CryoEM structure of a novel class of spore virulence factors on the foodborne outbreak strain Bacillus paranthracis NVH 0075-95

CryoEM structure of a novel class of spore virulence factors on the foodborne outbreak strain Bacillus paranthracis NVH 0075-95 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CryoEM structure of a novel class of spore virulence factors on the foodborne outbreak strain Bacillus paranthracis NVH 0075-95 Mike Sleutel, Ephrem Zegeye, Ann-Katrin LLarena, Brajabandhu Pradhan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4045273/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Bacterial endospores are remarkable examples of biological resilience, representing a dormant and heavily fortified differentiation form capable of withstanding physical and chemical stressors detrimental to vegetative cells. In pathogenic Bacillota, spores also form an infectious particle and can take up a central role in the environmental persistence and dissemination of disease. A poorly understood aspect of spore-mediated infection is the fibrous structures or ‘endospore appendages’ (ENAs) that have been seen to decorate the spores of pathogenic Bacilli and Clostridia. New methodological approaches are opening an unprecedented window on these long enigmatic structures. Using cryoID, Alphafold modelling and genetic approaches we identify a novel class of ultra-robust ENAs in the foodborne outbreak strain Bacillus paranthracis NVH 0075-95. We demonstrate that L-ENA are encoded by a rare three-gene cluster ( ena3 ) that contains all components for the self-assembly of ladder-like protein nanofibers of stacked heptameric rings, their anchoring to the exosporium, and their termination in a trimeric ‘ruffle’ made of a complement C1Q-like BclA paralogue. The role of ENA fibers in spore-spore interaction and the distribution of L-ENA operon as mobile genetic elements in predominantly pathogenic B. cereus s.l. strains, suggest that L-ENA fibers promote strain virulence by spore clustering and aggregation. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biophysics/Biopolymers in vivo Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Endospores formed by bacteria in the Bacillota (Fimicutes) phylum are among the most durable lifeforms found in nature. This cryptobiotic way of living can bridge long periods that are not conducive to survival of the vegetative state, waiting for environmental stimuli to trigger germination and terminate the state of hibernation. Their remarkable resilience can in part be traced back to the extraordinary robust layers that encase the genomic DNA, while maintaining the permeability for germinants that signal when conditions are again conducive for metabolically active life ( 1, 2 ). For the endospore forming bacteria of the genera Bacillus and Clostridium , the genome is housed within a partially dehydrated central core that is encapsulated by a thick peptidoglycan layer, referred to as the cortex. The cortex, in turn, is covered by at least four consecutive protein layers that make up the spore coat: i.e., the basement layer, the inner and outer coat and the crust, comprising about 70 different proteins ( 3, 4 ). Many species have an additional layer called the exosporium, which forms a flexible, glycosylated, para-crystalline sac-like structure that surrounds the spore, defining an interstitial volume between the crust and the exosporium. As the outermost spore layer, the exosporium mediates endospore´s connection to the environment and the infected host ( 5, 6 ). An important role in this interaction is played by the b acterial c ollagen l ike protein of A nthracis (BclA), which covers the spore surface as a dense decoration called the hairy nap ( 6 ). It plays an immune regulatory function by binding complement factor H (CHF) thereby inhibiting downstream complement activation ( 7 ). A poorly understood component of the spore surface are micrometer long filamentous, pilus-like structures seen in virulent Bacillus strains, dubbed endospore appendages (ENA). First reported and classified for Clostridium spores in the late 1960s ( 8-10 ), followed by B. cereus in the 1970s ( 11 ), ENAs can vary greatly in length, diameter and overall shape. They are present on spores of notable human and animal pathogens such as B. anthracis , B. cereus ( 11 ), C. botulinum ( 10 ) and C. sordellii ( 12 ), and seem mostly absent in saprophytic species, including B. subtilis . Pilus-like structures are well known from vegetative cells ( 13 ). In Gram-positive bacteria, pili are predominantly formed by the sortase-mediated covalent coupling and cell-wall anchoring of secreted LPxTG motif containing subunits via isopeptide bonds ( 14-16 ). These pili are primarily found in pathogenic species (i.e. Corynebacterium dyphtheriae , Staphylococcus spp. , Enterococcus spp. , Streptococcus spp. and Bacillus spp.), where they serve an adhesive function, by binding to host tissues and/or mediating self-contact in multicellular communities or biofilms. Endospores represent an important dissemination form in pathogenic Bacillaceae , so similar to pili on vegetative cells, ENAs may mediate cell-cell and cell-tissue interaction. Despite observations for more than six decades, their extreme physico-chemical robustness made ENAs resist traditional proteomic identification methods (N-terminal sequencing; peptide fingerprinting) ( 17 ) preventing the genetic study of their function. This stalemate was recently broken by the virtue of cryoID, the direct molecular identification of proteinaceous structures through 3D reconstruction by cryo-electron transmission microscopy (cryoEM) (18, 19) . Spores of the food poisoning outbreak strain Bacillus paranthracis NVH 0075-95 were found to display two structurally distinct ENAs, based on the typical patterning seen in nsTEM images dubbed ‘staggered’ and ‘ladder’-like or ‘S’- and ‘L-ENA’, respectively (19, 20) . CryoID studies of S-ENA revealed it represents a novel pilus superfamily composed of two major subunits, Ena1A and Ena1B. Both proteins consist of a single DUF3992 domain comprising a b-jellyroll fold with an N-terminal extension. Ena1A and 1B subunits self-organize into micrometers long helical ultra-structures by lateral b-sheet augmentation, fortified by lateral and longitudinal inter-molecular disulfide bridges. Strikingly, S-ENAs terminate into 4 to 6 flexible, 2nm diameter, tip-fibrillae (hereafter referred to as ruffles ) for which a genetic identification or structural elucidation remains absent. Phylogenomic analysis showed that ena1/2 clusters are ubiquitous in pathogenic Bacilli (>90% occurrence in B. cereus s.l.) suggesting that S-ENAs could be hitherto overlooked virulence factors. Biomechanical studies suggest S-ENAs are implicated in self-adherence and the clustering of spores, albeit by an unknown mechanism ( 21 ). As mentioned above, the spores of NVH 0075-95 are decorated with a second type of appendages, dubbed L-ENA. They are shorter (typically < 1µm), 7nm diameter ENAs that are tethered to the exosporium layer and are decorated with a single ruffle at their distal terminus which has been identified as a major driver in spore-spore aggregation (Jonsmoen et al. , in preparation). In this contribution, we use a combined cryoEM - genome mining approach to identify the major structural subunit of L-ENA (hereafter referred to as Ena3A) and solve the helical fiber ultrastructure to a resolution of 3.3 Å. We show that ena3A is embedded in a three-gene cluster that holds all components for L-ENA assembly and anchoring to the exosporium. Results L-ENA fibers constitute a novel sub-class of disulfide cross-linked endospore appendages. The endospores of the food-poisoning outbreak strain B. paranthracis NVH 0075-95 are decorated with two types of ENA fibers, i.e., S-ENA and L-ENA (Fig.1a-b). Close inspection of the exosporium layer reveals that L-ENA fibers protrude from the brush-like hairy nap layer (Fig.1b) seemingly tethered to the exosporium. L-ENA fibers have an apparent diameter of 8nm and exhibit a ladder-like uranyl staining pattern in nsTEM with a typical recurring distance of 4.6nm between consecutive ladder segments. L-ENAs consistently terminate in a single 2nm diameter tip-fibrillum or ruffle, that consists of a stalk region (45nm in length) and a terminal knob domain (Fig.1c). In preparation of cryoEM data collection, we produced a sample enriched in ENA via shear-induced dislodging from the spore surface followed by a series of purification steps as described in ( 18 ). Next, a 1568 movie cryoEM dataset (60 frames, 0.766Å/pixel) was collected on a JEOL CRYO ARM 300 microscope, yielding a 5.8Å resolution (FSC=0.143 criterion) reconstructed cryoEM-volume of L-ENA fibers with C7 symmetry (see Fig.1e and Supporting Figure 1). The refined helical parameters are a twist of 17.04°, and a rise of 43.82Å. We attribute the limited resolution of the final reconstruction to (i) the sparsity of images that contained L-ENA fibers (414 micrographs, i.e., 26% of the total dataset), and (ii) the innate flexibility of the L-ENA fibers (discussed below). The limited resolution precluded us to de novo build an atomic model of L-ENA but allowed us to identify the fold of the constituent monomers. Indeed, an exploratory rigid body docking exercise using Ena1B as template model (extracted from PDB: 7A02) taught us that the L-ENA subunits appeared structurally homologous to Ena1A/B and therefore likely represented members of the DUF3992 family. A careful search of the NVH 0075-95 genome (GCA_027945115.1) with HMMsearch 3.0 ( 22 ) using a hidden Markov model generated from a dataset of Ena1/2 protein sequences as query led to the identification of a fourth gene outside the ena1a-c cluster encoding for a DUF3992 domain-containing protein. As such, we reasoned that WP_017562367 represented the candidate L-ENA subunit and ordered its coding sequence as a synthetic gene, and cloned it into pET28a for cytoplasmic expression in E. coli C43 (DE3), similar to the approach taken for recombinant S-ENA production ( 18 ). To probe for the presence of putative L-ENA fibers, the insoluble fraction (after lysozyme treatment and 1% SDS extraction) of the E. coli lysate was analyzed using nsTEM. The obtained micrographs confirmed the presence of micron-sized, 7nm diameter fibers with a characteristic ladder-like pattern at 4.6nm intervals (Fig.1f). Notably, the recEna3A fibers did not have any ruffles on their termini. This result served as an initial confirmation that WP_017562367 is the major subunit of L-ENAs found on B. paranthracis NVH 0075-95. To reflect the fact that WP_017562367 is a member of a new ena gene cluster, we propose to name it ena3a , following the convention used for the ena1a-c (e.g., B. cereus type S-ENA ) and ena2a-c (e.g., B. thuringiensis type S-ENA ) ( 18 ). Given the relative ease of production and purification of recEna3A fibers in comparison to the L-ENA purification from the natural source, we proceeded to collect a 10886 movies cryoEM dataset of vitrified recEna3A fibers (Supporting Figure 2) leading to a 3.3 Å global resolution (FSC=0.143 criterion) cryoEM volume after helical refinement using C7 symmetry in CryoSPARC v4.0.3 (Fig.2). The refined helical parameters are a twist of 18.5°, and a rise of 44.97Å. The recEna3A cryoEM volume reveals an axial stacking of heptameric Ena3A rings that rotate 18.5° clockwise relative to each other (Fig.2a). Each ring is composed of seven Ena3A molecules that interface laterally via b-sheet augmentation. The rings encircle a central, hollow volume which we refer to as the ring lumen (Fig.2b). Consecutive rings interlock with each other using the N-terminal extensions of the upper ring docking into the lumen of the ring below (Fig.2c). We refer to the first 14 N-terminal residues as the N-terminal connector (i.e., Ntc) in analogy to the N-termini of Ena1A/B subunits that interlock from above with subunits i-9 and i-10 of the neighboring helical staircase ( 18 ). Note that the orientation of the L-ENA fiber in Fig. 2 is such that the top rings represent the distal, ruffled end of the fiber, and that the bottom rings form the pointy end of the fibers, proximal to the spore surface. Close inspection of the cryoEM map revealed covalent cross-links between neighboring Ena3A subunits at three distinct locations (Fig.2d). After manual building of the final L-ENA model, we identified these contacts to be 3 types of inter-molecular disulfide bridges. Two disulfide bridges (i.e., Cys22-Cys82 and Cys14-Cys15) serve to reinforce the lateral contacts within a single ring, whereas one disulfide bridge (i.e., Cys8-Cys21) confers longitudinal coupling between the lumen and the Ntcs of consecutive rings. Given the heptameric nature of the structure, each ring segment will contain 21 inter-molecular disulfide bridges that are organized along three concentric rings centered on the fiber axis (Fig.2e). Next we tested the stability of recombinant L-Ena fibers by subjecting the fibers to a series of physical (autoclaving) and/or chemical treatments (8M urea, SDS, proteinase K, formic acid). Following the respective treatments, we performed nsTEM imaging of the recovered fibers and determined the 2D class average to gauge the intactness of the fibers (Supporting Figure 4). Remarkably, the 2D class average images of all treatments (apart from the 100% formic acid) are very similar to the 2D class averages obtained from ex vivo purified fibers. We were not able to produce 2D class averages for the 100% formic acid (FA) treated sample, likely indicating that the Ena3A monomers had (partially) unfolded. Interestingly, the FA-treated fibers did not depolymerize, suggesting that disulphide bonds had not been reduced. We conclude that the extensive hydrogen bonding between the Ena3A subunits combined with the covalent cross-links underlies the physico-chemical robustness of the L-ENA fibers. L-ENA marries this extreme stability with a remarkable flexibility as judged from the regions of high local curvature in the nsTEM micrographs and 2D class average images obtained during cryoEM processing (Fig.1f). To illustrate the point further, we manually selected and extracted curved L-ENA fiber segments from the motion-corrected micrographs and performed 2D classification (Supporting Figure 3a). To resolve the underlying structural heterogeneity, we performed a 3D variability analysis using CryoSPARC (Supporting Figure 3b). The resulting volume series (processed in ChimeraX 1.4 ( 23, 24 ) and exported as Supporting Movie 1) provides further molecular insights into the L-ENA flexibility. Ena3A rings exhibit a rocking motion normal to the fiber axis with the respective hinge points centered on the Ntcs. Hence, L-ENA flexibility can be traced back to (i) the spatial separation between consecutive rings thereby providing the possibility for local ring displacement without introducing steric clashes, (ii) combined with the intrinsic flexibility of the N-terminal connectors. Ena3A subunits consist of a typical jellyroll fold ( 25 ) comprised of two juxtaposed β-sheets containing strands BIDG and CHEF (Fig.3). As mentioned earlier, the jellyroll domain is preceded by a flexible 14-residue Ntc that mediates inter-ring coupling. The backbone (420 atoms) root-mean squared displacement (RMSD) between Ena1B and Ena3A is 3.7Å even though the sequence identity is only 28.4% (Supporting Figure 5a-c). Despite the structural similarities at the fold level, S- and L-ENA have a markedly different quaternary architecture (Figure 3a-c). Ena1B subunits interact laterally via b-sheet augmentation (Supporting Figure 5d-e) with a non-zero (i.e., 3.2Å) vertical offset, making a 28° angle relative to the fiber axis. This in turn leads to a helical stacking of Ena1B monomers yielding an S-ENA fiber with a diameter of 110Å. Ena3A subunits form a similar dimer interface (see the residues marked with an asterisk in Supporting Figure 5b) that follows the same register between the interfacing strands G and C (Supporting Figure 5f-g). Contrary to Ena1B though, neighboring Ena3A subunits lie within the same plane (i.e., no vertical offset) thereby producing closed ring-like structures. We attribute this difference in axial displacement to the relative tilts that the subunits make with respect to the fiber axis, i.e., 28° and 14° for Ena1B and Ena3A, respectively (Fig. 3c and f). L-ENA fiber biogenesis therefore likely proceeds via docking and covalent locking (via the Cys8-Cys21 disulfide) of fully formed rings, whereas S-ENA fibers elongate via integration of successive monomers. Distribution and expression of the ena3A gene cluster Inspection of the GCA_027945115 genome reveals that ena3A (PGS39_28750) is embedded in a three-gene cluster on the NZ_CP116205 plasmid, hereafter referred to as the ena3 gene cluster (Figure 4). In this cluster, ena3A is preceded by the genes PGS39_28740 and PGS39_28745. For reasons discussed below, we suggest naming these two genes, i.e. exsL and l-bclA , respectively. The ena3 gene cluster is rare: it was found in only 62 organisms through a remote search of the entire NCBI RefSeq non-redundant protein database using cblaster (CAGECAT v. 1.0). Assemblies of sufficient quality were downloaded and appended to a representative database of genomes of the Bacillus genus. The proportion of genomes carrying an ena3 gene cluster was indeed low; only 9.5% (n=62/656) of the B. cereus s. l. genomes, of which 51 were B. cereus (n=51/126) , four B. thuringiensis (n=4/52), one B. anthracis (n=1/63), one B. paranthracis (n=1/4), one B. toyonensis (n=1/204) , two B. mobilis (n=2/5) , and two Bacillus sp. (2/4) (Supporting Figure 6). Most genomes had one copy of the gene cluster (n=53), while nine strains had paralogs, carrying two (n=5, B. cereus (3) , Bacillus sp. (2)), three (n=2, B. cereus ), four (n=1, B. thuringiensis ) and five (n=1, a B. thuringiensis ) copies. The ena3 gene cluster was not found in B. subtilis or other saprophytic Bacilli (see Supporting Data) . Thirteen genomes with an L-ENA gene cluster were complete and closed, and the genomic location of the gene cluster could be inspected. The ena3 gene cluster was located either on the chromosome (n=7 genomes), on plasmids (n=1 genome) or were found as paralogs on one or more plasmids and on the chromosome (n=5 genomes). Many of the isolates carrying the ena3 gene cluster were from outbreaks of bloodborne infections in hospitals in Italy and Japan ( 26, 27 ). The ena3 gene cluster of NVH 0075-95 is flanked by an incomplete topoisomerase upstream and a complete Tn3 family transposase upstream. This gene synteny was found in only two other strains in the cblaster/clinker analysis: B. cereus AFS093282 (NZ_NVMQ01000017.1) and B. pacificus strain BC444B (accession NZ_JAOPRQ010000006.1). In other strains, the L-ENA gene cluster was flanked by frameshifted versions of Tn3 family transposases, tyrosine- type recombinase/integrase and site-specific integrases. A few strains had a shorter Tn3 family transposase upstream of the L-ENA gene clusters. Taken together, the L-ENA gene cluster is/or has been located on a transposon and is part of the mobilome of the B. cereus s.l. group, accounting for the polypheletic distribution of this gene cluster in the population. To determine the expression of L-ENA genes ( exsL , l- bclA and ena3A ), NVH0075-95 was cultured in sporulation medium for 16 hours, and cDNA prepared from culture samples collected at four-hour intervals after inoculation (4, 8, 12 and 16 hours) and analyzed by PCR. Using primers that specifically amplify open reading frames across exsL → l-bclA and l-bclA → ena3A (Figure 4a), we detected a ~587 bp PCR product across l-bclA → ena3A , but not for exsL → l-bclA (Figure 4b) . This suggests that l-bclA and ena3A are expressed bicistronically. Notably, no l-bclA-ena3A transcript was detected during the first 8 hrs of cultivation, which represents the vegetative growth phase (Fig. 4b). Consistent with the PCR result, the data from a qPCR analysis indicated that the L-ENA genes are overexpressed exclusively during the sporulation phase (12 and 16 hours) (Fig. 4c). Notably, a ~13000, ~1200 and ~40-fold increase in the expression of ena3A , l-bclA and exsL , respectively, was evident in the samples collected at 12 hours after inoculation. The ena3 gene cluster consists of the L-ENA subunit, the exosporium anchoring protein ExsL and the complement C1Q-like ruffle protein L-BclA Ena3A is found in an operon with exsL and l-bclA . In pursuit of the biological roles of exsL and l-bclA , we made individual knockouts strains (Δ exsL and Δ l - bclA ) and investigated their respective endospores by nsTEM (Fig. 5). ExsL depleted spores are devoid of L-ENA fibers coupled to the exosporium but are otherwise morphologically identical to wild-type NVH 0075-95 spores. Careful inspection of the grid areas lead to the identification of detached L-ENAs in the spore supernatant, with ruffles present, suggesting that ExsL mediates the connection of L-ENA to the exosporium. Conversely, Δ l-bclA spores have L-ENAs present on the exosporium, but the fibers lack the distal ruffle. We do note that the ruffles remained present on the S-ENA fibers, suggesting that l-bclA specifically encodes for the L-ENA ruffle protein. As expected, ena3a - spore samples were completely devoid of L-ENA fibers, be it spore attached or detached. Complementation of the Δ exsL , Δ l-bclA and Δ ena3A mutants with a low copy plasmid (pHT315) containing the respective genes, restored the corresponding spore phenotypes to that of the wild type strain (Fig . 5). A primary sequence analysis of ExsL (WP_048548726.1) using Interpro ( 28 ) shows that it is composed of an N-terminal spore coat protein Z (PF10612) domain and a C-terminal Ena core (DUF3992) domain (see the domain organization and corresponding AF2 prediction in Fig.6b). To gain further insights into the molecular mechanisms of L-ENA anchoring and ruffle formation, we performed Alphafold2 (AF2) modelling ( 29-31 ). First, we tested for the plausibility of a Ena3A-ExsL complex formation. In Supporting Figure 7 we show the AF2 multimer v1.2 model of an Ena3A-ExsL dimer, which had an overall pLDDT score of 82.6 and a ptmscore of 0.73, which is in support of the hetero-dimer hypothesis. As expected, Ena3A interfaces with ExsL via its C-terminal Ena-core domain in a manner that mimics the Ena3A dimer interface found in the L-ENA structure (Supporting Figure 7e-f). This is somewhat surprising given the low sequence identity (17.4%) between the Ena3A and the ExsL Ena-core domain (i.e. residues 157-262). Inspection of the pairwise sequence alignment (Supporting Figure 6d) between Ena3A and ExsL learns that key Ena3A residues (C82, S84 and T86) involved in lateral subunit contacts are conserved in ExsL (C224, S226 and T228). In fact, AF2 predicts a disulfide bridge between ExsL cys224 and Ena3A cys22, which mimics the intra-ring L-ENA S-S bridge between cys22 and cys82 (Supporting Figure 7e-f; Fig. 2d-e). Despite the low sequence identity in Ena3A and the ExsL Ena-core, the Ena3A-Ena3A and Ena3A-ExsL contacts are highly equivalent (Supporting Figure 7e-f), in line with the general mechanism of b-sheet augmentation, which is primarily driven by shape complementarity and backbone H-bonding and is relatively insensitive to the amino acid sequence in the paring b-strands ( 32 ). In addition, we looked at the AF2 prediction of a putative ExsL-ExsY dimer. ExsY (WCA20099.1) is one of the major constituents of the B. cereus exosporium and shares structural similarities to the C-terminal CotZ domain of the exsL gene in the ena3 gene cluster, albeit at low sequence identity, i.e., 30.8% (Supporting Figure 8a,b,f). Our assumption based on the D exsL phenotype and the ExsL-Ena3A AF2 model was that ExsL could also be a potential binding partner of ExsY and, in doing so, act as an exosporium-embedded anchor point for L-ENA fibers. Indeed, the AF2 multimer model of a ExsL-ExsY dimer (with a relatively high pLDDT score of 80.2 and a ptmscore of 0.71) shows an ExsL-ExsY coupling via the N-terminal ExsL 1-156 domain with ExsY, lending further credence to the supposition that ExsL serves as a connective bridge between the paracrystalline exosporium and L-ENA. In analogy to the ExsL-Ena3A dimer, the predicted ExsL-ExsY contact mimics the homomeric ExsY contacts (Supporting Figure 8c,d,e,g) that we observe in a high confidence AF2 multimer prediction of an ExsY hexamer (pLDDT=81.3; ptmscore=0.86), i.e., the base building unit of the exosporium lattice. In turn, L-BclA (WP_271292911.1) contains an N-terminal Collagen triple helix repeat domain (PF01391) followed by a BclA C-terminal domain (PF18573; part of the C1q_TNF clan (Pfam CL0100)). BclA is the major glycoprotein of the hairy nap found to cover the exosporium of most B. cereus s.l. species ( 33, 34 ). Interestingly, this same architecture is found in vertebrate complement component C1q ( 35 ) (uniprot: P02745). In Supporting Figure 9a-b, we present the AF2 multimer model of an L-BclA 94-336 homotrimer that consists of the C-terminal trimerization domain and a segment of the collagen-like triple helix. The corresponding pLDDT value of 94 and ptmscore of 0.9 support the accuracy of the predicted fold, as well as the supposed trimeric stochiometry in analogy to the crystal structure (PDB: 1WCK) of BclA-C of B. anthracis . Based on this model, we predict the lateral dimension of the C-terminal domain to be approximately 4nm, and for the triple collagen helix we project an average length of 2.8 Å per residue (see discussion below; Supporting Figure 9a). The all-atom RMSD between L-BclA-CTD and BclA-C is 3.55 Å despite the low sequence identity of 22.1%, demonstrative of significant structural homology (Supporting Figure 9c). Tan and Turnbough showed that the attachment of BclA to the basal layer protein BxpB of the exosporium is dependent on (i) proteolytic removal of the first 20 residues from the N-terminus and (ii) the presence of an N-terminal submotif -hereafter referred to as the exosporium leader sequence (interpro: IPR0212010; Supporting Figure 9d) - in front of the collagen-like region ( 36 ). Inspection of the N-terminal region of L-BclA demonstrates the absence of an exosporium leader sequence, which indicates that this collagen-like protein is likely not targeted to the exosporium. A notable feature of collagen-like proteins is that the number of residues that comprise the collagen-stalk region in the primary sequence translates proportionally to the axial dimension of the folded, trimeric entity simply due to the extended nature of the collagen fold ( 34 ). To that end, we searched the GCA_027945115.1 genome for the ‘local’ orthologue of BclA (uniprot: Q81JD7) and compared its primary sequence to L-BclA (Supporting Figure 9e). BclA NVH 0075-95 (WCA20088.1) has a collagen-like region of 125 residues, whereas the corresponding domain of L-BclA spans 173 residues. Based on the calibration discussed in Supporting Figure 9a, this translates into a predicted fully extended length of a folded trimeric entity of 39 nm and 52 nm for BclA and L-BclA, respectively. These predictions are in agreement with experimental measurements (average height of the hairy nap: ~36nm and average length of L-ENA ruffle: ~50nm) from nsTEM micrographs (Supporting Figure 9f). To further understand the topology of the L-ENA/L-BclA complex, we performed a single round of 2D classification based on particles that were manually picked from the cryoEM dataset that was collected on ex vivo isolated ENA fibers, focusing on L-ENA termini that were decorated with ruffles (Supporting Figure 9g). This class average shows that the ruffle docks into the lumen of the Ena3A ring at the apex of the L-ENA fiber, i.e. mimicking the inter-ring docking mechanism that exists along the body of the fiber. We therefore compared the Ntc of Ena3A to the N-terminus of L-BclA (Supporting Figure 9d). Although no particular sequence homology could be detected, both sequences contain a double cysteine motif at positions 8 and 13, respectively. As cysteine 8 mediates coupling of the Ena3A Ntc to the ring lumen, we speculate that the L-BclA C 13 C 14 motif could be involved in a similar coupling mechanism. Discussion Although prominently present on the surfaces of many endospores ( 37 ) and described since the 1970s for Bacillus ( 11 ), the genetic identity and biological role of ENAs have remained poorly understood ( 19 ). Using cryoID, Alphafold modelling and genetic approaches we were able to identify (this work and ( 20 )) two members of a novel class (DUF3992-based) of ultra-robust ENAs found on the surface of Bacillus paranthracis spores. We showed that the endospores of the food-poisoning outbreak strain B. paranthracis NVH 0075-95 are decorated with two types of filamentous protein structures, i.e., the endospore appendages S- and L-ENA. Moreover, we have shown that both S- and L-ENA fibers are decorated with respectively, 3-5 or a single tip fibrillae (or ruffles) at their terminus. While the genetic identification of the S-ENA ruffles is still undergoing, the work presented here identifies L-ENA ruffles as L-BclA, a new member of the family of bacterial collagen-like proteins (CLPs). It is well established that Firmicutes endospores display bacterial CLPs on their surface ( 38-40 ), but the biological function remains unclear for most of these proteins ( 40 ). The most well-known CLP within the Bacillus clade is BclA ( 33 ), the major glycoprotein ( 41 ) of the hairy nap ( 42 ) that is targeted to the exosporium via an N-terminal exosporium leader sequence ( 36 ). BclA has been shown to recruit complement factor H (CFH) to the spore surface ( 7 ), and its recruitment onto the exosporium facilitates complement C3 degradation, which in turn inhibits downstream complement activation, and ultimately promotes spore persistence. Here we have shown that contrary to BclA, L-BclA is not coupled to the exosporium - this is reflected in the absence of an exosporium leader-sequence at the N-terminus of L-BclA. Based on the morphological similarities between S- and L-ENA tip-fibrillae (i.e. a ~2nm wide flexible fibrillum terminating in a knob-like domain) in combination with the l-bclA deletion phenotype (L-ENA ruffle: absent, S-ENA: present), we hypothesize that the tip fibrillum of S-ENA is likely to be encoded by a separate CLP also lacking in an exosporium leader sequence, and are searching for putative ‘S-BclA’ candidates among a list of CLP genes we identified in the B. paranthracis NVH0075-95 genome. This suggests that each ENA subtype is decorated by its own cognate ruffle protein hinting at a functional diversification across the ENA subclasses. Similar specialization of bacterial pili towards a specific target or class of surfaces has been reported for e.g. sortase-mediated and chaperone-user pili ( 43 ). Virulent strains can carry multiple pilus operons, most of which display a single, functionally diverse adhesin subunit at the tip of the pilus ( 44 ). The strategic spatial positioning of pilus-presented adhesins promotes efficient attachment and there is a high probability that the ruffles of Ena’s exert related functions. Following on the first structural and molecular characterization of ENAs ( 18 ), Jonsmoen et al. showed that ENAs are involved in spore aggregation ( 21 ). While the molecular underpinnings of the spore-spore coupling mechanism remained unclear at the time, we recently identified L-BclA as the ruffle species that is involved in establishing inter-spore interactions (Jonsmoen et al., in preparation). Biofilm formation in the Bacillus cereus group sensu lato is well recognized ( 45 ) and instantiates as floating pellicles, submerged biofilms attached to abiotic surfaces and on living tissues ( 46 ). Spores tend to accumulate towards the later stages of biofilm development and can -depending on the strain- constitute up to 90% of the total biofilm cell count ( 47 ). Our structural data allows us to frame a molecular model for the development of L-ENA mediated spore interaction networks. Although the exact role of spores in biofilms is still an area of active research, it can reasonably be expected that spore-spore interactions will contribute to virulence by affecting (i) biofilm resilience through fortification (e.g. reinforced core protected from stressors), (ii) development of a spore reservoir with sustained spore shedding, and (iii) establishment of spore clumps representing the minimal infectious dose (i.e. strength in numbers). Given that the ena3a gene cluster is predominantly found in pathogenic Bacilli we anticipate that L-ENA fibers could be considered as a novel class of secondary effect virulence factors through their impact on spore aggregation, and by extension biofilm formation. What remains unclear is whether the tip fibrillum (L-BclA) recognizes a specific target on the spore surface, be it a sugar, protein or proteoglycan or whether the interaction is based on aspecific interaction, similar to the hydrophobic CsuE adhesin found on the archaic Csu pili of Acinetobacter baumanni that is also implicated in the early stages of microcolony development leading up to biofilm formation (48) . From a structural perspective, L-ENA fibers are remarkable products of macromolecular self-assembly, who’s assemblage and surface display require a mere three genes: exsL , ena3A and l-bclA . Each Ena3A subunit is cross-linked to its neighbor via 3 disulphide bridges, which totals to 21 covalent connections for each heptameric unit. Consequently, L-ENA fibers are able to withstand extreme physico-chemical stressors (heat, strong acids, chaotropes, detergent, and desiccation) and are among the most stable protein structures found in nature. In its biological context, however, a protein fiber will only be as strong as its weakest link. It is therefore expected that these high tensile strength ENAs require an anchoring mechanism of comparable durability. The phenotype of D exsL spores (i.e. spores with detached L-ENA) combined with the AF2 modelling of the putative ExsL-Ena3A and ExsL-ExsY heteromers suggests that the anchoring complex is directly integrated into the exosporium 2D lattice. The exosporium of the B. anthracis / cereus / thuringiensis group is a flexible, disulphide cross-linked, protein 2D lattice that is predominantly composed of ExsY hexameric units ( 49 ). The AF2 model for the ExsL-ExsY dimer suggests that ExsL could be embedded within the ExsY lattice by mimicking the homotypic ExsY contacts, including the formation of an inter-molecular disulphide bridge. Similarly, the AF2 model for an ExsL-Ena3A dimer is reminiscent of the Ena3A contacts found in the L-ENA cryoEM model – this in turn also includes one of the intra-ring Ena3A disulphide bridges. Based on this, we hypothesize that ExsL acts as a bridging moiety that anchors the L-ENA pilus onto the spore surface by maintaining an unbroken chain of disulphide cross-links across the various contact points (i.e., ExsY → ExsL; ExsL → Ena3A). The experimental determination of the respective stoichiometries and the elucidation of the complex epitopes will be the subject of further study. Materials and Methods Negative stain transmission electron microscopy Negative stain TEM (nsTEM) imaging of bacterial spores and recEna3A filaments was done using formvar/carbon-coated copper grids (Electron Microscopy Sciences) with a 400-hole mesh. The grids were glow-discharged (ELMO; Agar Scientific) with 4mA plasma current for 45 seconds. 3 μl of a bacterial spore suspension or recEna3A protein solution was applied onto the glow-discharged grids and left to adsorb for 1 minute. The solution was dry blotted, followed by three washes with 15 μl Milli-Q. Next, 15 μl drops of 2% uranyl acetate were applied three times for 10 seconds, 2 seconds, and 1 minute respectively, with a blotting step in between each application. The excess uranyl acetate was then dry blotted with Whatman type 1 paper. All grids were screened with a 120 kV JEOL 1400 microscope equipped with LaB6 filament and TVIPS F416 CCD camera. Cryo-electron transmission microscopy QUANTIFOIL® holey Cu 400 mesh grids with 2‐µm holes and 1‐µm spacing were glow discharged in vacuum using plasma current of 5 mA for 1 minute (ELMO; Agar Scientific). 3 µl of 0.6 mg/ml graphene oxide (GO) solution was applied onto the grid and incubated 1 min for absorption at room temperature. Excess GO was blotted using a Whatman grade 1 filter paper and left to dry under ambient conditions. For cryo‐plunging, 3 µl of fiber suspension was applied on the GO‐coated grids at 100% humidity and room temperature in a Gatan CP3 cryo‐plunger. After 1 minute of absorption, the grid was machine‐blotted with Whatman grade 2 filter paper for 3.5 seconds from both sides and plunge frozen into liquid ethane at -176°C. Grids were stored in liquid nitrogen until data collection. Two datasets were collected for ex vivo and recEna3A appendages with slight changes in the collection parameters. High‐resolution cryoEM movies were recorded on a JEOL CRYO ARM 300 microscope equipped with omega energy filter and a K2 or K3 direct electron detector run in counting mode. For the ex vivo Ena, the microscope was equipped with a K2 summit detector and had the following settings: 300 keV, 100 mm aperture, 30 frames/image, 62.5 e−/Å 2 , total exposure 2.315‐second exposure, and 0.82 Å/pixel. The recEna3A dataset was recorded using a K3 detector, at a pixel size of 0.782 Å/pix, and a total exposure of 64.66 e −/ Å 2 accrued over 61 frames/movie. Generation of deletion mutants and complementation constructs Deletion mutants in B. paranthracis NVH0075-95 were generated following the marker less gene replacement method described previously (Janes and Stibitz, 2006; Pradhan et al., 2021). The gene replacement constructs contained the start and the stop codons of the respective genes to be deleted flanked by upstream and downstream homologous sequences. The upstream and downstream homologous sequences were 806 bp and 677 bp for ena3A , 727 bp and 750 bp for l- bclA , and 701 bp and 728 bp for exsL, respectively. Synthetic constructs (Synbio Technologies LLC) were cut out from the pUC57-Amp vector by digesting with EcoRI, and the gene replacement DNA fragment were cloned into EcoRI digested pMAD-I-SceI shuttle plasmid (Lindbäck et al., 2012). The gene replacement constructs, i.e., pMAD-I- Sce I-Δ ena3 and pMAD-I- Sce I-Δ bcla , and pMAD-I- Sce I-Δ exsL were then used to transform NVH0075-95 or the Δ ena1ABC triple mutant (Pradhan et al., 2021). Details of competent cell preparation, transformation, and screening are described (Zegeye and Aspholm, 2022; Pradhan et al., 2021). For complementation experiments, DNA fragment containing a 300 bp region upstream of the start codon of the l- bclA-ena3A operon and open reading frames of the respective genes was ordered (Synbio Technologies LLC) or amplified by PCR and cloned in the low copy number plasmid pHT315 at EcoRI restriction site. Culturing and RNA extraction B. paranthracis NVH0075-95 and B. paranthracis NVH0075-95 Δ enaABC/ Δ ena3A quadruple mutant (control) were cultured in sporulation medium as previously described (Pradhan et al., 2021) with a slight modification. Here, we used nutrient broth (oxoid) in place of bacto medium (Difco). A sample of 20 ml culture was withdrawn every four hours from three independent cultures, centrifuged, and cell pellets frozen immediately at -80 °C until RNA extraction. RNA was extracted using Purelink RNA minikit (Ambion, Life Technologies) according to the manufacturer’s protocol with a slight modification. Briefly, cell pellets were thawed on ice followed by addition of 200 µl of lysozyme solution (10 mM Tris-HCl (pH 8), 0.1 mM EDTA, 10 mg/ml lysozyme) and incubation for 5 minutes at RT. A volume of 1 µl of 10% SDS was added to the cell suspension and vortexed, after which 150 µl was transferred to new RNAse-free tubes. Following addition of 350 µl of lysis buffer, the cells were lysed by bead beating (FastPrep®-24, MP Biomedicals) for a total of two minutes at Speed 6, with 1 minute cooling on ice every 30 seconds. The lysates were centrifuged for 5 minutes (2600 xg), and the supernatants were transferred to new RNAse-free tubes. The remaining steps were carried out as described in the manufacturer’s protocol. RNA concentration was measured using Nanodrop 1000 (Thermo Scientific), and 1.5 µg RNA was treated with Turbo DNAase (Ambion) in 20 µl reaction volume following the manufacturer’s instruction. Quantitative real-time PCR (qRT-PCR) and PCR Complementary DNA (cDNA) was synthesized from 500 ng DNAse-treated RNA using the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer’s instructions. qRT-PCR was carried out using the PowerUp™ SYBR™ Green Master Mix kit (Applied Biosystems) and the AriaMx Real-Time PCR system (Agilent Technologies) following the manufacturers’ instructions. The expression of exsL , l-bclA , ena3A and rpoB (housekeeping gene) was analyzed from cDNA of each of three independent cultures sampled after 4, 8, 12 and 16 hours after inoculation and the relative gene expression ratio was calculated according to the Pfaffl formula (Pfaffl, 2001). Primers were used at 300 nM concentration, and their efficiencies were calculated from the slope of the standard curve using the formula (10 -1/slope -1) x 100, and were found to be between 95% and 101%. No template was added to the negative control samples. The Ct values obtained for the 4-hour samples were used as the calibrator sample in the data analysis, and rpoB served as an internal control gene. Each cDNA sample was analyzed in triplicate, and the experiment was repeated independently. The cycling conditions of the qRT-PCR were: 50 °C/2 minutes, 95°C/2 minutes; and 40 cycles of 95°C/15 seconds, 60°C/1 minute. To determine if exsL , bclA , and ena3A are co-expressed, standard PCR was conducted using the cDNAs as templates. Primers that span across the indicated genes (Table S4), and DreamTaq PCR master mix (Thermo Scientific™) were used. Purified genomic DNA (gDNA) from NVH0075-95 and cDNA from NVH0075-95 Δ enaABC/ Δ ena3A (4 h and 16 h) were used as positive and negative controls, respectively. Sequencing strain B. paranthracis NVH 0075-95 To achieve a closed, updated, and high-quality genome of B. paranthracis NVH 0075-95, the strain was subjected to hybrid assembly of long- and short read sequences. Genomic DNA from NVH 0075-95 was prepared from bacteria grown over night on blood agar plates. All sequencing was performed by Novogene Co. (London, UK) using a combination of Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing platform with a with a SMRTbell template library and an Illumina NovaSeq 6000 platform (2 x 150 bp) with an insert size of 300 bp. A closed genome was achieved using the assembly pipeline Unicycler v. 0.4.8.0+galaxy3 ( 50 ) conducting a short-read-first hybrid assembly. Genome contiguity, completeness and correctness was assess using Quast v. 5.0.2+galaxy3 ( 51 ), Bandage 0.8.1+galaxy3 ( 52 ), BUSCO v. 5.3.2+galaxy0 (mode genome, gene predictor prodigal, lineage dataset bacillales_odb10) ( 53 ) and post-assembly correctios using Pilon (( 54 )) wrapped in the assembler Unicycler v. 0.4.8.0+galaxy3. Species validation was done on the Type Strain Genome Server (TYGS) ( 55, 56 ). Plasmids and contigs were predicted with PlasFlow v. 1.1.0 (threshold >0.6, remaining default settings) ( 57 ), and the genome was annotated using the online NCBI Procaryotic Genome Annotation Pipeline (PGAP) ( 58-60 ). Data is available at NCBI under accession number GCA_027945115.1. Distribution of L-ena Cblaster (( 61 )) a python toolkit for detecting collocated genes, was run remotely through the web application CAGECAT v.1.0 (settings: -max gap 200 bp, n of unique query seq=3, min coverage 50%, min identity 30%, maximum e-value 0.01) on the NCBI RefSeq non-redundant protein database, using amino acid sequence of WP_017562367.1 (Ena3A), WP_048548723.1 (BclA), WP_048548726.1 (ExsL) and WP_058548727.1 (Tn3 family transposase) as query. The genomic region spanning WP_017562367.1, WP_048548723.1, WP_048548726.1 is hereafter referred to as “the gene cluster”. Furthermore, to investigate the proportion of organisms with the gene cluster within the B. cereus s.l. group, publicly available genomes yielding hits in the cblaster search (Appendix Table) were downloaded from NCBI RefSeq database (n = 656, NCB (https://www.ncbi.nlm.nih.gov/refseq/; Table EV1) and appended to a representative database of assemblies of B. ceresu s.l. group (Supplementary Table 1). In addition, 136 B. subtilis were included for comparison. Assemblies were quality checked using QUAST, and only genomes of correct size (~4.9 – 6.2 Mb) and a GC content of ~35% were included in the downstream analysis. Pairwise tBLASTn searches were performed (BLAST+ v. 2.11.0 (( 62 )), e-value 1e-10, max_hspr 1, default settings) to search for homo- and orthologs of the following query protein sequences from strain NVH 0075-95: WP_017562367.1, WP_048548723.1 and WP_048548726.1. Proteins were considered as orthologs or homologs when they matched the query protein with high coverage (> 70%) and moderate sequence identity (> 30%), and when the whole gene cluster was present in the genome with corresponding synteny as the NVH 0075/95 strain. Genomic location (chromosome or plasmid) was inspected manually for complete and closed genomes. Mashtree v. 0.57 (( 63 )) was used to infer whole genome clustering for the B. cereus s.l. group, using the accurate option (--min-depth 0). Clustering and metadata (hits for query proteins) were visualized in Microreact web browser (( 64 )). Declarations Author contributions MS, MA and HR designed the project. MS performed cryogenic freezing, nsTEM and cryo-EM imaging and data processing with assistance from MF. EDZ conducted the gene expression studies, designed and generated the knockout strains and complementation constructs, and prepared the spores for analysis. MS wrote the manuscript with contributions from all authors. Competing Interests Statement The authors declare no competing interests. Data Availability Statement The ex vivo L-ENA and the recombinant Ena3A cryo-EM maps were deposited to EMDB under entry IDs EMD-17579 and EMD-17627, respectively. The atomic model for recEna3A was deposited to the PDB under ID 8PDZ. Acknowledgements We thank Dirk Reiter at the VIB-VUB Facility for Bio Electron Cryogenic Microscopy (BECM) and Yohannes Beyene Mekonnen at NMBU for technical assistance. This work was funded by VIB, NMBU, EOS Excellence in Research Program by FWO through grant G0G0818N to HR and G043021N to MS. MA recognizes the Grant from the Norwegian research council (NFR): 335029 - FORSKER22. References P. T. McKenney, A. Driks, P. Eichenberger, The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nat Rev Microbiol 11 , 33-44 (2013). Y. Gao et al. , Bacterial spore germination receptors are nutrient-gated ion channels. Science 380 , 387-391 (2023). P. T. McKenney, A. Driks, P. Eichenberger, The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nature Reviews Microbiology 11 , 33-44 (2013). A. O. Henriques, J. Charles P. Moran, Structure, Assembly, and Function of the Spore Surface Layers. Annu. Rev. Microbiol. 61 , 555-588 (2007). B. J. Panessa-Warren, G. T. Tortora, J. B. Warren, High resolution FESEM and TEM reveal bacterial spore attachment. Microsc Microanal 13 , 251-266 (2007). L. Kailas et al. , Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale. Proc Natl Acad Sci U S A 108 , 16014-16019 (2011). Y. Wang et al. , Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence. PLoS Pathog 12 , e1005678 (2016). P. D. Walker, The spore antigens of Clostridium sporogenes, Cl. bifermentans and Cl. sordellii. J Pathol Bacteriol 85 , 41-49 (1963). W. Hodgkiss, Z. J. Ordal, Morphology of the spore of some strains of Clostridium botulinum type E. J Bacteriol 91 , 2031-2036 (1966). W. Hodgkiss, Z. J. Ordal, D. C. Cann, The morphology and ultrastructure of the spore and exosporium of some Clostridium species. J Gen Microbiol 47 , 213-225 (1967). Y. Hachisuka, T. Kuno, Filamentous appendages of Bacillus cereus spores. Jpn J Microbiol 20 , 555-558 (1976). L. J. Rode, L. Pope, C. Filip, L. D. Smith, Spore appendages and taxonomy of Clostridium sordellii. J Bacteriol 108 , 1384-1389 (1971). M. Lukaszczyk, B. Pradhan, H. Remaut, in Bacterial Cell Walls and Membranes, A. Kuhn, Ed. (Springer International Publishing, Cham, 2019), pp. 369-413. A. P. A. Hendrickx, J. M. Budzik, S.-Y. Oh, O. Schneewind, Architects at the bacterial surface — sortases and the assembly of pili with isopeptide bonds. Nature Reviews Microbiology 9 , 166-176 (2011). S. K. Mazmanian, G. Liu, H. Ton-That, O. Schneewind, Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285 , 760-763 (1999). H. Ton-That, O. Schneewind, Assembly of pili on the surface of Corynebacterium diphtheriae. Mol Microbiol 50 , 1429-1438 (2003). S. Kozuka, K. Tochikubo, Properties and origin of filamentous appendages on spores of Bacillus cereus. Microbiol Immunol 29 , 21-37 (1985). B. Pradhan et al. , Endospore Appendages: a novel pilus superfamily from the endospores of pathogenic Bacilli. EMBO J 40 , e106887 (2021). E. D. Zegeye, B. Pradhan, A. K. Llarena, M. Aspholm, Enigmatic Pilus-Like Endospore Appendages of Bacillus cereus Group Species. Int J Mol Sci 22 , (2021). B. Pradhan et al. , Endospore Appendages: A novel pilus superfamily from the endospores of pathogenic Bacilli. The EMBO Journal 40 , e106887 (2021). U. L. Jonsmoen et al. , Endospore pili: Flexible, stiff, and sticky nanofibers. Biophysical Journal , (2023). J. Mistry, R. D. Finn, S. R. Eddy, A. Bateman, M. Punta, Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Research 41 , e121-e121 (2013). E. F. Pettersen et al. , UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci 30 , 70-82 (2021). T. D. Goddard et al. , UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci 27 , 14-25 (2018). J. S. Richardson, The anatomy and taxonomy of protein structure. Adv Protein Chem 34 , 167-339 (1981). R. Akamatsu et al. , Novel Sequence Type in Bacillus cereus Strains Associated with Nosocomial Infections and Bacteremia, Japan. Emerging Infectious Disease journal 25 , 883 (2019). A. Bianco et al. , Characterization of Bacillus cereus Group Isolates From Human Bacteremia by Whole-Genome Sequencing. Front Microbiol 11 , (2021). T. Paysan-Lafosse et al. , InterPro in 2022. Nucleic Acids Res 51 , D418-D427 (2023). J. Jumper et al. , Highly accurate protein structure prediction with AlphaFold. Nature 596 , 583-589 (2021). E. Richard et al. , Protein complex prediction with AlphaFold-Multimer. bioRxiv , 2021.2010.2004.463034 (2022). M. Mirdita et al. , ColabFold: making protein folding accessible to all. Nature Methods 19 , 679-682 (2022). H. Remaut, G. Waksman, Protein-protein interaction through beta-strand addition. Trends Biochem Sci 31 , 436-444 (2006). P. Sylvestre, E. Couture-Tosi, M. Mock, A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium. Mol Microbiol 45 , 169-178 (2002). P. Sylvestre, E. Couture-Tosi, M. Mock, Polymorphism in the collagen-like region of the Bacillus anthracis BclA protein leads to variation in exosporium filament length. J Bacteriol 185 , 1555-1563 (2003). S. Rety et al. , The crystal structure of the Bacillus anthracis spore surface protein BclA shows remarkable similarity to mammalian proteins. J Biol Chem 280 , 43073-43078 (2005). L. Tan, C. L. Turnbough, Jr., Sequence motifs and proteolytic cleavage of the collagen-like glycoprotein BclA required for its attachment to the exosporium of Bacillus anthracis. J Bacteriol 192 , 1259-1268 (2010). Y. Hachisuka, S. Kozuka, M. Tsujikawa, Exosporia and appendages of spores of Bacillus species. Microbiol Immunol 28 , 619-624 (1984). F. Díaz-González et al. , Protein composition of the outermost exosporium-like layer of Clostridium difficile 630 spores. Journal of Proteomics 123 , 1-13 (2015). K. McElroy, L. Mouton, L. Du Pasquier, W. Qi, D. Ebert, Characterisation of a large family of polymorphic collagen-like proteins in the endospore-forming bacterium Pasteuria ramosa. Res Microbiol 162 , 701-714 (2011). Y. Qiu, C. Zhai, L. Chen, X. Liu, J. Yeo, Current Insights on the Diverse Structures and Functions in Bacterial Collagen-like Proteins. ACS Biomater Sci Eng , (2021). E. Maes et al. , Glycosylation of BclA Glycoprotein from Bacillus cereus and Bacillus anthracis Exosporium Is Domain-specific. J Biol Chem 291 , 9666-9677 (2016). J. A. Bozue, S. Welkos, C. K. Cote, The Bacillus anthracis Exosporium: What's the Big "Hairy" Deal? Microbiol Spectr 3 , (2015). M. S. Conover et al. , Inflammation-Induced Adhesin-Receptor Interaction Provides a Fitness Advantage to Uropathogenic E. coli during Chronic Infection. Cell Host Microbe 20 , 482-492 (2016). M. Lukaszczyk, B. Pradhan, H. Remaut, The Biosynthesis and Structures of Bacterial Pili. Subcell Biochem 92 , 369-413 (2019). Y. Lin, R. Briandet, Á. T. Kovács, Bacillus cereus sensu lato biofilm formation and its ecological importance. Biofilm 4 , 100070 (2022). Y. Huang, S. H. Flint, J. S. Palmer, Bacillus cereus spores and toxins - The potential role of biofilms. Food Microbiol 90 , 103493 (2020). J. G. Wijman, P. P. de Leeuw, R. Moezelaar, M. H. Zwietering, T. Abee, Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion. Appl Environ Microbiol 73 , 1481-1488 (2007). N. Pakharukova et al. , Structural basis for Acinetobacter baumannii biofilm formation. Proc Natl Acad Sci U S A 115 , 5558-5563 (2018). C. Terry et al. , Molecular tiling on the surface of a bacterial spore - the exosporium of the Bacillus anthracis/cereus/thuringiensis group. Mol Microbiol 104 , 539-552 (2017). R. R. Wick, L. M. Judd, C. L. Gorrie, K. E. Holt, Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLOS Computational Biology 13 , e1005595 (2017). A. Mikheenko, A. Prjibelski, V. Saveliev, D. Antipov, A. Gurevich, Versatile genome assembly evaluation with QUAST-LG. Bioinformatics 34 , i142-i150 (2018). R. R. Wick, M. B. Schultz, J. Zobel, K. E. Holt, Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 31 , 3350-3352 (2015). M. Manni, M. R. Berkeley, M. Seppey, F. A. Simão, E. M. Zdobnov, BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. Molecular Biology and Evolution 38 , 4647-4654 (2021). B. J. Walker et al. , Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement. PLoS ONE 9 , e112963 (2014). J. P. Meier-Kolthoff, J. S. Carbasse, R. L. Peinado-Olarte, M. Göker, TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Research 50 , D801-D807 (2021). J. P. Meier-Kolthoff, M. Göker, TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 10 , 2182 (2019). P. S. Krawczyk, L. Lipinski, A. Dziembowski, PlasFlow: predicting plasmid sequences in metagenomic data using genome signatures. Nucleic Acids Research 46 , e35-e35 (2018). T. Tatusova et al. , NCBI prokaryotic genome annotation pipeline. Nucleic Acids Research 44 , 6614-6624 (2016). D. H. Haft et al. , RefSeq: an update on prokaryotic genome annotation and curation. Nucleic Acids Research 46 , D851-D860 (2017). W. Li et al. , RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation. Nucleic Acids Research 49 , D1020-D1028 (2020). C. L. M. Gilchrist et al. , cblaster: a remote search tool for rapid identification and visualization of homologous gene clusters. Bioinformatics Advances 1 , (2021). S. F. Altschul, W. Gish, W. Miller, E. W. Myers, D. J. Lipman, Basic local alignment search tool. Journal of Molecular Biology 215 , 403-410 (1990). L. S. Katz et al. , Mashtree: a rapid comparison of whole genome sequence files. J Open Source Softw 4 , (2019). S. Argimón et al. , Microreact: visualizing and sharing data for genomic epidemiology and phylogeography. Microbial Genomics 2 , (2016). Additional Declarations There is NO Competing Interest. Supplementary Files SupportingData.csv Supporting Data SupportingMovie1.avi Supporting Movie 1 SupplementaryFigsandTables.docx Cite Share Download PDF Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4045273","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":278236177,"identity":"ab0a5696-15a5-417b-8bc1-f6987797a238","order_by":0,"name":"Mike Sleutel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIie2PMQrCQBBFJwhrE7QdUPQEwgZBBPUuGwRtohZp0ikE1sYD6C08QmRgKsEjGG+wpYWFSSqrbEqLfcUMA/OYPwAOxz/SEmWdlsXLDIACbKYUa0W/nRsp8KOQ30QZHUWYwwcH8plmtNC0hV6a1SoTEiQ9jWPJQtFGUwx9VhalrdE7YHhlX1J0p/CAkbQpx3cRbH/lrqFppeyMLRgXr6OS7ANBUl2pNUpliaHG4MIreTsl61j02RLswYExn/mwQ/TK33K27fbSvP5MifodhH3f4XA4HDa+pItHAO8VcGYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3247-2187","institution":"Vlaams Instituut voor Biotechnologie","correspondingAuthor":true,"prefix":"","firstName":"Mike","middleName":"","lastName":"Sleutel","suffix":""},{"id":278236178,"identity":"e88fb94b-36b8-4424-88d5-28e17ec052d9","order_by":1,"name":"Ephrem Zegeye","email":"","orcid":"","institution":"NMBU","correspondingAuthor":false,"prefix":"","firstName":"Ephrem","middleName":"","lastName":"Zegeye","suffix":""},{"id":278236179,"identity":"04371596-2824-4541-a78e-06ecb5bb8492","order_by":2,"name":"Ann-Katrin LLarena","email":"","orcid":"","institution":"NMBU","correspondingAuthor":false,"prefix":"","firstName":"Ann-Katrin","middleName":"","lastName":"LLarena","suffix":""},{"id":278236180,"identity":"74ff3d1e-5ce7-400c-a187-d548879da48b","order_by":3,"name":"Brajabandhu Pradhan","email":"","orcid":"","institution":"KUL","correspondingAuthor":false,"prefix":"","firstName":"Brajabandhu","middleName":"","lastName":"Pradhan","suffix":""},{"id":278236181,"identity":"dbab261a-ceca-4d73-9643-c919df5c0aef","order_by":4,"name":"Marcus Fislage","email":"","orcid":"","institution":"VUB","correspondingAuthor":false,"prefix":"","firstName":"Marcus","middleName":"","lastName":"Fislage","suffix":""},{"id":278236182,"identity":"6fdf022d-8f67-44ba-abb5-ec994428e305","order_by":5,"name":"Kristin O´Sullivan","email":"","orcid":"","institution":"NMBU","correspondingAuthor":false,"prefix":"","firstName":"Kristin","middleName":"","lastName":"O´Sullivan","suffix":""},{"id":278236183,"identity":"448de504-5f3b-4fef-a9d5-17b46081df0f","order_by":6,"name":"Nani Van Gerven","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Nani","middleName":"Van","lastName":"Gerven","suffix":""},{"id":278236184,"identity":"97a42ca5-edf5-452f-9f37-636664747c6b","order_by":7,"name":"Marina Aspholm","email":"","orcid":"","institution":"NMBU","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"","lastName":"Aspholm","suffix":""},{"id":278236185,"identity":"98a085b7-11f6-42fe-8084-bde7c164f6a4","order_by":8,"name":"Han Remaut","email":"","orcid":"https://orcid.org/0000-0002-9775-4102","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Remaut","suffix":""}],"badges":[],"createdAt":"2024-03-08 14:11:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4045273/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4045273/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-51804-w","type":"published","date":"2024-08-29T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52982935,"identity":"53232b87-3e1f-45a2-b434-245d5cd03446","added_by":"auto","created_at":"2024-03-19 10:36:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2536691,"visible":true,"origin":"","legend":"\u003cp\u003eNegative stain transmission electron microscopy images of the endospores of the food-poisoning outbreak strain \u003cem\u003eB. paranthracis\u003c/em\u003e NVH 0075-95: (a) Composite montage of a single endospore with a central spore body and the surrounding exosporial sack, (b) High-magnification image of the exosporium decorated with the hairy nap and two types of endospore appendages (L- and S-ENA), (c) L-ENA fibers anchored to the exosporium, exhibit a ‘ladder-like’ pattern (4.6nm intervals), are terminally decorated with single tip-fibrillae, i.e., ruffles. Ruffles consist of a 45nm long stalk that terminates into a globular head domain, (d) cryoEM image of isolated ENA fibers, (e) reconstructed cryoEM-volume of L-ENA fibers (5.8Å at FSC=0.143) with rigid body docking of 7 copies of Ena1B\u003csub\u003e19-117\u003c/sub\u003e, (f) nsTEM image of recEna3A produced in and purified from the cytoplasm of \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/617fa311e6dc70e24253ded4.png"},{"id":52983471,"identity":"4c2a37c6-6207-4ccb-af7e-506a08e32e4a","added_by":"auto","created_at":"2024-03-19 10:44:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1022932,"visible":true,"origin":"","legend":"\u003cp\u003eCryoEM volume of recEna3A L-ENA fibers: (a) Helical ultrastructure of L-ENA determined at 3.32 Å global resolution (cut-off criterion at FSC=0.143 Å\u003csup\u003e-1\u003c/sup\u003e) revealing an axial stacking of heptameric Ena3A rings that rotate 18.5° clockwise relative to each other. Two neighboring Ena3A monomers are colored in blue and cyan, (b) top-view of a single ring with a highlight of the b-sheet augmentation between the blue and cyan subunit, (c) zoom-in of a single ring - for clarity, two subunits were removed from the ring highlighted with an asterisk in (a) - showing the docking of the N-terminal connectors that covalently tether the ring docked above, (d) Highlight of the lateral intra-ring contacts: two types of disulfide bridges (Cys14-Cys15; Cys22-Cys82) exist within two neighboring subunits. In turn, each subunit connects to a subunit in the ring below via their Ntc (Cys8-Cys21; inset in stick representation), (e) On axis top-view of the L-ENA fiber model in cartoon with disulfide bridges shown in red; (f) 2D class averages of straight and curved L-ENA segments covering 4 and 9 rings, respectively,\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/216988dc1fc481c21f3d5a60.png"},{"id":52982943,"identity":"41fab917-6bff-4958-b128-7a7aba9da04b","added_by":"auto","created_at":"2024-03-19 10:36:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1236478,"visible":true,"origin":"","legend":"\u003cp\u003eStructural comparison between the S- and L-ENA fiber architectures: (a) Side and (b) on-axis view of an S-ENA fiber (pdb-id: 7A02) composed of Ena1B subunits. Helical parameters: Rise: 3.22Å, Twist: 31.0°, Pitch: 38Å), (c) Dimeric contacts of Ena1B subunits via β-sheet augmentation at the interface between the G and C strands. Subunits are tilted 28° with respect to the fiber axis. This out-of-plane interaction leads to a helical stacking of Ena1B monomers, (d) Side and (e) on-axis view of an L-ENA fiber (pdb-id: 8PDZ) composed of Ena3A subunits. Helical parameters: Rise = pitch: 45Å, Twist: 18.5°, C7), (f) Dimeric contacts of Ena3A subunits via β-sheet augmentation at the interface between the G and C strands. Although subunits are tilted 14° with respect to the fiber axis, their contacts remain in-plane yielding a heptameric ring.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/8a7de1d21d30b6ff4b67ac6f.png"},{"id":52983472,"identity":"8100671b-5de6-45fd-80b2-820b4b5e1aba","added_by":"auto","created_at":"2024-03-19 10:44:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":328966,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of L-ENA genes is concomitant with sporulation. (a) Chromosomal organization of \u003cem\u003eexsL\u003c/em\u003e, \u003cem\u003el-bclA\u003c/em\u003e and \u003cem\u003eena3A\u003c/em\u003e, and primers used in standard PCR using cDNA template; (b) The L-ENA genes \u003cem\u003el-bclA\u003c/em\u003e and \u003cem\u003eena3A\u003c/em\u003e form an operon. Agarose gel electrophoresis (1%) of PCR products conducted on cDNA from NVH0075-95 culture (4, 8, 12 and 16 h). Primer combinations and expected product sizes are shown in (a). Genomic DNA (gDNA) from NVH0075-95 and cDNA prepared from its isogenic\u003cem\u003e \u003c/em\u003eΔ\u003cem\u003eena1ABC\u003c/em\u003e/Δ\u003cem\u003eena3A \u003c/em\u003emutant were used as positive and negative controls for the PCR, respectively; (c) Expression of L-ENA genes relative to \u003cem\u003erpoB\u003c/em\u003e during vegetative growth and sporulation. Error bars represent the standard deviation of three independent experiments, each with three technical replicates.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/f9b7a7b16ca99e60d7d01aa3.png"},{"id":52982944,"identity":"579b89d2-5616-4bb3-a57a-f703dceea3b7","added_by":"auto","created_at":"2024-03-19 10:36:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":887643,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic read-out of L-ENA mutants: nsTEM micrographs of the respective L-ENA operon deletion mutants.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/fb81c3eb8e978f945dc154a2.png"},{"id":52982940,"identity":"a973977d-3234-402d-8df6-20084dee8fab","added_by":"auto","created_at":"2024-03-19 10:36:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":111476,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic organization of the L-ENA gene cluster: the \u003cem\u003eena3A\u003c/em\u003e gene is embedded in a three gene cluster, preceded on the CP116205.1 plasmid by two genes here annotated as \u003cem\u003eexsL\u003c/em\u003e and \u003cem\u003el-bclA\u003c/em\u003e, (b) Domain organization of the L-ENA proteins as identified by Interpro (10.1093/nar/gkac993). ExsL: consists of an N-terminal spore coat protein Z/Y domain and a C-terminal Ena core domain (DUF3992), L-BclA: consists of an N-terminal collagen-like domain and a C-terminal BclA-C domain, Ena3A: consists of a single Ena core domain (DUF3992). AlphaFold2 predictions are shown in cartoon representation, colour coded according to the pLDDT score.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/53174fe9a7468b62f8c74522.png"},{"id":52982938,"identity":"2d44f034-91d4-43b2-91bf-da62fe888182","added_by":"auto","created_at":"2024-03-19 10:36:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":314277,"visible":true,"origin":"","legend":"\u003cp\u003eMesoscale model of an L-ENA fiber projecting from the exosporium: L-ENA fibers are helical, protein ultrastructures composed of heptameric Ena3A rings that stack axially. At the tip that is distal from the spore, the L-ENA fiber terminates into a bacterial collagen-like ruffle protein, i.e., L-BclA. The L-ENA fiber protrudes through the hairy nap layer while being tethered to the exosporium via a dedicated anchoring protein ExsL. ExsL is expected to couple to or be integrated in the 2D crystal lattice of the exosporium. Note that for clarity, the L-ENA fibers are depicted shorter here.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/ba0dc5c79014452312b73054.png"},{"id":63611275,"identity":"fe07c7bf-a5e3-480e-aa74-2821a39b27f9","added_by":"auto","created_at":"2024-08-30 07:16:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8943500,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/f4e8448f-7a73-4cd8-b375-c8ab0bbe0a64.pdf"},{"id":52982937,"identity":"1d0bb646-2fd5-4b5a-a219-a1f91b53e1ed","added_by":"auto","created_at":"2024-03-19 10:36:59","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":74324,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting Data\u003c/p\u003e","description":"","filename":"SupportingData.csv","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/41bfa6bc631423f97e27fdd7.csv"},{"id":52982941,"identity":"4ba2ab84-48bd-4438-bb11-5cb440a39fa4","added_by":"auto","created_at":"2024-03-19 10:36:59","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11120504,"visible":true,"origin":"","legend":"Supporting Movie 1","description":"","filename":"SupportingMovie1.avi","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/a82cd42a41627fbd241f14d2.avi"},{"id":52982945,"identity":"0a2dc2e8-9d82-4a7a-8e99-63fc2bc8cbd1","added_by":"auto","created_at":"2024-03-19 10:36:59","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7756906,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigsandTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4045273/v1/75e3bafae296fbabc0996cc5.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"CryoEM structure of a novel class of spore virulence factors on the foodborne outbreak strain Bacillus paranthracis NVH 0075-95","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndospores formed by bacteria in the Bacillota (Fimicutes) phylum are among the most durable lifeforms found in nature. This cryptobiotic way of living can bridge long periods that are not conducive to survival of the vegetative state, waiting for environmental stimuli to trigger germination and terminate the state of hibernation. Their remarkable resilience can in part be traced back to the extraordinary robust layers that encase the genomic DNA, while maintaining the permeability for germinants that signal when conditions are again conducive for metabolically active life (\u003cem\u003e1, 2\u003c/em\u003e). For the endospore forming bacteria of the genera \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eClostridium\u003c/em\u003e, the genome is housed within a partially dehydrated central core that is encapsulated by a thick peptidoglycan layer, referred to as the cortex. The cortex, in turn, is covered by at least four consecutive protein layers that make up the spore coat: i.e., the basement layer, the inner and outer coat and the crust, comprising about 70 different proteins (\u003cem\u003e3, 4\u003c/em\u003e). Many species have an additional layer called the exosporium, which forms a flexible, glycosylated, para-crystalline sac-like structure that surrounds the spore, defining an interstitial volume between the crust and the exosporium. As the outermost spore layer, the exosporium mediates endospore\u0026acute;s connection to the environment and the infected host (\u003cem\u003e5, 6\u003c/em\u003e). An important role in this interaction is played by the \u003cu\u003eb\u003c/u\u003eacterial \u003cu\u003ec\u003c/u\u003eollagen \u003cu\u003el\u003c/u\u003eike protein of \u003cu\u003eA\u003c/u\u003enthracis (BclA), which covers the spore surface as a dense decoration called the hairy nap (\u003cem\u003e6\u003c/em\u003e). It plays an immune regulatory function by binding complement factor H (CHF) thereby inhibiting downstream complement activation (\u003cem\u003e7\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eA poorly understood component of the spore surface are micrometer long filamentous, pilus-like structures seen in virulent \u003cem\u003eBacillus\u003c/em\u003e strains, dubbed endospore appendages (ENA). First reported and classified for \u003cem\u003eClostridium \u003c/em\u003espores in the late 1960s (\u003cem\u003e8-10\u003c/em\u003e), followed by \u003cem\u003eB.\u003c/em\u003e \u003cem\u003ecereus\u003c/em\u003e in the 1970s (\u003cem\u003e11\u003c/em\u003e), ENAs can vary greatly in length, diameter and overall shape. They are present on spores of notable human and animal pathogens such as \u003cem\u003eB. anthracis\u003c/em\u003e, \u003cem\u003eB. cereus \u003c/em\u003e(\u003cem\u003e11\u003c/em\u003e), \u003cem\u003eC. botulinum \u003c/em\u003e(\u003cem\u003e10\u003c/em\u003e) and \u003cem\u003eC. sordellii \u003c/em\u003e(\u003cem\u003e12\u003c/em\u003e), and seem mostly absent in saprophytic species, including \u003cem\u003eB. subtilis\u003c/em\u003e. Pilus-like structures are well known from vegetative cells (\u003cem\u003e13\u003c/em\u003e). In Gram-positive bacteria, pili are predominantly formed by the sortase-mediated covalent coupling and cell-wall anchoring of secreted LPxTG motif containing subunits via isopeptide bonds (\u003cem\u003e14-16\u003c/em\u003e). These pili are primarily found in pathogenic species (i.e. \u003cem\u003eCorynebacterium dyphtheriae\u003c/em\u003e, \u003cem\u003eStaphylococcus spp.\u003c/em\u003e, \u003cem\u003eEnterococcus spp.\u003c/em\u003e, \u003cem\u003eStreptococcus spp.\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e spp.), where they serve an adhesive function, by binding to host tissues and/or mediating self-contact in multicellular communities or biofilms. Endospores represent an important dissemination form in pathogenic \u003cem\u003eBacillaceae\u003c/em\u003e, so similar to pili on vegetative cells, ENAs may mediate cell-cell and cell-tissue interaction.\u003c/p\u003e\n\u003cp\u003eDespite observations for more than six decades, their extreme physico-chemical robustness made ENAs resist traditional proteomic identification methods (N-terminal sequencing; peptide fingerprinting) (\u003cem\u003e17\u003c/em\u003e) preventing the genetic study of their function. This stalemate was recently broken by the virtue of cryoID, the direct molecular identification of proteinaceous structures through 3D reconstruction by cryo-electron transmission microscopy (cryoEM) \u003cem\u003e(18, 19)\u003c/em\u003e. Spores of the food poisoning outbreak strain \u003cem\u003eBacillus paranthracis\u003c/em\u003e NVH 0075-95 were found to display two structurally distinct ENAs, based on the typical patterning seen in nsTEM images dubbed \u0026lsquo;staggered\u0026rsquo; and \u0026lsquo;ladder\u0026rsquo;-like or \u0026lsquo;S\u0026rsquo;- and \u0026lsquo;L-ENA\u0026rsquo;, respectively \u003cem\u003e(19, 20)\u003c/em\u003e. CryoID studies of S-ENA revealed it represents a novel pilus superfamily composed of two major subunits, Ena1A and Ena1B. Both proteins consist of a single DUF3992 domain comprising a b-jellyroll fold with an N-terminal extension. Ena1A and 1B subunits self-organize into micrometers long helical ultra-structures by lateral b-sheet augmentation, fortified by lateral and longitudinal inter-molecular disulfide bridges. Strikingly, S-ENAs terminate into 4 to 6 flexible, 2nm diameter, tip-fibrillae (hereafter referred to as \u003cem\u003eruffles\u003c/em\u003e) for which a genetic identification or structural elucidation remains absent. Phylogenomic analysis showed that \u003cem\u003eena1/2\u003c/em\u003e clusters are ubiquitous in pathogenic Bacilli (\u0026gt;90% occurrence in \u003cem\u003eB. cereus \u003c/em\u003es.l.) suggesting that S-ENAs could be hitherto overlooked virulence factors. Biomechanical studies suggest S-ENAs are implicated in self-adherence and the clustering of spores, albeit by an unknown mechanism (\u003cem\u003e21\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eAs mentioned above, the spores of NVH 0075-95 are decorated with a second type of appendages, dubbed L-ENA. They are shorter (typically \u0026lt; 1\u0026micro;m), 7nm diameter ENAs that are tethered to the exosporium layer and are decorated with a single ruffle at their distal terminus which has been identified as a major driver in spore-spore aggregation (Jonsmoen \u003cem\u003eet al.\u003c/em\u003e, in preparation). In this contribution, we use a combined cryoEM - genome mining approach to identify the major structural subunit of L-ENA (hereafter referred to as Ena3A) and solve the helical fiber ultrastructure to a resolution of 3.3 \u0026Aring;. We show that \u003cem\u003eena3A\u003c/em\u003e is embedded in a three-gene cluster that holds all components for L-ENA assembly and anchoring to the exosporium. \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eL-ENA fibers constitute a novel sub-class of disulfide cross-linked endospore appendages.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe endospores of the food-poisoning outbreak strain \u003cem\u003eB. paranthracis\u003c/em\u003e NVH 0075-95 are decorated with two types of ENA fibers, i.e., S-ENA and L-ENA (Fig.1a-b). Close inspection of the exosporium layer reveals that L-ENA fibers protrude from the brush-like hairy nap layer (Fig.1b) seemingly tethered to the exosporium. L-ENA fibers have an apparent diameter of 8nm and exhibit a ladder-like uranyl staining pattern in nsTEM with a typical recurring distance of 4.6nm between consecutive ladder segments. L-ENAs consistently terminate in a single 2nm diameter tip-fibrillum or ruffle, that consists of a stalk region (45nm in length) and a terminal knob domain (Fig.1c). In preparation of cryoEM data collection, we produced a sample enriched in ENA via shear-induced dislodging from the spore surface followed by a series of purification steps as described in (\u003cem\u003e18\u003c/em\u003e). Next, a 1568 movie cryoEM dataset (60 frames, 0.766\u0026Aring;/pixel) was collected on a JEOL CRYO ARM 300 microscope, yielding a 5.8\u0026Aring; resolution (FSC=0.143 criterion) reconstructed cryoEM-volume of L-ENA fibers with C7 symmetry (see Fig.1e and Supporting Figure 1). The refined helical parameters are a twist of 17.04\u0026deg;, and a rise of 43.82\u0026Aring;. We attribute the limited resolution of the final reconstruction to (i) the sparsity of images that contained L-ENA fibers (414 micrographs, i.e., 26% of the total dataset), and (ii) the innate flexibility of the L-ENA fibers (discussed below). The limited resolution precluded us to \u003cem\u003ede novo\u003c/em\u003e build an atomic model of L-ENA but allowed us to identify the fold of the constituent monomers. Indeed, an exploratory rigid body docking exercise using Ena1B as template model (extracted from PDB: 7A02) taught us that the L-ENA subunits appeared structurally homologous to Ena1A/B and therefore likely represented members of the DUF3992 family. A careful search of the NVH 0075-95 genome (GCA_027945115.1) with HMMsearch 3.0 (\u003cem\u003e22\u003c/em\u003e) using a hidden Markov model generated from a dataset of Ena1/2 protein sequences as query led to the identification of a fourth gene outside the \u003cem\u003eena1a-c\u003c/em\u003e cluster encoding for a DUF3992 domain-containing protein. As such, we reasoned that WP_017562367 represented the candidate L-ENA subunit and ordered its coding sequence as a synthetic gene, and cloned it into pET28a for cytoplasmic expression in \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eC43 (DE3), similar to the approach taken for recombinant S-ENA production (\u003cem\u003e18\u003c/em\u003e). To probe for the presence of putative L-ENA fibers, the insoluble fraction (after lysozyme treatment and 1% SDS extraction) of the \u003cem\u003eE. coli\u003c/em\u003e lysate was analyzed using nsTEM. The obtained micrographs confirmed the presence of micron-sized, 7nm diameter fibers with a characteristic ladder-like pattern at 4.6nm intervals (Fig.1f). Notably, the recEna3A fibers did not have any ruffles on their termini. This result served as an initial confirmation that WP_017562367 is the major subunit of L-ENAs found on \u003cem\u003eB. paranthracis\u003c/em\u003e NVH 0075-95. To reflect the fact that WP_017562367 is a member of a new \u003cem\u003eena\u003c/em\u003e gene cluster, we propose to name it \u003cem\u003eena3a\u003c/em\u003e, following the convention used for the \u003cem\u003eena1a-c\u003c/em\u003e (e.g., \u003cem\u003eB. cereus\u0026nbsp;\u003c/em\u003etype\u003cem\u003e\u0026nbsp;S-ENA\u003c/em\u003e) and \u003cem\u003eena2a-c\u003c/em\u003e (e.g., \u003cem\u003eB. thuringiensis\u0026nbsp;\u003c/em\u003etype\u003cem\u003e\u0026nbsp;S-ENA\u003c/em\u003e) (\u003cem\u003e18\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eGiven the relative ease of production and purification of recEna3A fibers in comparison to the L-ENA purification from the natural source, we proceeded to collect a 10886 movies cryoEM dataset of vitrified recEna3A fibers (Supporting Figure 2) leading to a 3.3 \u0026Aring; global resolution (FSC=0.143 criterion) cryoEM volume after helical refinement using C7 symmetry in CryoSPARC v4.0.3 (Fig.2). The refined helical parameters are a twist of 18.5\u0026deg;, and a rise of 44.97\u0026Aring;. The recEna3A cryoEM volume reveals an axial stacking of heptameric Ena3A rings that rotate 18.5\u0026deg; clockwise relative to each other (Fig.2a). Each ring is composed of seven Ena3A molecules that interface laterally via b-sheet augmentation. The rings encircle a central, hollow volume which we refer to as the ring lumen (Fig.2b). Consecutive rings interlock with each other using the N-terminal extensions of the upper ring docking into the lumen of the ring below (Fig.2c). We refer to the first 14 N-terminal residues as the N-terminal connector (i.e., Ntc) in analogy to the N-termini of Ena1A/B subunits that interlock from above with subunits i-9 and i-10 of the neighboring helical staircase (\u003cem\u003e18\u003c/em\u003e). Note that the orientation of the L-ENA fiber in Fig. 2 is such that the top rings represent the distal, ruffled end of the fiber, and that the bottom rings form the pointy end of the fibers, proximal to the spore surface. Close inspection of the cryoEM map revealed covalent cross-links between neighboring Ena3A subunits at three distinct locations (Fig.2d). After manual building of the final L-ENA model, we identified these contacts to be 3 types of inter-molecular disulfide bridges. Two disulfide bridges (i.e., Cys22-Cys82 and Cys14-Cys15) serve to reinforce the lateral contacts within a single ring, whereas one disulfide bridge (i.e., Cys8-Cys21) confers longitudinal coupling between the lumen and the Ntcs of consecutive rings. Given the heptameric nature of the structure, each ring segment will contain 21 inter-molecular disulfide bridges that are organized along three concentric rings centered on the fiber axis (Fig.2e).\u003c/p\u003e\n\u003cp\u003eNext we tested the stability of recombinant L-Ena fibers by subjecting the fibers to a series of physical (autoclaving) and/or chemical treatments (8M urea, SDS, proteinase K, formic acid). Following the respective treatments, we performed nsTEM imaging of the recovered fibers and determined the 2D class average to gauge the intactness of the fibers (Supporting Figure 4). Remarkably, the 2D class average images of all treatments (apart from the 100% formic acid) are very similar to the 2D class averages obtained from \u003cem\u003eex vivo\u003c/em\u003e purified fibers. We were not able to produce 2D class averages for the 100% formic acid (FA) treated sample, likely indicating that the Ena3A monomers had (partially) unfolded. Interestingly, the FA-treated fibers did not depolymerize, suggesting that disulphide bonds had not been reduced. We conclude that the extensive hydrogen bonding between the Ena3A subunits combined with the covalent cross-links underlies the physico-chemical robustness of the L-ENA fibers.\u003c/p\u003e\n\u003cp\u003eL-ENA marries this extreme stability with a remarkable flexibility as judged from the regions of high local curvature in the nsTEM micrographs and 2D class average images obtained during cryoEM processing (Fig.1f). To illustrate the point further, we manually selected and extracted curved L-ENA fiber segments from the motion-corrected micrographs and performed 2D classification (Supporting Figure 3a). To resolve the underlying structural heterogeneity, we performed a 3D variability analysis using CryoSPARC (Supporting Figure 3b). The resulting volume series (processed in ChimeraX 1.4 (\u003cem\u003e23, 24\u003c/em\u003e) and exported as Supporting Movie 1) provides further molecular insights into the L-ENA flexibility. Ena3A rings exhibit a rocking motion normal to the fiber axis with the respective hinge points centered on the Ntcs. Hence, L-ENA flexibility can be traced back to (i) the spatial separation between consecutive rings thereby providing the possibility for local ring displacement without introducing steric clashes, (ii) combined with the intrinsic flexibility of the N-terminal connectors.\u003c/p\u003e\n\u003cp\u003eEna3A subunits consist of a typical jellyroll fold (\u003cem\u003e25\u003c/em\u003e) comprised of two juxtaposed \u0026beta;-sheets containing strands BIDG and CHEF (Fig.3). As mentioned earlier, the jellyroll domain is preceded by a flexible 14-residue Ntc that mediates inter-ring coupling. The backbone (420 atoms) root-mean squared displacement (RMSD) between Ena1B and Ena3A is 3.7\u0026Aring; even though the sequence identity is only 28.4% (Supporting Figure 5a-c). Despite the structural similarities at the fold level, S- and L-ENA have a markedly different quaternary architecture (Figure 3a-c). Ena1B subunits interact laterally via b-sheet augmentation (Supporting Figure 5d-e) with a non-zero (i.e., 3.2\u0026Aring;) vertical offset, making a 28\u0026deg; angle relative to the fiber axis. This in turn leads to a helical stacking of Ena1B monomers yielding an S-ENA fiber with a diameter of 110\u0026Aring;. Ena3A subunits form a similar dimer interface (see the residues marked with an asterisk in Supporting Figure 5b) that follows the same register between the interfacing strands G and C (Supporting Figure 5f-g). Contrary to Ena1B though, neighboring Ena3A subunits lie within the same plane (i.e., no vertical offset) thereby producing closed ring-like structures. We attribute this difference in axial displacement to the relative tilts that the subunits make with respect to the fiber axis, i.e., 28\u0026deg; and 14\u0026deg; for Ena1B and Ena3A, respectively (Fig. 3c and f). L-ENA fiber biogenesis therefore likely proceeds via docking and covalent locking (via the Cys8-Cys21 disulfide) of fully formed rings, whereas S-ENA fibers elongate via integration of successive monomers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution and expression of the \u003cem\u003eena3A\u0026nbsp;\u003c/em\u003egene cluster\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInspection of the GCA_027945115 genome reveals that \u003cem\u003eena3A\u003c/em\u003e (PGS39_28750) is embedded in a three-gene cluster on the NZ_CP116205 plasmid, hereafter referred to as the \u003cem\u003eena3\u003c/em\u003e gene cluster (Figure 4). In this cluster, \u003cem\u003eena3A\u003c/em\u003e is preceded by the genes PGS39_28740 and PGS39_28745. For reasons discussed below, we suggest naming these two genes, i.e. \u003cem\u003eexsL\u003c/em\u003e and \u003cem\u003el-bclA\u003c/em\u003e, respectively. The \u003cem\u003eena3\u0026nbsp;\u003c/em\u003egene cluster is rare: it was found in only 62 organisms through a remote search of the entire NCBI RefSeq non-redundant protein database using cblaster (CAGECAT v. 1.0). Assemblies of sufficient quality were downloaded and appended to a representative database of genomes of the \u003cem\u003eBacillus\u003c/em\u003e genus. The proportion of genomes carrying an \u003cem\u003eena3\u003c/em\u003e gene cluster was indeed low; only 9.5% (n=62/656) of the \u003cem\u003eB. cereus s. l.\u003c/em\u003e genomes, of which 51 were \u003cem\u003eB. cereus\u0026nbsp;\u003c/em\u003e(n=51/126)\u003cem\u003e,\u0026nbsp;\u003c/em\u003efour \u003cem\u003eB. thuringiensis\u0026nbsp;\u003c/em\u003e(n=4/52), one \u003cem\u003eB. anthracis\u0026nbsp;\u003c/em\u003e(n=1/63), one \u003cem\u003eB. paranthracis\u0026nbsp;\u003c/em\u003e(n=1/4), one\u003cem\u003e\u0026nbsp;B. toyonensis\u0026nbsp;\u003c/em\u003e(n=1/204)\u003cem\u003e,\u0026nbsp;\u003c/em\u003etwo \u003cem\u003eB. mobilis\u0026nbsp;\u003c/em\u003e(n=2/5)\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand two \u003cem\u003eBacillus\u0026nbsp;\u003c/em\u003esp. (2/4) (Supporting Figure 6). Most genomes had one copy of the gene cluster (n=53), while nine strains had paralogs, carrying two (n=5, \u003cem\u003eB. cereus\u0026nbsp;\u003c/em\u003e(3)\u003cem\u003e, Bacillus\u0026nbsp;\u003c/em\u003esp. (2)), three (n=2, \u003cem\u003eB. cereus\u003c/em\u003e), four (n=1, \u003cem\u003eB. thuringiensis\u003c/em\u003e) and five (n=1, a \u003cem\u003eB. thuringiensis\u003c/em\u003e) copies. The \u003cem\u003eena3\u0026nbsp;\u003c/em\u003egene cluster was not found in \u003cem\u003eB. subtilis\u003c/em\u003e or other saprophytic Bacilli (see Supporting Data)\u003cem\u003e.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThirteen genomes with an L-ENA gene cluster were complete and closed, and the genomic location of the gene cluster could be inspected. The \u003cem\u003eena3\u003c/em\u003e gene cluster was located either on the chromosome (n=7 genomes), on plasmids (n=1 genome) or were found as paralogs on one or more plasmids and on the chromosome (n=5 genomes). Many of the isolates carrying the \u003cem\u003eena3\u0026nbsp;\u003c/em\u003egene cluster were from outbreaks of bloodborne infections in hospitals in Italy and Japan (\u003cem\u003e26, 27\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eena3\u003c/em\u003e gene cluster of NVH 0075-95 is flanked by an incomplete topoisomerase upstream and a complete Tn3 family transposase upstream. This gene synteny was found in only two other strains in the cblaster/clinker analysis: \u003cem\u003eB. cereus\u003c/em\u003e AFS093282 (NZ_NVMQ01000017.1) and \u003cem\u003eB. pacificus\u003c/em\u003e strain BC444B (accession NZ_JAOPRQ010000006.1). In other strains, the L-ENA gene cluster was flanked by frameshifted versions of Tn3 family transposases, tyrosine- type recombinase/integrase and site-specific integrases. A few strains had a shorter Tn3 family transposase upstream of the L-ENA gene clusters. Taken together, the L-ENA gene cluster is/or has been located on a transposon and is part of the mobilome of the \u003cem\u003eB. cereus s.l.\u0026nbsp;\u003c/em\u003egroup, accounting for the polypheletic distribution of this gene cluster in the population.\u003c/p\u003e\n\u003cp\u003eTo determine the expression of L-ENA genes (\u003cem\u003eexsL\u003c/em\u003e, \u003cem\u003el-\u003c/em\u003e\u003cem\u003ebclA\u003c/em\u003e and \u003cem\u003eena3A\u003c/em\u003e), NVH0075-95 was cultured in sporulation medium for 16 hours, and cDNA prepared from culture samples collected at four-hour intervals after inoculation (4, 8, 12 and 16 hours) and analyzed by PCR. Using primers that specifically amplify open reading frames across \u003cem\u003eexsL\u003c/em\u003e\u0026rarr;\u003cem\u003el-bclA\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;l-bclA\u003c/em\u003e\u0026rarr;\u003cem\u003eena3A\u0026nbsp;\u003c/em\u003e(Figure 4a), we detected a ~587 bp PCR product across \u003cem\u003el-bclA\u003c/em\u003e\u0026rarr;\u003cem\u003eena3A\u003c/em\u003e, but not for\u003cem\u003e\u0026nbsp;exsL\u003c/em\u003e\u0026rarr;\u003cem\u003el-bclA\u0026nbsp;\u003c/em\u003e(Figure 4b)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThis suggests that \u003cem\u003el-bclA\u0026nbsp;\u003c/em\u003eand \u003cem\u003eena3A\u003c/em\u003e are expressed bicistronically. Notably, no \u003cem\u003el-bclA-ena3A\u003c/em\u003e transcript was detected during the first 8 hrs of cultivation, which represents the vegetative growth phase (Fig. 4b). Consistent with the PCR result, the data from a qPCR analysis indicated that the L-ENA genes are overexpressed exclusively during the sporulation phase (12 and 16 hours) (Fig. 4c). Notably, a ~13000, ~1200 and ~40-fold increase in the expression of \u003cem\u003eena3A\u003c/em\u003e,\u003cem\u003e\u0026nbsp;l-bclA\u0026nbsp;\u003c/em\u003eand \u003cem\u003eexsL\u003c/em\u003e, respectively, was evident in the samples collected at 12 hours after inoculation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe \u003cem\u003eena3\u003c/em\u003e gene cluster consists of the L-ENA subunit, the exosporium anchoring protein ExsL and the complement C1Q-like ruffle protein L-BclA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEna3A\u003c/em\u003e is found in an operon with \u003cem\u003eexsL\u003c/em\u003e and \u003cem\u003el-bclA\u003c/em\u003e. In pursuit of the biological roles of \u003cem\u003eexsL\u003c/em\u003e and \u003cem\u003el-bclA\u003c/em\u003e, we made individual knockouts strains (\u0026Delta;\u003cem\u003eexsL\u003c/em\u003e and \u0026Delta;\u003cem\u003el\u003c/em\u003e-\u003cem\u003ebclA\u003c/em\u003e) and investigated their respective endospores by nsTEM (Fig. 5). ExsL depleted spores are devoid of L-ENA fibers coupled to the exosporium but are otherwise morphologically identical to wild-type NVH 0075-95 spores. Careful inspection of the grid areas lead to the identification of detached L-ENAs in the spore supernatant, with ruffles present, suggesting that ExsL mediates the connection of L-ENA to the exosporium. Conversely, \u0026Delta;\u003cem\u003el-bclA\u003c/em\u003e spores have L-ENAs present on the exosporium, but the fibers lack the distal ruffle. We do note that the ruffles remained present on the S-ENA fibers, suggesting that \u003cem\u003el-bclA\u0026nbsp;\u003c/em\u003especifically encodes for the L-ENA ruffle protein. As expected, \u003cem\u003eena3a\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e spore samples were completely devoid of L-ENA fibers, be it spore attached or detached. Complementation of the \u0026Delta;\u003cem\u003eexsL\u003c/em\u003e, \u0026Delta;\u003cem\u003el-bclA\u003c/em\u003e and \u0026Delta;\u003cem\u003eena3A\u003c/em\u003e mutants with a low copy plasmid (pHT315) containing the respective genes, restored the corresponding spore phenotypes to that of the wild type strain (Fig\u003cstrong\u003e. 5).\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA primary sequence analysis of ExsL (WP_048548726.1) using Interpro (\u003cem\u003e28\u003c/em\u003e) shows that it is composed of an N-terminal spore coat protein Z (PF10612) domain and a C-terminal Ena core (DUF3992) domain (see the domain organization and corresponding AF2 prediction in Fig.6b). To gain further insights into the molecular mechanisms of L-ENA anchoring and ruffle formation, we performed Alphafold2 (AF2) modelling (\u003cem\u003e29-31\u003c/em\u003e). First, we tested for the plausibility of a Ena3A-ExsL complex formation. In Supporting Figure 7 we show the AF2 multimer v1.2 model of an Ena3A-ExsL dimer, which had an overall pLDDT score of 82.6 and a ptmscore of 0.73, which is in support of the hetero-dimer hypothesis. As expected, Ena3A interfaces with ExsL via its C-terminal Ena-core domain in a manner that mimics the Ena3A dimer interface found in the L-ENA structure (Supporting Figure 7e-f). This is somewhat surprising given the low sequence identity (17.4%) between the Ena3A and the ExsL Ena-core domain (i.e. residues 157-262). Inspection of the pairwise sequence alignment (Supporting Figure 6d) between Ena3A and ExsL learns that key Ena3A residues (C82, S84 and T86) involved in lateral subunit contacts are conserved in ExsL (C224, S226 and T228). In fact, AF2 predicts a disulfide bridge between ExsL cys224 and Ena3A cys22, which mimics the intra-ring L-ENA S-S bridge between cys22 and cys82 (Supporting Figure 7e-f; Fig. 2d-e). \u0026nbsp;Despite the low sequence identity in Ena3A and the ExsL Ena-core, the Ena3A-Ena3A and Ena3A-ExsL contacts are highly equivalent (Supporting Figure 7e-f), in line with the general mechanism of b-sheet augmentation, which is primarily driven by shape complementarity and backbone H-bonding and is relatively insensitive to the amino acid sequence in the paring\u0026nbsp; b-strands (\u003cem\u003e32\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eIn addition, we looked at the AF2 prediction of a putative ExsL-ExsY dimer. ExsY (WCA20099.1) is one of the major constituents of the \u003cem\u003eB. cereus\u003c/em\u003e exosporium and shares structural similarities to the C-terminal CotZ domain of the \u003cem\u003eexsL\u003c/em\u003e gene in the \u003cem\u003eena3\u003c/em\u003e gene cluster, albeit at low sequence identity, i.e., 30.8% (Supporting Figure 8a,b,f). Our assumption based on the \u003cem\u003eD\u003c/em\u003e\u003cem\u003eexsL\u0026nbsp;\u003c/em\u003ephenotype and the ExsL-Ena3A AF2 model was that ExsL could also be a potential binding partner of ExsY and, in doing so, act as an exosporium-embedded anchor point for L-ENA fibers. Indeed, the AF2 multimer model of a ExsL-ExsY dimer (with a relatively high pLDDT score of 80.2 and a ptmscore of 0.71) shows an ExsL-ExsY coupling via the N-terminal ExsL\u003csub\u003e1-156\u003c/sub\u003e domain with ExsY, lending further credence to the supposition that ExsL serves as a connective bridge between the paracrystalline exosporium and L-ENA. In analogy to the ExsL-Ena3A dimer, the predicted ExsL-ExsY contact mimics the homomeric ExsY contacts (Supporting Figure 8c,d,e,g) that we observe in a high confidence AF2 multimer prediction of an ExsY hexamer (pLDDT=81.3; ptmscore=0.86), i.e., the base building unit of the exosporium lattice.\u003c/p\u003e\n\u003cp\u003eIn turn, L-BclA (WP_271292911.1) contains an N-terminal Collagen triple helix repeat domain (PF01391) followed by a BclA C-terminal domain (PF18573; part of the C1q_TNF clan (Pfam CL0100)). BclA is the major glycoprotein of the hairy nap found to cover the exosporium of most B. cereus s.l. species (\u003cem\u003e33, 34\u003c/em\u003e). Interestingly, this same architecture is found in vertebrate complement component C1q (\u003cem\u003e35\u003c/em\u003e) (uniprot: P02745). In Supporting Figure 9a-b, we present the AF2 multimer model of an L-BclA\u003csub\u003e94-336\u003c/sub\u003e homotrimer that consists of the C-terminal trimerization domain and a segment of the collagen-like triple helix. The corresponding pLDDT value of 94 and ptmscore of 0.9 support the accuracy of the predicted fold, as well as the supposed trimeric stochiometry in analogy to the crystal structure (PDB: 1WCK) of BclA-C of \u003cem\u003eB. anthracis\u003c/em\u003e. Based on this model, we predict the lateral dimension of the C-terminal domain to be approximately 4nm, and for the triple collagen helix we project an average length of 2.8 \u0026Aring; per residue (see discussion below; Supporting Figure 9a). The all-atom RMSD between L-BclA-CTD and BclA-C is 3.55 \u0026Aring; despite the low sequence identity of 22.1%, demonstrative of significant structural homology (Supporting Figure 9c). Tan and Turnbough showed that the attachment of BclA to the basal layer protein BxpB of the exosporium is dependent on (i) proteolytic removal of the first 20 residues from the N-terminus and (ii) the presence of an N-terminal submotif -hereafter referred to as the exosporium leader sequence (interpro: IPR0212010; Supporting Figure 9d) - in front of the collagen-like region (\u003cem\u003e36\u003c/em\u003e). Inspection of the N-terminal region of L-BclA demonstrates the absence of an exosporium leader sequence, which indicates that this collagen-like protein is likely not targeted to the exosporium.\u003c/p\u003e\n\u003cp\u003eA notable feature of collagen-like proteins is that the number of residues that comprise the collagen-stalk region in the primary sequence translates proportionally to the axial dimension of the folded, trimeric entity simply due to the extended nature of the collagen fold (\u003cem\u003e34\u003c/em\u003e). To that end, we searched the GCA_027945115.1 genome for the \u0026lsquo;local\u0026rsquo; orthologue of BclA (uniprot: Q81JD7) and compared its primary sequence to L-BclA (Supporting Figure 9e). BclA\u003csub\u003eNVH 0075-95\u003c/sub\u003e (WCA20088.1) has a collagen-like region of 125 residues, whereas the corresponding domain of L-BclA spans 173 residues. Based on the calibration discussed in Supporting Figure 9a, this translates into a predicted fully extended length of a folded trimeric entity of 39 nm and 52 nm for BclA and L-BclA, respectively. These predictions are in agreement with experimental measurements (average height of the hairy nap: ~36nm and average length of L-ENA ruffle: ~50nm) from nsTEM micrographs (Supporting Figure 9f). To further understand the topology of the L-ENA/L-BclA complex, we performed a single round of 2D classification based on particles that were manually picked from the cryoEM dataset that was collected on \u003cem\u003eex vivo\u003c/em\u003e isolated ENA fibers, focusing on L-ENA termini that were decorated with ruffles (Supporting Figure 9g). This class average shows that the ruffle docks into the lumen of the Ena3A ring at the apex of the L-ENA fiber, i.e. mimicking the inter-ring docking mechanism that exists along the body of the fiber. We therefore compared the Ntc of Ena3A to the N-terminus of L-BclA (Supporting Figure 9d). Although no particular sequence homology could be detected, both sequences contain a double cysteine motif at positions 8 and 13, respectively. As cysteine 8 mediates coupling of the Ena3A Ntc to the ring lumen, we speculate that the L-BclA C\u003csub\u003e13\u003c/sub\u003eC\u003csub\u003e14\u003c/sub\u003e motif could be involved in a similar coupling mechanism.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough prominently present on the surfaces of many endospores (\u003cem\u003e37\u003c/em\u003e) and described since the 1970s for \u003cem\u003eBacillus \u003c/em\u003e(\u003cem\u003e11\u003c/em\u003e), the genetic identity and biological role of ENAs have remained poorly understood (\u003cem\u003e19\u003c/em\u003e). Using cryoID, Alphafold modelling and genetic approaches we were able to identify (this work and (\u003cem\u003e20\u003c/em\u003e)) two members of a novel class (DUF3992-based) of ultra-robust ENAs found on the surface of \u003cem\u003eBacillus paranthracis\u003c/em\u003e spores. We showed that the endospores of the food-poisoning outbreak strain \u003cem\u003eB. paranthracis\u003c/em\u003e NVH 0075-95 are decorated with two types of filamentous protein structures, i.e., the endospore appendages S- and L-ENA. Moreover, we have shown that both S- and L-ENA fibers are decorated with respectively, 3-5 or a single tip fibrillae (or ruffles) at their terminus. While the genetic identification of the S-ENA ruffles is still undergoing, the work presented here identifies L-ENA ruffles as L-BclA, a new member of the family of bacterial collagen-like proteins (CLPs). It is well established that Firmicutes endospores display bacterial CLPs on their surface (\u003cem\u003e38-40\u003c/em\u003e), but the biological function remains unclear for most of these proteins (\u003cem\u003e40\u003c/em\u003e). The most well-known CLP within the \u003cem\u003eBacillus \u003c/em\u003eclade is BclA (\u003cem\u003e33\u003c/em\u003e), the major glycoprotein (\u003cem\u003e41\u003c/em\u003e) of the hairy nap (\u003cem\u003e42\u003c/em\u003e) that is targeted to the exosporium via an N-terminal exosporium leader sequence (\u003cem\u003e36\u003c/em\u003e). BclA has been shown to recruit complement factor H (CFH) to the spore surface (\u003cem\u003e7\u003c/em\u003e), and its recruitment onto the exosporium facilitates complement C3 degradation, which in turn inhibits downstream complement activation, and ultimately promotes spore persistence. Here we have shown that contrary to BclA, L-BclA is not coupled to the exosporium - this is reflected in the absence of an exosporium leader-sequence at the N-terminus of L-BclA. Based on the morphological similarities between S- and L-ENA tip-fibrillae (i.e. a ~2nm wide flexible fibrillum terminating in a knob-like domain) in combination with the \u003cem\u003el-bclA\u003c/em\u003e deletion phenotype (L-ENA ruffle: absent, S-ENA: present), we hypothesize that the tip fibrillum of S-ENA is likely to be encoded by a separate CLP also lacking in an exosporium leader sequence, and are searching for putative \u0026lsquo;S-BclA\u0026rsquo; candidates among a list of CLP genes we identified in the \u003cem\u003eB. paranthracis \u003c/em\u003eNVH0075-95 genome. This suggests that each ENA subtype is decorated by its own cognate ruffle protein hinting at a functional diversification across the ENA subclasses. Similar specialization of bacterial pili towards a specific target or class of surfaces has been reported for e.g. sortase-mediated and chaperone-user pili (\u003cem\u003e43\u003c/em\u003e). Virulent strains can carry multiple pilus operons, most of which display a single, functionally diverse adhesin subunit at the tip of the pilus (\u003cem\u003e44\u003c/em\u003e). The strategic spatial positioning of pilus-presented adhesins promotes efficient attachment and there is a high probability that the ruffles of Ena\u0026rsquo;s exert related functions.\u003c/p\u003e\n\u003cp\u003eFollowing on the first structural and molecular characterization of ENAs (\u003cem\u003e18\u003c/em\u003e), Jonsmoen \u003cem\u003eet al.\u003c/em\u003e showed that ENAs are involved in spore aggregation (\u003cem\u003e21\u003c/em\u003e). While the molecular underpinnings of the spore-spore coupling mechanism remained unclear at the time, we recently identified L-BclA as the ruffle species that is involved in establishing inter-spore interactions (Jonsmoen et al., in preparation). Biofilm formation in the \u003cem\u003eBacillus cereus \u003c/em\u003egroup \u003cem\u003esensu lato \u003c/em\u003eis well recognized (\u003cem\u003e45\u003c/em\u003e) and instantiates as floating pellicles, submerged biofilms attached to abiotic surfaces and on living tissues (\u003cem\u003e46\u003c/em\u003e). Spores tend to accumulate towards the later stages of biofilm development and can -depending on the strain- constitute up to 90% of the total biofilm cell count (\u003cem\u003e47\u003c/em\u003e). Our structural data allows us to frame a molecular model for the development of L-ENA mediated spore interaction networks. Although the exact role of spores in biofilms is still an area of active research, it can reasonably be expected that spore-spore interactions will contribute to virulence by affecting (i) biofilm resilience through fortification (e.g. reinforced core protected from stressors), (ii) development of a spore reservoir with sustained spore shedding, and (iii) establishment of spore clumps representing the minimal infectious dose (i.e. strength in numbers). Given that the \u003cem\u003eena3a\u003c/em\u003e gene cluster is predominantly found in pathogenic \u003cem\u003eBacilli\u003c/em\u003e we anticipate that L-ENA fibers could be considered as a novel class of secondary effect virulence factors through their impact on spore aggregation, and by extension biofilm formation. What remains unclear is whether the tip fibrillum (L-BclA) recognizes a specific target on the spore surface, be it a sugar, protein or proteoglycan or whether the interaction is based on aspecific interaction, similar to the hydrophobic CsuE adhesin found on the archaic Csu pili of \u003cem\u003eAcinetobacter baumanni \u003c/em\u003ethat is also implicated in the early stages of microcolony development leading up to biofilm formation \u003cem\u003e(48)\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFrom a structural perspective, L-ENA fibers are remarkable products of macromolecular self-assembly, who\u0026rsquo;s assemblage and surface display require a mere three genes: \u003cem\u003eexsL\u003c/em\u003e, \u003cem\u003eena3A\u003c/em\u003e and \u003cem\u003el-bclA\u003c/em\u003e. Each Ena3A subunit is cross-linked to its neighbor via 3 disulphide bridges, which totals to 21 covalent connections for each heptameric unit. Consequently, L-ENA fibers are able to withstand extreme physico-chemical stressors (heat, strong acids, chaotropes, detergent, and desiccation) and are among the most stable protein structures found in nature. In its biological context, however, a protein fiber will only be as strong as its weakest link. It is therefore expected that these high tensile strength ENAs require an anchoring mechanism of comparable durability. The phenotype of D\u003cem\u003eexsL\u003c/em\u003e spores (i.e. spores with detached L-ENA) combined with the AF2 modelling of the putative ExsL-Ena3A and ExsL-ExsY heteromers suggests that the anchoring complex is directly integrated into the exosporium 2D lattice. The exosporium of the \u003cem\u003eB. anthracis\u003c/em\u003e/\u003cem\u003ecereus\u003c/em\u003e/\u003cem\u003ethuringiensis\u003c/em\u003e group is a flexible, disulphide cross-linked, protein 2D lattice that is predominantly composed of ExsY hexameric units (\u003cem\u003e49\u003c/em\u003e). The AF2 model for the ExsL-ExsY dimer suggests that ExsL could be embedded within the ExsY lattice by mimicking the homotypic ExsY contacts, including the formation of an inter-molecular disulphide bridge. Similarly, the AF2 model for an ExsL-Ena3A dimer is reminiscent of the Ena3A contacts found in the L-ENA cryoEM model \u0026ndash; this in turn also includes one of the intra-ring Ena3A disulphide bridges. Based on this, we hypothesize that ExsL acts as a bridging moiety that anchors the L-ENA pilus onto the spore surface by maintaining an unbroken chain of disulphide cross-links across the various contact points (i.e., ExsY \u0026rarr; ExsL; ExsL \u0026rarr; Ena3A). The experimental determination of the respective stoichiometries and the elucidation of the complex epitopes will be the subject of further study.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eNegative stain transmission electron microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNegative stain TEM (nsTEM) imaging of bacterial spores and recEna3A filaments was done using formvar/carbon-coated copper grids (Electron Microscopy Sciences) with a 400-hole mesh. The grids were glow-discharged (ELMO; Agar Scientific) with 4mA plasma current for 45 seconds. 3 \u0026mu;l of a bacterial spore suspension or recEna3A protein solution was applied onto the glow-discharged grids and left to adsorb for 1 minute. The solution was dry blotted, followed by three washes with 15 \u0026mu;l Milli-Q. Next, 15 \u0026mu;l drops of 2% uranyl acetate were applied three times for 10 seconds, 2 seconds, and 1 minute respectively, with a blotting step in between each application. The excess uranyl acetate was then dry blotted with Whatman type 1 paper. All grids were screened with a 120 kV JEOL 1400 microscope equipped with LaB6 filament and TVIPS F416 CCD camera.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-electron transmission microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQUANTIFOIL\u0026reg; holey Cu 400 mesh grids with 2‐\u0026micro;m holes and 1‐\u0026micro;m spacing were glow discharged in vacuum using plasma current of 5 mA for 1 minute (ELMO; Agar Scientific). 3 \u0026micro;l of 0.6 mg/ml graphene oxide (GO) solution was applied onto the grid and incubated 1 min for absorption at room temperature. Excess GO was blotted using a Whatman grade 1 filter paper and left to dry under ambient conditions. For cryo‐plunging, 3 \u0026micro;l of fiber suspension was applied on the GO‐coated grids at 100% humidity and room temperature in a Gatan CP3 cryo‐plunger. After 1 minute of absorption, the grid was machine‐blotted with Whatman grade 2 filter paper for 3.5 seconds from both sides and plunge frozen into liquid ethane at -176\u0026deg;C. Grids were stored in liquid nitrogen until data collection. Two datasets were collected for \u003cem\u003eex vivo\u003c/em\u003e and recEna3A appendages with slight changes in the collection parameters. High‐resolution cryoEM movies were recorded on a JEOL CRYO ARM 300 microscope equipped with omega energy filter and a K2 or K3 direct electron detector run in counting mode. For the \u003cem\u003eex vivo\u003c/em\u003e Ena, the microscope was equipped with a K2 summit detector and had the following settings: 300 keV, 100 mm aperture, 30 frames/image, 62.5 e\u0026minus;/\u0026Aring;\u003csup\u003e2\u003c/sup\u003e, total exposure 2.315‐second exposure, and 0.82 \u0026Aring;/pixel. The recEna3A dataset was recorded using a K3 detector, at a pixel size of 0.782 \u0026Aring;/pix, and a total exposure of 64.66 e\u003csup\u003e\u0026minus;/\u003c/sup\u003e\u0026Aring;\u003csup\u003e2\u003c/sup\u003e accrued over 61 frames/movie.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of deletion mutants and complementation constructs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeletion mutants in \u003cem\u003eB. paranthracis \u003c/em\u003eNVH0075-95 were generated following the marker less gene replacement method described previously (Janes and Stibitz, 2006; Pradhan et al., 2021). The gene replacement constructs contained the start and the stop codons of the respective genes to be deleted flanked by upstream and downstream homologous sequences. The upstream and downstream homologous sequences were 806 bp and 677 bp for \u003cem\u003eena3A\u003c/em\u003e, 727 bp and 750 bp for \u003cem\u003el-\u003c/em\u003e\u003cem\u003ebclA\u003c/em\u003e, and 701 bp and 728 bp for \u003cem\u003eexsL, \u003c/em\u003erespectively. Synthetic constructs (Synbio Technologies LLC) were cut out from the pUC57-Amp vector by digesting with EcoRI, and the gene replacement DNA fragment were cloned into EcoRI digested pMAD-I-SceI shuttle plasmid (Lindb\u0026auml;ck et al., 2012). The gene replacement constructs, i.e., pMAD-I-\u003cem\u003eSce\u003c/em\u003eI-\u0026Delta;\u003cem\u003eena3\u003c/em\u003e and pMAD-I-\u003cem\u003eSce\u003c/em\u003eI-\u0026Delta;\u003cem\u003ebcla\u003c/em\u003e, and pMAD-I-\u003cem\u003eSce\u003c/em\u003eI-\u0026Delta;\u003cem\u003eexsL\u003c/em\u003e were then used to transform NVH0075-95 or the \u0026Delta;\u003cem\u003eena1ABC\u003c/em\u003e triple mutant (Pradhan et al., 2021). Details of competent cell preparation, transformation, and screening are described (Zegeye and Aspholm, 2022; Pradhan et al., 2021). \u003c/p\u003e\n\u003cp\u003eFor complementation experiments, DNA fragment containing a 300 bp region upstream of the start codon of the \u003cem\u003el-\u003c/em\u003e\u003cem\u003ebclA-ena3A operon\u003c/em\u003e and open reading frames of the respective genes was ordered (Synbio Technologies LLC) or amplified by PCR and cloned in the low copy number plasmid pHT315 at EcoRI restriction site. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulturing and RNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eB. paranthracis\u003c/em\u003e NVH0075-95\u003cem\u003e \u003c/em\u003eand \u003cem\u003eB. paranthracis \u003c/em\u003eNVH0075-95\u003cem\u003e \u003c/em\u003e\u0026Delta;\u003cem\u003eenaABC/\u003c/em\u003e\u0026Delta;\u003cem\u003eena3A\u003c/em\u003e quadruple mutant (control) were cultured in sporulation medium as previously described (Pradhan et al., 2021) with a slight modification. Here, we used nutrient broth (oxoid) in place of bacto medium (Difco). A sample of 20 ml culture was withdrawn every four hours from three independent cultures, centrifuged, and cell pellets frozen immediately at -80 \u0026deg;C until RNA extraction. \u003c/p\u003e\n\u003cp\u003eRNA was extracted using Purelink RNA minikit (Ambion, Life Technologies) according to the manufacturer\u0026rsquo;s protocol with a slight modification. Briefly, cell pellets were thawed on ice followed by addition of 200 \u0026micro;l of lysozyme solution (10 mM Tris-HCl (pH 8), 0.1 mM EDTA, 10 mg/ml lysozyme) and incubation for 5 minutes at RT. A volume of 1 \u0026micro;l of 10% SDS was added to the cell suspension and vortexed, after which 150 \u0026micro;l was transferred to new RNAse-free tubes. Following addition of 350 \u0026micro;l of lysis buffer, the cells were lysed by bead beating (FastPrep\u0026reg;-24, MP Biomedicals) for a total of two minutes at Speed 6, with 1 minute cooling on ice every 30 seconds. The lysates were centrifuged for 5 minutes (2600 xg), and the supernatants were transferred to new RNAse-free tubes. The remaining steps were carried out as described in the manufacturer\u0026rsquo;s protocol. RNA concentration was measured using Nanodrop 1000 (Thermo Scientific), and 1.5 \u0026micro;g RNA was treated with Turbo DNAase (Ambion) in 20 \u0026micro;l reaction volume following the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR (qRT-PCR) and PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplementary DNA (cDNA) was synthesized from 500 ng DNAse-treated RNA using the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer\u0026rsquo;s instructions. qRT-PCR was carried out using the PowerUp\u0026trade; SYBR\u0026trade; Green Master Mix kit (Applied Biosystems) and the AriaMx Real-Time PCR system (Agilent Technologies) following the manufacturers\u0026rsquo; instructions. The expression of \u003cem\u003eexsL\u003c/em\u003e, \u003cem\u003el-bclA\u003c/em\u003e, \u003cem\u003eena3A\u003c/em\u003e and \u003cem\u003erpoB\u003c/em\u003e (housekeeping gene) was analyzed from cDNA of each of three independent cultures sampled after 4, 8, 12 and 16 hours after inoculation and the relative gene expression ratio was calculated according to the Pfaffl formula (Pfaffl, 2001). Primers were used at 300 nM concentration, and their efficiencies were calculated from the slope of the standard curve using the formula (10\u003csup\u003e-1/slope\u003c/sup\u003e-1) x 100, and were found to be between 95% and 101%. No template was added to the negative control samples. The Ct values obtained for the 4-hour samples were used as the calibrator sample in the data analysis, and \u003cem\u003erpoB\u003c/em\u003e served as an internal control gene. Each cDNA sample was analyzed in triplicate, and the experiment was repeated independently. The cycling conditions of the qRT-PCR were: 50 \u0026deg;C/2 minutes, 95\u0026deg;C/2 minutes; and 40 cycles of 95\u0026deg;C/15 seconds, 60\u0026deg;C/1 minute. \u003c/p\u003e\n\u003cp\u003eTo determine if \u003cem\u003eexsL\u003c/em\u003e, \u003cem\u003ebclA\u003c/em\u003e, and \u003cem\u003eena3A\u003c/em\u003e are co-expressed, standard PCR was conducted using the cDNAs as templates. Primers that span across the indicated genes (Table S4), and DreamTaq PCR master mix (Thermo Scientific\u0026trade;) were used. Purified genomic DNA (gDNA) from NVH0075-95\u003cem\u003e \u003c/em\u003eand cDNA\u003cem\u003e \u003c/em\u003efrom\u003cem\u003e \u003c/em\u003eNVH0075-95\u003cem\u003e \u003c/em\u003e\u0026Delta;\u003cem\u003eenaABC/\u003c/em\u003e\u0026Delta;\u003cem\u003eena3A \u003c/em\u003e(4 h and 16 h) were used as positive and negative controls, respectively. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing strain \u003cem\u003eB. paranthracis\u003c/em\u003e NVH 0075-95\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo achieve a closed, updated, and high-quality genome of \u003cem\u003eB. paranthracis \u003c/em\u003eNVH 0075-95, the strain was subjected to hybrid assembly of long- and short read sequences. Genomic DNA from NVH 0075-95 was prepared from bacteria grown over night on blood agar plates. All sequencing was performed by Novogene Co. (London, UK) using a combination of Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing platform with a with a SMRTbell template library and an Illumina NovaSeq 6000 platform (2 x 150 bp) with an insert size of 300 bp. \u003c/p\u003e\n\u003cp\u003eA closed genome was achieved using the assembly pipeline Unicycler v. 0.4.8.0+galaxy3 (\u003cem\u003e50\u003c/em\u003e) conducting a short-read-first hybrid assembly. Genome contiguity, completeness and correctness was assess using Quast v. 5.0.2+galaxy3 (\u003cem\u003e51\u003c/em\u003e), Bandage 0.8.1+galaxy3 (\u003cem\u003e52\u003c/em\u003e), BUSCO v. 5.3.2+galaxy0 (mode genome, gene predictor prodigal, lineage dataset bacillales_odb10) (\u003cem\u003e53\u003c/em\u003e) and post-assembly correctios using Pilon ((\u003cem\u003e54\u003c/em\u003e)) wrapped in the assembler Unicycler v. 0.4.8.0+galaxy3. Species validation was done on the Type Strain Genome Server (TYGS) (\u003cem\u003e55, 56\u003c/em\u003e). Plasmids and contigs were predicted with PlasFlow v. 1.1.0 (threshold \u0026gt;0.6, remaining default settings) (\u003cem\u003e57\u003c/em\u003e), and the genome was annotated using the online NCBI Procaryotic Genome Annotation Pipeline (PGAP) (\u003cem\u003e58-60\u003c/em\u003e). Data is available at NCBI under accession number GCA_027945115.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of \u003cem\u003eL-ena\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCblaster ((\u003cem\u003e61\u003c/em\u003e)) a python toolkit for detecting collocated genes, was run remotely through the web application CAGECAT v.1.0 (settings: -max gap 200 bp, n of unique query seq=3, min coverage 50%, min identity 30%, maximum e-value 0.01) on the NCBI RefSeq non-redundant protein database, using amino acid sequence of WP_017562367.1 (Ena3A), WP_048548723.1 (BclA), WP_048548726.1 (ExsL) and WP_058548727.1 (Tn3 family transposase) as query. The genomic region spanning WP_017562367.1, WP_048548723.1, WP_048548726.1 is hereafter referred to as \u0026ldquo;the gene cluster\u0026rdquo;. Furthermore, to investigate the proportion of organisms with the gene cluster within the \u003cem\u003eB. cereus s.l.\u003c/em\u003e group, publicly available genomes yielding hits in the cblaster search (Appendix Table) were downloaded from NCBI RefSeq database (n = 656, NCB (https://www.ncbi.nlm.nih.gov/refseq/; Table EV1) and appended to a representative database of assemblies of \u003cem\u003eB. ceresu s.l. \u003c/em\u003egroup (Supplementary Table 1). In addition, 136 \u003cem\u003eB. subtilis \u003c/em\u003ewere included for comparison. Assemblies were quality checked using QUAST, and only genomes of correct size (~4.9 \u0026ndash; 6.2 Mb) and a GC content of ~35% were included in the downstream analysis. Pairwise tBLASTn searches were performed (BLAST+ v. 2.11.0 ((\u003cem\u003e62\u003c/em\u003e)), e-value 1e-10, max_hspr 1, default settings) to search for homo- and orthologs of the following query protein sequences from strain NVH 0075-95: WP_017562367.1, WP_048548723.1 and WP_048548726.1. Proteins were considered as orthologs or homologs when they matched the query protein with high coverage (\u0026gt; 70%) and moderate sequence identity (\u0026gt; 30%), and when the whole gene cluster was present in the genome with corresponding synteny as the NVH 0075/95 strain. Genomic location (chromosome or plasmid) was inspected manually for complete and closed genomes. Mashtree v. 0.57 ((\u003cem\u003e63\u003c/em\u003e)) was used to infer whole genome clustering for the \u003cem\u003eB. cereus s.l.\u003c/em\u003e group, using the accurate option (--min-depth 0). Clustering and metadata (hits for query proteins) were visualized in Microreact web browser ((\u003cem\u003e64\u003c/em\u003e)).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMS, MA and HR designed the project. MS performed cryogenic freezing, nsTEM and cryo-EM imaging and data processing with assistance from MF. EDZ conducted the gene expression studies, designed and generated the knockout strains and complementation constructs, and prepared the spores for analysis. MS wrote the manuscript with contributions from all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eex vivo\u003c/em\u003e L-ENA and the recombinant Ena3A cryo-EM maps were deposited to EMDB under entry IDs EMD-17579 and EMD-17627, respectively. The atomic model for recEna3A was deposited to the PDB under ID 8PDZ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dirk Reiter at the VIB-VUB Facility for Bio Electron Cryogenic Microscopy (BECM) and Yohannes Beyene Mekonnen at NMBU for technical assistance. This work was funded by VIB, NMBU, EOS Excellence in Research Program by FWO through grant G0G0818N to HR and G043021N to MS. MA recognizes the Grant from the Norwegian research council (NFR): 335029 - FORSKER22.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eP. T. McKenney, A. Driks, P. Eichenberger, The Bacillus subtilis endospore: assembly and functions of the multilayered coat. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 33-44 (2013).\u003c/li\u003e\n\u003cli\u003eY. Gao\u003cem\u003e et al.\u003c/em\u003e, Bacterial spore germination receptors are nutrient-gated ion channels. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e380\u003c/strong\u003e, 387-391 (2023).\u003c/li\u003e\n\u003cli\u003eP. T. McKenney, A. Driks, P. Eichenberger, The Bacillus subtilis endospore: assembly and functions of the multilayered coat. \u003cem\u003eNature Reviews Microbiology\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 33-44 (2013).\u003c/li\u003e\n\u003cli\u003eA. O. Henriques, J. Charles P. Moran, Structure, Assembly, and Function of the Spore Surface Layers. \u003cem\u003eAnnu. Rev. Microbiol.\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 555-588 (2007).\u003c/li\u003e\n\u003cli\u003eB. J. Panessa-Warren, G. T. Tortora, J. B. Warren, High resolution FESEM and TEM reveal bacterial spore attachment. \u003cem\u003eMicrosc Microanal\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 251-266 (2007).\u003c/li\u003e\n\u003cli\u003eL. Kailas\u003cem\u003e et al.\u003c/em\u003e, Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 16014-16019 (2011).\u003c/li\u003e\n\u003cli\u003eY. Wang\u003cem\u003e et al.\u003c/em\u003e, Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e1005678 (2016).\u003c/li\u003e\n\u003cli\u003eP. D. Walker, The spore antigens of Clostridium sporogenes, Cl. bifermentans and Cl. sordellii. \u003cem\u003eJ Pathol Bacteriol\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 41-49 (1963).\u003c/li\u003e\n\u003cli\u003eW. Hodgkiss, Z. J. Ordal, Morphology of the spore of some strains of Clostridium botulinum type E. \u003cem\u003eJ Bacteriol\u003c/em\u003e \u003cstrong\u003e91\u003c/strong\u003e, 2031-2036 (1966).\u003c/li\u003e\n\u003cli\u003eW. Hodgkiss, Z. J. Ordal, D. C. Cann, The morphology and ultrastructure of the spore and exosporium of some Clostridium species. \u003cem\u003eJ Gen Microbiol\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 213-225 (1967).\u003c/li\u003e\n\u003cli\u003eY. Hachisuka, T. Kuno, Filamentous appendages of Bacillus cereus spores. \u003cem\u003eJpn J Microbiol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 555-558 (1976).\u003c/li\u003e\n\u003cli\u003eL. J. Rode, L. Pope, C. Filip, L. D. Smith, Spore appendages and taxonomy of Clostridium sordellii. \u003cem\u003eJ Bacteriol\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 1384-1389 (1971).\u003c/li\u003e\n\u003cli\u003eM. Lukaszczyk, B. Pradhan, H. Remaut, in \u003cem\u003eBacterial Cell Walls and Membranes,\u003c/em\u003e A. Kuhn, Ed. (Springer International Publishing, Cham, 2019), pp. 369-413.\u003c/li\u003e\n\u003cli\u003eA. P. A. Hendrickx, J. M. Budzik, S.-Y. Oh, O. Schneewind, Architects at the bacterial surface \u0026mdash; sortases and the assembly of pili with isopeptide bonds. \u003cem\u003eNature Reviews Microbiology\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 166-176 (2011).\u003c/li\u003e\n\u003cli\u003eS. K. Mazmanian, G. Liu, H. Ton-That, O. Schneewind, Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e285\u003c/strong\u003e, 760-763 (1999).\u003c/li\u003e\n\u003cli\u003eH. Ton-That, O. Schneewind, Assembly of pili on the surface of Corynebacterium diphtheriae. \u003cem\u003eMol Microbiol\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 1429-1438 (2003).\u003c/li\u003e\n\u003cli\u003eS. Kozuka, K. Tochikubo, Properties and origin of filamentous appendages on spores of Bacillus cereus. \u003cem\u003eMicrobiol Immunol\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 21-37 (1985).\u003c/li\u003e\n\u003cli\u003eB. Pradhan\u003cem\u003e et al.\u003c/em\u003e, Endospore Appendages: a novel pilus superfamily from the endospores of pathogenic Bacilli. \u003cem\u003eEMBO J\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, e106887 (2021).\u003c/li\u003e\n\u003cli\u003eE. D. Zegeye, B. Pradhan, A. K. Llarena, M. Aspholm, Enigmatic Pilus-Like Endospore Appendages of Bacillus cereus Group Species. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eB. Pradhan\u003cem\u003e et al.\u003c/em\u003e, Endospore Appendages: A novel pilus superfamily from the endospores of pathogenic Bacilli. \u003cem\u003eThe EMBO Journal\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, e106887 (2021).\u003c/li\u003e\n\u003cli\u003eU. L. Jonsmoen\u003cem\u003e et al.\u003c/em\u003e, Endospore pili: Flexible, stiff, and sticky nanofibers. \u003cem\u003eBiophysical Journal\u003c/em\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eJ. Mistry, R. D. Finn, S. R. Eddy, A. Bateman, M. Punta, Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, e121-e121 (2013).\u003c/li\u003e\n\u003cli\u003eE. F. Pettersen\u003cem\u003e et al.\u003c/em\u003e, UCSF ChimeraX: Structure visualization for researchers, educators, and developers. \u003cem\u003eProtein Sci\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 70-82 (2021).\u003c/li\u003e\n\u003cli\u003eT. D. Goddard\u003cem\u003e et al.\u003c/em\u003e, UCSF ChimeraX: Meeting modern challenges in visualization and analysis. \u003cem\u003eProtein Sci\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 14-25 (2018).\u003c/li\u003e\n\u003cli\u003eJ. S. Richardson, The anatomy and taxonomy of protein structure. \u003cem\u003eAdv Protein Chem\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 167-339 (1981).\u003c/li\u003e\n\u003cli\u003eR. Akamatsu\u003cem\u003e et al.\u003c/em\u003e, Novel Sequence Type in \u0026lt;em\u0026gt;Bacillus cereus\u0026lt;/em\u0026gt; Strains Associated with Nosocomial Infections and Bacteremia, Japan. \u003cem\u003eEmerging Infectious Disease journal\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 883 (2019).\u003c/li\u003e\n\u003cli\u003eA. Bianco\u003cem\u003e et al.\u003c/em\u003e, Characterization of Bacillus cereus Group Isolates From Human Bacteremia by Whole-Genome Sequencing. \u003cem\u003eFront Microbiol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eT. Paysan-Lafosse\u003cem\u003e et al.\u003c/em\u003e, InterPro in 2022. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, D418-D427 (2023).\u003c/li\u003e\n\u003cli\u003eJ. Jumper\u003cem\u003e et al.\u003c/em\u003e, Highly accurate protein structure prediction with AlphaFold. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e596\u003c/strong\u003e, 583-589 (2021).\u003c/li\u003e\n\u003cli\u003eE. Richard\u003cem\u003e et al.\u003c/em\u003e, Protein complex prediction with AlphaFold-Multimer. \u003cem\u003ebioRxiv\u003c/em\u003e, 2021.2010.2004.463034 (2022).\u003c/li\u003e\n\u003cli\u003eM. Mirdita\u003cem\u003e et al.\u003c/em\u003e, ColabFold: making protein folding accessible to all. \u003cem\u003eNature Methods\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 679-682 (2022).\u003c/li\u003e\n\u003cli\u003eH. Remaut, G. Waksman, Protein-protein interaction through beta-strand addition. \u003cem\u003eTrends Biochem Sci\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 436-444 (2006).\u003c/li\u003e\n\u003cli\u003eP. Sylvestre, E. Couture-Tosi, M. Mock, A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium. \u003cem\u003eMol Microbiol\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 169-178 (2002).\u003c/li\u003e\n\u003cli\u003eP. Sylvestre, E. Couture-Tosi, M. Mock, Polymorphism in the collagen-like region of the Bacillus anthracis BclA protein leads to variation in exosporium filament length. \u003cem\u003eJ Bacteriol\u003c/em\u003e \u003cstrong\u003e185\u003c/strong\u003e, 1555-1563 (2003).\u003c/li\u003e\n\u003cli\u003eS. Rety\u003cem\u003e et al.\u003c/em\u003e, The crystal structure of the Bacillus anthracis spore surface protein BclA shows remarkable similarity to mammalian proteins. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e280\u003c/strong\u003e, 43073-43078 (2005).\u003c/li\u003e\n\u003cli\u003eL. Tan, C. L. Turnbough, Jr., Sequence motifs and proteolytic cleavage of the collagen-like glycoprotein BclA required for its attachment to the exosporium of Bacillus anthracis. \u003cem\u003eJ Bacteriol\u003c/em\u003e \u003cstrong\u003e192\u003c/strong\u003e, 1259-1268 (2010).\u003c/li\u003e\n\u003cli\u003eY. Hachisuka, S. Kozuka, M. Tsujikawa, Exosporia and appendages of spores of Bacillus species. \u003cem\u003eMicrobiol Immunol\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 619-624 (1984).\u003c/li\u003e\n\u003cli\u003eF. D\u0026iacute;az-Gonz\u0026aacute;lez\u003cem\u003e et al.\u003c/em\u003e, Protein composition of the outermost exosporium-like layer of Clostridium difficile 630 spores. \u003cem\u003eJournal of Proteomics\u003c/em\u003e \u003cstrong\u003e123\u003c/strong\u003e, 1-13 (2015).\u003c/li\u003e\n\u003cli\u003eK. McElroy, L. Mouton, L. Du Pasquier, W. Qi, D. Ebert, Characterisation of a large family of polymorphic collagen-like proteins in the endospore-forming bacterium Pasteuria ramosa. \u003cem\u003eRes Microbiol\u003c/em\u003e \u003cstrong\u003e162\u003c/strong\u003e, 701-714 (2011).\u003c/li\u003e\n\u003cli\u003eY. Qiu, C. Zhai, L. Chen, X. Liu, J. Yeo, Current Insights on the Diverse Structures and Functions in Bacterial Collagen-like Proteins. \u003cem\u003eACS Biomater Sci Eng\u003c/em\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eE. Maes\u003cem\u003e et al.\u003c/em\u003e, Glycosylation of BclA Glycoprotein from Bacillus cereus and Bacillus anthracis Exosporium Is Domain-specific. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e291\u003c/strong\u003e, 9666-9677 (2016).\u003c/li\u003e\n\u003cli\u003eJ. A. Bozue, S. Welkos, C. K. Cote, The Bacillus anthracis Exosporium: What\u0026apos;s the Big \u0026quot;Hairy\u0026quot; Deal? \u003cem\u003eMicrobiol Spectr\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, (2015).\u003c/li\u003e\n\u003cli\u003eM. S. Conover\u003cem\u003e et al.\u003c/em\u003e, Inflammation-Induced Adhesin-Receptor Interaction Provides a Fitness Advantage to Uropathogenic E. coli during Chronic Infection. \u003cem\u003eCell Host Microbe\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 482-492 (2016).\u003c/li\u003e\n\u003cli\u003eM. Lukaszczyk, B. Pradhan, H. Remaut, The Biosynthesis and Structures of Bacterial Pili. \u003cem\u003eSubcell Biochem\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 369-413 (2019).\u003c/li\u003e\n\u003cli\u003eY. Lin, R. Briandet, \u0026Aacute;. T. Kov\u0026aacute;cs, Bacillus cereus sensu lato biofilm formation and its ecological importance. \u003cem\u003eBiofilm\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 100070 (2022).\u003c/li\u003e\n\u003cli\u003eY. Huang, S. H. Flint, J. S. Palmer, Bacillus cereus spores and toxins - The potential role of biofilms. \u003cem\u003eFood Microbiol\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 103493 (2020).\u003c/li\u003e\n\u003cli\u003eJ. G. Wijman, P. P. de Leeuw, R. Moezelaar, M. H. Zwietering, T. Abee, Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion. \u003cem\u003eAppl Environ Microbiol\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 1481-1488 (2007).\u003c/li\u003e\n\u003cli\u003eN. Pakharukova\u003cem\u003e et al.\u003c/em\u003e, Structural basis for Acinetobacter baumannii biofilm formation. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, 5558-5563 (2018).\u003c/li\u003e\n\u003cli\u003eC. Terry\u003cem\u003e et al.\u003c/em\u003e, Molecular tiling on the surface of a bacterial spore - the exosporium of the Bacillus anthracis/cereus/thuringiensis group. \u003cem\u003eMol Microbiol\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 539-552 (2017).\u003c/li\u003e\n\u003cli\u003eR. R. Wick, L. M. Judd, C. L. Gorrie, K. E. Holt, Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. \u003cem\u003ePLOS Computational Biology\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e1005595 (2017).\u003c/li\u003e\n\u003cli\u003eA. Mikheenko, A. Prjibelski, V. Saveliev, D. Antipov, A. Gurevich, Versatile genome assembly evaluation with QUAST-LG. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, i142-i150 (2018).\u003c/li\u003e\n\u003cli\u003eR. R. Wick, M. B. Schultz, J. Zobel, K. E. Holt, Bandage: interactive visualization of de novo genome assemblies. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 3350-3352 (2015).\u003c/li\u003e\n\u003cli\u003eM. Manni, M. R. Berkeley, M. Seppey, F. A. Sim\u0026atilde;o, E. M. Zdobnov, BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 4647-4654 (2021).\u003c/li\u003e\n\u003cli\u003eB. J. Walker\u003cem\u003e et al.\u003c/em\u003e, Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e112963 (2014).\u003c/li\u003e\n\u003cli\u003eJ. P. Meier-Kolthoff, J. S. Carbasse, R. L. Peinado-Olarte, M. G\u0026ouml;ker, TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, D801-D807 (2021).\u003c/li\u003e\n\u003cli\u003eJ. P. Meier-Kolthoff, M. G\u0026ouml;ker, TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 2182 (2019).\u003c/li\u003e\n\u003cli\u003eP. S. Krawczyk, L. Lipinski, A. Dziembowski, PlasFlow: predicting plasmid sequences in metagenomic data using genome signatures. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, e35-e35 (2018).\u003c/li\u003e\n\u003cli\u003eT. Tatusova\u003cem\u003e et al.\u003c/em\u003e, NCBI prokaryotic genome annotation pipeline. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 6614-6624 (2016).\u003c/li\u003e\n\u003cli\u003eD. H. Haft\u003cem\u003e et al.\u003c/em\u003e, RefSeq: an update on prokaryotic genome annotation and curation. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, D851-D860 (2017).\u003c/li\u003e\n\u003cli\u003eW. Li\u003cem\u003e et al.\u003c/em\u003e, RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, D1020-D1028 (2020).\u003c/li\u003e\n\u003cli\u003eC. L. M. Gilchrist\u003cem\u003e et al.\u003c/em\u003e, cblaster: a remote search tool for rapid identification and visualization of homologous gene clusters. \u003cem\u003eBioinformatics Advances\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eS. F. Altschul, W. Gish, W. Miller, E. W. Myers, D. J. Lipman, Basic local alignment search tool. \u003cem\u003eJournal of Molecular Biology\u003c/em\u003e \u003cstrong\u003e215\u003c/strong\u003e, 403-410 (1990).\u003c/li\u003e\n\u003cli\u003eL. S. Katz\u003cem\u003e et al.\u003c/em\u003e, Mashtree: a rapid comparison of whole genome sequence files. \u003cem\u003eJ Open Source Softw\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eS. Argim\u0026oacute;n\u003cem\u003e et al.\u003c/em\u003e, Microreact: visualizing and sharing data for genomic epidemiology and phylogeography. \u003cem\u003eMicrobial Genomics\u003c/em\u003e\u003cstrong\u003e2\u003c/strong\u003e, (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4045273/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4045273/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacterial endospores are remarkable examples of biological resilience, representing a dormant and heavily fortified differentiation form capable of withstanding physical and chemical stressors detrimental to vegetative cells. In pathogenic Bacillota, spores also form an infectious particle and can take up a central role in the environmental persistence and dissemination of disease. A poorly understood aspect of spore-mediated infection is the fibrous structures or ‘endospore appendages’ (ENAs) that have been seen to decorate the spores of pathogenic Bacilli and Clostridia. New methodological approaches are opening an unprecedented window on these long enigmatic structures. Using cryoID, Alphafold modelling and genetic approaches we identify a novel class of ultra-robust ENAs in the foodborne outbreak strain \u003cem\u003eBacillus paranthracis \u003c/em\u003eNVH 0075-95. We demonstrate that L-ENA are encoded by a rare three-gene cluster\u003cem\u003e \u003c/em\u003e(\u003cem\u003eena3\u003c/em\u003e) that contains all components for the self-assembly of ladder-like protein nanofibers of stacked heptameric rings, their anchoring to the exosporium, and their termination in a trimeric ‘ruffle’ made of a complement C1Q-like BclA paralogue. The role of ENA fibers in spore-spore interaction and the distribution of L-ENA operon as mobile genetic elements in predominantly pathogenic \u003cem\u003eB. cereus\u003c/em\u003e s.l. strains, suggest that L-ENA fibers promote strain virulence by spore clustering and aggregation.\u003c/p\u003e","manuscriptTitle":"CryoEM structure of a novel class of spore virulence factors on the foodborne outbreak strain Bacillus paranthracis NVH 0075-95","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-19 10:36:54","doi":"10.21203/rs.3.rs-4045273/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6522e8b0-da7d-46ae-ad26-0eced1ed1f25","owner":[],"postedDate":"March 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29305861,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":29305862,"name":"Biological sciences/Biophysics/Biopolymers in vivo"}],"tags":[],"updatedAt":"2024-08-30T07:15:59+00:00","versionOfRecord":{"articleIdentity":"rs-4045273","link":"https://doi.org/10.1038/s41467-024-51804-w","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-08-29 04:00:00","publishedOnDateReadable":"August 29th, 2024"},"versionCreatedAt":"2024-03-19 10:36:54","video":"","vorDoi":"10.1038/s41467-024-51804-w","vorDoiUrl":"https://doi.org/10.1038/s41467-024-51804-w","workflowStages":[]},"version":"v1","identity":"rs-4045273","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4045273","identity":"rs-4045273","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}