embedded in vitreous ice using a Vitrobot freeze plunger (FEI) and those from B. thuringiensis kurstaki HD1 were embedded in 1% glucose or 1% glucose/1% uranyl formate on carbon-coated 400-mesh EM grids. Samples were mounted on a liquid nitrogen-cooled Oxford Instruments CT3500 Cryotrans cold stage in a Philips CM200 FEG transmission electron microscope operating at 200 kV. Low-dose images (27) were collected at a nominal magnification of 66,000 with an exposure of ∼10 electrons/Ų in 1 s and recorded on Kodak SO-163 film.

Image Processing.

The developed micrographs were digitized on a Zeiss SCAI scanner. Digitized images were imported into 2dx_Image (34) and subjected to rounds of crystal unbending (27). For each image, possible plane group symmetries were determined using ALLSPACE within the 2dx suite. Fourier amplitudes and phases generated for individual images from 2dx_Image were refined to a common phase origin using ORIGTILTK within the MRC software suite (27). SCALEIMAMP was used to determine B-factors for amplitudes (26), and processed images were merged using the MRC suite of programs. Merged phases and amplitudes were averaged with AVRGAMPHS and used to generate figure-of-merit weighted projection maps (35).

CD Spectroscopy of Exosporium.

A B. cereus exosporium suspension was examined in a demountable Suprasil quartz cell (Hellma) with a path length of 0.01 cm. Three repeats of the spectrum were measured at 10 °C over the wavelength range 190–280 nm with a step size of 1 nm using an Aviv 62ds spectropolarimeter with a detector acceptance angle of ≥90° (essential for samples such as exosporium that scatter light). The data were processed using CDtool software (36). Sample scans were averaged and the baseline scans of buffer were subtracted. The resulting spectrum was smoothed using a Savitsky–Golay filter. The protein concentration was determined using the minimum NRMSD method (37). Secondary structure content was analyzed using Selmat3 analysis software with the SP175 reference dataset (38).

Atomic Force Microscopy.

An exosporium suspension was applied to an ∼200-Å-thick carbon film supported on a copper Maxtaform finder grid (Agar Scientific). The grid was imaged under ambient conditions in Tapping mode using a Dimension 3100 AFM with Nanoscope IV controller (Veeco Instruments). High-resolution carbon tips (∼1-nm tip radius/DP15/HI′Res-C; MikroMasch) and silicon tips (<10-nm tip radius; Olympus) of typical spring constant 40 N/m and resonant frequency of ∼300 KHz were used for imaging. Additionally, “torsional tapping” measurements were carried out using a Veeco Multimode AFM with a Nanoscope IIIa controller, extender electronics, and signal access module as described in ref. 39. The T-shaped cantilevers (TL01 HiRes-C; MikroMasch) used had a torsional spring constant of ∼15–20 N/m and an ∼1-nm tip radius. Height and phase images were collected simultaneously. Phase images were generated by recording the phase lag of the cantilever oscillation relative to the drive signal given to the piezo driver and given contrast that is indicative of mechanical and adhesive properties. Under normal imaging conditions, dark areas are soft and/or sticky, whereas bright areas are stiff and less sticky.