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

Lekshmi Kailasa, Cassandra Terrya,1, Nicholas Abbotta, Robert Taylora, Nic Mullinb, Svetomir B. Tzokova, Sarah J. Todda, B. A. Wallacec, Jamie K. Hobbsb, Anne Moira, and Per A. Bullougha,2

aKrebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom; bDepartment of Physics and Astronomy, University of Sheffield, Sheffield S3 7RH, United Kingdom; and cDepartment of Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom

Edited by Richard M. Losick, Harvard University, Cambridge, MA, and approved August 5, 2011 (received for review June 14, 2011) of the family form highly resistant spores, carbohydrate (9). Proteins of the exosporium are likely to have which in the case of the pathogen B. anthracis act as the agents of intimate interactions with the environment and are also potential infection. The outermost layer, the exosporium, enveloping spores candidates for vaccines and ligands for spore detection (10, 11). of the B. cereus family as well as a number of Clostridia, plays roles in Two exosporium glycoproteins, BclA and BclB, have been spore adhesion, dissemination, targeting, and control. identified in B. anthracis (8, 12, 13). These contain a central tri- We have analyzed two naturally crystalline layers associated with meric collagen-like domain, a processed N-terminal domain an- the exosporium, one representing the “basal” layer to which the choring the protein to the basal layer, and a C-terminal head outermost spore layer (“hairy nap”) is attached, and the other likely domain whose structure has been elucidated to atomic resolution representing a subsurface (“parasporal”) layer. We have used elec- in the case of BclA (14). BclA filaments are the major components tron cryomicroscopy at a resolution of 0.8–0.6 nm and circular di- of the hairy nap (8, 13). ExsFA/BxpB and its homolog ExsFB are chroism spectroscopic measurements to reveal a highly α-helical required for attachment of BclA to the basal layer (15, 16). BclA, structure for both layers. The helices are assembled into 2D arrays ExsFA, and ExsY form high-molecular weight complexes, ExsY of “cups” or “crowns.” High-resolution atomic force microscopy of being required for the complete assembly of the exosporium (17– the outermost layer showed that the open ends of these cups face 19). The exosporium is likely to contain a number of other struc- the external environment and the highly immunogenic collagen-like tural proteins (many reviewed in ref. 4) as well as loosely bound fibrils of the hairy nap (BclA) are attached to this surface. Based on proteins such as immune inhibitor A (20) and arginase (21). our findings, we present a molecular model for the spore surface and The exosporium is likely to play multiple roles in the interaction propose how this surface can act as a semipermeable barrier and of the spore with its environment. For example, in B. anthracis, a matrix for binding of molecules involved in defense, germination BclA–integrin interaction promotes spore uptake by macrophages control, and other interactions of the spore with the environment. in vitro (22). The exosporium contributes protection against macrophage-mediated damage (21), whereas enzymes in the electron crystallography | cryoelectron microscopy | two-dimensional crystal exosporium (23) moderate responses to germinants. However, although the exosporium may have a role in natural acterial endospores are uniquely resistant survival structures infections, a BclA-containing exosporium is not required for an- Bthat result from a complex differentiation process. The thrax infection in animal models (24). The adherent properties dormancy, resistance (1), and widespread dispersal of bacterial exhibited by the exosporium (25) point to a role in attachment endospores mean that they are ubiquitous in the environment. to surfaces; this has important implications for development of Spores of a few species can be very effective infectious agents, decontamination protocols. remaining dormant in the environment until the opportunity for A substantial part of the basal layer of the exosporium from infection arises, in addition to being resistant to host defenses members of the B. cereus family takes the form of a natural 2D after infection. Formation of spores involves a tightly controlled crystal (7). Ball et al. have isolated fragments of this layer (type II sequence of steps of gene expression and cellular morphological crystals) from B. cereus, B. anthracis, and B. thuringiensis, de- change. Although these steps have been well-studied (2), the scribing the 3D structure to ∼30 Å resolution (3). The basic structure of the fully assembled spore remains poorly understood architecture of the crystalline basal layer, including unit cell at the molecular scale. Coupled with previous genetic and dimensions, is identical in all three species and the following morphological studies, our recent work on electron crystallog- description applies to all of them. The layer forms a network of raphy of spore proteins suggests that spores may present an at- “crown”-like structures, which were cautiously proposed to be tractive model system for exploring the molecular mechanisms trimeric although the density showed strong indications of higher driving the formation of complex supramolecular structures (3). symmetry. The open ends of the crowns are linked together to 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). Author contributions: L.K., C.T., B.A.W., A.M., and P.A.B. designed research; L.K., C.T., Some species, including, but by no means limited to, pathogens N.A., R.T., N.M., S.B.T., S.J.T., B.A.W., and P.A.B. performed research; L.K., C.T., N.A., R.T., N.M., S.B.T., B.A.W., J.K.H., A.M., and P.A.B. analyzed data; and L.K., B.A.W., such as Bacillus cereus and Clostridium botulinum (food-borne A.M., and P.A.B. wrote the paper. pathogens), B. thuringiensis (an insect pathogen), and B. anthracis The authors declare no conflict of interest. (animal and human pathogen), possess a further outermost layer This article is a PNAS Direct Submission. called the “exosporium” (7), a loosely fitting shell surrounding the 1Present address: Medical Research Council Prion Unit and Department of Neurodegen- coat. The volume between the spore coat and exosporium, the erative Disease, University College London Institute of Neurology, Queen Square, Lon- “interspace,” contains proteins but has not been well-studied. In don WC1N 3BG, United Kingdom. B. cereus and the closely related B. anthracis and B. thuringiensis, 2To whom correspondence should be addressed. E-mail: p.bullough@sheffield.ac.uk. “ ” fi the exosporium is made up of a basal layer supporting a la- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mentous “hairy nap” (8) and is composed of proteins, lipid, and 1073/pnas.1109419108/-/DCSupplemental.

16014e16019 | PNAS | September 20, 2011 | vol. 108 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1109419108 Downloaded by guest on September 24, 2021 form tunnels connecting one surface of the basal layer with the maps calculated from crystals embedded in a glucose/uranyl other (figure 5 in ref. 3). In some spores, there is evidence of formate mixture, we have been able to superimpose the 6-Å map another crystalline layer, a “parasporal” layer (type I crystals), onto a projection of the negatively stained protein assembly, located within the interspace of the spore. This layer does not whose approximate envelope is indicated by the filled gray circles appear to envelop the spore completely; it follows the same ar- (Fig. 1A). This allowed us to identify which threefold symmetry chitectural theme as the basal layer, but the crowns appear axis corresponds to the central symmetry axis of the trimeric smaller and more closely packed (3). The parasporal layer may crown. The envelope of the trimeric crown encloses a number of represent a different conformation of the same complex found in very dense circular features, which have the appearance of pro- the basal layer, and/or it may be an intermediate assembly state jected α-helices; we have circled the six densest features in an on the way to forming a mature exosporium basal layer. arbitrary cluster in magenta with their trimeric partners in blue. Our aim is to move on to the next stage in visualizing the detailed molecular architecture of the exosporium, to map Basal Layer Crystals: Projection Structure at 8 Å Resolution. Electron identified proteins onto the structure, and to understand how micrographs of exosporium fragments from B. cereus 10876 em- these various proteins determine function and architecture. A bedded in ice were recorded and 16 of them were processed. A few key questions are: (i) Which surface revealed in the B. cereus representative Fourier transform computed after unbending is family basal layer structure of Ball et al. (3) corresponds to the shown in Fig. S1B. Fourier phases were consistent with p6 plane ii group symmetry (Table S3). Table S4 shows that the mean phase spore surface exposed to the environment? ( ) Where and how is fi the hairy nap attached to this outer surface? (iii) Where are the error is signi cantly less than that expected for random phases “core” structural proteins located in the exosporium? These are (45°) to a resolution of 8 Å, and we have truncated all subsequent calculations to this resolution. Temperature factors applied to likely to be the proteins resistant to all washing regimes and fi 2 making up the crystalline lattice. (iv) Where are the more easily individual lms ranged from 546 to 1,000 Å . The calculated removed “accessory” proteins such as enzymes located? (v) What projection map is shown in Fig. 1B. The unit cell has dimensions of a b ∼ is the relation between the crystalline basal and parasporal lay- = = 80 Å. There is only one unique sixfold symmetry axis, so we can match this to the previously determined structure in neg- ers? To address these questions, we need higher resolution and ative stain (3), which has its sixfold axis centered on the crown to exploit noncrystallographic techniques to visualize the less (although only p3 symmetry had been imposed in this previous regular components of the spore surface. In this study, we have study). The approximate maximum extent of the projected density used electron cryomicroscopy (cryo-EM) to reveal the internal fi of a negatively stained crown is indicated by lled gray circles in structural details of the crystalline layers and circular dichroism Fig. 1B. The six highest unique density features have been circled (CD) spectroscopy to confirm the high helical content; atomic fi in magenta with their symmetry partners in blue; some or all of force microscopy (AFM) has revealed the ne details of the these could correspond to projections of α-helices. surfaces facing both the environment and the spore interior. CD Spectroscopy of Exosporium. The CD spectrum recorded from Results an exosporium preparation of B. cereus (Fig. 2) had the appear- Parasporal Layer Crystals: Projection Structure at 6 Å Resolution. ance of a protein with a high α-helical content, with a positive peak Electron micrographs of exosporium fragments from B. thur- at 193 nm and approximately equal magnitude negative peaks at ingiensis kurstaki HD1 embedded in glucose were recorded and 210 and 224 nm. Secondary structural analyses indicated a helical six of them were processed. A representative Fourier transform content of 75% with no β-sheet, 20% “other” (random coil), and is displayed in Fig. S1A. Phases were consistent with p3 symmetry ∼5% collagen. The low normalized root-mean-square deviation (Table S1). After phase origin alignment and averaging, the (NRMSD) value (0.07) indicated a strong correspondence be- mean phase error was significantly less than that expected for tween the data and the calculated secondary structure. random phases (90°) to a resolution of 6 Å, and we have trun- cated all subsequent calculations to this resolution (Table S2). AFM of Basal Layer Crystals. The height and phase images from Individual images were corrected for fall-off in the envelope of a folded fragment of B. cereus 10876 exosporium (Fig. 3 A and B) the contrast transfer function (26); temperature factors for in- show the two surfaces. Fig. 3B shows patches of ordered crys- dividual films ranged from 158 to 521 Å2. The projection map talline lattice on one side (indicated by the dashed white arrows) calculated from the averaged Fourier terms is shown in Fig. 1A. and disordered surface (indicated by the solid white arrows) on The unit cell has dimensions of a = b = ∼67 Å. By comparing the other side of the folded exosporium. The crystalline surface this map with the 3D map in negative stain (3) and also with appears to be made up of an array of “dome”-shaped structures.

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.

Kailas et al. PNAS | September 20, 2011 | vol. 108 | no. 38 | 16015 Downloaded by guest on September 24, 2021 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. 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 archi- tecture of the exosporium in B. cereus, B. anthracis, and B. thuringiensis is identical (3), meaning that we can directly com- pare 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 crys- tallographic 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 Fig. 2. CD spectrum of exosporium fragments from B. cereus. in Fig. 1 A and B) that are entirely consistent with end-on pro- jection views of α-helices and appear remarkably similar to he- lices seen in projection maps calculated from integral membrane Additional representative AFM images can be seen in Fig. S2. proteins (27); these features have diameters of ∼10 Å and are The ordered surface is more clearly illustrated in an ultra-high- packed with minimum center-to-center spacings on the order of resolution image of B. cereus exosporium obtained using tor- 10 Å, as expected for close-packed helical bundles. The CD sional tapping that reveals a studded surface of hexagonally spectrum of exosporium from B. cereus (Fig. 2) indicated a very close-packed units of ∼8-nm diameter (Fig. 3 C and D). The high α-helical content of ∼75%, which is qualitatively consistent exosporium in B. thuringiensis 4D11 strain retains the same basal with the observations by EM. Notably, the CD spectrum also layer architecture as B. cereus, but has little or no nap; therefore, indicated ∼5% polyproline (collagen-like) helix content; this is the outer surface of the basal layer can be visualized by AFM likely to arise from BclA and possibly other proteins of the hairy without obstruction from the hairy nap (Fig. 4). The height nap. We are unsure of the precise molecular composition of the images from the B. thuringiensis 4D11 (Fig. 4C) exosporium two crystal types, but a very approximate molecular mass of 100 fragments show that the crystalline lattice on one side of the B. kDa was estimated for the hexameric crown found in basal layer thuringiensis exosporium appears to be made up of hexagonally crystals (3). If we assume a 75% helical content, then each crown close-packed concave “cups” forming a honeycomb-like pattern. would contain ∼650 amino acids in a helical conformation Fig. 4B shows that the B. thuringiensis exosporium has two dif- equivalent to an ∼100-nm length of helix, which could span the ferent crystalline surfaces on opposite sides of the basal layer thickness of the crystal (as determined by AFM measurements; see below) about 20–25 times, equivalent to roughly three to four

A B A B

C D C D

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 Fig. 4. AFM height images of exosporium fragments from B. thuringiensis (C and D).] Dark to bright variation in color represents a change in height of 100 4D11. [Scale bars, 500 nm (A), 50 nm (B), and 10 nm (C and D).] Dark to bright nm (A) and 10 nm (C) and a change in phase of 40° (B) and 8° (D). Images A and variation in color represents a change in height of 80 nm (A) and 15 nm (B). B were taken in normal tapping mode using high-resolution diamond-like B is a magnified image of the square area depicted in A. C denotes the area carbon tips, and C and D were taken in torsional tapping mode using T-shaped indicated by the white arrow, and D represents the area indicated by cantilevers. The ordered surface is indicated by the dashed white arrows, and the black arrow in B. Images were taken in torsional tapping mode using the disordered surface is indicated by the solid white arrows in B. T-shaped cantilevers.

16016 | www.pnas.org/cgi/doi/10.1073/pnas.1109419108 Kailas et al. Downloaded by guest on September 24, 2021 crystal-spanning helices per subunit. If we examine Fig. 1B,wesee dome-shaped structures and the other side is highly disordered. one exceptionally dense feature (marked with an asterisk) with This disordered side is most likely to represent the hairy nap other less dense features (circled) clustered nearby at a center– collapsed onto the external surface of the spore. Therefore, the center distance of ∼10 Å. Other, less dense (circled) features ordered array of domes must correspond to the interior-facing nearby could also correspond to shorter, tilted, or more mobile side of the basal layer. The domes appear to have a diameter helices. Similar clusters of dense helix-like features can be seen in ranging between ∼7.5 and 9 nm that fits the unit cell dimensions the projection of parasporal layer crystals, but we have not been of the basal layer crystals determined by EM. In contrast to B. able to find any broad match of density features in the two maps. cereus, the AFM images from the B. thuringiensis 4D11 samples Ball et al. (3) had proposed that the type I structure originated show that both surfaces of the basal layer are well-ordered (Fig. from a parasporal layer located in the interspace between the 4), with one surface showing the same dome structures as seen in exosporium and the spore coat. It was suggested that this layer B. cereus. However, the other surface shows a honeycomb-like might represent an immature or nascent form of the type II pattern with concave cups. This cup view must correspond to the crystal, which makes up the true exosporium basal layer. The views into the crowns described by Ball et al. (3) for both B. unambiguous threefold and sixfold symmetries now determined cereus and B. thuringiensis strains and also inferred for B. for the parasporal and basal layer crowns, respectively, show that anthracis. Because this B. thuringiensis strain is found to have at the very least, the two forms must represent different assembly little or no nap and the AFM images from the B. cereus strain states. Although we can show that the two forms have features in show the inner surface to be made of domes, the side having the common such as the overall crown-like architecture and proba- concave cups must correspond to the external face of the basal ble clusters of α-helices traversing the layer, there are clearly layer. From this, we can infer that the crowns described in ref. 3 differences in detail. These differences may arise from both open out toward the external face of the spore. conformational and compositional differences within the sub- The use of high-resolution AFM to complement the EM work units. A further notable feature is that the density in parasporal has provided insights into the detailed architecture of the exo- layer crystals appears much more tightly packed than in the basal sporium. AFM studies had previously been done on spore sur- layer crystals, consistent with its smaller unit cell. Of more cer- faces (28–30), but the exosporium appeared to be featureless tain origin are the type II crystals, which make up a substantial (28) or to have a granular texture (30), as the hairy nap present part of the exosporium basal layer to which the hairy nap is at- on the exosporium would not have revealed the underlying or- tached. Because one surface of the basal layer must be involved dered structure of the basal layer. Because we had fragmented in the interaction with the hairy nap and the other surface is the exosporium as well as looked at B. cereus family strains with MICROBIOLOGY likely to interact with proteins deeper within the spore, the as- and without the hairy nap (but with identical basal layer archi- signment of the relative orientation of the crystalline basal lattice tecture), we were able to image both the outer and inner side of is essential if we are to model a 3D structure of the spore and to the crystalline basal layer and this has revealed the well-ordered understand how other spore proteins and factors from the ex- crystalline lattice. In our studies with fragmented exosporium, we ternal environment interact with the crystalline layers. We found have calculated the thickness of the exosporium in B. cereus to be from negative-stain electron micrographs that B. cereus 10876 ∼10 nm, whereas that in B. thuringiensis 4D11 is ∼4 nm. As this exosporium has a very clear hairy nap whereas B. thuringiensis B. thuringiensis sample was apparently devoid of the hairy nap, 4D11 has little or no nap. It should be noted, however, that the we can conclude that the crystalline basal lattice is ∼4-nm thick basic basal layer architecture is identical for all B. cereus, B. and the collapsed hairy nap is contributing to ∼6 nm in height on anthracis, and B. thuringienis strains examined, and in general B. the B. cereus basal layer. thuringiensis strains do have a hairy nap (3). The AFM images Fig. 5 shows a proposed model of the exosporium of the Ba- from the B. cereus samples (Fig. 3 and Fig. S2) suggest that one cillus cereus family, bringing together data from negative-stain side of the basal layer is made up of a well-ordered array of electron microscopy, cryo-EM, and AFM. One primary struc-

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.

Kailas et al. PNAS | September 20, 2011 | vol. 108 | no. 38 | 16017 Downloaded by guest on September 24, 2021 tural role for the basal layer is to act as a platform to which the small enough to exclude typical proteolytic enzymes but large hairy nap is attached. Our comparative AFM analysis of a B. enough to allow entry of spore germinants. The barrier may not cereus strain with nap and a very closely related B. thuringiensis be identically structured around the whole spore surface because strain without nap has established that the nap is attached to the a specialized “cap” structure directs the escape of the germi- surface corresponding to the crown openings. The positions of nating cell enclosed within the exosporium (31). (ii) The exo- the nap anchor points on the surface are unknown, but are most sporium is made up of interlinked cups opening to the external likely to be on the outer perimeter of the crowns. In Fig. 5, we environment of the spore. (iii) These cups are constructed in have placed BclA fibrils attached near the threefold connectors, large part from α-helices traversing some or all of the thickness but they could be elsewhere on this outer surface. This could be of the crystalline part of the basal layer; the bottom of the cups an attractive hypothesis, because these connections have three- could be closed off or might have a small depression or opening, fold symmetry and could then dock specifically onto the three- which would be revealed only with higher-resolution data. (iv) stranded collagen-like fibers found in the proteins of the nap The threefold symmetric linkers between the cups form open such as BclA (14) in a specific fashion. However, there is evi- caps over the relatively empty volume between crowns, and there dence that one BclA trimer is linked to only one EsxFA/BxpB could be docking sites for the hairy nap fibers in the vicinity of monomer (16), which would break such symmetry. At the base of these or elsewhere on the crown perimeter. The docking sites the hairy nap fibrils there is likely to be a complex of ExsF/BxpB may be formed by a complex between BclA (and/or potentially and ExsY proteins, which would make up part of the observed other glycoprotein homologs), ExsFA/BxpB, and ExsY and/or basal layer crystal structure. There are likely to be other less their cognate homologs ExsFB and CotY. (v) The cups could act abundant glycoprotein fibers, such as BclB, attached to the same as a matrix to sequester various additional proteins associated face; these have not been shown. with the exosporium such as arginase, alanine racemase, and so What are the protein–protein interactions that hold the forth. (vi) The tapering of the cups toward the interior of the crowns together in the lattice? This is an intriguing question, spore would facilitate the expected concave curvature of the because the exosporium is very flexible. ExsFA and BclA are inner surface of the exosporium as it wraps around the spore. clearly not essential to hold the lattice together, because exo- (vii) The exosporium is highly deformable, which may allow sporium from ΔexsFA and ΔbclA, mutants maintains the crystal a larger surface-to-surface contact area than would be achieved lattice (8, 15). We speculate that ExsY and CotY could be good with the more tightly wrapped spore coat. This could be an ad- candidates for major components of the lattice because a ΔexsY vantage for attachment and spore dispersal in the environment. strain of B. cereus makes only a small terminal cap of exosporium This deformability may come partly through flexible cross-links and a ΔexsY ΔcotY strain is completely devoid of exosporium (17, between cups and/or partly through weak boundaries or defects 18, 31). It is possible that the main bulk of the exosporium between crystalline domains that would allow bending and contains ExsY whereas the cap contains CotY, which is 85% folding. To test the structural aspects of this model, we require identical to ExsY (17). CotY could confer subtly different higher-resolution structural information in three dimensions. structural properties on the cap compared with the rest of the exosporium. The projection structure of the basal layer crystals Materials and Methods shows that the crystalline part of the basal layer is made up of Exosporium Preparation. Crystalline fragments of exosporium were prepared hexameric assemblies linked together. The putative helices as described in ref. 23, which included a French press step and salt and de- clustered around the sixfold symmetry axis (Fig. 1B) could have tergent washes. A detailed description of the spore preparation and exo- roles in lining the wall of the crown cavity, whereas the putative sporium isolation is given in SI Materials and Methods. Basal layer crystals peripheral helices could have roles in defining pore size and were from B. cereus American Type Culture Collection (ATCC) 10876 and also linking individual crowns together through helix–helix and B. thuringiensis 4D11. Parasporal layer crystals were from B. thuringiensis other interactions. kurstaki HD1. The exosporium clearly forms a semipermeable barrier, be- Electron Microscopy. Crystals from B. cereus ATCC 10876 were embedded in cause it fully encloses the spore. The projection map shown in vitreous ice using a Vitrobot freeze plunger (FEI) and those from B. thur- Fig. 1B shows relatively low density at the threefold crystallo- ingiensis kurstaki HD1 were embedded in 1% glucose or 1% glucose/1% graphic symmetry positions, which coincide with the “linkers” uranyl formate on carbon-coated 400-mesh EM grids. Samples were moun- revealed in the 3D negative-stain map. With the caveat that ted on a liquid nitrogen-cooled Oxford Instruments CT3500 Cryotrans cold there is always additional uncertainty in interpretation of fea- stage in a Philips CM200 FEG transmission electron microscope operating at tures on symmetry axes due to the enhanced noise level, this is 200 kV. Low-dose images (27) were collected at a nominal magnification of consistent with the large stain-filled volume that is seen to be 66,000 with an exposure of ∼10 electrons/Å2 in 1 s and recorded on Kodak capped by the threefold linker (3). Centered on these threefold SO-163 film. positions in the unstained projection (Fig. 1B), we have drawn a circle (dotted in red) whose circumference just grazes the three Image Processing. The developed micrographs were digitized on a Zeiss SCAI scanner. Digitized images were imported into 2dx_Image (34) and subjected prominent symmetry-related putative helices. This circle is less to rounds of crystal unbending (27). For each image, possible plane group than 20 Å across, suggesting a minimum diameter for the cavity symmetries were determined using ALLSPACE within the 2dx suite. Fourier capped by the linker; this cavity is connected to the three pores amplitudes and phases generated for individual images from 2dx_Image distributed around the threefold axis (Fig. 5). A 20-Å constric- were refined to a common phase origin using ORIGTILTK within the MRC tion is entirely consistent with pore sizes estimated from per- software suite (27). SCALEIMAMP was used to determine B-factors for meation experiments (32). A passage of this size is clearly large amplitudes (26), and processed images were merged using the MRC suite of enough to allow entry of the germinants alanine or programs. Merged phases and amplitudes were averaged with AVRGAMPHS inosine (33) but not degradative enzymes or antibodies; equally, and used to generate figure-of-merit weighted projection maps (35). it would act as a barrier to the loss of proteins from the in- terspace between coat and exosporium. CD Spectroscopy of Exosporium. A B. cereus exosporium suspension was ex- Fig. 5 summarizes the main conclusions of this paper, although amined 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 it should be noted that in different strains the basal layer wavelength range 190–280 nm with a step size of 1 nm using an Aviv 62ds thickness may vary depending on whether the layer has a single spectropolarimeter with a detector acceptance angle of $90° (essential for tier or is made up of multiple layers stuck together: (i) The samples such as exosporium that scatter light). The data were processed exosporium forms a semipermeable barrier enclosing the spore using CDtool software (36). Sample scans were averaged and the baseline and breached by passages as narrow as ∼20 Å in diameter and scans of buffer were subtracted. The resulting spectrum was smoothed using

16018 | www.pnas.org/cgi/doi/10.1073/pnas.1109419108 Kailas et al. Downloaded by guest on September 24, 2021 a Savitsky–Golay filter. The protein concentration was determined using the ∼15–20 N/m and an ∼1-nm tip radius. Height and phase images were col- minimum NRMSD method (37). Secondary structure content was analyzed lected simultaneously. Phase images were generated by recording the phase using Selmat3 analysis software with the SP175 reference dataset (38). 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 Atomic Force Microscopy. An exosporium suspension was applied to an ∼200- properties. Under normal imaging conditions, dark areas are soft and/or Å-thick carbon film supported on a copper Maxtaform finder grid (Agar sticky, whereas bright areas are stiff and less sticky. Scientific). The grid was imaged under ambient conditions in Tapping mode using a Dimension 3100 AFM with Nanoscope IV controller (Veeco Instru- ACKNOWLEDGMENTS. We thank Prof. Pete Artymiuk, Dr. Pu Qian, Dr. John ments). High-resolution carbon tips (∼1-nm tip radius/DP15/HI9Res-C; Mik- Olsen, and Mr. Chris Glover (University of Sheffield) for helpful discussions roMasch) and silicon tips (<10-nm tip radius; Olympus) of typical spring and suggestions with the drawing of the schematic diagram, and Dr. Christa constant 40 N/m and resonant frequency of ∼300 KHz were used for imag- Weber (University of Sheffield) for assistance with the use of high-resolution ing. Additionally, “torsional tapping” measurements were carried out using AFM tips. The authors (L.K., C.T., R.T., S.B.T., S.J.T., B.A.W., A.M., and P.A.B.) a Veeco Multimode AFM with a Nanoscope IIIa controller, extender elec- thank the Biotechnology and Biological Sciences Research Council and (N.M. tronics, and signal access module as described in ref. 39. The T-shaped can- and J.K.H.) the Medical Research Council and Engineering and Physical tilevers (TL01 HiRes-C; MikroMasch) used had a torsional spring constant of Sciences Research Council for funding.

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