Molecular tiling on the surface of a bacterial spore – the exosporium of the Bacillus anthracis/cereus/thuringiensis group

Summary Bacteria of the genera Bacillus and Clostridium form highly resistant spores, which in the case of some pathogens act as the infectious agents. An exosporium forms the outermost layer of some spores; it plays roles in protection, adhesion, dissemination, host targeting in pathogens and germination control. The exosporium of the Bacillus cereus group, including the anthrax pathogen, contains a 2D‐crystalline basal layer, overlaid by a hairy nap. BclA and related proteins form the hairy nap, and require ExsFA (BxpB) for their localization on the basal layer. Until now, the identity of the main structural protein components of the basal layer was unknown. We demonstrate here that ExsY forms one of the essential components. Through heterologous expression in Escherichia coli, we also demonstrate that ExsY can self‐assemble into ordered 2D arrays that mimic the structure of the exosporium basal layer. Self‐assembly is likely to play an important role in the construction of the exosporium. The ExsY array is stable to heat and chemical denaturants, forming a robust layer that would contribute to overall spore resistance. Our structural analysis also provides novel insight into the location of other molecular components anchored onto the exosporium, such as BclA and ExsFA.


. A proportion of exosporium crystal fragments are solubilised and broken into small spheroidal particles after SDS treatment.
Small 'spheroidal particles' 10 -45 nm in dimension plus some small crystalline fragments that do not pellet during centrifugation are observed in the soluble fraction after incubation of B. cereus 10876 exosporium crystals with 1% SDS and analysis by negative stain electron microscopy. Scale bar, 100 nm.
(A) Large amorphous aggregates and small globular particles were found after treatment with 8M urea, 2% SDS, 2 M DTT and 95° C heating for 20 minutes. (B) Computer Fourier transform of (A) indicates the loss of an ordered lattice. Scale bar, 100 nm. Internal phase residuals were determined from spots of IQ1-1Q5 to 20 Å resolution (Crowther et al., 1996, Valpuesta et al., 1994. The values marked with * are acceptable candidates for the symmetry as the experimental phase residual is better than that expected, based on the signal-to-noise ratio. Internal phase residuals were determined from spots of IQ1-1Q5 to 20 Å resolution (Crowther et al., 1996, Valpuesta et al., 1994. The values marked with * are acceptable candidates for the symmetry as the experimental phase residual is better than that expected, based on the signal-to-noise ratio. Lattice lines showing the phase variation along the z* axis in ° (top panels) and amplitude variation in arbitrary units (lower panels). Horizontal axis displays reciprocal distance from the origin of the lattice line. Standard error of fitted amplitude and phase values is represented in the error bars.

Supplementary Methods
Endospore preparation. Vegetative cells were grown in nutrient broth and spores prepared using CCY media as previously described (Todd et al., 2003, Stewart et al., 1981. Spores were harvested when the culture contained >95% free spores followed by 10 washes in sterile ice-cold water to remove debris and vegetative cells. Washed spore pellets were resuspended in 50 mM Tris-HCl, 0.5 mM EDTA pH 7.5 at 20-50 mg ml -1 dry weight, and stored at -20 °C. Isolation of exosporium fragments. Exosporium fragments were isolated using the French press method ('unwashed') and washed with salt and detergent buffers ('fully washed') as described by Terry et al., (2011). The unwashed exosporium was first washed in TEP buffer (50 mM Tris-HCl pH 7.2, 10 mM EDTA, 1 mM PMSF) containing 0.5 M KCl and 1% (v/v) glycerol. 1 M NaCl was used in the second wash followed by TEP buffer containing 0.1% (w/v) SDS. TEP buffer was used as the final wash. The exosporium was recovered after each wash step by ultracentrifugation at 145,000 x g for 2 hours. B. thuringiensis 4D11 exosporium used for image processing was isolated from spores grown with gentle agitation and sonicated to remove the exosporium. Protein concentration was determined using a BCA™ Protein Assay Kit (Pierce).
Mouse monoclonal anti-BclA antibodies (Terry et al., 2011) were used at a concentration of concentration of 5 µg ml -1 . Rabbit polyclonal anti-ExsK antibodies (Severson et al., 2009) were used at a final concentration of 6 µg ml -1 . Polyclonal anti-ExsFA antibodies (Sylvestre et al., 2005) were used at a concentration of 1/2000. We used as negative controls either mouse anti-human B cell CD23 IgG primary antibodies (Dako) or anti-Goat IgG whole molecule produced in rabbit (Sigma-Aldrich). Polyclonal antibody preparations were cleaned using an Immobilized E. coli Lysate Kit (Thermo Scientific) to remove any contaminating anti-E. coli antibodies.
Expression and purification of ExsY crystals. One Shot ® TOP10 Electrocomp TM E. coli (Invitrogen TM ) was used to maintain all plasmids used in this study. The exsY gene from B.
cereus ATCC 10876, with the last two amino acids deleted (V153 and K154) to replicate the exsY gene from B. anthracis, was cloned into NdeI-and XhoI-digested pET28a, generating both N-and C-terminal poly-histidine tags and a construct with tags at both termini (2His 6 -ExsY). An untagged exsY construct was produced by cloning into NdeI-XhoI digested pCOLADuet-1 vector. ExsY protein over-expression was carried out in E. coli strain BL21(DE3)pLysS and grown in LB media containing the appropriate antibiotic marker. Cells were grown at 37 °C with vigorous shaking until an OD 600 of 0.6 before inducing with 1 mM IPTG for 4 hours and collected by centrifugation. Cells were resuspended in urea buffer (8 M urea, 150 mM NaCl, 25 mM Tris-HCl, pH 8.0) and disrupted by sonication. Crystals of histagged ExsY were collected by batch purification using NiNTA Agarose beads (Qiagen) and eluted using 300 mM imidazole in urea buffer. Crystals were isolated by ultracentrifugation of eluate at 39,000 x g and resuspended in urea buffer.
Electron microscopy. 5 µl of spores at a concentration of ~ 4 mg ml -1 and 3 µl of exosporium at ~ 0.6 mg ml -1 were loaded onto carbon coated grids and stained with 0.75 % uranyl formate as previously described (Ball et al., 2008). Samples were examined on a Phillips CM100 transmission electron microscope at an accelerating voltage of 100 kV.
Digital images were collected on a 1K x 1K Gatan Multiscan 794 CCD camera. Fragments of