Architecture and self-assembly of the Clostridium sporogenes/botulinum spore surface illustrate a general protective strategy across spore formers

Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives have similar spore architectures incorporating protective proteinaceous envelopes. We reveal in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other Clostridial relatives, forms a hexagonally symmetric molecular filter. A cysteine-rich protein, CsxA, when expressed in E. coli, self-assembles into a highly thermally stable structure identical to native exosporium. Like exosporium, CsxA arrays require harsh reducing conditions for disassembly. We conclude that in vivo, CsxA self-organises into a highly resilient, disulphide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar in Bacillus spores, despite lack of protein homology. In both cases, intracellular disulphide formation is favoured by the high lattice symmetry. We propose that cysteine-rich proteins identified in distantly related spore formers may adopt a similar strategy for intracellular assembly of robust protective structures.


Introduction 17
Spores formed by bacteria of the genera Clostridium and Bacillus provide a uniquely 18 effective means of surviving environmental stress (1); they act as the infectious agent in 19 pathogens such as Bacillus anthracis, Clostridium botulinum and Clostridium difficile. In 20 the anaerobic Clostridia they are essential to survival in air. Despite the early evolutionary 21 divergence of the genera Clostridium and Bacillus, the overall process of spore formation 22 is strikingly similar, and a large number of the genes responsible for regulation and 23 morphogenesis in sporulation are conserved. However, proteins making up the spore 24 outer layers are much less conserved (2). These layers include a complex protein coat, 25 and in some species, such as the pathogens B. anthracis and C. botulinum (but not B. 26 observed on folded fragments, but if the imaging force was increased a punctate lattice 1 with similar unit cell dimensions to the ordered areas in dry fragments became apparent 2 (Fig. S4). Presumably at higher imaging forces the AFM tip was able to penetrate to the 3 more ordered anchoring zone of the hairy nap filaments. 4

5
The 3D structure of self-assembled recombinant CsxA matches that of the native 6 exosporium array 7 The C. sporogenes exosporium protein CsxA is found exclusively in the very high 8 molecular weight material extracted from C. sporogenes exosporium, strongly suggesting 9 that it has a structural role in the basal layer (20). The CsxA proteins of Group I C. 10 botulinum are highly similar, with amino acid identities ranging from 77% (strain A3 Loch 11 Maree) to 100% (strain Prevot 594) (20), so data may be extrapolated more widely across 12 the group. The C. sporogenes csxA gene was cloned and expressed in E. coli, with a C-13 images. The unit cell parameters (a = b =111 ± 2 Å and γ = 120.3°± 0.4°, p6 symmetry 20 (inferred from CryoEM, Table S2)) and projection structure are identical to that of native 21 exosporium (Fig. S1B) The CsxA 2D crystals were better ordered than the native exosporium and more amenable 3 to higher resolution analysis. The unit cell dimensions in vitreous ice were a = b = 103 ± 2 4 Å and γ = 120 ± 2°. Analysis of high resolution Fourier phases indicated p6 symmetry, thus 5 unambiguously determining the hexameric nature of the protein assembly (Table S2). We 6 calculated a projection map from averaged amplitudes and phases from 14 images. Phase 7 measurements were significant to ~9 Å resolution (Table S4). The projection map (Fig. 4B) 8 reveals a sixfold symmetric ring of protein density with outer diameter of ~115 Å. The ring 9 encloses a less dense core (centre of Fig. 4B). The possible approximate envelope of one 10 subunit is outlined and it is notable that the closest points of contact between subunits are 11 within the hexameric ring and in the vicinity of the threefold symmetry axes that connect 12 rings; this is consistent with the lower resolution 3D reconstruction (Fig. 2). to that seen in native exosporium (Fig. 3F), meaning that we can confidently assign it to 20 the internal surface of the basal layer. The other (external) surface displayed a lattice of 21 hexameric assemblies with petal-like lobes (Fig. 5B). When samples were dried and 22 imaged in air, we observed little difference in the overall arrangement of the 110 Å lattice 23 of pits on the honeycomb face except that the threefold linkers were less pronounced, as 24 in dehydrated native exosporium (Figs. 3B, 5C). On the 'petal' face, instead of a ~110 Å 25 lattice, we observed an array of pits with apparent ~50 Å spacing (Fig. 5D). However, the 26 Fourier transform showed weak first order spots indicating the true unit cell was still ~110 1 Å. The overall sheet thickness of crystals decreased from ~65 Å in water to ~40 Å in air, 2 regardless of which surface was exposed to the AFM tip. A cycle of dehydration followed 3 by rehydration on crystals displaying the 'petal' face, showed the structural change to be 4 reversible (Fig. S6). The mechanism and functional implications of this change remain to 5 be determined. However, extrapolating to the native exosporium, both hydrated and 6 dehydrated basal layer structures are likely to represent in vivo states, reflecting the 7 different environments which spores would experience, such as dry to water-saturated 8 soils or surfaces, and inside infected hosts or predators. CsxA crystals were exposed to a variety of denaturing conditions (Table S5). As complete 2 disassembly of CsxA crystals requires boiling in the presence of 2M DTT, it is likely that 3 disulphide bonding between some or all of CsxA's 25 cysteines plays a critical role in 4 holding together the crystal lattice. The projection map of ice-embedded CsxA (Fig. 4B) 5 suggests that the six monomers within a single hexameric ring are closely packed and 6 may be connected by multiple disulphide bonds. However, the packing between rings 7 appears less tight and it is likely that cross links occur only in the vicinity of the threefold 8 symmetric bridge (Fig. 4B, triangle). This could explain why DTT treatment of CsxA 9 crystals at room temperature leads to increased crystal disorder but not complete 10 disassembly (Fig. S7). 11

12
The CsxA protein is essential for formation of the exosporium 13 A csxA mutant of C. sporogenes was constructed in the more genetically amenable strain 14 ATCC15579. Spores of this mutant no longer have a typical 110 Å lattice exosporium, 15 consistent with the interpretation that CsxA is the core structural component of the 16 outermost exosporium basal layer. Instead, spores appeared partially wrapped in thin 17 broken sheets of material, fragments of which were frequently sloughed off the spores 18 ( Fig. 1C). These sheets formed 2D crystals, but with a trigonal rather than hexagonal unit 19 cell of ~65 Å on a side (Fig. S8). This loose proteinaceous layer may derive either from 20 the coat or from some additional exosporium sub-layer normally more tightly associated 21 with the spore core (23). The CsxA protein is conserved across C. sporogenes and related Group I C. botulinum 3 species (20), and CsxA homologues are also present in a wide variety of other Clostridium 4 species (Fig. S9). In C. sporogenes strain ATCC 15579, and others, the CsxA protein is 5 encoded in a gene cluster between zapA and a gene encoding a U32 family peptidase 6 ( Fig S10). In ATCC 15579, the cluster also encodes a glycosyl transferase and proteins can be found encoded within 20 kb of each other across many spore formers 24 (Supplementary data set 1); a number of these cysteine-rich proteins are annotated as 25 spore coat proteins and others are potential candidates for spore coat and/or exosporium 1 proteins. 2 3 Distantly related spore formers use different proteins but adopt similar design principles to 4 build the spore envelope 5 The representatives of the Bacillales and Clostridiales that we have studied (B. with a crystalline basal layer enveloping the spore core and decorated by a more 9 disordered 'hairy nap' (Fig. 1A,B) (25); in the cases of B. cereus and C. sporogenes, a 10 disordered outer surface and ordered inner surface are observed by AFM (Fig. 3) (7, 20). 11 In B. cereus/anthracis, C. botulinum/sporogenes and other Clostridia, the basal layer of 12 the exosporium has a regular tiling pattern of interlinked sixfold symmetric oligomers 13 (compare Fig. 2, 3, S1 and S2 with (7)). In B. cereus the core components are ExsY and 14 CotY (8); these are cysteine-rich and analogous to, but not homologous to CsxA. The 15 lattice spacings in the Clostridia tested were higher (~110 to ~127 Å versus ~80 Å in B. 16 cereus/anthracis), and the C. sporogenes basal layer reveals an apparently more 17 permeable structure, with pores of ~55 Å, compared to ~20 Å (25) (Fig. S2). Although the 18 effective diameters for diffusion would be smaller than those physically measured in the Cysteine-rich proteins are emerging as a characteristic feature of the proteomes of spores 12 (8, 13, 20, 29-32). We have now identified a variety of spore proteins, some with very 13 different sequences, that have the common properties of being cysteine-rich and capable 14 of self-organization into extended 2D ordered arrays resembling natural assemblies found 15 in the native spore. It is notable that the identical crystal packing symmetry that we see in 16 CsxA assemblies has been found in the unrelated cysteine-rich proteins, CotY from B. 17 subtilis spore coat (31), and ExsY and CotY from B. cereus exosporium (8). Arrays of 18 these proteins isolated from an E. coli expression host also require harsh reducing 19 conditions and boiling for complete disassembly. This robust cross-linking of arrays is 20 likely to reflect the situation in the native spore -harsh denaturing and reducing conditions 21 are required for complete disintegration of the hexagonal lattice of C. botulinum and B. 22 cereus exosporium (8, 33). Whilst the cellular milieu of the native mother cell, where the 23 exosporium is assembled, or of the heterologous E. coli host is generally considered 24 'reducing' and intracellular disulphide bonding is rare, the ordered lattice and high 25 symmetry of the respective proteins could provide sufficient avidity to overcome this and 1 still drive cooperative intracellular disulphide cross-linking (Fig. 6). 2 3 As we have done previously for ExsY (8), for CsxA we can describe a hierarchy of units of 4 assembly in the protein lattice, from monomer through hexamer to the extended cross-5 linked 2D hexagonal array. In both cases this array of cysteine rich proteins can then be 6 decorated by filamentous appendages including proteins with CLR domains (Fig. 6). We 7 propose that the many cysteine-rich coat and exosporium proteins identified in the It may not be a general requirement for self-assembly of cysteine-rich proteins that they 17 form the extensive crystalline arrays that we have described; some may be only locally 18 ordered. Nevertheless, extensive crystalline layers of unidentified proteins are associated 19 with spore coats and exosporium-like layers in a wide range of spore forming species (36-20 44). 21 22

Role of the exosporium in outgrowth 23
We have shown that those parts of the in situ exosporium that are accessible for diffraction 24 analysis adopt the basal layer structure identical to that of recombinant CsxA crystals 25 (Figs. 1A and 4A). However, it is possible that the spore is not completely enclosed by a 26 uniform and continuous CsxA lattice. Although low resolution scanning electron 1 micrographs suggest a mostly uniform exosporium fully enveloping the C. sporogenes 2 spore there does appear to be a relatively weak structure (sporiduct) at one pole through 3 which the outgrowing cell emerges (45). This may be made up of different proteins and 4 could incorporate a 'cap' as seen in B. anthracis (46, 47). The exosporium does not 5 appear to tear beyond this region, indicating that the CsxA lattice is relatively resistant to 6 any pressure from the emerging vegetative cell. 7 8

Conclusion 9
We have achieved the first three dimensional molecular reconstruction of a Clostridial 10 spore surface. The C. sporogenes spore is enveloped by an exosporium built with a 11 paracrystalline basal layer, the core component of which is the cysteine-rich protein, CsxA. 12 This is the first example of a Clostridial protein capable of self assembly to form a 13 paracrystalline cross-linked spore layer; a phenomenon that we previously demonstrated 14 in unrelated spore surface proteins of the Bacillales (8, 31). We have demonstrated how 15 apparently unrelated proteins from different species can assemble to form similar highly 16 symmetric tiled arrays. The proteins are cysteine-rich and self-assemble to form highly 17 resilient spore layers. We propose that diverse cysteine-rich proteins identified in the 18 genomes of a broad range of spore formers may adopt a similar strategy for assembly. 19 20

Materials and methods 21
A full account of the methods used is in Supplementary Information. A csxA mutant of C. sporogenes strain ATCC15579 was generated using the Clostron 5 system as previously described (27). C. sporogenes strain NCIMB 701792 (NCDO1792) 6 was not used for mutational studies as this strain is erythromycin resistant and not 7 amenable to Clostron mutation. 8 9

Expression of csxA in E. coli 10
The csxA gene was amplified by PCR from genomic DNA of NCIMB 701792, and cloned 11 into pET21a, forming a C-terminal His 6 fusion. After 3h of induction in BL21(DE3) pLysS, 12 cells were harvested, sonicated and the CsxA 2D crystals recovered by binding to Ni-NTA 13 agarose beads, washing with buffer, and elution with 1M imidazole. 14 15 CsxA crystals were examined by negative stain EM (see below) after incubation under a 16 variety of combined conditions including 95 °C heat treatment, 8M urea and 2M DTT-see 17 Table S5. Exosporium fragments were bound to poly-d-lysine coated cover slides at pH 4. 2D 23 crystals of CsxA were bound to freshly cleaved mica. Samples were either imaged in water 24 or washed, dried and imaged in air. Imaging in air was performed using a JPK 25 NanoWizard Ultra AFM in AC mode with TESPA V2 cantilevers in a home built vibration 26 and acoustic isolation system. Imaging in water was performed using a Bruker Dimension 1 FastScan AFM in Tapping Mode with FastScan D cantilevers. Images were processed and 2 analyzed using JPK DP software, Gwyydion and NanoScope Analysis. 3 4 Acknowledgements 5 We thank Chris Hill for help with EM of thin sections. All electron microscopy work was 6 carried out in the University of Sheffield's Faculty of Science Electron Microscopy Facility. 7 We also thank Paul Kemp-Russell and Simon Dixon who fabricated the acoustic enclosure 8 for AFM. JKH and NM gratefully acknowledge the Imagine: Imaging Life initiative at the 9 University of Sheffield and the EPSRC for financial support through its Programme Grant 10 scheme (Grant No. EP/I012060/1). PAB and TKJ gratefully acknowledge the Wellcome 11 Trust for financial support. ADS was in receipt of a White Rose BBSRC DTP studentship. Transmission electron micrograph of a spore negatively stained with uranyl formate. The 5 thin exosporium layer surrounds the dense spore core and is often extended at one pole. 6 (B) A high magnification image from an area of exosporium displaying a 'hairy nap' fringe 7