Auto-crosslinking sporesilk fibers promote endospore and Cry toxin clustering

Domain: biology
Published: 2026-03-11
Editorial outcome: major_revision

Abstract
Bacillus thuringiesis infects and kills insect larvae via its spores and associated entomotoxin-containing parasporal bodies (PSBs). We show that endospores and PSBs of B. thuringiensis Sv. Israëlensis are covered in a ‘sporesilk’ matrix that consist of 8 nm wide fibers with a double helical symmetry, formed by protofilaments of stacked alphahelical hairpins. These alpha endospore appendages (‘A-ENA’) are stabilized by up to ten autocatalytic and proximity-induced intermolecular isopeptide bonds per subunit, forming a continuous covalent polymer with remarkable chemical and physical robustness. We show A-ENA functions as B. thuringiensis virulence factor, increasing insecticidal activity by clustering spores and PSBs into infectious clumps. Moreover, we demonstrate the recombinant production and self-assembly of A-ENA nanofibers, and show that the exogenic addition of A-ENA fibers to B. thuringiensis strains natively lacking sporesilks results in spore-PSB clustering an d gain of virulence, enabling the rational, non-GMO functionalization of these biological pest control agents.

Introduction
Bacillus thuringiensis (Bt) is an insecticidal, Gram-positive, aerobic endospore-forming bacterium of the BaAcillus cereus sensu lato group, found in soil, dust, and on plant surfaces1. Bt spores are used as established biopesticides in agriculture, private residential use and vector-borne disease control due to their remarkable capacity to kill larvae of various insect pests from the Coleoptera, Lepidoptera, Hymenoptera and Diptera orders, including Aedes and Anopheles mosquitos - vectors for zika2, dengue3 and malaria4. Since Bt strains target specific insect groups with minimal impact on humans and non-target organisms (e.g. wildlife, pollinators and other beneficial insects), they can form the basis of biopesticides that offer a safe and eco-friendly alternative to non-discriminatory, environmentally persistent and potentially toxic insecticidal chemicals5,6.

Bt adopts its entomopathogenic lifestyle by the characteristic production of vegetative insecticidal toxins (Vip)7,8 or delta-endotoxins (Cry and Cyt) during sporulation7,8. Bt toxins exhibit a distinctive composition unique to each strain, resulting in a remarkable selectivity for specific insect genera. The toxins are produced as pro-toxins that assemble into pure or mixed crystalline condensates referred to as parasporal bodies or crystals (PSB)9 (Fig. 1a,b). Upon ingestion of Bt spores, the associated pro-toxin crystals dissolve and exert a poreforming activity in the insect gut, followed by intestinal sepsis, starvation and host death. In the process, germinated vegetative Bt cells colonize the insect until available nutrients in the carcass become depleted, then inducing sporulation to ensure further dissemination and survival of adverse conditions until the spores are taken up by e.g. a new host to restart the cycle.

Bt was originally registered as a General Use in 1961 but is now found in over 1000 registered American pesticide products (National Pesticide Information Center. NPIC Product Research Online (NPRO): ‘Bacillus thuringieEnsis’. http://npic. orst.edu/NPRO/), 157 of which are specific to Bacillus thuringiensis subsp. israelensis (bti; (National Pesticide Information Center. NPIC Product Research Online (NPRO): ‘israelensis’. http://npic. orst.edu/NPRO/)). Bti is a naturally occurring soil bacterium that is used as a larvicide in aquatic and wetland areas w here black fly and mosquito-borne disease control is deployed10. Typical commerciaLl formulations (Aquabac©, Teknar©, Bactimos© , and Vectobac© ) consist of a mixture of spores and extracellular PSBs that are produced through a simple fermentTation process that leverages the natural growth cycle of Bti where nutrient starvation and high cell density trigger sporulation. These conditions mimic the natural scenario of Bacillus biofilm formation which consists of a complex network of vegetative cells, spores, PSBs and extracellular matrix (ECM) components (e.g. for Bacillus subtilis: TasA fibers, eDNA, poly-γ-glutamic acid, exopolysaccharide)11. Because most studies on Bti tend to focus on PSB composition, larvicidal activity and specificity, the composition of the extracellular milieu of a spore dominated population remains poorly understood despite obvious academic and industrial interest.

Here we show that Bti forms spore clumps characterized by a pervasive extrasporal matrix (ESM) that is constructed of 8nm diameter protein nanofibers (dubbed A-ENA; see nomenclature below) that function as molecular tethers between spores and PSBs. Using cryoEM we solve the helical ultrastructure of the A-ENA fibers and show that they are composed of an 11kDa two-helix bundle protein that is extensively covalently cross-linked to its nearest neighbors via iso-peptide bonds. We demonstrate that A-ENA subunits spontaneously and efficiently polymerize in E. coli through a dock-and-lock self-assembly mechanism wherein subunits first rendezvous driven by hydrophobics, followed by enzyme-free, autocatalytic iso-peptide bond formation, yielding robust, micrometer-long covalent, flexible nanofibers. We show that loss of A-ENA breaks up the association of PSBs to Bti spores and attenuates their killing activity towards Chironomus aprilinus larvae. Remarkably, the addition of recombinant A-ENA fibers to Bacillus thuringiensis subsp. Kurstaki (Btk; natively a-ena-) enhanced spore clustering and toxin retention, resulting in an increased killing activity towards the agricultural pest Trichoplusia ni, commonly known as the cabbage looper. Our results show that A-ENA (i) is a virulence factor that exerts its function by ensuring close proximity of the infectious particle (spore) to its toxic payload (PSB) and (ii) is shown to function outside its native Bti context due to its aspecific mode of binding to spore and PSB surfaces, and is therefore expected to see more widespread use in other Bt systems that are naturally devoid of A-ENA but otherwise characterized by the presence of extrac ellular PSBs.

Results
BTI spores and PSBs are surrounded by a proteinaceous fiber matrix
To investigate the mesoscopic structure of a Bacillus thuringiensis subsp. israelensis AM65-52 spore preparation, we dissolved freshly sporulated (72h, LB agar, 30°C) Bti cultures into miliQ and analyzed the bacterial suspension using light (Fig. 1c,d) and negative stain electron transmission microscopy (nsTEM; Fig. 1e). The suspension showed the presence of clustered microcolonies that were composed primarily of bacterial spores and parasporal bodies (PSBs), with vegetative cells representing only a small minority (<1%) of the population (Fig. 1c,d). Strikingly, both the spores and PSBs were engulfed in a dense extrasporal matrix composed of a fibrous mesh that permeated the entire sample (Fig. 1e, unstained). The fibers were shown to emanate from both the spore and PSB surface, and were also seen to mediate direct connections between endospores and across endospores and PSBs (Fig. 1e; Supplementary Figure 1a-e). We found numerous cases of large fiber bundles (100nm diameter) composed of dozens of individual fibers, making connection to the PSBs and the spore surface. Closer inspection of the fiber matrix revealed nanofibers of ~8nm diameter and with a distinct double helical patterning in 2D class averages (Fig. 2a,b; Supplementary Figure 1e). Bti PSBs comprise polycrystalline condensates of insecticidal proteins (Cry11Aa1, Cyt1Aa1, Cry4Aa1 and Cry4Ba1) that are packaged in a spherical parasporal sack, and that dissolve under the elevated pH in the insect gut (pH 8-10 in Lepidoptera)12. Even when incubated at pH 11 we found that Bti spore preparations retained a clumped organization, with spores embedded in a dense nanofiber matrix (Supplementary Figure 1f-j). Remarkably, parasporal sacks remained tightly entangled in the fiber network, but lost their condensate content. Close inspections showed nanofibers tightly associated with the surface of the parasporal sacks. Based on these observations, we hypothesized that the particulate, crystalline nature of the toxins and the fiber-mediated tethering of spores and PSBs ensures the cytotoxic payload remains at a high localized concentration and closely associated with the infectious particles (spore), thus ensuring their co-transmission during host-pathogen interactions.

A-ENA is a unique family type of Endospore appendages
Intrigued by the existence of this fiber net, we proceeded with a cryo electron microscopy (cryoEM) analysis of the TBti exosporal matrix. Freshly resuspended Bti spores were deposited with no further processing onto a quantifoil holey carbon grid and plunge-frozen in liquid ethane, and imaged at 60k magnification using a JEOL CRYO ARM 300 microscope equipped with omega energy filter and a K3 direct electron detector. Similarly to nsTEM, cryoEM revealed a dense network of fibers in proximity to the vitrified endospores (Fig. 2c). Fragments of isolated fibers were boxed and subjected to 2D alignment and classification. The resulting 2D class averages and Fourrier-Bessel indexing using PyHi13 confirmed the double helical character of the protein filaments (Figure 2d). Using cryosparc v4.4.1, we refined the fiber ultrastructure to a global resolution of 2.1Å, with a helical rise and twist of 5.37Å and 186.4° (Figure 2e; Supplementary Figure 2). The cryoEM map revealed a right-handed two-start helix with parallel protofilaments that showed clear continuous main chain density for axially stacked alpha-helical subunits (Fig. 3a,b,d). The high-resolution cryoEM reconstruction showed identifiable side chain densities that allowed for the de novo building and sequence assignment of an initial atomic model of the constituent subunits (Figure 2f). A standard protein BLAST search using the manually built protein sequence yielded protein Q8KNV8 (https://www.uniprot.org/) as the top-ranking candidate for the major subunit. Q8KNV8 is a 101 amino acids protein with no known protein family membership as judged from InterProScan (https://www.ebi.ac.uk/interpro/). Based on the lack of any sequence or structural homology or resemblance to known pili, functional amyloids or the recently characterized endospore appendages (i.e. S- and L-ENA (DUF3992)14,15, it is clear that the Bti fibers constitute a unique family of protein nanofibers referred to as alpha helical endospore appendages, or A-ENA in short.

The genetic locus corresponding to Q8KNV8 is ATN07_33990, and is found on the 128kb pBtoxis-like plasmid16,17 (pAM65-52-4-128K; RefSeq: NZ_CP013279.1) that carries all the insecticidal -endotoxins. We identify a local three gene cluster (a-ena) consisting of ATN07_33990 (a-ena), ATN07_33 985 and ATN07_33980 (a-ena1) (Fig. 2e; Supplementary Figure 2). ATN07_33985, also referred to as pBt031, encodes a putative two-domain N-acetylmuramoyl-L-alanine amidase with a catalytic cell wall hydrolase domain (Pfam: PF01520)18 and a sporulation related repeat domain (Pfam: PF05036), and has previously been suggested to be involved in mother cell wall lysis similar to the sporulation specific autolysin CwlB in B. subtilis17. Looking at the broader genomic context, the a-ena cluster is integrated into a region encoding for numerous -endotoxins, i.e. upstream are located Cyt2Ba (ATN07_33970) and Cry8Ea (ATN07_33980). Downstream of a-ena, there are three other toxin genes, i.e. Cyt1A (ATN07_34010), Cry2A/C (ATN07_34015) and Cry11a (ATN07_34020). Of final note, the a-ena gene cluster is flanked by ATN07_34000, which has been annotated as a Tn3 family transposase (uniprot: A0A160LKE1) (Fig. 2g).

In the a-ena gene cluster, ATN07_33980 encodes for protein Q8KNV7 which shares 74.0% seq id to Q8KNV8, and is predicted to fold into a two-helix bundle that is structurally homologous to Q8KNV8 (the all-atom RMSD using Alphafold database entries AF-Q8KNV7-F1 and AF-Q8KNV8-F1 is 0.216 Å). Based on this similarity, we refer to Q8KNV8 and Q8KNV7 as A-ENA and A-ENA1. LC-MS/MS de novo sequencing of isolated fibers detected the presence of all three proteins of the local a-ena gene cluster, i.e. A-ENA, A-ENA1 and the N-acetylmuramoyl-L-alanine amidase. To resolve the ambiguity concerning the subunit composition of the ex vivo fibers we carefully inspected the reconstructed cryoEM volume and manually compared the A-ENA and A-ENA1 sequences at key sites with subunit-specific side-chain type (Supplementary Figure 2e). Based on that analysis, we unambiguously identified A-ENA as the major subunit of the fibers, but do not rule out that A-ENA1 is present in the fibers at low substoichiometric levels.

A-ENA forms cross- fibrils crosslinked via isopeptide bonds
Through careful inspection of the A-ENA cryoEM map, we identified multiple sites of continuous density bridging adjacent A-ENA protomers within and across protofilaments (Fig. 3, Supplementary Figure 3). After the atomic A-ENA model was built, we identified these sites as intermolecular isopeptide bonds (IPB) composed of a nucleophilic IPB ‘donor’ (i.e. G2 N-terminus, K42, K76, K77 or K84; Figure 3a, k) and an electrophilic IPB ‘acceptor’ (i.e. E29, E44, N64, E78, or Q82; Figure 3a, k). Adjacent each IPB site, we find glutamic acid residues favorably positioned to act as acid-base catalyst (e.g. E28 Figure 3k, Supplementary Figure 3c), similar to what is seen for intramolecular IPBs in bacterial pili19,20. In the latter, subunit folding brings the triads of IPB donor (K), acceptor (D, E, N or Q) and acid-base catalyst (E or D) in close proximity resulting in autocatalytic IPB formation. In A-ENA, however, IPB donor and acceptor are located on separate subunits, and a knobs-in-holes stacking of adjacent A-ENA subunits results in the formation of proximity-induced intermolecular IPBs (Supplementary Figure 3c, Supplementary Figure 4). In this way, five different IPB pairs (IPB1-5: G2-Q44; K42-N64; K77-E78; K84-E29 and K76-Q82) are formed twice per A-ENA subunit, i.e. ones as donor (→) and ones as acceptor (), totaling ten IPBs per protomer (Figure 3; Table 1; Supplementary Figure 3). Of these, six IPBs connect consecutive subunits within a single protofilament (IPB 2, 3 and 4; as donor towards N-pole: i→i-1, and as acceptor towards C-pole: ii+1). Two additional axial IPBs (IPB1) are formed between the subunits i→i-5 as donor, and ii+5 as acceptor (Fig. 3e). Finally, IPB5 forms two isopeptide bonds across the two protofibrils, (i→i’-1) as donor, and (ii’) as acceptor (Fig. 3f). Whilst IPB2-5 are formed by knobs-in-holes interactions of alpha helices 1 and 2 of consecutive protomers within (IPB2-4) and adjacent protomers across (IPB5) the protofilaments, IPB1 connects distant subunits, realized by a long N-terminal latch (NTL) constructed of residues 2 to 14 and that is docked via residues M3, P4, I6, P7, L10 and I12 into a hydrophobic groove that runs axially along the A-ENA fiber surface spanning 4 subunits.

A-ENA self-assembly and autocatalytic isopeptide bond formation
Most bacterial pili and filaments require a dedicated assembly machinery21, and known polymers with intermolecular isopeptide bonds such as poly-ubiquitin or sortase-mediated pili employ a transpeptidation reaction that requires enzymatic accessory proteins19. In A-ENA, however, the proximity-induced IPB triads as a result of subunits stacking suggests they may form autocatalytically (Supplementary Figure 3). To evaluate the requirement of accessory proteins, A-ENA and A-ENA1 were recombinantly expressed in the cytoplasm of E. coli BL21. Purified fractions enriched towards either A-ENA or A-ENA1 were shown to contain abundant protein fibers, which both yielded 2D class averages that appeared identical to those obtained from the ex vivo material (Fig. 3g,h,i; Supplementary Figure 2b,c). Testament to the remarkable stability of these fibers is the 30min incubation step at 100°C in the presence of 2% (w/v) sodium dodecyl sulfate (SDS) that we employed to purify the target fibers form cellular proteins. Similarly, A-ENA fibers were recalcitrant to treatment with chaotropic agents (8M urea), exposure to strong acidic (50% (v/v) formic acid) or alkaline conditions (2M NaOH), as well as wet (i.e. autoclaving) or desiccated (200C oven) high temperature treatment (Error! Reference source not found.). This remarkable stability suggested that IPBs were present in the recombinant fibers as well. We confirmed that hypothesis by solving the cryoEM structures of the highly similar recA-ENA and rec-A-ENA1 fibers to 2.40Å (rise:5.47 Å, twist: 186.36°) and 2.63 Å (rise:5.31 Å, twist: 186.50°) resolution, respectively (Supplementary Figure 2). Indeed, rec-A-ENA fibers exhibited all 5 IPB types observed in the ex vivo A-ENA fibers, totaling 10 IPBs per protomer. Although rec-A-ENA1 fibers are near isomorphous to A-ENA (Supplementary Figure 2d), they lacked both the type 1 and 5 IPBs, leading to 6 confirmed IPBs per protomer, with no observable impact on the fiber stability (Fig. 3j,k). This was unexpected because the IPB-associated residues are conserved in A-ENA1, and the NTL sequence is identical to the A-ENA sequence apart from N5 (A-ENA: T5; Supplementary Figure 2e). Possibly, an increased susceptibility to proteolytic attack of the NTL and/or reduced processing of N-terminal methionine (i.e. to liberate the G2 N-terminus) may impair IPB formation for recombinant A-ENA1 in E. coli. Regardless of these minor differences between A-ENA and A-ENA1, these results clearly show that IPB formation is auto-catalytic and independent of e.g. intervention of a transglutaminase22. Moreover, the efficient polymerization in E. coli (yield 1.8g A-ENA fiber / L culture) demonstrates that A-ENA biogenesis is not reliant on external factors (e.g. chaperone, usher, protease, …) and that A-ENA self-assembly is fully encoded in its primary sequence. A multiple sequence alignment of A-ENA like sequences shows the strong conservation of the IPB donor/acceptor pairs, as well as their cognate acid-base catalysts (Supplementary Figure 3a,b). Different to intermolecular IPBs that form by transpeptidation19, A-ENA IPB triads are not part of recognition sequences, but rather take up predefined positions in the alpha 1 and alpha 2 helical wheels, such that packing of the A-ENA subunits brings the IPB partners in close proximity (Supplementary Figure 3a,b,c). Looking at the A-ENA axial protomer/protomer interface (e.g. i→i-1), we find that the buried interfaces are predominantly hydrophobic (mainly small hydrophobic chains apart from IPB residues; Supplementary Figure 4), and there are no obvious complementary charged surface patches, suggesting protomer docking is predominantly driven by the hydrophobic effect (Supplementary Figure 4). There is exceptional complementarity between the N- and C-pole surfaces of helices  and  . This leads to a tight ‘knobs-in-holes’ packing that drives the pre-orientation of the nucleophile/electrophile pair and its acid-base catalyst, and results in their electrostatic activation by the exclusion of solvent from the cross- interfaces, essentially priming the residues for autocatalytic IPB formation.

A-ENA and A-ENA1 expression is uniquely associated with the spore extracellular matrix
The close association of A-ENA fibers with spores and parasporal bodies (Fig. 1 and Supplementary Figure 1), and localization of the a-ena and a-ena1 genes on the pAM65-52-4-128K plasmid and in close proximity to the Bti cry toxins (Fig. 2g) suggest A-ENA expression occurs during sporulation. Using the DBTSBS server23 we identify a putative recognition site (CACCTTTGGAATAGCTGTTACATATAATA; p-value 0.01) for the late stage mother cell sporulation transcription factor SigK 46bp upstream of a-ena, in agreement with the predicted timing of expression given the extracellular, spore-associated localization of A-ENA. In agreement with this analysis, expression of a plasmid-encoded mcherry reporter gene under control of the hypothesized a-ena promotor sequence was found to be uniquely associated with sporulating cells, and absent from vegetative cells (Supplementary Figure 6a). No known sigma factor recognition motifs were found upstream of a-ena1 or ATN07_33980 (using a p-value cut-off of 0.01), although separation of the forward oriented a-ena and a-ena1 by the reverse oriented ATN07_33985 would suggest the a-ena genes to have independent promotors. To further test this hypothesis and evaluate the timing, (co-)occurrence and localization of A-ENA and A-ENA1 fibers, we modified a-ena and a-ena1 with C-terminally fused Spy and Snoop-tags, thus enabling their selective labelling with Spy-catcher-GFP and SnoopCatcher-mCherry conjugates, respectively. In agreement with the P promotor a-ena activity seen from the mcherry reporter (Supplementary Figure 6a), the presence of A-ENASpyTag was restricted to sporulating cells and released spores (Supplementary Figure 6b,c). Similarly, A-ENA1SnoopTag was spore-associated and not observed for vegetative cells. However, whilst AT-ENASpyTag fibers were ubiquitous throughout the spore population, A-ENA1SnoopTag only showed sporadic labelling (i.e <1% of spores), suggesting a phase variable expression that is independent of a-ena.

A-ENA promotes spore clustering and retention of PSBs
To shed light on the biological function of A-ENA, a-ena was selectively inactivated by introduction of a premature stop codon in the Bti pAM65-52-4-128K plasmid using CRISPR/Cas9 technology. Selective inactivation of a-ena versus a-ena1 (74% sequence identity) was achieved by means of an unique guide RNA target sequence and confirmed using PCR and Sanger sequencing (Supplementary Figure 6d,e). Spores of the resulting Bti a-ena lost recognition by a polyclonal serum raised against recombinant A-ENA (Supplementary Figure 6f), and lacked discernable sporesilk fibers when inspected by nsEM (Supplementary Figure 6d). Conversely, inactivation of a-ena1 did not result in loss of spore-associated A-ENA fibers (Supplementary Figure 6d). Thus a-ena expression is essential and sufficient for A-ENA production in the extrasporal matrix, whilst A-ENA1 is only a sporadic, or a minor, dispensable component of the A-ENA-like fibers. Using time-lapse light microscopy, we found no discernable difference in growth rate or sporulation efficiency for WT and a-ena Bti (Supplementary Figure 6g). To evaluate if a-ena expression would alter PSB content, OD-normalized sporeprep fractions were treated at pH11 and analysed by SDS-PAGE (Fig. 4b, Supplementary Figure 7a). SDS-PAGE showed bands at ~28, ~70 and ~130 kDa, identified, respectively, as the Cyt1A, Cry11Aa1 and Cry4Aa1/Cry4Ab1 toxins by MS fingerprinting, as well as minor bands around 55 and 38 kDa found to represent Cry11Aa1 breakdown products (Supplementary Figure 7a). A quantitative comparison of the released bands showed sporeprep inputs of Bti WT and a-ena mutant showed no discernable difference in the of amounts of Cry toxins.

Our microscopy experiments reveal Bti spore preparations consist of microcolonies with clustered spores and PSBs, embedded in a dense extrasporal matrix of A-ENA sporesilks (Fig. 1, Supplementary Figure 1). During sporulation, the endospore and PSB are formed in the daughter and mother cell compartment, respectively (Fig. 1a)7. We therefore asked if A-ENA fibers are implicated in spore clustering and may serve to ensure spores and PSBs remain associated after mother cell lysis. To do so, spore preparation of Bti WT and a-ena were subjected to cushioned density centrifugation (1 hr at 100g or 10.000g) with three phases corresponding to a low density buffer or supernatant (‘SN’) layer with a =1.00g/ml (low), a medium density layer of 40% Histodenz with a = 1.21 g/ml (medium), and a high density layer of 60% Histodenz with a =1.32 g/ml (high). Sedimentation fractions were qualitatively followed by light microscopy (Fig. 4a), and quantitatively monitored by SDS-PAGE of the high pH-released fraction to follow PSBs (Fig. 4b) and by OD to track the fractionation of spores (Fig. 4c). At low g force, PSBs of WT Bti were primarily retained in the low fraction (i.e. >95%), where they were found as agglutinated spore – PSB clusters. These low density spore-PSB clusters retained about 50% of spores (based on OD600), with the other half found as smaller spore clusters in the medium fraction (with sparse PSBs). In the a-ena mutant, however, no agglutinated spore-PSB clusters were observed, and the majority of PSBs migrated into the medium fraction as individual, dispersed particles. Similarly, a-ena spores were found as individual particles that migrated into medium fraction. At high g force, PSBs in both WT and a-ena Bti fully migrated into the medium fraction, WT as smaller spore-PSB clusters, a-ena as dispersed particles. In both cases, about 30% of spores migrated into the high fraction as individual particles. These fractionations indicate the individual PSBs and the majority of spores to have a density between 1.21 - 1.32 g/ml. In the WT Bti, the extrasporal matrix of A-ENA nanofibers agglutinates spores and PSBs into clumped aggregates with reduced density. With an average volume of ~0.68 µm3, spores experience around ~0.8 and ~80 pN gravitational force at 100 and 10.000 g, respectively. Whilst at 1 pN force a fraction of the spores gets dislodged from the low density spore clumps, higher forces (up to 80pN) are required to break up the spore clumps into smaller fragments that migrate into the medium fraction. In absence of A-ENA, sporse and PSBs fractionate as individual particles, even at low g force.

Spurred on by this striking result, we also followed a gain-of-function approach, where we introduced a-ena in Btk, which naturally lacks pAM65-52-4-128K or A-ENA homologs. To do so, a-ena was cloned with its native promotor into shuttle vector pAS (i.e. modified pJOE8999 by removal of cas9), forming pAS_a-ena. -A-ENA dot blot analysis the respective spore preparation confirmed a lack of A-ENA in Btk WT, and showed the recombinant expression of A-ENA in Btk pAS_a-ena (Supplementary Figure 6f). Light microscopy and nsTAEM imaging of Btk WT showed mostly dispersed spores and PSBs (Fig. 4e, Supplementary Figure 7c), with the presence of spore-attached S-ENA fibers (Supplementary Figure 7d; Ref(Pradhan, 2021 #87), but a lack of A-ENA-like fibers or an extensive extrasporal matrix as seen for Bti (Supplementary Figure 7d). In sharp contrast, Btk pAS_a-ena showed a rich network of A-ENA fibers, resulting in the clustering of Btk spores into multibody clumps (Fig. 4e, Supplementary Figure 7c,d). Moreover, typical bipyramidal Btk toxin crystals (here also referred to as PSB were seen to be enclosed in the Btk pAS_a-ena spore clumps. Large A-ENA bundles could be seen to grace the PSB crystal surface as well as making contact with neighbouring spores, seemingly functioning as a molecular tether mimicking the spore-PSB association seen in Bti. Similar to Bti, we found no significant difference in the PSB formation for WT and Btk pAS_a-ena (Supplementary Figure 7a). Remarkably, a very similar A-ENA dependent spore and PSB clustering can be seen by the exogenous addition of recombinant A-ENA nanofibers (‘+A-ENA’) purified from E. coli (Fig. 4e, Supplementary Figure 7c,d), showing the agglutinating activity does not require A-ENA expression by the sporulating cells, and can be induced post-sporulation. When WT Btk, pAS_a-ena and +A-ENA spore preps were subjected to cushioned density centrifugation, we observed that the spore – PSB agglutination induced by recombinant expression, or exogenous addition of A-ENA results in the increased retention of spores and PSBs in the low density fraction (Supplementary Figure 7b,e). Even at high g-forces, as much as 50% of PSBs were retained in a low density agglutinated spore clump fraction. Moreover, whereas WT Btk spores and PSB separated over a 70% sucrose cushion, as previously reported (, in Btk pAS_a-ena PSBs would co-pellet with the spores, demonstrating the encapsulation in A-ENA matrix ensured a robust spore – PSB association (Supplementary Figure 7f).

A-ENA as a virulence factor
The genetic association of a-ena and Cry toxin genes on a plasmid and the observation that A-ENA facilitates spore – PSB union suggested to us that A-ENA may represent a virulence factor for Bt insecticidal activity. We therefore compared the survival of Chironomus aprilinus (family Chironomidae) larvae in the presence of buffer or equivalent spore preparations of Bti WT and a-ena at a dose of approximately OD 0.004. Mortality was monitored over 6 days following a single addition of spores to the aqueous medium. The results showed an increase in the MDK (mean duration of killing 80% of the population) from 3 to 4 days for Bti WT and A-ENA KO, respectively. We next evaluated if recombinant A-ENA would result in a gain of virulence in strains naturally lacking sporesilks. To do so, we exposed two week old cabbage looper larvae (Trichoplusia ni (family Noctuidae); a common pest of cruciferous crops, also referred to as cabbage loopers, and susceptible to Btk24 to Btk WT, Btk pAS_a-ena and Btk +A-ENA. Resuspended spore preparations were deposited on the solid feeding medium at a dose of 8 L/cm2 of OD 0.06. Whereas the recombinant expression or exogenous addition of A-ENA in Btk resulted in an MDK80 of 4 days, WT Btk did not show more than 50% lethality after six days of exposure (Fig. 4g). To ensure that any observed effects were not due to the toxic properties of A-ENA fibers, we included Bti WT in the experiment. As expected from its known killing profile, Bti WT had no effect on the survival rate of T. ni larvae. Moreover, exposure of the larvae to A-ENA fibers showed no killing activity (Fig. 4g). Thus addition of A-ENA fibers provides a convenient approach for the non-recombinant increase of the spore – toxin clustering in Bt strains lacking native sporesilks, and results in improved pesticidal activity.

Phylogenetic analysis suggests biological functions beyond spore/PSB coupling
Phylogenetic analysis of A-ENA prevalence across the Firmicutes phylum shows that A-ENA homologues are found across both the Clostridia and Bacillales clades (Figure 5a). Due to the genetic association between A-ENA and cyt/cry-toxins in Bti, we focused on the A-ENA prevalence across the Bacillus cereus group sensu lato. A-ENA is found in 496 of 5976 (8%) tested genomes of the Btyper database, and although found in all GTDB Bacillus species, it is predominantly found in B. thuringiensis (156 genomes), B. mycoides (124 genomes), B. wiedmanni (96 genomes) and to a lesser extent in B. anthracis/paranthracis/cereus (39 genomes) (Figure 5b). Moreover, out of the 496 genomes containing A-ENA, 351 (71%) genomes contained one or more type of Bt-toxin (Fig. 5 outer ring). If we compar that to the Bt-toxin prevalence across the Btyper database, only 1071 genomes (18%) were Bt positive (2=1022.93, p<0.0001), indicating A-ENA and Bt-toxin occurrence are correlated. The correlated occurrence of A-ENA and Bt-toxin persists when controlling for shared strain ancestry using phylogenetic linear regression analysis (A-ENA -> Bt-toxin =38,57, AIC=1589 and a log-odds coefficient =2.23±0.26; Bt-toxin -> A-ENA =11,73, AIC=3173 and =2.61±0.28), reinforcing the notion that the main biological function of A-ENA is related to the presence of toxins, and by extension parasporal bodies.

Interestingly, 350 genomes (70%) carried more than 2 A-ENA-like genes (as is the case for Bti) with 83 (17%) genomes harboring 5 or more homologues (e.g. the Bacillus cereus sensu stricto strain AFS036381 has 19 A-ENA-like genes) (Supplementary Figure 8a). We performed a gene cluster analysis and found that many A-ENA-like genes are organized in (putative) operons and/or gene clusters (Supplementary Figure 8b). Looking at these gene clusters in further detail, we discern two types of A-ENA homologues, (i) those constructed of a single A-ENA domain (shown in orange) and (ii) those carrying an additional domain (A-ENA fusion, shown in blue). After performing an interproscan analysis on the dataset of A-ENA homologue sequences, we identify 5 classes of passenger domain types (Supplementary Figure 8c,d). The gene architecture of the most abundant class (type 1) consists of an A-ENA domain, followed by a collagen-like region and a C1q domain. Other less frequently observed passenger domain types are SdrD_B (PF17210) / OmcB (PF01345) or 4Fe-4S cluster domain (PF13370). We recently showed that collagen-like proteins can form trimeric tip fibrillae (ruffles) on the termini of S- and L-type ENA fibers found on Bacillus cereus and showed that these ruffles are involved in spore-spore clustering 14,15. We reasoned that a similar mechanism could be at play for the A-ENA/C1q homologues and modelled a heterohexamer of WP_277491740.1 (putative A-ENA ruffle) of a Bacillus cereus strain SIBC65 isolated from raw bulk milk in complex with WP_000075910.1 (single A-ENA domain) which is the presumed major subunit of an A-ENA type pilus (Supplementary Figure 9a,b). AF3 predicts an A-ENA / ruffle complex wherein the A-ENA domains of the ruffle dock onto the single domain A-ENA protomers thereby mimicking the homotypic A-ENA contacts in the fiber (Supplementary Figure 9c). The residues involved in IPB formation are conserved in both WP_277491740.1 and WP_000075910.1 (Supplementary Figure 9d), suggesting that the ruffle is seamlessly integrated into the fiber or covalently tethered to the terminus of the A-ENA fiber (Supplementary Figure 9e). Clues to a putative function of A-ENA ruffles can be found for Pasteuria ramosa, an endoparasitic bacterium of aquatic microcrustaceans (e.g. water flea Daphnia). Recently, Huessy et al demonstrated that the collagen-like protein Pcl7 of P. ramosa functions as an adhesin to mediate spore attachment to the epithelium of the oesophagus of the host Daphnia25. Here we show that although Pcl7 is a CLP lacking an N-terminal A-ENA domain, it is embedded in a 3 gene operon flanked by two additional CLPs (ORFs 01218, 01220) one of which (01218) has an N-terminal A-ENA domain (Supplementary Figure 9f,g). We also identify another 3 gene cluster (ORFs 00694, 00695 and 00696) which encode for 3 A-ENA-like genes. AF3 predicts a heterohexameric structure of the three CLPs in complex with the 3 putative A-ENA major subunits that is topologically similar to the ruffle described above (Supplementary Figure 9h,i). These observations, while suggestive, remain speculative and do not constitute direct functional evidence. They provide a basis for future experimental investigation into potential roles of A-ena beyond spore/psb coupling.

Methods
Ethical statement
All insect experiments were performed in accordance with relevant guidelines and regulations, where invertebrates are exempt from ethical approval under European and Belgian legislation [https://www.ecopa.eu/wp-content/uploads/belgian_legislation.pdf].

Cryo-EM sample preparation, data collection and helical reconstruction
Bacillus thuringiensis serovar israelensis was inoculated in 10mL LB and grown over night at 37°C under shaking conditions. Next, 500µl of the preculture was spread out onto a square LB agar plate and incubated for 72h at 30°C under stagnant conditions. The resulting confluent growth was harvested and resuspended in miliQ, centrifuged for 5min at 20.000 rcf, the supernatant was discarded and the spore pellet was resuspended in miliQ. The resulting washed spore suspension was used as is for cryoEM grid preparation.

High-resolution cryo-EM datasets were collected using Quantifoil™ R2/1 300 copper mesh holey carbon grids. Grids were glow-discharged at 5 mA plasma current for 30 s in an ELMO (Agar Scientific) glow-discharger. A Gatan CP3 cryo-plunger set at −176 °C and relative humidity of 90% was used to prepare the cryo-samples. 3 μL sample of the spore suspension or of the purified recombinant fibers was applied on the holey grid and incubated for 60 seconds. The sample was double sided blotted using Whatman type 2 paper for 3 s and plunge-frozen into precooled liquid ethane at −176 °C. High-resolution movies were recorded at 300 kV on a JEOL Cryoarm300 microscope equipped with an in-column Ω energy filter (operated at slit width of 14 eV) automated with SerialEM 3.0.850. The movies were captured with a K3 direct electron detector run in counting mode at a magnification of 60K with a calibrated pixel size of 0.71 Å/pix, and a total exposure of 60 e/Å2 over 60 frames. A total of 2817, 3771 and 4160 movies were collected on the ex vivo fibers, the recombinant A-ENA and the recombinant A-ENA1 fibers, respectively. Raw tiffs were imported into Cryosparc v4.5.3 Ref31 for further processing. Movies were motion-corrected using Patch Motion Correction and defocus values were determined using Patch CTF. Exposures were curated and fiber segments were picked using the filament tracer and extracted with a box size of 150 x 150 pixels at a pixel resolution of 1.42 Å/pix. After several rounds of 2D classification, curated particles were used in a helical refinement job using initial estimates for the helical rise and twist values derived from the 2D class average images. After an initial round of helical refinement, particles were recentred and re-extracted at 0.766 Å/pix (300 x 300pix) and used as input for a second round of helical refinement, followed by local and global CTF refinement, and a final helical refinement. The resulting high-resolution volume and particle stack were used for reference-based motion correction after particle duplicate removal (310k particles) followed by a final helical refinement. Reported map statistics were based on these maps for the recombinant A-ENA and A-ENA1 structures. For the ex vivo dataset, we additionally performed reference based motion correction followed by one more round of helical refinement. Figures were created using maps that were sharpened by cryosparc using a B-factor of -77.6Å2, -116.9 Å2, -114.5Å2 for ex vivo, rec A-ENA and rec A-ENA1, respectively, automatically determined by cryosparc.

For ex vivo A-ENA, the initial atomic model was built either manually de novo or using ModelAngelo32 without providing an input sequence (build_no_seq). PSI-blast analysis using the sequence from the built model as a query, identified Q8KNV8 (A-ENA) as the top ranking hit. A second round of model building was performed with ModelAngelo now using the A-ENA sequence as input (build) which was iteratively refined in real-space using WinCoot 0.9.8.96 Ref33 and Phenix34. For model building in the recombinant A-ENA map, a single protomer of the ex vivo structure was docked and iteratively real-space refined in WinCoot 0.9.8.96. Next, the map was populated with remaining chains by applying helical symmetry in ChimeraX 1.9 (sym command) and refined in Phenix 1.21.2. For model building in the recombinant A-ENA1 map, we used ModelAngelo32 with A-ENA1 sequence provided) and refined the model as outlined above. MolProbity35 was used to evaluate the quality of all the filament models using the webserver http://molprobity.biochem.duke.edu/. The refinement statistics are shown in Supplementary Table 1. Determination of the absolute helical hands of the maps were determined from the hand of an -helix. Alternatively, we also performed ModelAngelo test runs on either hand which would lead to either a highly segmented model consisting of many chains, or a filament model wherein chains would fully map onto individual protomers The ‘ModelAngelo’ hand assignment agreed with hand-assignment following the handedness of the -helices.

Bacterial strains and growth conditions. Escherichia coli Stellar™ was used for cloning and was grown in Luria-Bertani (LB) broth or agar plates at 37°C supplemented with 50 µg/ml kanamycin or 100 µg/ml ampicillin when required. For protein production, E. coli BL21 (DE3) was grown in TB broth supplemented with 100 µg/ml ampicillin and placed at 30°C for A-ENA expression after induction. E. coli Dam-/Dcm- (New England Biolabs) was used to obtain unmethylated plasmids in order to increase the efficiency of transformation in Bt. Bti and Btk were grown in LB at 30°C with 150 rpm shaking and supplemented with 25 µg/ml kanamycin when required. For sporulation, 1 ml of overnight culture was plated on LB media and placed at 30°C for a week. Spores were scrapped and diluted in miliQ water and stored at 4°C. The strains used in this study are listed in Supplementary Table 2.

Cloning for recombinant protein expression in E. coli
Cloning of untagged A-ENA, A-ENA1 and variants were produced in this study with the purpose of cytoplasmic overexpression in E. coli. Water-boiled colonies of Bti were used as a source of gDNA. As a general cloning strategy, all PCR fragments containing the coding sequence were cloned into a linearized pASK-IBA3plus vector (using primers 321 and 322) by Gibson assembly using NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs).

The cloning of mCherry:SnoopCatcher (plasmid A185) was carried out using Gibson assembly. The mCherry gene was amplified from a plasmid containing mCherry in the lab collection, using primers p693 and p694. The SnoopCatcher fragment was synthetically ordered as a GeneBlock from IDT. Both fragments were subsequently cloned into the linearized pASK-IBA3plus vector. Cloning of sfGFP:SpyCatcher (plasmid A142) was done by amplifying sfGFP from a plasmid containing sfGFP in the lab collection, using primers p503 and p504. The SpyCatcher fragment was synthetically ordered as a GeneBlock from IDT. Both fragments were subsequently cloned into the linearized pASK-IBA3plus vector. All primers and plasmids used in this study are listed in Supplementary Table 2.

Cloning for a-ena-STOP and Δa-ena1 using CRISPR/Cas9 based deletion plasmid
Plasmid A266 and A190 were used to knockout a-ena and a-ena1 respectively from B. thuringiensis serovar isrealensis. Both plasmids are derived from the plasmid pJOE8999 36 purchased from Bacillus Genetic Stock Center (BGSC). Oligonucleotides for sgRNA construction were designed using the online sgRNA design tool Cas-Designer37. First, pJOE8999 was linearized with primers p551 + p552. A266 homology regions were cloned with p588+p591 and three STOP codon mutations were introduced with oligos p961+p962 whereas p709+p710 and p711+p712 were used to construct the homology regions of A-ENA1 (A190). In both cases, we used NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs) to insert homology regions. Sequencing and insertion were verified with the pair of oligos p525+p526. Insertion of sgRNA was done by PCR using oligos p594+p595 (for A266) and p713+p714 (for A190). Primers p531+p579 were used to sequence sgRNA region. Verification of the A-ENA STOP mutant was done by PCR and sequencing using oligos p963+p6 51 and p964 respectively. Verification of the A-ENA1 mutant was performed by PCR with oligos p717+p718. Plasmids used in this study are listed in the Supplementary Table 2.

Construction and isolation of B. thuringiensis serovar isrealensis mutants
The recombinant pJOE8999-based plasmids A266 and A190 described above, were introduced by electroporation into B. thuringiensis serovar isrealensis competent cells. Briefly, B. thuringiensis cells were made electrocompetent by growing in BHI + 0.5% glycerol at 37 ºC until OD reached 0.6. Cells were harvested by centrifugation (5 min at 15 000 xg). Cells were washed 3 times with ice-cold sterile electroporation buffer (1 mM HEPES, 10% glycerol, pH 7.0) and flash-frozen in liquid nitrogen. Up to 1µg of unmethylated plasmid was transformed into 200 µl of competent cells using a 0.2 cm gap cuvette (Biorad) and electroporated using the MicroPulser Electroporator device (Biorad). Cells were transferred into 1ml of recovery buffer (BHI with 10% glycerol, 0.4% glucose, and 10 mM MgCl2) for 1h at 30 ºC before plating in a 25 µg/ml kanamycin plate. Next day, a single colony was inoculated into 10 ml of liquid BHI supplemented with 25 µg/ml kanamycin and incubated with 180 rpm shaking for 3h at 37 ºC. Then, Mannose (Sigma) was added to a final concentration of 0.4% w/v in order to induce the expression of the Cas9 nuclease. After 3 hours, serial dilution of the culture was plated on LB agar plates with 25 µg/ml kanamycin + 0.4% w/v Mannose and incubated at 37 ºC overnight. Screening of the knockout was done using colony PCR, WT B. thuringiensis serovar isrealensis was used as a control. Positive colonies were sequenced-verified using the same oligos (Eurofins). To curate the pJOE8999-based plasmid, mutant strains were passaged three times in the absence of antibiotics and plate in LB plates at 42 ºC overnight. The absence of the plasmid was checked by striking the same colony in LB plates with or without Kanamycin. Strains used in this study are listed in Supplementary Table 2.

Cloning for recombinant protein expression in B. thuringiensis
For ectopic recombinant expression of the different constructs in B. thuringiensis we modified the pJOE8999 plasmid36 by removal of the coding sequence for the Cas9 nuclease and its promoter by PCR using oligos p622+p623, resulting in the ‘pAS’ shuttle expression vector. Complementation of a-ena stop and a-ena1 was achieved by expressing these constructs in the pAS vector. the a-ena region, including its promoter, ORF, and terminator, was amplified using primers p683 and p684. Colony PCR and sequencing (using primers p631 and p671) confirmed successful insertion, resulting in plasmid A176. Plasmid A176 was subsequently used to introduce the SpyTAGsequence via primers p681 and p682, yielding plasmid A180.

For a-ena1, the region was amplified using primers p695 and p696, generating plasmid A186. This plasmid was then used as a template to introduce the SnoopTAG sequence using primers p697 and p698, resulting in plasmid A187.

To construct a dual-expression vector for a-enaspytag and a-ena1snooptag under their native promoters, plasmid A180 was linearized using primers p699 and p700. The a-enasnooptag fragment, amplified from plasmid A187 using primers p701 and p702, was then cloned into the linearized A180 backbone. This final step produced plasmid A188. To monitor A-ENA expression, we replaced the a-ena coding sequence (CDS) with mCherry. This was achieved by linearizing plasmid A176 using primers p737 and p738, followed by cloning the mCherry CDS amplified with primers p714 and p742 using plasmid A185 as the template. This process resulted in the assembly of plasmid A197. Plasmids generated are listed in the Supplementary Table 2.

Protein expression and purification
sfGFP:spyCatcher and mCherry:Snoopcatcher were expressed in E.coli BL21 (DE3) in Terrific Broth (TB) supplemented with 100 µg/mL of Ampicillin at 37 °C and induced with 200 µg/L anhydrotetracycline when OD reached 0.5. At this point, the temperature was set at 23 °C and cells were left to express overnight. Next day, cells were harvested by centrifugation (20 min at 5,000g) and pellets were kept at -20°C. Frozen pellets were resuspended in 100 ml of lysis buffer (50 mM Hepes pH 8, 300 mM NaCl, 1 mM MgCl2, DNase, lysozyme and EDTA-free protease inhibitor cocktails (ROCHE)) and lysed by sonication at 4°C. The lysate was centrifuged for 25 min at 30000g and 4°C. The cleared supernatant was loaded onto a 5ml Ni-NTA affinity chromatography column (HisTrap FF crude, GE Healthcare). The column was washed with 5 column volumes with Buffer A (300 mM NaCl, 10 mM Hepes pH 8, 10 mM Imidazole) and finally, His-tagged proteins were eluted with a linear gradient of Buffer B (300 mM NaCl, 10 mM Hepes pH8, 0.5 M Imidazole). Eluted protein was concentrated and loaded onto a Superdex 200 16/60 size-exclusion column (GE Life Sciences) that was equilibrated with SEC buffer (25 mM Hepes pH 8 and 150 mM NaCl) at 4°C. The peak corresponding to the protein was concentrated, flash-frozen in small aliquots in liquid nitrogen and stored at −80°C. After purification, the samples were run on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate their purity.

A-ENA and A-ENA1 were expressed in E. coli BL21 (DE3) in Terrific Broth (TB) supplemented with 100 µg/mL of Ampicillin at 37 °C and induced with 200 µg/L anhydrotetracycline when OD reached 0.5. At this point, the temperature was set at 18 °C and cells were left to express overnight. Cells were harvested by centrifugation (20 min at 5,000g) and incubated with A-ENA lysis buffer (25 mM Hepes pH8, 2.5 mM EDTA, 250 mM NaCl and 20 mg of Lysozyme) at 37 °C for 2 hours under shaking conditions. Next, 1% final SDS concentration was added and the resuspended pellet was boiled for 30 min. A-ENA fibers were pelleted down by centrifugation at 30,000g for 45 minutes. Pellet was resuspended with a homogenizer in 30 ml deionized H2O and centrifuged for 45 minutes at 30,000g. This washing step was repeated 4 times. After the last centrifugation step, the pellet was resuspended with 3 ml of deionized H2O.

Spore preparation and Fractionation
Sporulation of Bti and Btk was triggered by nutrient exhaustion on LB-agar plates supplemented with 25 µg/mL of kanamycin when required. A single colony was inoculated in LB media (supplemented with 25 µg/mL of kanamycin when required) and grown overnight at 30°C. Next day, 200 µl of the cell suspension was plated on 245 x 245 mm LB plates (with or without antibiotic). Plates were incubated at 30°C for 7 days under aerobic conditions. Spores were harvested by scraping the LB plates with a cell scraper and resuspended in deionized H2O, spore stock was stored at 4°C. To fractionate Bti and Btk spores, the spore suspension was gently vortexed and then a discontinuous Histodenz gradient was prepared by layering 500 µl of 40% (w/v) Histodenz over 500 µl of 60% (w/v) Histodenz in 2 mL microcentrifuge tubes. A total of 500 µl of spore stock (OD₆₀₀ = 42) was carefully layered on top. Two tubes were prepared per strain: one for SDS-PAGE analysis and the other for optical density (OD) measurements and imaging. Samples were centrifuged at 10,000 × g or 100 × g for 1 hour using a tabletop swinging-bucket rotor (S-24-11-AR, TEppendorf).

After centrifugation, three fractions (supernatant (SN), 40%, and 60% )were carefully collected into separate tubes. Each fraction was washed 3 times by adding 500 µl of water, vortexed, and centrifuged at 12,000 × g for 5 minutes. The supernatant was removed, and the pellet was resuspended in 100 µl of DX buffer (10 mM CHES pH 9.6, 8M Urea, 1% SDS, 1× protease inhibitor tablet (Roche), 2 mM DTT). Samples were boiled for 10 minutes with securely closed lids and subsequently centrifuged at maximum speed for 10 minutes to pellet insoluble spores. A total of 50 µl of the supernatant was mixed with 12.5 µl of blue loading dye, and 5 µl of the final mixture was loaded onto an SDS-PAGE gel for protein analysis. Uncropped images for the SDS-PAGE and Western blots can be found in the online source data accompanying the paper.

For OD measurements and imaging, the SN, 40% Histodenz, and 60% Histodenz fractions were collected into separate tubes. Each fraction was washed as descibed above. The supernatant was removed, and the pellet was resuspended in 100 µl of water. OD₆₀₀ measurements were performed by diluting 50 µl of the sample into 950 µl of water (1:20 dilution) before spectrophotometric analysis.

Phase contrast and fluorescence microscopy
For imaging non-labeled spores, a single colony of Bti or Btk was inoculated into 10 mL of LB medium with the appropriate antibiotic when required and grown overnight at 30°C. The next day, 250 µL of the culture was plated onto LB agar, with or without antibiotics, and incubated for 5 days at 30°C to allow sporulation. The resulting spore lawn was harvested and resuspended in 5 mL of water. A 3 µL aliquot of the spore suspension was transferred onto an LB-agar strip for microscopy. For time-lapse experiments comparing sporulation, t = 0 was defined as the moment cells were plated on the LB agar plate. At each time point, a sample was collected using a sterile loop, resuspended in 30 µL of PBS, and imaged. For dual labeling with SpyCatcher:GFP and SnoopCatcher:mCherry, a strain expressing A-ENA:SpyTag and A-ENA1:SnoopTag under their native promoters was induced to sporulate as described above. After 5 days, the spore lawn was harvested, and the OD₆₀₀ was adjusted to 1.

Both SpyCatcher:GFP and SnoopCatcher:mCherry were added to a final concentration of 50 µM and incubated overnight with gentle rotation. The following day, the spore mixture was washed three times with PBS and mounted on a 1% agar slide for imaging.

In all cases, Images were acquired in phase contrast and fluorescence mode using a Leica DMi8 inverted microscope (Leica) equipped with a 100×/1.32 oil objective (Leica).

Immunodetection
Due to the high degree of sequence identity of surface exposed residues between A-ENA and A-ENA1 we anticipated that it would not be feasible to raise polyclonal antibodies that would be specific to either A-ENA or A-ENA1. Given that our cryoEM analysis indicated that ex vivo fibers are predominantly composed of A-ENA, we opted to solely raise antisera against rec A-ENA. For this, a polyclonal mouse antiserum raised against purified rec-A-ENA fibers was ordered from Davids Biotechnologie GmbH (Regensburg, Germany). The following 63 day immunization schedule was followed: immunizations on days 0, 14, 28, 42 and 56. To detect A-ENA on spore samples, we diluted the spore suspension to OD =1 and blotted 2 µl onto a nitrocellulose membrane. After 5 minutes of drying at room temperature (RT), the membrane was blocked with 5% (w/v) skimmed milk for 30 minutes. The membrane was incubated with A-ENA antibody mix (dilution 1:1000 in TBS-Tween buffer (Tris-HCl pH8 10 mM; NaCl 150 mM; Tween20 0,05% (vol/vol)) and incubated at RT for 1h. After 3 washes of 5 minutes each, the membrane was incubated in the secondary antibody mix (dilution 1:1000) for an extra hour at RT. After 3 washes of 5 minutes each, the membrane was imaged with the LI-COR Odyssey M using the LI-COR Acquisition Software v 2.0.0.86.

A-ENA stability assay
To test the chemical stability of recombinant A-ENA fibers, fibers were harvested from a concentrated stock solution via centrifugation (20.000rcf, 1h, supernatans discarded). The resulting pellets after centrifugation of 100µl aliquots were resuspended in various conditions: 100µl 2% (w/v) SDS, 8M urea, 2M NaOH or 100 (v/v) formic acid and incubated as indicated on Supplementary Figure 5 prior to nsTEM sample preparation.

To test the physical robustness of recombinant A-ENA fibers, a 100µl aliquot of the fiber stock solution was desiccated at 200°C for 15min in an open glass vial in an oven. The resulting dried material was rehydrated in 100µl miliQ prior to nsTEM imaging.

Additionally, a 100µl aliquot was autoclaved for 20min at 121°C. Next, 3µl aliquots of the treated samples were deposited onto Cu-mesh formvar grids, washed twice with 20µl miliQ and incubated for 1min with 2% (w/v) uranyl acetate and blotted dry with Whatmann 2 filter paper. Micrographs were collected with a 120 kV JEOL 1400 microscope equipped with LaB6 filament and TVIPS F416 CCD camera operated at 60.000x magnification.

Monitoring the entomopathogenic activity of different Bt strains
To evaluate the virulence of Bti strains either expressing or lacking A-ENA (wild-type vs. a-ena knockout), we employed the insect model Chironomus aprilinus. Fresh C. aprilinus larvae were procured from Zooschatz (Berlin, Germany) under batch number 1223153. Bti strains were cultured on LB plates at 30°C for one week, after which the lawn of spores and PSB were harvested and resuspended in deionized water, and OD was measured. To determine the mortality rate, a minimum of 39 C. aprilinus larvae were included in each experimental group, with a final volume of 1 ml of sea salt solution supplemented with a vitamin premix provided by the supplier. Experiments were carried out in 12 well plates with a single individual per well. Spore suspension was added to reach a final OD of 0.02. Phosphate saline buffer (PBS) was used as a negative control. Larvae survival was monitored daily over a 6-day period. The experiment was conducted in biological triplicates under room temperature conditions.

For the virulence assessment of Btk strains, we used the insect model Trichoplusia ni. Larvae or eggs were obtained from Frontier Agricultural Sciences (Newark, USA). Btk wild-type and A-ENA-expressing strains (pA-ENA) were sporulated on LB plates at 30°C for one week. Spores and PSBs were harvested and processed as described for Bti strains. To determine the mortality rate, a minimum of 16 two week old T. ni larvae per group were individually placed on 2.5 × 2.5 cm pieces of solid food provided by the supplier. Each food piece was spread with a 50 µl aliquot of the harvested spore suspension at an OD₆₀₀ of 0.06. As a negative control, Bti (which has no toxic effect on T. ni) was included. Larval survival was monitored daily for one week. For experiments involving recombinant A-ENA, it was added to Btk WT samples at a final concentration of 4.18 mg/ml and incubated for at least 3 hours under slow rotation. As an additional control, purified recombinant A-ENA was tested at the same final concentration (4.18 mg/ml) under identical conditions. The study was performed in biological triplicates at room temperature

Search for orthologs and homologs of A-ENA
To probe for the presence of A-ENA across the Firmicutes phylum, all publicly available Genbank genomes (9880 genomes) belonging to the Bacillus/Clostridium group (taxid 1239; assembly-level complete) were downloaded from the NCBI database. Homo- and orthologs of A-ENA were searched in all assemblies with hmmsearch from the HMMER 3.4 software package38 (881 genomes) using a hidden Markov model (Supplementary Data 1) that was generated with hmmbuild using a manually curated multiple sequence alignment of A-ENA sequences obtained from a blastp39 web-search using Q8KNV8 as a query.

For this, for this, the top 1000 blast hits were downloaded and filtered to remove redundant sequences above 75% sequence identity to reduce bias. The remaining sequences were aligned using Muscle (protein alignment preset) in Jalview 2.11.4.1. Columns with ≥50% occupancy were retained, and collagen-like triplet repeat regions located C-terminal to the A-ENA domain were manually removed to isolate the core A-ena sequence. The final multiple sequence alignment (270 sequences) was used as input for hmmbuild to generate the A-ENA hmm profile. The resulting hmm file has been included as a supporting document for peer evaluation. AlphaFold2 models (either retrieved from the AlpfaFold database (Varadi, 2024 #103) or predicted using localcolabfold40 of the corresponding blast sequences were manually inspected and compared to the A-ENA cryoEM structure to remove any non-A-ENA sequences. The inclusion threshold for hmmsearch was set to an E-value of 1e-7. The phylogenetic tree of the Firmicutes phylum was generated using phyloTv2 (https://phylot.biobyte.de/) using the NCBI taxonomy IDs of the corresponding assemblies as node identifiers, and imported into iTOL41 for visualization using the online server (https://itol.embl.de/).

For the more detailed search of A-ENA homo- and orthologs across the Bacillus cereus group sensu lato, the BtyperDB v1 database (5976 genomes) was downloaded from https://www.btyper.app/, and hmmsearch was run against all assemblies with an E-value threshold of 1e-7 (496 genomes). The phylogenetic tree described in BtyperDB42 and the corresponding meta-data table was kindly provided by Laura M. Caroll. Tree Figures were generated using https://microreact.org/. Gene cluster analysis was performed using cblaster 1.3.18 Ref43 using default settings. Protein domain organization of A-ENA homologs with C-terminal fusions was performed with Interproscan 5.67-99.0 Ref44.

Statistical analysis A-ENA and Bt-toxin occurrence.
To evaluate if A-ENA and Bt-toxin occurrence is correlated, a chi-square test of independence was run in R 4.5.1, using a categorical contingency table of A-ENA and Bt-toxin occurrence in the 5976 B. cereus genomes in the BtyperDB v1 database42. To test for the phylogenetic non-independence, we performed a phylogenetic logistic regression using the ‘phyloglm’ function (model: Bt_toxin_presence ~ A_ENA_presence; method: logistic_MPLE - Maximum Penalized Likelihood Estimation) from the R package phylolm. The analysis incorporated a pruned phylogenetic tree (as input we used the Btyper-derived Newick tree that was used to construct Fig. 5b) and binary trait data for A-ENA and Bt-toxin gene presence. We obtain an =70,259, AIC=2680 and a log-odds coefficient =1.61±0.15. To probe for tree scaling effects, we rescaled the tree using ‘tree_rescaled’ (method: Grafen; mean tip height 1) and reran the analysis in both the forward (A_ENA_presence ~ Bt_toxin_presence) and reverse (Bt_toxin_presence ~ A_ENA_presence) direction, resulting in the reported statistics.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a strong and well-designed study that provides compelling evidence for A-ENA as a virulence factor in Bacillus\nthuringiensis (Bt). The structural and functional work is thorough, and the integration of cryoEM, molecular biology, and\ninsect bioassays makes for a solid and convincing story. What stands out most is the finding that A-ENA\nphysically links spores and parasporal bodies (PSBs) into biofilm-like clusters, enhancing virulence by keeping the infectious particle and\ntoxic payload together. This has clear implications for biopesticide applications, especially in aquatic environments or other\nsettings where dispersal might limit the effectiveness of traditional Bt formulations. The fact that A-ENA can be added\nexogenously or expressed in Bt strains that lack it—and still increase virulence—makes it especially interesting from a\npractical standpoint.\nThe insect experiments are well executed and appropriate for this line of research. The authors use two relevant insect\nmodels: Chironomus aprilinus (Family Chironomidae), midge fly larvae, and Trichoplusia ni (Family Noctuidae),\nrepresenting both aquatic and terrestrial pest control contexts. The use of OD600 to standardize spore concentrations is\ncommon practice, and the doses fall within typical ranges for larval mortality assays. The six-day exposure window is also\nreasonable and consistent with prior studies. Importantly, the controls are well designed, and the gain- and loss-of-function\nexperiments are clearly presented. The only area that could be improved is the\nstatistical treatment of the survival data. While the MDK₈₀ values are useful, the\ndifferences in mortality between treatments would be better supported with standard\nstatistical analyses, such as log-rank (Mantel–Cox) tests for survival curves, p-values for pairwise comparisons, and\nconfidence intervals around MDK₈₀ estimates. Including these would help quantify the strength of the observed effects and\nallow readers to better assess the reliability and reproducibility of the results.\nThere are a few other small points that could further improve the manuscript. First, the authors do not provide the\nfamily names or authorities for the invertebrate species used/ mentioned. This is standard practice in entomology and should be\nadded at first mention. Second, the common name “cabbage looper” is only used late in the discussion; I suggest introducing\nthe common name earlier in the text and using both scientific and common names consistently throughout. Third, the authors\nincubated Bti spores at pH 11 to mimic gut dissolution of parasporal bodies, but this seems quite high compared to the gut\npH of most insect larvae, which typically ranges from 6 to 8 depending on species. It would be helpful for the authors to\njustify or contextualize this choice, particularly for readers outside the microbiology community. Lastly, the\nmanuscript refers to the “promiscuity” of A-ENA binding interactions—a term that carries anthropomorphic and sometimes sexist connotations.\nA more neutral alternative such as “non-specific,” “broad-spectrum,” or “versatile binding” would avoid unintended\nimplications and better reflect the scientific intent.\nOverall, the methodology is solid, and the experimental detail is sufficient to allow others to replicate the work. The\nconclusions are well supported by the data. The broader significance is clear: this paper advances our understanding of how\nBt maintains spore–toxin proximity, and it introduces a novel strategy for enhancing\nvirulence without genetic modification of the toxins themselves. I see this being of interest not just to microbial pathogenesis researchers, but also to those working in\napplied pest control and biopesticide development.", "reviewer_2": "The manuscript describes the occurrence, in B. thuringiensis serovar israelensis, of a network of proteinaceous fibers\nproduced during sporulation and encasing spores and parasporal bodies in a gel-like matrix. The clustering of spores and\ncrystals appear to promote the virulence of BT israelensis or Bt kurstaki against their insect targets.\nThe observations and the methodologies used to identify the protein involved in the formation of the fibers appear quite\nconvincing and the findings reported here are of value. However, these findings raise a number of questions which are not\ncurrently addressed in the manuscript.\nFirst, the protein A-ENA polymerises spontaneously, is not secreted, and the fibers are quite stable : therefore, it is expected\nthat fibers forms intracellularly. Have they been observed within the cell ? Are they present in E coli expressing the\nrecombinant protein ? If yes, how is prepared the monomer ?\nSecond, these fibers have not been described previously in Bti : are they specific to the strain (which is not named) used in\nthis study ?\nThird, while it makes sense to cluster spores and toxins in Bti, which are diluted in water upon the insect death, this is not so\ntrue for Btk, where crystals and spores are in close proximity on leaves when the insect dies.\nFourth, cultures were made on LB-agar. Are these fibers also observed when sporulation is conducted in liquid cultures ?\nOther remarks :\nAbstract and throughout the manuscript : the fibers, spores and parasporal bodies are described as forming a biofilm-like\nstructure. Biofilms are living structures adherent to a surface, which is not the case for the structure described here, since\nspores are metabolically inert and the fibers-spores-parasporal bodies are not described as adherent.\nResults :\nDiscussion : ‘in Bacillus sphaericus crystals are withing the exosporium’ – this is also the case for Bacillus thuringiensis\nserovar finitimus.\nDiscussion : ‘…to grace the PSB crystal surface as well as making contact with neighbouring spores, seemingly functioning\nas a molecular tether mimicking the spore-PSB…’, in Bti crystals are within a membraneous body, whereas in Btk\ncrystals are free in the cytoplasm : does it mean that A-ENA fibers would stick to any protein ?", "reviewer_3": "In the final results section of the manuscript, the authors present the distribution of A-ENA homologs across the B. cereus\ngroup and Firmicutes phylogeny, revealing an association with Bt toxins. Many homologs are identified as fusion proteins\ncontaining additional domains, and structural predictions suggest potential functions beyond spore–parasporal body (PSB)\ncoupling. This analysis provides a strong foundation and raises intriguing hypotheses about A-ENA functional evolution.\nHowever, improvements in scope, statistical rigor, methodological transparency, figure design, and cautious interpretation\nare needed to substantiate broader functional claims and ensure reproducibility.\nMajor concerns:\n1. The current analysis is limited to species distribution and gene/domain co-occurrence. To gain deeper insight into A-ENA\nfunctional diversification, a phylogenetic tree based on A-ENA domain sequences (including both fused and non-fused\nforms) should be constructed. Reducing redundancy using CD-HIT or MMseqs2 will help control for genome\noverrepresentation and improve resolution. This approach could clarify whether specific A-ENA gene clades are associated\nwith Bt toxins or with distinct fusion domains (e.g., C1q, redox), and whether similar functions have independently emerged\nacross multiple gene clades.\n2. The assertion that A-ENA functions “beyond spore/PSB coupling” is currently based solely on bioinformatic inferences.\nSuch functional claims require experimental validation or significantly stronger predictive modeling. It is recommended to\nrephrase these statements to reflect their hypothetical nature and avoid overstating conclusions based only on domain\narchitecture or phylogenetic co-occurrence.\n3. The claim that A-ENA presence correlates with Bt-toxin genes lacks statistical support. A phylogenetically informed test is\nrecommended to account for lineage effects and sampling bias.\nMinor concerns:\n1. The authors mention using curated HMMs to identify A-ENA homologs but do not provide sufficient detail on their\nconstruction. To ensure reproducibility, the manuscript should clarify how the HMMs were built, how domains were filtered or\nvalidated. Or, the HMM profiles should be included or made available for peer evaluation.\n2. The current analysis focuses on Firmicutes (e.g., Bacillales and Clostridia). To more fully understand the evolutionary and\nfunctional breadth of A-ENA, the search should be expanded to all prokaryotes (not only limited to NCBI TaxID: 1239).\n3. Missing software version information. Please report the software names and versions used throughout the pipeline.\n4. Figure order inconsistency. Please swap Figure 5a and 5b for narrative consistency with the Results and Methods\nsections.\n5. Figures 5 and Extended Data Figure 7 use inconsistent color schemes to represent species.\n6. Extended Data Figure 7d lacks a legend.\n7. In Extended Data Figure 7d, the domain architecture (e.g., in panel d) and 3D structure (e.g., in panel c) are not visually\naligned. Please use consistent color-coding and labels for each domain across both the linear cartoon and 3D structure.", "reviewer_4": "This is a carefully conducted study that will be of considerable interest to the bacterial spore community. The presence of the\nappendages has been noted over many years, but their function was largely unknown. This manuscript makes considerable\nprogress towards addressing their structure and function. In particular, the structural work is excellent; the functional analysis\nis slightly more speculative in places, although a general role in biofilm formation seems very plausible. My main concern is\nover the identity of the so-called parasporal bodies and their interaction with the fibres- this will be discussed below.\nMain concerns:-\n1. Extended data Fig. 1g. There is no hard evidence that this object has anything to do with parasporal bodies. Indeed, the\nresult shown in panel (h) casts even more doubt on this interpretation. It could easily be some other kind of cellular debris\ncarried over in the preparation. It is also not clear what the authors mean by “treatment” in the legend. The authors refer to\n“bipyramidal toxin crystals” but nowhere in the paper do they show convincing images showing that such a thing exists. This\nseems surprising.\n2. Discussion. A major conclusion is that the role of the fibres is to produce an extracellular matrix holding spores and\nparasporal bodies together. Does B. sphaericus have these fibres? If so, it would seem that interaction with the parasporal\nbodies is not the only role.\n3. The authors state that “the effects of the physical spore-PSB coupling will be more pronounced in turbulent and/or larger\nreaction volumes (here we work in 3mL) where the dilution factor and subsequent probability of spore-PSB decoupling\nwould be larger for the A-ENA mutant strain.” The authors should either test this or make it clear that this is only speculation.\nMinor issues:-\n4. The introduction is rather long and the second paragraph could be omitted.\n5. Fig. 2e. The colouring should be explained and it would be good to have the rise and twist indicated on the figure.\n6. Fig. 3a. The labelling of nucleo- and electrophilic residues is a bit unconventional. How about electronegative and\nelectropositive?\n7. Fig. 3d. It would be good to have a panel showing the buried interface.\n8. Fig. 3e is hard to follow. The residue numbers should be indicated. Use of a ball and stick representation might make the\nlinks more obvious.\n9. Fig. 3f is hard to make sense of without showing neighbouring subunits in the helix. What is the purpose of the\nbackground surface rendering? The residue numbers should be indicated.\n10. Fig. 4a. Why are the middle panels labelled as parasporal body fraction? They seem to show mixed populations.\n11. Fig. 4d. The panels don’t correspond to the legend.\n12. Fig. 4g. The identical headers to each panel are confusing.\n13. Extended data Figs. It would be easier for the reader if there was just one figure per page.\n14. Extended data Fig.1a-c. Each panel should be described separately in the legend. The legend is confusing as it stands.\n15. Extended dats Fig.1d. What is the inset?\n16. Extended data Fig. 2. The panels and labels are too small.\n17. Extended data Fig. 4 needs scale bars."}, "author_response": "We thank the reviewer for his/her appreciation of our study and for the thoughtful\nsuggestions for the manuscript. In response, we have added the family names for the two\ninvertebrate species at their first mention, and now introduce Trichoplusia ni as the\ncabbage looper earlier in the text.\nWe now also include the statistical analyses of the insect larvae experiments, reporting\nthe log-rank p-values comparing samples populations with and without A-ENA (i.e. Bti\nWT vs. Bti A-ENA, Btk WT vs. Btk pA-ENA and Btk WT versus Btk +A-ENA), and including\nthe 95% confidence intervals for the different populations.\nRegarding the use of pH 11 conditions to dissolve parasporal bodies inside the Bti spore\nbiofilms, we used pH 11 to ensure timely dissolution of the PSBs and test the integrity of\nthe extrasporal matrix, but did not mean to imply that the pH 11 condition mimics the pH\nin the insect mid gut. To avoid any confusions, we have rephrased the section to:\n“Bti PSBs comprise polycrystalline condensates of insecticidal proteins (Cry11Aa1,\nCyt1Aa1, Cry4Aa1 and Cry4Ba1) that are packaged in a spherical parasporal sack,\nand that dissolve under the elevated pH in the insect gut (pH 8-10 in Lepidoptera)\n(Rudd et al., 2023). Even when incubated at pH 11 we found that Bti spore\npreparations retained a clumped organization, with spores embedded in a dense\nnanofiber matrix (Extended Data Fig. 1f-j).”\nFinally, we acknowledge the reviewer’s point about the term “promiscuity.” We have\nreplaced it with more neutral and scientifically precise “non-specific”.\nWe thank the reviewer for his/her insightful comments and suggestions. Detailed\nresponses to the specific points are listed below.\nFirst, the protein A-ENA polymerises spontaneously, is not secreted, and the fibers are\nquite stable: therefore, it is expected that fibers forms intracellularly. Have they been\nobserved within the cell ? Are they present in E. coli expressing the recombinant protein\n? If yes, how is prepared the monomer ?\nNative A-ENA expression is absent from vegetative cells, and occurs in the mother cell as\nindicated by the use of the A-ENA promoter driving the expression of the mCherry\nfluorescent protein (Ext. Data Fig. 6). In our TEM images, A-ENA fibers first become visible\nupon mother cell lysis and the release of the endospore. Unfortunately, the cell wall of\nthe sporulating cells is too dense to directly observe A-ENA fibers in situ. At no time are\nA-ENA monomers detectible by SDS-PAGE – Western Blotting, suggesting that nascent\nmonomers are readily incorporated into SDS- and heat-stable A-ENA fibers.\nIn our BTK experiments, where A-ENA is over-expressed from a plasmid (still under native\nA-ENA promotor), we observe dense clusters of A-ENA that retain the shape of the\nmother cell (Ext Data Figure 7d), suggesting that the fibers are indeed expressed and\nassembled intracellularly. A similar phenomenon is observed in E. coli recombinantly\nexpressing A-ENA in the cytoplasm. Again, no A-ENA monomers are detectible, with\nexpressed A-ENA entirely present in the form of SDS- and heat-resistant nanofibers. On\nSDS PAGE, such fibers are stuck in the stacking gel, on cryoEM grids, these fibers proof\nisomorphous to ex vivo A-ENA fibers form Bti, including the formation of the isopeptide\nbonds. Gently, detergent-based lysis of E. coli in absence of lysozyme results in bundled\nA-ENA fibers trapped inside the E. coli peptidoglycan sacculus.\nTogether, all observations indicate that A-ENA monomers readily self-assemble into iso-\npeptide bound polymers inside the producing cell.\nSecond, these fibers have not been described previously in Bti: are they specific to the\nstrain (which is not named) used in this study ?\nThis is an excellent question. The reviewer is correct that these fibers have not previously\nbeen reported in Bti. We apologise for not mentioning the strain identifier in the\nmanuscript: we are working with strain Bacillus thuringiensis serovar israelensis AM65-\n52. We have now included the full strain name at its first mention in the manuscript.\nWe have not experimentally investigated other Bti strains, but looked at all the published\nBti genomes from NCBI (https://www.ncbi.nlm.nih.gov/datasets/genome/?taxon=1430)\nand searched for A-ENA genes using hmmsearch. 10 out of 13 genomes carry the A-ENA\nand A-ENA-1 gene, 3 out of 13 genomes did not have hits above the default hmmsearch\nthreshold (1e-5) and can be expected to be A-ENA deficient.\nThird, while it makes sense to cluster spores and toxins in Bti, which are diluted in water\nupon the insect death, this is not so true for Btk, where crystals and spores are in close\nproximity on leaves when the insect dies.\nWe agree and hypothesize this may be the reason A-ENA genes are not natively found in\nBtk strains. For a terrestrial species, a matrix-based association of spores and\nparasporal crystals may give comparatively little selective advantage than in aquatic\nspecies. In that sense, the gain of function observation of A-ENA expression or addition\nin Btk is non-intuitive at first glance, and requires further study to be fully elucidated. We\nspeculate that the observed clustering of spores Btk toxins might lead to (i) a higher\npeak loads when ingested by insects and/or (ii) higher local concentrations of dissolved\ntoxins in the digestive tract of the cabbage looper. Either or both of these could lead to\nthe moderate increase in the observed virulence.\nFourth, cultures were made on LB-agar. Are these fibers also observed when\nsporulation is conducted in liquid cultures ?\nYes, A-ENA fibers are also observed when sporulation is conducted in liquid LB cultures.\nHowever, we found that sporulation tends to occur more slowly and less uniform across\nthe cell population in liquid compared to agar plates. In liquid media, we consistently\nobserved formation of a primarily vegetative cell biofilm prior to sporulation, and a much\nlarger fraction of cells that remain in vegetative state. In both solid and liquid medium\ncultures we observe the formation of A-ENA fibers and spore PSB clustering. To maximise\nsporulation we work with solid medium cultures.\nOther remarks :\nAbstract and throughout the manuscript : the fibers, spores and parasporal bodies are\ndescribed as forming a biofilm-like structure. Biofilms are living structures adherent to a\nsurface, which is not the case for the structure described here, since spores are\nmetabolically inert and the fibers-spores-parasporal bodies are not described as\nadherent.\nWe agree with the reviewer that the comparison of the multispore clusters to bacterial\nbiofilms may not be optimal. Although the exact definition of ‘bacterial biofilm’ varies\nbetween sources, they generally comprise vegetative cells that are grouped into\nmulticellular communities usually held together by an extracellular matrix. Although the\nspore aggregates we observe can still contain remaining vegetative cells, they do not\nrepresent a specialized organization form of metabolically active cells.\nTo avoid any such confusion or ambiguity, we now refer to the matrix-embedded spore –\nPSB aggregates as ‘spore clumps’. We have adopted this terminology throughout the\nmanuscript and figure, and removed any mention of biofilm-like spore aggregates.\nResults :\nDiscussion : ‘in Bacillus sphaericus crystals are withing the exosporium’ – this is also\nthe case for Bacillus thuringiensis serovar finitimus.\nWe thank the reviewer for pointing this out, we now also mention and reference B.t.\nfinitimus in the discussion. We also searched 4 genome Refseq assemblies of Bacillus\nthuringiensis serovar finitimus (Btf) and were not able to find any a-ena homologues.\nThis fits in with the hypothesis / rationale that A-ENA’s predominant biological function\nis to couple PSBs to spores. Based on the fact that the Btf crystals reside in the\ninterstitial space of the Btf spores, it makes logical sense that a-ena would be absent in\nthe Btf genome.\nDiscussion : ‘…to grace the PSB crystal surface as well as making contact with\nneighbouring spores, seemingly functioning as a molecular tether mimicking the spore-\nPSB…’, in Bti crystals are within a membraneous body, whereas in Btk crystals are free in\nthe cytoplasm: does it mean that A-ENA fibers would stick to any protein ?\nInspection of the A-ENA structures shows a surface topography and amino acid\ncomposition that does not point to any likely binding pockets. This shows the A-ENA\nsurface to be largely hydrophilic with no specific charged or hydrophobic pockets.\nMoreover, the genomic organization does not point to any minor subunits that could\ntake up the role as adhesive subunits as is often seen in various pilus families.\nMoreover, the recombinant A-ENA fibers produced in E. coli and used to induce spore –\ncrystal clustering in Btk (Ext. Data Fig 7d), encompass the A-ENA protein only and are\ndevoid of any other Bacillus thuringiensis gene product.\nYet, as rightfully pointed out by the reviewer, A-ENA fibers bind surfaces with very\ndifferent molecular compositions, comprising the exosporium surfaces of Bti and Btk\nspores, the parasporal sack enclosing the Bti Cry toxins, or the cry toxin crystals directly\nin the case of Btk. There is no indication that these different surfaces would have any\ncommon motif or composition.\nInstead, we postulate that the interaction between A-ENA and the spore, PSB and\ncrystal surfaces is largely mediated by weak, non-specific polar contacts (constituting\nhydrogen bond formation and Van der Waals contacts). When present in high\nmultivalency, (A-ENA fibers have a repeat length of 5 Å), such weak non-specific\ncontacts can still result in a macroscopic interaction with high avidity. The high flexibility\nand micrometer length scale of A-ENA fibers are likely to help in the clustering and\nentanglement of micrometer-sized particulates such as spores, PSBs and Cry toxin\ncrystals. We note that A-ENA preparations can have an intrinsic propensity to form fiber\nentanglements. Micrometer-sized particles are likely to get physically entrapped into\nsuch entanglements, further aided by a high valency of weak, non-specific polar\ninteractions.\nWe predict and have preliminary (non-published) results that indicate that A-ENA\nnanofiber entanglements will also entrap and cluster non-spore-derived particulate\nmaterials.\nWe thank the reviewer for his/her enthusiasm on the discovery of A-ENA subunits as a\nnovel nanofiber scaffold in bacterial spores, and share the curiosity on the extent of the\n(functional?) diversity amongst A-ENA like sequences, particularly A-ENA subunits with\nadditional domain decorations.\nAlthough we agree that gaining deeper insights into the functional diversification of A-\nENA genes, and the connection to taxonomic distribution would be highly interesting and\ninformative, we deliberately refrained from doing in this first manuscript reporting A-ENA\ndiscovery. As it stands, the manuscript already conveys a multiple of different messages,\naddressing a multitude of audiences: (1) primary A-ENA discovery and cryoID molecular\nidentification, (2) atomic structure the fiber encompassing an unprecedented cross-\nalpha stacked protein nanofiber with autocatalytically cross-linked monomers and\nprotofibrils, resulting in remarkable chemical and physical stability, (3) the microscopic\nmechanism of spore - PSB clumping, and its influence in Bt virulence, (4) recombinant\nproduction of a self-assembling protein nanofiber, and its use for induced spore – PSB\nclumping and the gain-of-function activity this has on Bt virulence.\nOur phylogenetic analysis demonstrates A-ENA like sequence are widespread, and our\nstructural analysis of A-ENA like sequences show these may putatively diversify in\nfunction by incorporating additional folded domains expected to decorate the length of\nthe A-ENA fibers. To balance the different points made in the paper and the diverse\naudiences they address, we feel a more in depth analysis of the functional diversification\nof A-ENA like sequences and their occurrences amongst different taxa is best done in a\nseparate, dedicated study. We find this hugely exciting and envision this to be coupled\nwith experimental validation of selected A-ENA like derivatives.\nWe agree that no definitive proof is provided towards any functional diversification of A-\nENA like proteins beyond spore/PSB coupling. We have carefully rephrased the section\nto make clear this is merely a tentative hypothesis and outlook based on a molecular\nmodelling of A-ENA like sequences with added domains. This is meant more to arouse\ninterest rather than to lay claim of such broader A-ENA function.\nWe agree our initial report lacked a rigorous statistical support for A-ENA – Bt toxin co-\noccurrence. We more carefully address this point in our revised manuscript.\nTo test for A-ENA / Bt-toxin correlation, we first performed a Chi-square analysis to test\nfor independence using the following contingency table.\nA-ENA Present Bt-toxin Absent Bt-toxin Present Total\nYes 145 351 496\nNo 4760 720 5480\nTotal 4905 1071 5976\nWe obtain Chi² = 1022.93, p < 0.0001, which we interpret as strong evidence of\nassociation between A-ENA and Bt-toxin presence. But as rightfully pointed out by the\nreviewer, this correlation might be spurious if one doesn't account for shared\nevolutionary history, i.e. closely related species may share traits due to common\nancestry, not because of a functional relationship.\nTo address the reviewer’s concern regarding phylogenetic non-independence, we\nperformed a phylogenetic logistic regression using the ‘phyloglm’ function (model:\nBt_toxin_presence ~ A_ENA_presence; method: logistic_MPLE - Maximum Penalized\nLikelihood Estimation) from the R package phylolm. The analysis incorporated a pruned\nphylogenetic tree (as input we used the Btyper-derived Newick tree that was used to\nconstruct Fig. 5b) and binary trait data for A-ENA and Bt-toxin gene presence. We obtain\nan =70,259, AIC=2680 and a log-odds coefficient =1.61±0.15. The estimated\nphylogenetic signal  is high, which could indicate weak lineage effects. However, we\nfound that the mean tip height of the tree is 0.00056, indicating that the branch lengths\nare very short -> which could (artefactually) contribute to the weak phylogenetic signal.\nTo probe for tree scaling effects, we rescaled the tree using ‘tree_rescaled’ (method:\nGrafen; mean tip height 1) and reran the analysis in both the forward (A_ENA_presence ~\nBt_toxin_presence) and reverse (Bt_toxin_presence ~ A_ENA_presence) direction:\nDirection Log-odds Std. Error  AIC\ncoefficient\nA_ENA → 2.23 0.26 38.57 1589\nBt_toxin\n(Forward)\nBt_toxin → 2.61 0.28 11.73 3173\nA_ENA\n(Reverse)\nFrom this we conclude that species with Bt-toxin are 9.3x more likely to have A-ENA than\nspecies without Bt-toxin, after accounting for shared ancestry. And conversely, species\nwith A-ENA are 13.6x more likely to have Bt-toxin than species without A-ENA, again\ncontrolling for phylogeny. We also note that the -values have decreased significantly\nafter rescaling.\nIn summary, we infer that the co-occurrence of A-ENA and Bt-toxins is not strongly\nimpacted by shared ancestry, but likely reflects a true functional linkage. This analysis is\nin line with a purely qualitative assessment of the phylogenetic tree and mapping of the\nbinary trait data, in that the different areas of A-ENA and toxin co-occurrence are\ndispersed over distant branches, and with numerous gaps in between.\nWe apologize for not explaining our methodology more thoroughly in the manuscript. We\nnow provide the A_ENA.hmm file as a Supporting Document, and we have added a\nMaterials and Methods paragraph regarding the construction of the hmm profile. In brief,\nwe performed a blastp search using Q8KNV8 as a query sequence. The top 1000 hits were\ndownloaded, filtered down to 75 percent sequence identity to remove redundant\nsequences and lower sequencing bias. The remaining sequences were aligned in Jalview\n2.11.4.1 using Muscle (preset: Protein Alignment). Columns with an occupancy of 50%\nor higher were retained, and any columns C-terminal to the A-ENA domain that\ncorresponded to fusion sequences (for example collagen-like triplet repeats) were\nremoved manually so as to only retain the core A-ENA sequences. The resulting MSA (270\nsequences) was used as input in hmmbuild to construct the HMM A-ENA profile.\nWe also like to mention that in the interim we have been contacted by Alex Bateman at\nthe EMBL-EBI. They informed us that based on our preprint, they were building a Pfam\nprofile for the A-ENA domain with accession code PF26595 (“A-ENA”; Alpha-helical\nendospore appendage domain) which will appear via the InterPro website in the near\nfuture. Although we agree that for the sake of reproducibility it is important we share our\ncustom HMM profile in the SI of the manuscript, we also refer the readers to the PFAM A-\nENA entry for future and more convenient access.\nThank you for this valuable suggestion. We performed a blastp search using the A-ENA\nsequence as a query, excluding Firmicutes (taxid:1783272) from the “Organism”\nidentifier. The blastp search yielded 8 putative A-ENA homologues in various Polyangium\nspecies. Trimeric AF3 predictions showed close structural resemblance to the axial\npacking of A-ENA fibers seen in Bti. Members of the Polyangiaceae family belong to the\nMyxococcota phylum, which are Gram-negative “fruiting gliding” bacteria that aggregate\ninto multicellular structures that contain differentiated spores. We did not find A-ENA\nhomologues in one of its most well-studied representatives Myxococcus xanthus, so we\ncan’t really infer from literature if candidate A-ENA fibers would have been observed in\nprior studies.\nThese have been added.\nThis has been changed.\nThis has been added.\nSince panel d shows AF3-predicted structural models, we opt to color according to\npLDDT confidence score. We have now made this explicit in the legend and added the\ncolor scheme in the figure panel. For clarity, we have added the domain labels in panel\nd, and removed color from panel e, now use patterned fills to differentiate the domains.\nWe that the reviewer for his/her general appreciation of the manuscript and the\nsuggestions for further improvement.\nWe apologize for the confusion and appreciate the opportunity to clarify these\nobservations. The structures referred to in Extended Data Fig. 1e–i correspond to the\nsame sample shown in panel 1d, which contains only spores and PSBs following alkaline\ntreatment at pH 11, as described in the first paragraph of the Results section. After\ntreatment, we observed that the overall sample morphology remained consistent, with\nthe key difference being the apparent loss of content from the PSBs. Based on this, we\ninterpret the remaining structures as empty PSB envelopes. This interpretation is\nsupported by five observations: (1) the empty shell structures are not present in the\nsample prior pH11 treatment, (2) they retain the same size and shape as the untreated\nspore sample, with the exception of loosing their content, (3) the sample was purified\nusing Histodenz, which removes vegetative cells and cellular debris prior to pH 11\ntreatment, and (4) the empty shells maintain the same spatial association with A-ENA\nfibers and spores as observed before treatment and (5) the spatial association with\nspores, the size, and relative density of the structures are as seen in the time lapse\nsporulation images, showing them form in the mother cell. Based on these combined\nobservations, the dense pre-pH11 structures, and the empty post-pH11 shells can be\nannotated as parasporal bodies and parasporal sacks, respectively.\nWe altered the figure legends to increase their clarity. We also rearranged the figure and\nadded an additional panel to allow a better comparison of a wider field of view of spore\npreparation prior to and after treatment with pH11 and urea.\nRegarding the mention of bipyramidal toxin crystals, we acknowledge that this shape is\nspecific to the B. thuringiensis subsp. kurstaki (Btk) strain. These are clearly visible in\nExtended Data Fig. 7d and in the additional image provided below. We would like to clarify\nthat the PSBs shown in Extended Data Fig. 1 correspond to the B. thuringiensis subsp.\nisraelensis (Bti) strain, which is known to produce PSBs with a spherical morphology.\nThis is an interesting comment. Using A-ENA as a query sequence, we searched for A-\nENA homologues in Lysinibacillus sphaericus (previously known as Bacillus sphaericus)\ngenomes using blastp. That search led to the identification of 8 putative A-ENA\nhomologues. Notably, 4 of these have a C-terminal domain fused to the A-ENA like\nsequence (WP_185903352.1; WP_185903353.1; TQR39880.1; WP_221887655.1),\nsuggesting they may be minor subunits with an additional functional domain.\nIf we focus on WP_142506947.1, it is a 100aa protein that is predicted to fold in a highly\nsimilar manner to A-ENA. Further inspection of the genome NZ_SADV01000001.1, leads\nto the identification of WP_221887655.1 and WP_185903353.1, both of which are\npredicted (based on Interpro analysis and AF3 modelling) to consist of an N-terminal A-\nENA domain, followed by a collagen-like region and terminating in a C-terminal C1q-like\ndomain, similar to A-ENA like sequences seen also in Bacillus cereus SIBC65 and\nPasteuria ramosa (Ext. Data Fig. 8). Similar to the latter two, Alphafold3 modelling suggest\nthese A-ENA fusion proteins (WP_221887655.1 and WP_185903353.1) co-assemble with\nthe main A-ENA subunit (WP_142506947.1) to result in C1Q-like decorated A-ENA like\nfibers. As presented in our discussion, we hypothesize that A-ENA-like proteins and\nfibers, particularly operons including A-ENA fusion proteins, may adopt functions other\nthan spore – PSB associations. We like to point out that functional diversification is not\nuncommon in pilus-like structures. Closely related chaperone-usher pilus pathways, can\nadopt host-adhesive functions in one species, whilst taking up a biofilm-associated\nfunction in another. Or similarly, Type 4-like pili can have a functional diversification\nranging from specific host adhesion, bacterial competence, biofilm formation to\nbacterial motility (Lukaszczyk et al. 2019; doi: 10.1007/978-3-030-18768-2_12).\nIndeed, our statement regarding the potential effects of a scale-up of the reaction\nvolume was speculative. We have now more clearly indicated in the main text that this\nremains a hypothesis\nThe second paragraph introduces Bti as species and biopesticide. We would prefer to\nleave this paragraph for reader less familiar with the Bt field.\nWe have explained the colouring in the figure legend and added the rise and twist in the\nfigure\nIn the figure we point to the three residues involved in isopeptide bond formation, which\noccurs through nucleophilic attack. In addition to the acid/base catalyst, the labelling\nannotates the nucleophilic and electrophilic residue implicated in the reaction.\nElectronegative and electropositive could be confused to refer to electrostatic\ninteractions.\nThe buried surfaces are shown in Ext Data Figure 4a and b. For extra clarity, we have\nadded an additional reference to these panels in the main text, where discussing the\nrole of buried surfaces during subunit packing.\nWe acknowledge that the detail of the isopeptide bonds is hard to see from Fig. 3e. The\npanel is meant to give the overview of where the different isopeptide bonds are located\nin the fiber, requiring a zoomed out figure. For the atomic detail of the respective\nisopeptide bonds, the reader is referred to Ext. Data Fig. 3. We have rearranged the\nfigure to better highlight the atomic detail of the IPB modules, and now explicitly point to\nExt. Data Fig 3 in the Fig3 figure legend.\n“(e) Overview of IPB location with cartoon representation of the subunits in protofibril f\n(i+1 to i-5), and illustrated as sticks are the residues involved in the intra-protofibrillar\nIPBs between i/i+1, i/i-1 and i/i-5 (donating and accepting IPB in full and dotted circles,\nresp.; see Ext. Data Fig. 3 for close-up views of the individual IPB modules)”\nSimilar to panel e, panel f gives the overview of the now interfibrillar IPB location. For\natomic detail of the IPB module, the reader is referred to Ext. Data Fig. 3. The semi-\ntransparent surface rendering illustrates the tight packing of protofibrils and the solvent\nburied nature of the IPB. This is consistent with the illustration in panel e.\nThe labels meant to indicate the dominant species in the fractions, although we\nacknowledge this may result in confusion. We have removed the labels from Fig. 4a\nThank you for pointing this out, this has been corrected.\nWe have altered the panel.\nWe have now added page breaks between all figures.\nWe have now reworked the figure legend, and also added an additional panel (a) that\nshows the zoomed out nsTEM view of the spore – PSB preparations deposited on non-\nglow discharged formvar grids. This gives a better appreciation of scale, and allows\nbetter comparison of morphology and position of the PSBs pre (a) and post (b) pH11\ntreatment to dissolve the cry toxins (see point 1 of the reviewer).\nThe inset represents a 2D class average of the A-Ena fibers. We have added this\ninformation to the figure legend\nImage is now in landscape to provide larger panels.\nThese have been added."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for the thorough and constructive revision. The changes you’ve made address my core concerns and improve\nclarity for readers across microbiology and entomology.\nStatistical treatment of survival data\nI appreciate the addition of log-rank (Mantel–Cox) tests and 95% CIs comparing survival with and without A-ENA for the\nspecified strain pairs. This substantially strengthens the inference around treatment effects. As a minor enhancement, please\n(a) report the exact test statistics and p-values in the figure legends or a dedicated statistics table, (b) note the number of\nbiological replicates and total larvae per group for each curve, and (c) state whether any multiple-comparison adjustment\nwas applied for pairwise contrasts. If feasible, providing hazard ratios (with CIs) alongside the log-rank results would help\nreaders quantify effect sizes.\nInsect nomenclature and audience clarity\nThank you for adding family names at first mention and for introducing Trichoplusia ni as “cabbage looper” earlier in the text.\nThese adjustments will support a broad audience.\nContext for high-pH treatments\nYour revised language makes clear that pH 11 was used to ensure timely PSB dissolution and to probe matrix integrity,\nrather than to mimic gut conditions; the added gut-pH context for Lepidoptera is helpful. To make this unmistakable to non-\nspecialists, consider adding one explicit sentence in Methods stating that the pH 11 condition is an experimental stress and\nnot intended as a physiological surrogate. The current phrasing already goes most of the way there.\nTerminology (“promiscuity”)\nReplacing “promiscuity” with “non-specific” is the right call and reads as both accurate and professional.", "reviewer_2": "The authors responded satisfactorily to my comments and remarks.", "reviewer_3": "The authors have addressed the large majority of my comments with substantive revisions and added analyses.The main\nitem left for future work—a deeper, de‑redundancy‑aware phylogenetic analysis of A‑ENA sequences—is reasonable to\ndefer and is not essential to evaluate the present study. I support publication.", "reviewer_4": "I am now generally happy with the authors' changes. However, I would ask that they make the evidence for the full and\nempty PSBs very clearly laid out as in their response to reviewers. They should also make it clear further work could be\ndone in the future to confirm the identity of the PSBs by labelling, mutational analysis or other additional means."}, "author_response": "We thank all reviewers for their helpful comments and suggestions throughout the review\nprocess. We have included the last suggestions of Reviewer 1 and 2 in the final version of\nthe manuscript. A point-by-point response is outlined below.\nReviewer 1:\nStatistical treatment of survival data\nI appreciate the addition of log-rank (Mantel–Cox) tests and 95% CIs comparing survival with and\nwithout A-ENA for the specified strain pairs. This substantially strengthens the inference around\ntreatment effects. As a minor enhancement, please (a) report the exact test statistics and p-values in\nthe figure legends or a dedicated statistics table, (b) note the number of biological replicates and total\nlarvae per group for each curve, and (c) state whether any multiple-comparison adjustment was applied\nfor pairwise contrasts. If feasible, providing hazard ratios (with CIs) alongside the log-rank results would\nhelp readers quantify effect sizes.\nAuthor response:\nDone. P-values are present in the image panels and figure legends, and HR and 95% CI are now\nincluded for all pairwise comparisons.\nContext for high-pH treatments\nYour revised language makes clear that pH 11 was used to ensure timely PSB dissolution and to probe\nmatrix integrity, rather than to mimic gut conditions; the added gut-pH context for Lepidoptera is helpful.\nTo make this unmistakable to non-specialists, consider adding one explicit sentence in Methods stating\nthat the pH 11 condition is an experimental stress and not intended as a physiological surrogate. The\ncurrent phrasing already goes most of the way there.\nAuthors response:\nDone, such sentence is added to the methods.\nReviewer 4:\nHowever, I would ask that they make the evidence for the full and empty PSBs very clearly laid out as\nin their response to reviewers. They should also make it clear further work could be done in the future\nto confirm the identity of the PSBs by labelling, mutational analysis or other additional means\nAuthors response:\nDone, such sentence is added to the figure legend of Ext Data Figure 1, where the parasporal sacks\nare shown in detail."}]

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