The structure of the Vibrio alginolyticus flagellar filament suggests molecular mechanism for the rotation of sheathed flagella

The structure of the Vibrio alginolyticus flagellar filament suggests molecular mechanism for the rotation of sheathed flagella | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The structure of the Vibrio alginolyticus flagellar filament suggests molecular mechanism for the rotation of sheathed flagella Julien Bergeron, Kailin Qin, Rosa Einenkel, Weilong Zhao, Joseph Atherton, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6983709/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Apr, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract In several pathogenic bacteria, including Vibrio species, the filament of the bacterial flagellum is encased by a membranous sheath, an extension of the bacterial outer membrane. It has been proposed that having sheathed flagella permit bacteria to evade an immune response against flagellar components, suggesting a role in virulence. However, the molecular details of the interaction between sheath and filament, and how it impacts filament rotation and assembly, have remained largely uncharacterized. Here, we combine single-particle cryo-electron microscopy, cryo-electron tomography, and genetic analyses to resolve the molecular architecture and biogenesis of the sheathed flagellum in Vibrio alginolyticus . We show that the flagellar filament forms a canonical 11-stranded supercoil made of the flagellin FlaD2 and enveloped by a bilayered sheath. We observed that the filament surface is highly electronegative, suggesting that electrostatic repulsion between filament and sheath may reduce friction and supports high-speed flagellar rotation. We also show that the filament cap protein FliD possesses a unique domain in sheathed flagella, that may coordinate sheath assembly with filament elongation. Collectively, this structural insight into the structure of the Vibrio alginolyticus flagellum suggests a molecular mechanism for the rotation of sheathed flagella. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Microbiology/Bacteriology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Many bacteria swim through liquid environments using the flagellum. By rotating like a propeller, the flagellum drives bacterial motility and directional movement in many bacterial species 1 . In human gastrointestinal pathogens, such as Vibrio cholerae ( V. cholerae ) , Salmonella enterica ( S. enterica ) , Campylobacter jejuni ( C. jejuni ), the flagellum also plays an important role in invasion and colonization to human tissue 2 . The flagellum consists of a membrane-spanning basal body that houses the export apparatus and motor proteins, and an extracellular appendage formed by the hook, the hook-filament junction, the filament, and a cap at the distal end 3,4 . The filament, typically ~10 µm long in most bacteria, is composed of tens of thousands of flagellin subunits – either a single flagellin protein or multiple flagellin variants – which are secreted through the flagellum-specific type-III secretion system 5 . In recent years, cryo-EM structures of flagellum filaments have been reported, in many bacterial species. They demonstrate a common architecture, consisting of a 11-stranded filament of the flagellin protein, with slight conformational changes to each subunit allowing the formation of a long-range supercoil. The flagellin consists of two conserved internal domains, D0 and D1, that form the secretion lumen; and several external domains, D2-D5, of which the number and arrangement vary between bacterial species and strains. At the tip of the filament, the cap complex, consisting of five copies of the protein FliD, is responsible for flagellin folding and insertion in the growing filament. In several bacteria, including the human pathogens Vibrio , Helicobacter , and Bdellovibrio , the flagellar filament is surrounded by a membranous sheath, thought to be an extension of the outer membrane 6 . Accordingly, the filament sheath is presumed to be an asymmetric bilayer, containing primarily glycerophospholipids (GPLs) in its inner-leaflet, and lipopolysaccharides (LPS) in its outer-leaflet 7,8 , with lipoproteins embedded in. Flagellar sheaths have been proposed to serve multiple, and possibly species-specific, functions 9 . In Helicobacter pylori , the sheath has been suggested to protect flagellin from acidic conditions and to contribute to adherence, while in Vibrio species, sheaths reduce recognition of flagellin by the host innate immune receptor TLR5 and play a role in outer membrane vesicle (OMV) release 10,11 . Rotation of the sheathed filament has been linked to the production of OMVs, which can mediate host signaling, deliver virulence factors, and provide protection against antimicrobial peptides or bacteriophages. More generally, the sheath has also been hypothesized to shield flagella from flagellotropic phages, which exploit unsheathed filaments for infection 9 . Despite these proposed roles, direct mechanistic insight into the rotation of sheathed flagella has remained limited, with two theoretical models proposed: One where the sheath membrane rotates together with the flagellum filament, and one where only the filament rotates, within a flexible sheath. Low-resolution cryo-ET analyses of sheathed flagella basal body complexes have revealed that these flagella possess additional periplasmic structures, called H-ring and T-ring, thought to promote sheath formation. Deletion of the entire ring structures leads to the formation of periplasmic flagella beneath the PG layer 12 . Some periplasmic filaments penetrate the cell envelop at the region far from the basal body, with and without the sheath. These observations suggest the HT-ring and filament alone plays a role in outer membrane remodeling during sheath biogenesis. Moreover, the identification of an extracellular O-ring at the base of the V. alginolyticus sheath suggests a further architectural element, although this feature is not conserved across all sheathed species 6 . Despite these observations, the molecular mechanisms underlying sheath assembly remain largely unknown. Here, we combine single-particle cryo-EM, cryo-ET, and genetic approaches to investigate the structure and assembly of sheathed flagella in Vibrio . We determine high-resolution structures of both sheathed (3.6 Å) and unsheathed (3.2 Å) filaments and identify FlaD2 as the major polar flagellin of V. alginolyticus . Electrostatic analyses reveal strong repulsion between the filament and sheath, consistent with a mechanism that reduces friction during rapid rotation. Furthermore, sequence and functional analyses reveal that the FliD protein possesses an additional domain, which may anchor the flagellum tip to the sheath end. Together, these findings provide structural and mechanistic insights into the adaptations that underlie the rotation mechanism of sheathed flagella. Results Single-particle analysis of the V. alginolyticus sheathed flagellum filament It has not been shown whether the filament formed direct interactions with the membrane, in sheathed flagella. To address this, we isolated the sheathed filament from Vibrio alginolyticus ( V. alginolyticus ), a pathogen affecting a variety of marine animals and humans, causing otitis and wound infection 13,14 . V. alginolyticus encodes a single, polar, sheathed flagellum that drives swimming in liquid environments. In addition, it produces peritrichous, non-sheathed lateral flagella that enable swarming and surface-associated motility under various environmental conditions 15 . To characterize the structure of the V. alginolyticus sheathed flagellum, we engineered a mutant of this bacterium lacking the flhG gene, shown previously to lead to multi-flagellated bacteria 16 . We subsequently isolated flagellar filaments from this mutant strain using mechanical shearing, achieved by repeatedly forcing a cell suspension through a narrow-gauge needle. Cryo-EM analysis of the resulting sample confirmed that we successfully isolated the flagellar filament with an intact sheath ( Fig. 1a ). We also obtained sheath-free filaments, as well as filaments exhibiting membrane pearling, suggesting partial detachment of the sheath ( Fig. 1b ). Using this, we were able to obtain a map of the sheathed filament to 3.6 Å resolution by single-particle analysis ( Fig. 1c, 1d, Supplementary Fig. 1, Table S1 ). This map confirms that the filament adopts a 11-stranded supercoiled architecture, similar to that of other bacteria. The membrane is well-resolved, with diffused density for the GPL inner leaflet and the LPS outer leaflet clearly identifiable ( Fig. 1c, Supplementary Movie 1 ). Fluorescence microscopy using an outer membrane dye demonstrated that in both V. alginolyticus and V. cholerae , the filament sheath is composed of outer membrane material. This observation is consistent with previous reports 6,17 ‑19 . In contrast, staining with HADA, a fluorescent D-amino acid that labels peptidoglycan, revealed no signal within the sheath structure. This indicates that the filament sheath is an extension of the outer membrane but lacks an underlying peptidoglycan layer, distinguishing it from the envelope of the cell body ( Supplementary Fig. 2 ). FlaD2 is the main flagellin in V. alginolyticus V. alginolyticus encodes six flagellin homologues in its genome ( Fig. 2a , Supplementary Fig. 3 and Table S2 ), FlaB and FlaD1-FlaD5, ~44-99% identical in sequence (FlaD2/FlaD4 are 99% identical). Because of this, it was not immediately clear whether the map of the sheathed filament consisted of a mixture of the above flagellins, or was formed by predominantly one (or a subset of) these flagellins ( Fig. 2a ). We noted nonetheless that FlaD5 and FlaD1 possess 2-6 residue insertions in their D2 domain, respectively, not present in the other flagellin homologues ( Supplementary Fig. 3 ). In our cryo-EM map of the sheathed filament, there is no density for either of these loops, suggesting that they are not the main flagellins in our structure ( Fig. 2b, c ). Similarly, in our cryo-EM map, we noted the presence of large side-chain density in positions corresponding to residues Y300, H304, F315, H317 in FlaD2 and FlaD4 ( Fig. 2d ). In contrast, FlaB, FlaD5, FlaD1, and FlaD3, possess small amino-acids in these positions ( Supplementary Fig. 3 ). Collectively, this suggests that FlaD2/FlaD4 are the main flagellins in the filament map we have obtained. To verify this, we deleted the genes of all flagellin genes individually, and assessed the motility of the corresponding strains. Because V. alginolyticus encodes a lateral flagellum system capable of compensating for defects in polar flagellum-dependent motility 15 , all experiments were performed in a ∆ lafK background (Laf - ) lacking the lateral flagellar master regulator. Furthermore, a lateral and polar flagellar master regulator mutant was used as a non-motile control strain (∆ lafK ∆ flrA ). As shown in Fig. 2e, f, deletion of the flaD2 gene abolished bacterial motility similar to the non-motile control strain, whereas deletion of all the other flagellin homologues leads to only a mild (FlaD1) or no significant reduction of motility (FlaD3, FlaD4, FlaD5, FlaB). This confirms that FlaD2 is necessary to form a functional polar flagellum in V. alginolyticus . These observations are similar to V. cholerae , which also possesses multiple flagellin genes, but a single one ( flaA ) is absolutely necessary for motility in this bacterium ( Supplementary Fig. 4a ). Whether the various flagellins of V. alginolyticus are localized to different zones of the filament, similar to V. cholerae , remains to be verified 20 . Collectively, both the motility data and the cryo-EM map reported above suggest that FlaD2 is the main flagellin in the V. alginolyticus flagellar filament. Structural basis for the filament-membrane interface Based on the aforementioned results, we then built an atomic model of the V. alginolyticus filament, using the FlaD2 sequence, in the cryo-EM map of the sheathed filament ( Fig. 3a ). To further characterize the interaction between the filament and the membrane, we also built an atomic model of the outer membrane, using CHARMM-GUI 21 , which could be fitted in the corresponding density ( Fig. 3b ). In this model, the flagellin outward-most D2 domain faces the membrane inner leaflet, with several residues in proximity (~7-10 Å) to the density attributed to the GPL phosphate groups ( Fig. 3c ). Intriguingly, these are largely negatively-charged amino-acids, and the intact filament possesses a highly electronegative surface ( Fig. 3d ). As GPL headgroups are mainly negatively charged, this raises questions as to how the contacts between the sheath and the filament are maintained. At this distance, electrostatic forces still exert a large influence 22 , which suggests that there is repulsion force between the sheath and the filament. We postulate that this facilitates the rotation of the filament within the sheath, without any friction that would perturb rotation velocity. In support of this, we note that in most bacteria, the surface of the flagellum filament is largely charge-neutral ( Supplementary Fig. 5 ), supporting the notion that this charge of the V. alginolyticus filament is a species-specific adaptation permitting to facilitate rotation within a membranous sheath. Supercoiling of the V. alginolyticus flagellum filament Previous studies showed that the flagellum filament is supercoiled, with a range of supercoiling architectures observed, all corresponding to subtly different arrangements of the flagellin interactions in the 11 strands of the filament. Supercoiling had previously been reported also in sheathed flagella 20 , and indeed, 2D classes of filament particles with a larger box size ( Fig. 4a ) confirmed that the V. alginolyticus sheathed flagellum filament possesses a defined curvature. Nonetheless, due to the relatively low resolution of the structure of the corresponding filament reported above ( Fig. 3a , Supplementary Fig. 1 ), caused by the limited number of particles, we were not able to resolve the differences between flagellin subunits leading to this curvature. To address this, we determined the structure of the unsheathed filament, from the corresponding particles present in the aforementioned dataset, to 3.16 Å resolution ( Supplementary Fig. 1, Supplementary Fig. 6a ). In this structure, we were able to build 55 copies of the FlaD2 flagellin. Both structures (sheathed and unsheathed) are highly similar, and notably we observed no significant structural changes in the D2 domain ( Supplementary Fig. 6b ). This also confirmed that this structure corresponds primarily of FlaD2 filaments lacking the sheath, not of filaments formed by other polar flagellins, nor that of the lateral flagellum, for which the flagellin LafA shares only ~52% identity with FlaD2. Thus, the isolated unsheathed filaments likely originate from the polar flagellum, from unsheathed filaments in V. alginolyticus 6 or from filaments that lost their sheath during isolation. In the resulting structure, obtained without helical symmetry imposed, we can clearly distinguish convex and concave sides on the filament ( Fig. 4b ), in agreement with the 2D classes described above. Comparison of individual flagellin protofilaments revealed that each possess a distinct curvature, with the protofilament from the concave side significantly more curved ( Fig. 4c ). Close-up view of the longitudinal interfaces of protofilaments from the concave and convex sides reveal subtle differences in the interaction, ( Fig. 4d ), which when propagated across many subunits explain the difference in curvature. These observations confirm that the supercoiled nature of the flagellum filament is also conserved in sheathed flagella. The cap protein FliD possesses a unique domain, likely anchored to the sheath The flagellum cap protein FliD is required for filament formation in most bacteria, as it promotes flagellin folding during filament assembly. Our previous structural studies had revealed that FliD forms a pentameric structure at the tip of the filament, with domains D0-D1 buried in the filament structure, and D2-D3 forming the pentameric contacts at the filament tip 3,23 . Intriguingly, analysis of FliD sequences revealed that in both Vibrio spp and Helicobacter spp, FliD possesses a distinct architecture, with predicted unique domains (termed D4 in Vibrio , and D4-D5 in Helicobacter ) not found in other FliD orthologues ( Supplementary Fig. 7a) . Modeling of the V. alginolyticus D4 domain with AlphaFold3 24 revealed that it consists of two helical domains, located on top of the cap complex ( Supplementary Fig. 7b ). This allowed us to build a hybrid model of the V. alginolyticus filament-cap complex ( Fig. 5a, Supplementary Fig. 7c ), by combining this model to our previously-published structure of the S. enterica cap-filament complex, and the structure of the sheathed filament reported above. Notably, the diameter of the resulting cap complex is ~16.8 nm, larger than that of the filament ( Supplementary Fig. 7c ), which is similar to the width of the sheath inner leaflet. This suggests that the edge of the FliD D4 domain could be directly interacting with the sheath itself ( Supplementary Fig. 7d ). To verify this, we constructed mutant strains lacking the D4 domain in FliD ( fliD ∆D4 ). As shown on Fig. 5b, negative-stain TEM analysis of this mutant in V. cholerae revealed the presence of empty sheath extensions at the tip of the filament, not observed in WT bacteria. This observation suggests a role on the D4 domain of FliD in synchronizing filament assembly with sheath elongation. We note, however, that fliD ∆D4 strains retained swimming motility in soft-agar, in both V. alginolyticus and V. cholerae , in contrast to the respective ∆ fliD deletion mutants ( Supplementary Fig. 8 ), indicating that the D4 domain of FliD is not essential for filament assembly. To further investigate the architecture of the V. alginolyticus cap complex in-situ, we employed cryo-electron tomography (cryo-ET) to visualize filament tips in the V. alginolyticus ∆ flhG mutant described above. As shown in Fig. 5c and Supplementary Movie 2 , the distal tips of the flagellar filaments are clearly resolved in the tomograms and the surrounding sheath appears continuous and sealed, with no detectable gap or bulge at the filament tip. Structural docking of the filament–cap complex model into these tomograms supports the presence of an intact cap structure beneath the closed sheath ( Fig. 4d , Supplementary Movie 2 ). Collectively, these results indicate that while the FliD D4 domain is not essential for sheath formation or maintenance, it may contribute to the coordination between sheath elongation and filament growth. The molecular basis of this contribution remains to be clarified. Finally, we sought to characterize the architecture of the cap complex in the early stages of flagellum assembly. To this end, we engineered a V. alginolyticus ∆flhG ∆fliS mutant, in which secretion of flagellin and therefore motility is strongly reduced ( Supplementary Fig. 8a ) 25 , and used cryo-ET to gain structural insights into their flagellum architecture. As expected, flagella are significantly shorter in this mutant, yet the sheath is present and still closed at the tip ( Supplementary Fig. 9a ). Intriguingly, we observed a range of flagellar architectures, including ones where the cap complex is near the tip of the sheath (i), and ones where the filament is significantly shorter, with the cap complex visible below the tip of the sheath (ii). We also observed empty sheaths without a filament (iii). These results suggests that synchronization between filament assembly and sheath elongation occurs later during flagellum biogenesis, although we cannot rule out that this is an artifact caused by the ∆flhG ∆fliS mutation, which significantly disrupts flagellum assembly and regulation. Discussion In this study, we present the structure of the sheathed flagellum filament in V. alginolyticus , providing insights into the interface between the filament and the membranous sheath. Based on sequences of flagellin homologs and EM densities of filament, we demonstrate that FlaD2 is the main flagellin in this bacterium. Furthermore, electrostatic analyses indicate the filament surface is uniformly negatively charged, presumably strongly repulsing between the filament surface and the sheath, which may help reduce friction and thereby facilitate rapid rotation of the filament within the sheath. Finally, we report that the cap complex possesses a unique architecture in sheathed flagella, which is not required for filament assembly but may contribute to synchronizing it to sheath elongation. Based on these results, we propose a model whereby in sheathed flagella, there is no direct molecular interaction between the sheath and the filament during flagella rotation. Nonetheless, our data suggests that the cap complex is forming direct contact with the sheath inner layer during flagella assembly to synchronize filament growth with sheath elongation ( F ig. 6a ). A recent study examining the architecture of the sheathed flagellar motor of V. cholerae likewise proposes that sheath biogenesis is coordinated with ongoing flagellar assembly, specifically the coordination between HL-ring formation and hook elongation 26 . This complementary perspective, though focused on distinct regions of the flagellum, supports the broader view that sheath growth and flagellar assembly are mechanistically linked. In this model, we propose that charge repulsion “greases” the interface between sheath and filament, ensuring that the filament rotates within the sheath without friction ( Fig. 6b ). Together with the previously reported continuity with the outer membrane, and the lack of direct contacts between the filament and sheath, this observation provides molecular support for earlier models proposing that the filament rotates freely within a flexible sheath, rather than as a rigid filament-sheath complex 20,27 . Such a mechanism would allow the sheath to deform in response to filament motion while maintaining structural integrity. It remains to be determined how the sheath maintains its tubular structure, in the context of it surrounding a filament with repulsive electrostatic interactions. It has been shown that the outer leaflet of the bacterial OM is relatively rigid 28 , and it plausible that the LPS structure alone is sufficient to maintain a tubular sheath. Nonetheless, further molecular and computational studies will be required to confirm this hypothesis. We also note that rotation of sheathed flagella in Vibrio has been shown to promote the release of outer membrane vesicles (OMVs), which contribute to immune modulation, host colonization, and delivery of virulence factors 10,29 . Electrostatic repulsion could facilitate local membrane curvature and blebbing, consistent with the bubbling we observed in our cryo-EM datasets ( Fig. 1a,b ). Although speculative, these observations suggest a potential link between sheath architecture, filament-sheath interactions, and OMV biogenesis that warrants future investigation. Taken together, our structural and genetic analyses provide a framework for understanding how sheath architecture is supported in Vibrio and highlight the unique adaptations of sheathed flagellar filaments compared to unsheathed systems. These findings establish a basis for future work to dissect the molecular mechanisms of sheath formation and its impact on bacterial motility and virulence. Methods Bacterial strains and growth conditions All Vibrio alginolyticus , Vibrio cholerae and Escherichia coli ( E. coli ) strains used in this study are listed in Table S3 . The strains were derived from V. alginolyticus NCTC 10675 or V. cholerae V52 Δ vasK 30 . The wild-type V. alginolyticus NCTC 10675 was obtained from the National Collection (UK Health Security Agency, UK) and originally isolated by Miyamoto et al. 31 . Bacteria were cultured in lysogeny broth (LB) at 37 °C with constant shaking at 180 rpm. For V. alginolyticus , LB was supplemented with 2% NaCl (LBS). Antibiotics were added as required at the following final concentrations: chloramphenicol (6.25 µg/ml for V. alginolyticus , 12.5 µg/ml for V. cholerae and E. coli ), ampicillin (50 µg/ml), and streptomycin (100 µg/ml). Bacterial growth was monitored by measuring optical density at 600 nm (OD 600 ) using a spectrophotometer (Amersham Bioscience). Genetic Manipulations Chromosomal mutations were introduced into the genomes of V. alginolyticus and V. cholerae using the suicide plasmid pRE112, which carries the sacB gene for sucrose-based counterselection 32 . Approximately 600 bp of DNA up- and downstream of the target gene were amplified and assembled into pRE112 using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, catalogue number: E2621L), then transformed into E. coli DH5α λ-pir⁺ or E. coli CC118 λ-pir⁺ and after confirmation of correct assembly into E. coli SM10 λ-pir⁺. Conjugation was performed between donor E. coli SM10 λ-pir⁺ and recipient Vibrio strains. Recombinants resulting from single crossover events were selected on LB/LBS plates containing chloramphenicol, supplemented with ampicillin ( V. alginolyticus ) or streptomycin ( V. cholerae ). To select for the second crossover and excision of the plasmid backbone, cultures were grown for 4 h in LB/LBS, and plated on no-salt LB agar supplemented with 10% sucrose and streptomycin ( V. cholerae ) or on tryptic soy agar (TSA) supplemented with 10% sucrose and ampicillin ( V. alginolyticus ). Colonies were screened for loss of chloramphenicol resistance to confirm plasmid excision, and the desired chromosomal modifications were verified by PCR and Sanger sequencing. Lists of plasmids and oligonucleotides used in strain construction are provided in Table S4 and Table S5 . Isolation of flagellar filaments A single colony of wild-type V. alginolyticus was inoculated into 5 ml LB medium and cultured overnight at 30 °C. The next day, 250 mL LB medium was inoculated 1:100 with the overnight culture and grown to OD 0.5-0.8. Cells were harvested at 4,000 × g at 4 °C for 10 min. The cell pellet was resuspended in 40 mL ice-cold sucrose buffer (20mM Tris-HCl, 20% w/v sucrose, pH 8.0) on ice. Flagellar filaments were sheared off by passing the cell-suspension through a 23ga, 0.7 x 50 mm needle using a 20 mL syringe 10 times. To separate the cells from the sheared filaments, the cell suspension was centrifuged at 20,000 × g at 4 °C for 30 min, and the supernatant containing the sheared filaments was collected and centrifuged at 100,000 × g 4 °C for 1h. The filaments in the pellet were resuspended in 200 µL ice-cold sucrose solution (20mM Tris-HCl, 20% w/v sucrose, pH 8.0). Single-particle cryo-EM sample preparation, data acquisition, and processing Filament resuspension (3 μL) was applied to glow-discharged holey carbon grids (Quantifoil R2/2, 300 mesh). Samples were incubated for 30 s at 4 °C and 88% humidity before being blotted by Leica EM GP1 and then rapidly plunged into liquid ethane. Grids that were blotted for 5, 6, 7, 8 s were screened on 200 kV Glacios microscope (Thermo Fisher). The grids with good ice thickness were deposited to 300 kV Krios G3i microscope with a Gatan K3 direct electron detector (Thermo Fisher). Dataset was collected using a physical pixel size of 1.078 Å at the magnification of 81,000 ×. Finally, 21,747 movies were collected with a total dose rate of 40 e-/Å 2 , with 40 frame fractionations. All movies were collected over 40 frames with a defocus range of -0.9 μm to -2.7 μm. For all movies, motion-correction and CTF estimation were processed in CryoSPARC v4.7 33 using patch motion correction and patch CTF estimation respectively. A total of 21,634 movies were used. Filament tracer was performed for particle picking, with a filament diameter of 200 Å and a separation distance of 0.265-fold diameters. 3,059,499 particles were extracted with a box size of 500×500 pixels and subjected to 2D classification. Two rounds of 2D classification were performed, to remove junk particles and to separate sheathed and unsheathed filament, respectively. For the sheathed filament, a subset of 76,818 particles were subjected to a round of helical refinement, followed by a round of local CTF refinement. Two rounds of 3D classification were performed to remove particles that present abnormal sheaths. Finally, a local refinement with a tight mask was performed with 72,779 particles and a map with a resolution of 3.6 Å was obtained. For the unsheathed filament, a subset of 443,577 particles were subjected to a round of helical refinement, followed by local CTF refinement. A round of 3D classification was performed to remove junk particles. Finally, a local refinement with a tight mask was performed with 439,378 particles and a map with a resolution of 3.16 Å was obtained. Model building and refinement Atomic models for all V. alginolyticus NCTC 10675 flagellin homologs, namely FlaD1, FlaD2, FlaD3, FlaD4, FlaD5, were obtained from the AlphaFold database 24 , and fitted in the cryo-EM maps. Based on the density, we identified FlaD2 as the main flagellin in the structure (See results). Therefore, we used FlaD2 to build the atomic models for both sheathed and unsheathed filaments. 33 flagellin monomers (sheathed filament) or 55 flagellin monomers (unsheathed) were manually fitted into the reconstructed maps, and the complex was flexibly refined by ISOLDE 34 with secondary structure restraints gained from the AlphaFold model in UCSF ChimeraX 35 . Real-space refinement in PHENIX 36 with secondary structure, rotamer and Ramachandran restraints but without NCS restraints was next employed. Coot 37 was then used to correct rotamer outliers, side-chain clashes, and unattributed density. The final model was validated using the validation program in PHENIX 36 . The atomic model of the bacterial OM was generated with the membrane builder in CHARMM-GUI 21 , using a 1:1:1 ratio of Phosphatidylethanolamine:Phosphatidylglycerol: Cardiolipin for the inner leaflet, and LPS for the outer leaflet. Atomic models of the V. alginolyticus , V. cholerae and H. pylori FliD orthologues were obtained from the AlphaFold Database. A model of the V. alginolyticus cap-filament complex was generated by aligning five copies of the V. alginolyticus FliD model, and the structure of the V. alginolyticus filament (this study), onto the structure of the S. enterica filament-cap complex; this model was fitted in the tomogram of filament tips using ChimeraX 35 . Cryo-ET sample preparation, data collection and processing Overnight cultures of the respective strains were diluted 1:100 in 10 mL LBS and incubated for 1.5 h. Cells were centrifuged at 2,500 × g for 10 min and resuspended in ice-cold sucrose buffer (20mM Tris-HCl, 20% w/v sucrose, pH 8.0) in a gentle manner. The pipette tip was cut off to reduce flagella to be sheared off. 4 μL cell suspension was applied to glow-discharged lacey carbon grids (Agar scientific, 200 mesh). Samples were incubated for 30 s at 5 °C and 95% humidity before being blotted by Vitrobot Mark IV and then rapidly plunged into liquid ethane. Grids that were blotted for 8, 12, 16, 20 s were screened on 200 kV Glacios microscope (Thermo Fisher). Cryo-EM tomography data were collected using a Titan Krios TEM (Thermo Fisher) operated at 300 kV and equipped with a K3 direct camera. Tomograms were collected using the TOMO5 software (Thermo Fisher) with a pixel size of 2.1 Å at 46,000 x. 21 tilt series ranging from -50 to +50 degrees with an increment of 5 degree and a fixed defocus of –3.5 μm were collect for each position in dose-symmetric tilt scheme. A dose of 1.5 ~ 2.0 e − /Å −2 per tilt was set, resulting in a total dose of 30~40 e − /Å −2 . Relion 5 38 was used for Motion correction, CTF correction, tilt series alignment (AreTomo3), tomogram reconstruction, and tomogram denoising (cryoCARE). Motility assay Swimming motility was studied using tryptone broth-based soft agar swim plates containing 0.3% Bacto agar, supplemented with 2% NaCl for V. alginolyticus . Motility plates were inoculated with 2 µL of overnight culture and incubated at 37 °C for 6-8 h. Images were acquired by scanning the plates and the diameters of the swimming halos were measured using Fiji 39 . The swimming diameters of the mutant strains were normalized to those of the respective WT. Outer membrane and peptidoglycan staining Overnight cultures were diluted 1:100 into 5 mL M9 minimal medium supplemented with 0.4% glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 and 2% NaCl. For each strain, 500 µL aliquots were transferred into a new tube, while the remaining 4.5 mL culture was used to monitor growth at 37 °C, 180 rpm. To perform dual labelling, HADA (Tocris BioScience, catalogue number: 6647/5) was added to the culture to a final concentration of 500 µM. Cells were incubated for 4-5 h at 37 °C in the dark with shaking at 650 rpm. Thirty minutes before harvesting, FM1-43 (Invitrogen, catalogue number: T3163) was added to a final concentration of 15 µg/mL. After staining, 1 mL of fresh M9 medium was added to dilute the stains. Cells were pelleted by centrifugation at 2500 × g for 5 min, and the supernatant was discarded. Pellets were washed twice: first resuspended in 1 mL of fresh medium, pelleted again at 2500 × g for 2 min, and then resuspended in 500 µL of the same medium. The samples were applied to home-made flow cells prepared with poly-L-lysine (PLL)-coated coverslips that were prepared as described previously 40 . Briefly, coverslips were incubated with 0.1% PLL for 10 min, then air-dried, and subsequently fixed to an objective slide via two layers of pre-heated parafilm to create a chamber. The side of the coverslip incubated with PLL faced the objective slide. Cells were allowed to adhere for 10 min in the dark in an inverted position. Non-adherent cells were gently washed off by rinsing twice with 40 µl of M9 medium. Mounting medium (Fluoroshield, Sigma Aldrich, catalogue number: F6182) was added before imaging. Fluorescence microscopy was performed using a Ti-2 Nikon inverted microscope equipped with a CFI Plan Apochromat DM 60× Lambda oil Ph3/1.40 (Nikon) oil objective, an Orca Fusion BT camera (Hamamatsu), and a SPECTRA III LED light source (Lumencor). Z-stack images were acquired every 0.4 µm across a 1.6 µm range (5 slices). FM1-43 was excited with a 488 nm laser at 20% power and 100 ms exposure. HADA was excited with a 365 nm laser at 20% power and 100 ms exposure. Emission was collected using the GFP emission filter (499–530 nm; FF01-515/30, Em2) and DAPI emission filter (414–450 nm; FF01-432/36, Em1) from the LED-DA/FI/TR/Cy5/ Cy7-A Full Multiband Penta filter (Semrock, IDEX). Images were analysed using Fiji 39 . Statistical analyses Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, Inc., San Diego, CA), and values of P < 0.05 were considered statistically significant. Molecular modeling A homology model of the V. alginolyticus FliD pentamer was built with SwissModel, with the S. enterica structure (PDB ID: 9GNZ) as a template. The structure of the V. alginolyticus FliD D3-D4 region was modeled using the AlpfaFold3 server 24 , and this model was added to each FliD chain, aligned on the D3 domain. For the full filament-cap model, the FliD pentamer model was aligned to the S. enterica FliD-filament structure (PDB ID: 9GNZ), and the V. alginolyticus filament structure (See above) was aligned to the S. enterica filament in the same structure. Declarations Data Availability The coordinates and/or EM maps that are displayed in this paper have been deposited in PDB and/or EMDB databases with the following accession code: sheathed filament, PDB: 9RCD, EMDB: EMD-53917; unsheathed filament, PDB: 9RCB, EMDB: EMD-53912; tomogram of multiple flagella tips in ΔflhG : EMD-53992; tomogram of multiple short flagella in ΔflhG ΔfliS : EMD-53993. The raw micrographs for the purified flagella dataset has been deposited to the EMPIAR database, with the accession code EMPIAR-47487467. Acknowledgements K.Q. is supported by a PhD studentship from the China Scholarship Council. M.E. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement n 864971) and from the Max Planck Society as Max Planck Fellow. J.R.C.B. acknowledges funding from the BBSRC (BB/R009759/2) and HFSP program (RGY0080/2021). We thank members of the Erhardt, Bergeron labs for helpful discussions. We thank Christian Goosmann (Max Planck Institute for Infection Biology) for TEM grid preparations and observation of Vibrio cells. Cryo-EM grids were screened at the Imperial College London cryo-EM facility (funded by BBSRC grant BB/V019732/1), and data was collected at the LonCEM facility; we acknowledge Paul Simpson, and Nora Cronin, respectively, for support. Author Contributions J.R.C.B., M.E. and KQ conceptualized the research project and J.R.C.B. and M.E. ensured funding. R.E. generated chromosomal V. cholerae and V. alginolyticus mutants, performed and analysed motility assays and fluorescent microscopy experiments on mutants with the help of C.K.. K.Q. prepared negative-staining grids and collected TEM images for V. cholerae. K.Q. isolated the flagella of V. alginolyticus, prepared the EM grids, collected and processed the EM data. K.Q. reconstructed and refined the EM map of the sheathed and unsheathed filament, and built and refined their atomic models. For tomography on Vibrio flagella, K.Q. collected the tomography data, and W.Z. processed the data, with support from J.A.. J.R.C.B., K.Q. and R.E. wrote the first draft of the manuscript, with comments from all authors. K.Q. and R.E. prepared figures and K.Q. prepared movies. M.E. and J.R.C.B. reviewed and edited the manuscript. All authors reviewed the results and approved the final version of the manuscript. Competing interests The authors declare no competing interests. References Berg, H. 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Demonstration of lipopolysaccharide on sheathed flagella of Vibrio cholerae O:1 by protein A-gold immunoelectron microscopy. J. Bacteriol. 170 , 1488-1494 (1988). Geis, G., Suerbaum, S., Forsthoff, B., Leying, H. & Opferkuch, W. Ultrastructure and Biochemical Studies of the Flagellar Sheath of Helicobacter pylori . J. Med. Microbiol. 38 , 371-377 (1993). Chu, J., Liu, J. & Hoover, T. R. Phylogenetic Distribution, Ultrastructure, and Function of Bacterial Flagellar Sheaths. Biomolecules. 10 , (2020). Aschtgen, M.-S. et al. Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development. J. Bacteriol. 198 , 2156-2165 (2016). Yoon, S. S. & Mekalanos, J. J. Decreased potency of the Vibrio cholerae sheathed flagellum to trigger host innate immunity. Infect. Immun. 76 , 1282-1288 (2008). Zhu, S., Nishikino, T., Kojima, S., Homma, M. & Liu, J. The Vibrio H-Ring Facilitates the Outer Membrane Penetration of the Polar Sheathed Flagellum. J. 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Bacteriol. 202 , 10.1128/jb.00592-19 (2020). Zhuang, X.-Y. et al. Live-cell fluorescence imaging reveals dynamic production and loss of bacterial flagella. Mol. Microbiol. 114 , 279-291 (2020). Guo, W. et al. Structures of the sheathed flagellum reveal mechanisms of assembly and rotation in Vibrio cholerae . Nat. Microbiol. (2025). Wu, E. L. et al. CHARMM-GUI Membrane Builder toward realistic biological membrane simulations. J. Comput. Chem. 35 , 1997-2004 (2014). Zhou, H.-X. & Pang, X. Electrostatic Interactions in Protein Structure, Folding, Binding, and Condensation. Chem. Rev. 118 , 1691-1741 (2018). Einenkel, R. et al. The structure of the complete extracellular bacterial flagellum reveals the mechanism of flagellin incorporation. Nat. Microbiol. (2025). Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 630 , 493-500 (2024). Renault, T. T. et al. Bacterial flagella grow through an injection-diffusion mechanism. eLife. 6 , (2017). Guo, W. et al. Near-atomic in-situ architecture and membrane-coupled dynamics of the Vibrio cholerae sheathed flagellum. Preprint at bioRxiv. 2025.12.02.691832 (2025). Fuerst, J. A. Bacterial sheathed flagella and the rotary motor model for the mechanism of bacterial motility. J. Theor. Biol. 84 , 761-774 (1980). Shearer, J., Marzinek, J. K., Bond, P. J. & Khalid, S. Molecular dynamics simulations of bacterial outer membrane lipid extraction: Adequate sampling? J. Chem. Phys. 153 , 044122 (2020). Hampton, C. M. et al. The Opportunistic Pathogen Vibrio vulnificus Produces Outer Membrane Vesicles in a Spatially Distinct Manner Related to Capsular Polysaccharide. Front. Microbiol. 8 , (2017). Pukatzki, S. et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl. Acad. Sci. U.S.A. 103 , 1528-1533 (2006). Sakazaki, R., Iwanami, S. & Fukumi, H. Studies on the Enteropathogenic, Facultatively Halophilic Bacterium, Vibrio parahaemolyticus : I. Morphological, Cultural, and Biochemical Properties and Its Taxonomical Position. Jpn. J. Med. Sci. Biol. 16 , 161-188 (1963). Edwards, R. A., Keller, L. H. & Schifferli, D. M. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene. 207 , 149-157 (1998). Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods. 14 , 290-296 (2017). Croll, T. I. ISOLDE : a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D. 74 , 519-530 (2018). Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27 , 14-25 (2018). Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix . Acta Crystallogr. D. 75 , 861-877 (2019). Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot . Acta Crystallogr. D. 66 , 486-501 (2010). Burt, A. et al. An image processing pipeline for electron cryo-tomography in RELION-5. FEBS Open Bio. 14 , 1788-1804 (2024). Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 9 , 676-682 (2012). Erhardt, M. Fluorescent Microscopy Techniques to Study Hook Length Control and Flagella Formation. Methods Mol. Biol. 1593 , 37-46 (2017). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable1X2QinEinenkel.docx Supplementary tables 1 & 2 SupplementaryTable3QinEinenkel.xlsx Supplementary table 3 SupplementaryFigures20251204.pdf Supplementary Figures 1-9 QinEinenkelSupplementaryVideo1.mp4 Supplementary movie 1 QinEinenkelSupplementaryVideo2.mp4 Supplementary movie 2 Cite Share Download PDF Status: Published Journal Publication published 23 Apr, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6983709","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":560675721,"identity":"a98a3af0-5250-444e-8659-d878198b2edd","order_by":0,"name":"Julien Bergeron","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYBACxgbmhgNAOgEmICfBwNwAZkng1MKIqsVYAiiGVwsDVAFcS+IMQlqYZyQ2Hvi4gyGPX/r4xccVFXfSZ7YfbGD4UcOQOLMBhx0zEhsOzjzDUCzZl1NseObMs9zZPIkNjD3HGBJn43IWUMth3jaGxA1neNIkG9sO586TADqMt4EhcR4+LX+BWvaf4Un/CdSSLgfUwviXkBZGkC087McYgVoSpIFamEG24HRYz8OGg71tEsUSZ3iYJRvOHDac2QM0ROaYhDEu7xu2Jx/+8LPNJo+/h/3hx4aKw/ISxw8ffPimxkZ2xgEcWiBGgeKAxwAuegBfRMojmOwPcKoaBaNgFIyCkQ0Amo5ia8osCv4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2841-9511","institution":"King's College London","correspondingAuthor":true,"prefix":"","firstName":"Julien","middleName":"","lastName":"Bergeron","suffix":""},{"id":560675722,"identity":"4da73f59-716f-4784-8cfc-1653ba2a7306","order_by":1,"name":"Kailin Qin","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Kailin","middleName":"","lastName":"Qin","suffix":""},{"id":560675723,"identity":"ca8f69e6-cd99-403a-ba9c-7bbcece1f3a1","order_by":2,"name":"Rosa Einenkel","email":"","orcid":"https://orcid.org/0000-0002-6987-6225","institution":"Humboldt-Universität zu Berlin","correspondingAuthor":false,"prefix":"","firstName":"Rosa","middleName":"","lastName":"Einenkel","suffix":""},{"id":560675724,"identity":"c249dc2d-9394-4684-91a9-7c83bb18fe5a","order_by":3,"name":"Weilong Zhao","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Weilong","middleName":"","lastName":"Zhao","suffix":""},{"id":560675725,"identity":"844ee1c0-fa5d-486b-8abc-73393c34108f","order_by":4,"name":"Joseph Atherton","email":"","orcid":"https://orcid.org/0000-0002-6362-2347","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Atherton","suffix":""},{"id":560675726,"identity":"7db15c0a-f11a-45b1-b511-0eb62f24477e","order_by":5,"name":"Marc Erhardt","email":"","orcid":"https://orcid.org/0000-0001-6292-619X","institution":"Humboldt-Universität zu Berlin","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"","lastName":"Erhardt","suffix":""},{"id":560675727,"identity":"144ac993-eb7b-40be-a2f0-33352711000c","order_by":6,"name":"Caroline Kühne","email":"","orcid":"","institution":"Humboldt University of Berlin","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Kühne","suffix":""}],"badges":[],"createdAt":"2025-06-26 13:01:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6983709/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6983709/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-71203-7","type":"published","date":"2026-04-23T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":98435774,"identity":"4e534a73-46e6-4846-8db5-9ce6bbdedc82","added_by":"auto","created_at":"2025-12-17 16:54:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":225182,"visible":true,"origin":"","legend":"\u003cp\u003eCryo-EM structure of the sheathed filament of \u003cem\u003eV. alginolyticus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003ea, Cryo-electron micrograph of purified \u003cem\u003eV. alginolyticus\u003c/em\u003e filaments, with intact sheaths indicated with a white arrow, naked filaments with a black arrow, and pearling sheaths with a yellow arrow. b, Selected 2D class averages, representing the different types of filaments indicated above. c, 3D reconstruction of sheathed filaments (left), with cross-sections shown on the right. The sheath outer-leaflet is in blue, its inner leaflet is in yellow, and the filament is in green. 11 proto-filaments are clearly visible. d, Schematic representation of the sheathed flagellum filament structure, colored as in c.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/8cca8f1a613ed6b9fb20c2e2.jpg"},{"id":98316009,"identity":"3c874476-ca49-480b-8b3c-20b70be972b2","added_by":"auto","created_at":"2025-12-16 13:17:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the flagellin homologues in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. alginolyticus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e filament structure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Structural alignment of the AlphaFold model for all 6 \u003cem\u003eV. alginolyticus\u003c/em\u003e flagellin proteins. The position of the inserts used for identification is shown with red arrows. \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, Close-up view of the cryo-EM map of the \u003cem\u003eV. alginolyticus\u003c/em\u003e filament cryo-EM map, in the positions corresponding to inserts 1, 2 and 3, demonstrating that no density is present for these inserts. \u003cstrong\u003ed\u003c/strong\u003e, Close-up view of the cryo-EM map of the \u003cem\u003eV. alginolyticus\u003c/em\u003e filament cryo-EM map, in the positions corresponding to residues Y300, H304, F315 and H317, demonstrating the presence of bulky side-chains in these positions. \u003cstrong\u003ee\u003c/strong\u003e, Motility of \u003cem\u003eV. alginolyticus\u003c/em\u003e strains with individual flagellin genes deleted, in the context of a strain with a defect in the lateral flagella system (Laf\u003csup\u003e-\u003c/sup\u003e).\u0026nbsp;Only the \u003cem\u003eflaD2\u003c/em\u003e deletion results in abolished motility, suggesting that it is the main flagellin in this bacterium. \u003cstrong\u003ef\u003c/strong\u003e, Representative swimming halos for the strains\u0026nbsp;shown in \u003cstrong\u003ee\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test (GraphPad Prism). *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ns, non-significant. Laf-: lateral flagella system defect, Pof+: polar flagella system intact.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/1119fd6b6d298ce61878e889.jpg"},{"id":98435589,"identity":"f4dfbd22-ce14-4706-a449-35ea3c120ccc","added_by":"auto","created_at":"2025-12-17 16:54:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":208188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural basis for the filament-membrane interface. a\u003c/strong\u003e, Atomic model of the \u003cem\u003eV. alginolyticus \u003c/em\u003eflagellum filament, fitted into the cryo-EM derived map. Elements are colored as in Figure 2. \u003cstrong\u003eb\u003c/strong\u003e, Close-up view of the corresponding map, with a model of the bacterial outer membrane built in. \u003cstrong\u003ec\u003c/strong\u003e, Electrostatic surface representation (in kT/e) of the \u003cem\u003eV. alginolyticus\u003c/em\u003e flagellum filament. The surface is highly electronegative. \u003cstrong\u003ed\u003c/strong\u003e, Close-up view of the interface between FlaD2 and the sheath.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/cf8d2e8597fbac2f542e7956.jpg"},{"id":98316011,"identity":"c2146eee-29fc-49a1-9d5b-6e5e1c506a8a","added_by":"auto","created_at":"2025-12-16 13:17:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":145740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupercoiling of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. alginolyticus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eflagellum filament.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, selected 2D class averages, representing sheathed filaments with a defined curvature. \u003cstrong\u003eb\u003c/strong\u003e, Atomic model of the \u003cem\u003eV. alginolyticus\u003c/em\u003e unsheathed flagellum filament, fitted into the cryo-EM derived map showing convex and concave sides. \u003cstrong\u003ec\u003c/strong\u003e, Comparison of individual flagellin protofilaments revealing that the protofilament from the concave side is significantly more curved. \u003cstrong\u003ed\u003c/strong\u003e, Close-up view of the longitudinal interfaces of protofilaments from the concave and convex sides.​\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/1476e259ff86711ab009beda.jpg"},{"id":98316013,"identity":"16be1a6e-3a0f-407f-9a5f-4658c98c76c2","added_by":"auto","created_at":"2025-12-16 13:17:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":125424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA domain in the cap protein FliD anchors the flagellum filament to the sheath. a\u003c/strong\u003e, Atomic model the \u003cem\u003eV. alginolyticus\u003c/em\u003e filament-cap complex. \u003cstrong\u003eb\u003c/strong\u003e, Negative-stain TEM micrographs of \u003cem\u003eV. cholerae\u003c/em\u003e cells (i) and filament tips (ii), (iii) for the \u003cem\u003efliD\u003c/em\u003e\u003csub\u003eΔD4\u003c/sub\u003e mutant. The sheath expands past the filament tip, demonstrating a lack of coordination between filament and sheath assembly in this mutant. Arrows indicate the empty sheath (white) and the sheathed filament (black). \u003cstrong\u003ec\u003c/strong\u003e, Cryo-electron tomogram of filament tips in the hyper-flagellated strain (Δ\u003cem\u003eflhG\u003c/em\u003e) of \u003cem\u003eV. alginolyticus\u003c/em\u003e. Tips are indicated with a white box, with selected close-up view shown on the right. Arrows indicate the membrane outer later (blue), inner layer (yellow), filament (green) and cap (orange). Scale bar: 50 nm. \u003cstrong\u003ed\u003c/strong\u003e, Schematic representation of sheath tip architecture. The atomic model of the filament-cap complex was positioned at the correct location in the sub-tomogram, with the membrane leaflets segmented in blue (outer leaflet) and yellow (inner leaflet). The FliD D4 domain has a similar diameter like the inner leaflet.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/6530949fd76df6aeb00573d4.jpg"},{"id":98316015,"identity":"410b8e0e-be96-4a32-82dc-100dd6a0bf51","added_by":"auto","created_at":"2025-12-16 13:17:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the proposed sheathed filament structure, and molecular mechanism for its rotation. a, \u003c/strong\u003eSchematic representation of the sheathed flagellum of \u003cem\u003eV. alginolyticus\u003c/em\u003e showing the proposed interactions of FliD D4 domain with the inner leaflet of the sheath. \u003cstrong\u003eb\u003c/strong\u003e, Proposed molecular mechanism of filament rotation within the membranous sheath. Charge repulsion between the filament and the sheath facilitates the rapid rotation of the filament within the sheath.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/ed09aa4cddfa12a1dcfb12eb.jpg"},{"id":107696536,"identity":"27c700d4-95fc-4149-bca6-973772b7de1c","added_by":"auto","created_at":"2026-04-24 07:11:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1345014,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/02bfd2a3-1cd4-4560-9d8f-5aebe6ab891a.pdf"},{"id":98316017,"identity":"2e8a2a3e-79c1-4b9a-9f4c-7fa52d6aed55","added_by":"auto","created_at":"2025-12-16 13:17:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3845196,"visible":true,"origin":"","legend":"Supplementary tables 1 \u0026 2","description":"","filename":"SupplementaryTable1X2QinEinenkel.docx","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/d153899ad64d3b52d382ad36.docx"},{"id":98436800,"identity":"b049bd27-db35-46df-b554-ae3dab8bc72c","added_by":"auto","created_at":"2025-12-17 16:56:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":569469,"visible":true,"origin":"","legend":"Supplementary table 3","description":"","filename":"SupplementaryTable3QinEinenkel.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/8897fa3f87fb374e57e070bf.xlsx"},{"id":98316016,"identity":"1177500c-6a45-43c5-bbba-dbec8180c684","added_by":"auto","created_at":"2025-12-16 13:17:02","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1942364,"visible":true,"origin":"","legend":"Supplementary Figures 1-9","description":"","filename":"SupplementaryFigures20251204.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/621775a6c3fb087ed17c0599.pdf"},{"id":98316020,"identity":"2974dc31-c75c-4c96-89b3-0807573ae8f8","added_by":"auto","created_at":"2025-12-16 13:17:02","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":37725730,"visible":true,"origin":"","legend":"Supplementary movie 1","description":"","filename":"QinEinenkelSupplementaryVideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/a53636f8e1cf56ccfa275aad.mp4"},{"id":98316019,"identity":"e15b4a88-4f20-463b-b4ec-3680c41ea686","added_by":"auto","created_at":"2025-12-16 13:17:02","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15886493,"visible":true,"origin":"","legend":"Supplementary movie 2","description":"","filename":"QinEinenkelSupplementaryVideo2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6983709/v1/360e0c8e69238929ee4a8cc6.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The structure of the Vibrio alginolyticus flagellar filament suggests molecular mechanism for the rotation of sheathed flagella","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMany bacteria swim through liquid environments using the flagellum. By rotating like a propeller, the flagellum drives bacterial motility and directional movement in many bacterial species\u003csup\u003e1\u003c/sup\u003e. In human gastrointestinal pathogens, such as \u003cem\u003eVibrio cholerae\u0026nbsp;\u003c/em\u003e(\u003cem\u003eV. cholerae\u003c/em\u003e)\u003cem\u003e, Salmonella enterica\u0026nbsp;\u003c/em\u003e(\u003cem\u003eS. enterica\u003c/em\u003e)\u003cem\u003e, Campylobacter jejuni\u0026nbsp;\u003c/em\u003e(\u003cem\u003eC. jejuni\u003c/em\u003e), the flagellum also plays an important role in invasion and colonization to human tissue\u003csup\u003e2\u003c/sup\u003e. The flagellum consists of a membrane-spanning basal body that houses the export apparatus and motor proteins, and an extracellular appendage formed by the hook, the hook-filament junction, the filament, and a cap at the distal end \u003csup\u003e3,4\u003c/sup\u003e. The filament, typically ~10 \u0026micro;m long in most bacteria, is composed of tens of thousands of flagellin subunits \u0026ndash; either a single flagellin protein or multiple flagellin variants \u0026ndash; which are secreted through the flagellum-specific type-III secretion system\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn recent years, cryo-EM structures of flagellum filaments have been reported, in many bacterial species. They demonstrate a common architecture, consisting of a 11-stranded filament of the flagellin protein, with slight conformational changes to each subunit allowing the formation of a long-range supercoil. The flagellin consists of two conserved internal domains, D0 and D1, that form the secretion lumen; and several external domains, D2-D5, of which the number and arrangement vary between bacterial species and strains. At the tip of the filament, the cap complex, consisting of five copies of the protein FliD, is responsible for flagellin folding and insertion in the growing filament.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn several bacteria, including the human pathogens \u003cem\u003eVibrio\u003c/em\u003e, \u003cem\u003eHelicobacter\u003c/em\u003e, and \u003cem\u003eBdellovibrio\u003c/em\u003e, the flagellar filament is surrounded by a membranous sheath, thought to be an extension of the outer membrane\u003csup\u003e6\u003c/sup\u003e. Accordingly, the filament sheath is presumed to be an asymmetric bilayer, containing primarily glycerophospholipids (GPLs) in its inner-leaflet, and lipopolysaccharides (LPS) in its outer-leaflet\u003csup\u003e7,8\u003c/sup\u003e, with lipoproteins embedded in.\u003c/p\u003e\n\u003cp\u003eFlagellar sheaths have been proposed to serve multiple, and possibly species-specific, functions\u003csup\u003e9\u003c/sup\u003e. In \u003cem\u003eHelicobacter pylori\u003c/em\u003e, the sheath has been suggested to protect flagellin from acidic conditions and to contribute to adherence, while in \u003cem\u003eVibrio\u003c/em\u003e species, sheaths reduce recognition of flagellin by the host innate immune receptor TLR5 and play a role in outer membrane vesicle (OMV) release\u003csup\u003e10,11\u003c/sup\u003e. Rotation of the sheathed filament has been linked to the production of OMVs, which can mediate host signaling, deliver virulence factors, and provide protection against antimicrobial peptides or bacteriophages. More generally, the sheath has also been hypothesized to shield flagella from flagellotropic phages, which exploit unsheathed filaments for infection\u003csup\u003e9\u003c/sup\u003e. Despite these proposed roles, direct mechanistic insight into the rotation of sheathed flagella has remained limited, with two theoretical models proposed: One where the sheath membrane rotates together with the flagellum filament, and one where only the filament rotates, within a flexible sheath.\u003c/p\u003e\n\u003cp\u003eLow-resolution cryo-ET analyses of sheathed flagella basal body complexes have revealed that these flagella possess additional periplasmic structures,\u0026nbsp;called H-ring and T-ring,\u0026nbsp;thought to promote sheath formation. Deletion of the\u0026nbsp;entire ring structures leads to the formation of periplasmic flagella\u0026nbsp;beneath the PG layer\u003csup\u003e12\u003c/sup\u003e. Some periplasmic filaments penetrate the cell envelop at the region far from the basal body, with and without the sheath. These observations\u0026nbsp;suggest\u0026nbsp;the HT-ring and filament alone plays a role in outer membrane remodeling during sheath biogenesis. Moreover, the identification of an extracellular O-ring at the base of the \u003cem\u003eV. alginolyticus\u003c/em\u003e sheath suggests a further architectural element, although this feature is not conserved across all sheathed species\u003csup\u003e6\u003c/sup\u003e. Despite these observations, the molecular mechanisms underlying sheath assembly remain largely unknown.\u003c/p\u003e\n\u003cp\u003eHere, we combine single-particle cryo-EM, cryo-ET, and genetic approaches to investigate the structure and assembly of sheathed flagella in \u003cem\u003eVibrio\u003c/em\u003e. We determine high-resolution structures of both sheathed (3.6 \u0026Aring;) and unsheathed (3.2 \u0026Aring;) filaments and identify FlaD2 as the major polar flagellin of \u003cem\u003eV. alginolyticus\u003c/em\u003e. Electrostatic analyses reveal strong repulsion between the filament and sheath, consistent with a mechanism that reduces friction during rapid rotation. Furthermore, sequence and functional analyses reveal that the FliD protein possesses an additional domain, which may anchor the flagellum tip to the sheath end. Together, these findings provide structural and mechanistic insights into the adaptations that underlie the rotation mechanism of sheathed flagella.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSingle-particle analysis of the \u003cem\u003eV. alginolyticus\u003c/em\u003e sheathed flagellum filament\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has not been shown whether the filament formed direct interactions with the membrane, in sheathed flagella. To address this, we isolated the sheathed filament from \u003cem\u003eVibrio alginolyticus\u0026nbsp;\u003c/em\u003e(\u003cem\u003eV. alginolyticus\u003c/em\u003e), a pathogen affecting a variety of marine animals and humans, causing otitis and wound infection\u003csup\u003e13,14\u003c/sup\u003e. \u003cem\u003eV. alginolyticus\u003c/em\u003e encodes a single, polar, sheathed flagellum that drives swimming in liquid environments. In addition, it produces peritrichous, non-sheathed lateral flagella that enable swarming and surface-associated motility under various environmental conditions\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo characterize the structure of the \u003cem\u003eV. alginolyticus\u003c/em\u003e sheathed flagellum, we engineered a mutant of this bacterium lacking the \u003cem\u003eflhG\u003c/em\u003e gene, shown previously to lead to multi-flagellated bacteria\u003csup\u003e16\u003c/sup\u003e. We subsequently isolated flagellar filaments from this mutant strain using mechanical shearing, achieved by repeatedly forcing a cell suspension through a narrow-gauge needle. Cryo-EM analysis of the resulting sample confirmed that we successfully isolated the flagellar filament with an intact sheath (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). We also obtained sheath-free filaments, as well as filaments exhibiting membrane pearling, suggesting partial detachment of the sheath (\u003cstrong\u003eFig. 1b\u003c/strong\u003e). Using this, we were able to obtain a map of the sheathed filament to 3.6 Å resolution by single-particle analysis (\u003cstrong\u003eFig. 1c, 1d, Supplementary Fig. 1, Table S1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThis map confirms that the filament adopts a 11-stranded supercoiled architecture, similar to that of other bacteria. The membrane is well-resolved, with diffused density for the GPL inner leaflet and the LPS outer leaflet clearly identifiable (\u003cstrong\u003eFig. 1c, Supplementary Movie 1\u003c/strong\u003e). Fluorescence microscopy using an outer membrane dye demonstrated that in both \u003cem\u003eV. alginolyticus\u003c/em\u003e and \u003cem\u003eV. cholerae\u003c/em\u003e, the filament sheath is composed of \u0026nbsp;outer membrane material. This observation is consistent with previous reports\u003csup\u003e6,17\u003c/sup\u003e\u003csup\u003e‑19\u003c/sup\u003e. In contrast, staining with HADA, a fluorescent D-amino acid that labels peptidoglycan, revealed no signal within the sheath structure. This indicates that the filament sheath is an extension of the outer membrane but lacks an underlying peptidoglycan layer, distinguishing it from the envelope of the cell body (\u003cstrong\u003eSupplementary Fig. 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlaD2 is the main flagellin in \u003cem\u003eV. alginolyticus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e encodes six flagellin homologues in its genome (\u003cstrong\u003eFig. 2a\u003c/strong\u003e, \u003cstrong\u003eSupplementary Fig. 3\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and Table S2\u003c/strong\u003e), FlaB and FlaD1-FlaD5, ~44-99% identical in sequence (FlaD2/FlaD4 are 99% identical). Because of this, it was not immediately clear whether the map of the sheathed filament consisted of a mixture of the above flagellins, or was formed by predominantly one (or a subset of) these flagellins (\u003cstrong\u003eFig. 2a\u003c/strong\u003e). We noted nonetheless that FlaD5 and FlaD1 possess 2-6 residue insertions in their D2 domain, respectively, not present in the other flagellin homologues (\u003cstrong\u003eSupplementary Fig. 3\u003c/strong\u003e). In our cryo-EM map of the sheathed filament, there is no density for either of these loops, suggesting that they are not the main flagellins in our structure (\u003cstrong\u003eFig. 2b, c\u003c/strong\u003e). Similarly, in our cryo-EM map, we noted the presence of large side-chain density in positions corresponding to residues Y300, H304, F315, H317 in FlaD2 and FlaD4 (\u003cstrong\u003eFig. 2d\u003c/strong\u003e). In contrast, FlaB, FlaD5, FlaD1, and FlaD3, possess small amino-acids in these positions (\u003cstrong\u003eSupplementary Fig. 3\u003c/strong\u003e). Collectively, this suggests that FlaD2/FlaD4 are the main flagellins in the filament map we have obtained.\u003c/p\u003e\n\u003cp\u003eTo verify this, we deleted the genes of all flagellin genes individually, and assessed the motility of the corresponding strains. Because \u003cem\u003eV. alginolyticus\u003c/em\u003e encodes a lateral flagellum system capable of compensating for defects in polar flagellum-dependent motility\u003csup\u003e15\u003c/sup\u003e, all experiments were performed in a ∆\u003cem\u003elafK\u003c/em\u003e background (Laf\u003csup\u003e-\u003c/sup\u003e) lacking the lateral flagellar master regulator. Furthermore, a lateral and polar flagellar master regulator mutant was used as a non-motile control strain (∆\u003cem\u003elafK\u003c/em\u003e ∆\u003cem\u003eflrA\u003c/em\u003e). As shown in \u003cstrong\u003eFig. 2e, f,\u0026nbsp;\u003c/strong\u003edeletion of the \u003cem\u003eflaD2\u003c/em\u003e gene abolished bacterial motility similar to the non-motile control strain, whereas deletion of all the other flagellin homologues leads to only a mild (FlaD1) or no significant reduction of motility (FlaD3, FlaD4, FlaD5, FlaB). This confirms that FlaD2 is necessary to form a functional polar flagellum in \u003cem\u003eV. alginolyticus\u003c/em\u003e. These observations are similar to \u003cem\u003eV. cholerae\u003c/em\u003e, which also possesses multiple flagellin genes, but a single one (\u003cem\u003eflaA\u003c/em\u003e) is absolutely necessary for motility in this bacterium (\u003cstrong\u003eSupplementary Fig. 4a\u003c/strong\u003e). Whether the various flagellins of \u003cem\u003eV. alginolyticus\u003c/em\u003e are localized to different zones of the filament, similar to \u003cem\u003eV. cholerae\u003c/em\u003e, remains to be verified\u003csup\u003e20\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, both the motility data and the cryo-EM map reported above suggest that FlaD2 is the main flagellin in the \u003cem\u003eV. alginolyticus\u003c/em\u003e flagellar filament.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural basis for the filament-membrane interface\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the aforementioned results, we then built an atomic model of the \u003cem\u003eV. alginolyticus\u003c/em\u003e filament, using the FlaD2 sequence, in the cryo-EM map of the sheathed filament (\u003cstrong\u003eFig. 3a\u003c/strong\u003e). To further characterize the interaction between the filament and the membrane, we also built an atomic model of the outer membrane, using CHARMM-GUI\u003csup\u003e21\u003c/sup\u003e, which could be fitted in the corresponding density (\u003cstrong\u003eFig. 3b\u003c/strong\u003e). In this model, the flagellin outward-most D2 domain faces the membrane inner leaflet, with several residues in proximity (~7-10 Å) to the density attributed to the GPL phosphate groups (\u003cstrong\u003eFig. 3c\u003c/strong\u003e). Intriguingly, these are largely negatively-charged amino-acids, and the intact filament possesses a highly electronegative surface (\u003cstrong\u003eFig. 3d\u003c/strong\u003e). As GPL headgroups are mainly negatively charged, this raises questions as to how the contacts between the sheath and the filament are maintained. At this distance, electrostatic forces still exert a large influence\u003csup\u003e22\u003c/sup\u003e, which suggests that there is repulsion force between the sheath and the filament. We postulate that this facilitates the rotation of the filament within the sheath, without any friction that would perturb rotation velocity. In support of this, we note that in most bacteria, the surface of the flagellum filament is largely charge-neutral (\u003cstrong\u003eSupplementary Fig. 5\u003c/strong\u003e), supporting the notion that this charge of the \u003cem\u003eV. alginolyticus\u003c/em\u003e filament is a species-specific adaptation permitting to facilitate rotation within a membranous sheath.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupercoiling of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e flagellum filament\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies showed that the flagellum filament is supercoiled, with a range of supercoiling architectures observed, all corresponding to subtly different arrangements of the flagellin interactions in the 11 strands of the filament. Supercoiling had previously been reported also in sheathed flagella\u003csup\u003e20\u003c/sup\u003e, and indeed, 2D classes of filament particles with a larger box size (\u003cstrong\u003eFig. 4a\u003c/strong\u003e) confirmed that the \u003cem\u003eV. alginolyticus\u003c/em\u003e sheathed flagellum filament possesses a defined curvature.\u003c/p\u003e\n\u003cp\u003eNonetheless, due to the relatively low resolution of the structure of the corresponding filament reported above (\u003cstrong\u003eFig. 3a\u003c/strong\u003e, \u003cstrong\u003eSupplementary Fig. 1\u003c/strong\u003e), caused by the limited number of particles, we were not able to resolve the differences between flagellin subunits leading to this curvature. To address this, we determined the structure of the unsheathed filament, from the corresponding particles present in the aforementioned dataset, to 3.16 Å resolution (\u003cstrong\u003eSupplementary Fig. 1, Supplementary Fig. 6a\u003c/strong\u003e). In this structure, we were able to build 55 copies of the FlaD2 flagellin.\u003c/p\u003e\n\u003cp\u003eBoth structures (sheathed and unsheathed) are highly similar, and notably we observed no significant structural changes in the D2 domain (\u003cstrong\u003eSupplementary Fig. 6b\u003c/strong\u003e). This also confirmed that this structure corresponds primarily of FlaD2 filaments lacking the sheath, not of filaments formed by other polar flagellins, nor that of the lateral flagellum, for which the flagellin LafA shares only ~52% identity with FlaD2. Thus, the isolated unsheathed filaments likely originate from the polar flagellum, from unsheathed filaments in \u003cem\u003eV. alginolyticus\u003c/em\u003e\u003csup\u003e6\u003c/sup\u003e or from filaments that lost their sheath during isolation.\u003c/p\u003e\n\u003cp\u003eIn the resulting structure, obtained without helical symmetry imposed, we can clearly distinguish convex and concave sides on the filament (\u003cstrong\u003eFig. 4b\u003c/strong\u003e), in agreement with the 2D classes described above. Comparison of individual flagellin protofilaments revealed that each possess a distinct curvature, with the protofilament from the concave side significantly more curved (\u003cstrong\u003eFig. 4c\u003c/strong\u003e). Close-up view of the longitudinal interfaces of protofilaments from the concave and convex sides reveal subtle differences in the interaction, (\u003cstrong\u003eFig. 4d\u003c/strong\u003e), which when propagated across many subunits explain the difference in curvature.\u003c/p\u003e\n\u003cp\u003eThese observations confirm that the supercoiled nature of the flagellum filament is also conserved in sheathed flagella.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe cap protein FliD possesses a unique domain, likely anchored to the sheath\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe flagellum cap protein FliD is required for filament formation in most bacteria, as it promotes flagellin folding during filament assembly. Our previous structural studies had revealed that FliD forms a pentameric structure at the tip of the filament, with domains D0-D1 buried in the filament structure, and D2-D3 forming the pentameric contacts at the filament tip\u003csup\u003e3,23\u003c/sup\u003e. Intriguingly, analysis of FliD sequences revealed that in both \u003cem\u003eVibrio\u003c/em\u003e spp and \u003cem\u003eHelicobacter\u003c/em\u003e spp, FliD possesses a distinct architecture, with predicted unique domains (termed D4 in \u003cem\u003eVibrio\u003c/em\u003e, and D4-D5 in \u003cem\u003eHelicobacter\u003c/em\u003e) not found in other FliD orthologues (\u003cstrong\u003eSupplementary Fig. 7a)\u003c/strong\u003e. Modeling of the \u003cem\u003eV. alginolyticus\u003c/em\u003e D4 domain with AlphaFold3\u003csup\u003e24\u003c/sup\u003e revealed that it consists of two helical domains, located on top of the cap complex (\u003cstrong\u003eSupplementary Fig. 7b\u003c/strong\u003e). This allowed us to build a hybrid model of the \u003cem\u003eV. alginolyticus\u003c/em\u003e filament-cap complex (\u003cstrong\u003eFig. 5a, Supplementary Fig. 7c\u003c/strong\u003e), by combining this model to our previously-published structure of the \u003cem\u003eS. enterica\u003c/em\u003e cap-filament complex, and the structure of the sheathed filament reported above. Notably, the diameter of the resulting cap complex is ~16.8 nm, larger than that of the filament (\u003cstrong\u003eSupplementary Fig. 7c\u003c/strong\u003e), which is similar to the width of the sheath inner leaflet. This suggests that the edge of the FliD D4 domain could be directly interacting with the sheath itself (\u003cstrong\u003eSupplementary Fig. 7d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo verify this, we constructed mutant strains lacking the D4 domain in FliD (\u003cem\u003efliD\u003c/em\u003e\u003csub\u003e∆D4\u003c/sub\u003e). As shown on \u003cstrong\u003eFig. 5b,\u003c/strong\u003e negative-stain TEM analysis of this mutant in \u003cem\u003eV. cholerae\u003c/em\u003e revealed the presence of empty sheath extensions at the tip of the filament, not observed in WT bacteria. This observation suggests a role on the D4 domain of FliD in synchronizing filament assembly with sheath elongation. We note, however, that \u003cem\u003efliD\u003c/em\u003e\u003csub\u003e∆D4\u003c/sub\u003e strains retained swimming motility in soft-agar, in both \u003cem\u003eV. alginolyticus\u003c/em\u003e and \u003cem\u003eV. cholerae\u003c/em\u003e, in contrast to the respective ∆\u003cem\u003efliD\u003c/em\u003e deletion mutants (\u003cstrong\u003eSupplementary Fig. 8\u003c/strong\u003e), indicating that the D4 domain of FliD is not essential for filament assembly.\u003c/p\u003e\n\u003cp\u003eTo further investigate the architecture of the \u003cem\u003eV. alginolyticus\u003c/em\u003e cap complex in-situ, we employed cryo-electron tomography (cryo-ET) to visualize filament tips in the \u003cem\u003eV. alginolyticus\u003c/em\u003e ∆\u003cem\u003eflhG\u0026nbsp;\u003c/em\u003emutant described above.\u0026nbsp;As shown in \u003cstrong\u003eFig. 5c\u003c/strong\u003e and \u003cstrong\u003eSupplementary Movie 2\u003c/strong\u003e, the distal tips of the flagellar filaments are clearly resolved in the tomograms and the surrounding sheath appears continuous and sealed, with no detectable gap or bulge at the filament tip. Structural docking of the filament–cap complex model into these tomograms supports the presence of an intact cap structure beneath the closed sheath (\u003cstrong\u003eFig. 4d\u003c/strong\u003e, \u003cstrong\u003eSupplementary Movie 2\u003c/strong\u003e). Collectively, these results indicate that while the FliD D4 domain is not essential for sheath formation or maintenance, it may contribute to the coordination between sheath elongation and filament growth. The molecular basis of this contribution remains to be clarified.\u003c/p\u003e\n\u003cp\u003eFinally, we sought to characterize the architecture of the cap complex in the early stages of flagellum assembly. To this end, we engineered a \u003cem\u003eV. alginolyticus\u003c/em\u003e \u003cem\u003e∆flhG\u003c/em\u003e \u003cem\u003e∆fliS\u003c/em\u003e mutant, in which secretion of flagellin and therefore motility is strongly reduced (\u003cstrong\u003eSupplementary Fig. 8a\u003c/strong\u003e)\u003csup\u003e25\u003c/sup\u003e, and used cryo-ET to gain structural insights into their flagellum architecture. As expected, flagella are significantly shorter in this mutant, yet the sheath is present and still closed at the tip (\u003cstrong\u003eSupplementary Fig. 9a\u003c/strong\u003e). Intriguingly, we observed a range of flagellar architectures, including ones where the cap complex is near the tip of the sheath (i), and ones where the filament is significantly shorter, with the cap complex visible below the tip of the sheath (ii). We also observed empty sheaths without a filament (iii). These results suggests that synchronization between filament assembly and sheath elongation occurs later during flagellum biogenesis, although we cannot rule out that this is an artifact caused by the \u003cem\u003e∆flhG\u003c/em\u003e \u003cem\u003e∆fliS\u0026nbsp;\u003c/em\u003emutation, which significantly disrupts flagellum assembly and regulation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we present the structure of the sheathed flagellum filament in \u003cem\u003eV. alginolyticus\u003c/em\u003e, providing\u0026nbsp;insights into the interface between the filament and the membranous sheath.\u0026nbsp;Based on sequences of flagellin homologs and EM densities of filament, we demonstrate that FlaD2 is the main flagellin in this bacterium.\u0026nbsp;Furthermore, electrostatic analyses indicate\u0026nbsp;the filament surface is uniformly negatively charged, presumably\u0026nbsp;strongly\u0026nbsp;repulsing\u0026nbsp;between the filament surface and the sheath, which may help reduce friction and thereby facilitate rapid rotation of the filament within the sheath. Finally, we report that the cap complex possesses a unique architecture in sheathed flagella, which is not required for filament assembly but may contribute to synchronizing it to sheath elongation.\u003c/p\u003e\n\u003cp\u003eBased on these results, we propose a model whereby in sheathed flagella, there is no direct molecular interaction between the sheath and the filament during flagella rotation. Nonetheless, our data suggests that the cap complex is forming direct contact with the sheath inner layer during flagella assembly to synchronize filament growth with sheath elongation (\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eig. 6a\u003c/strong\u003e). A recent study examining the architecture of the sheathed flagellar motor of \u003cem\u003eV. cholerae\u003c/em\u003e likewise proposes that sheath biogenesis is coordinated with ongoing flagellar assembly, specifically the coordination between HL-ring formation and hook elongation\u003csup\u003e26\u003c/sup\u003e. This complementary perspective, though focused on distinct regions of the flagellum, supports the broader view that sheath growth and flagellar assembly are mechanistically linked.\u003c/p\u003e\n\u003cp\u003eIn this model, we propose that charge repulsion “greases” the interface between sheath and filament, ensuring that the filament rotates within the sheath without friction (\u003cstrong\u003eFig. 6b\u003c/strong\u003e). Together with the previously reported continuity with the outer membrane, and the lack of direct contacts between the filament and sheath, this observation provides molecular support for earlier models proposing that the filament rotates freely within a flexible sheath, rather than as a rigid filament-sheath complex\u003csup\u003e20,27\u003c/sup\u003e. Such a mechanism would allow the sheath to deform in response to filament motion while maintaining structural integrity.\u003c/p\u003e\n\u003cp\u003eIt remains to be determined how the sheath maintains its tubular structure, in the context of it surrounding a filament with repulsive electrostatic interactions. It has been shown that the outer leaflet of the bacterial OM is relatively rigid\u003csup\u003e28\u003c/sup\u003e, and it plausible that the LPS structure alone is sufficient to maintain a tubular sheath. Nonetheless, further molecular and computational studies will be required to confirm this hypothesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe also note that rotation of sheathed flagella in \u003cem\u003eVibrio\u003c/em\u003e has been shown to promote the release of outer membrane vesicles (OMVs), which contribute to immune modulation, host colonization, and delivery of virulence factors\u003csup\u003e10,29\u003c/sup\u003e. Electrostatic repulsion could facilitate local membrane curvature and blebbing, consistent with the bubbling we observed in our cryo-EM datasets (\u003cstrong\u003eFig. 1a,b\u003c/strong\u003e). Although speculative, these observations suggest a potential link between sheath architecture, filament-sheath interactions, and OMV biogenesis that warrants future investigation.\u003c/p\u003e\n\u003cp\u003eTaken together, our structural and genetic analyses provide a framework for understanding how sheath architecture is supported in \u003cem\u003eVibrio\u003c/em\u003e and highlight the unique adaptations of sheathed flagellar filaments compared to unsheathed systems. These findings establish a basis for future work to dissect the molecular mechanisms of sheath formation and its impact on bacterial motility and virulence.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial strains and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egrowth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll \u003cem\u003eVibrio alginolyticus\u003c/em\u003e, \u003cem\u003eVibrio cholerae\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) strains used in this study are listed in \u003cstrong\u003eTable S3\u003c/strong\u003e. The strains were derived from \u003cem\u003eV. alginolyticus\u003c/em\u003e NCTC 10675 or \u003cem\u003eV. cholerae\u003c/em\u003e V52 Δ\u003cem\u003evasK\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e. The wild-type \u003cem\u003eV. alginolyticus\u003c/em\u003e NCTC 10675 was obtained from the National Collection (UK Health Security Agency, UK) and originally isolated by Miyamoto et al.\u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBacteria were cultured in lysogeny broth (LB) at 37 °C with constant shaking at 180 rpm. For \u003cem\u003eV. alginolyticus\u003c/em\u003e, LB was supplemented with 2% NaCl (LBS). Antibiotics were added as required at the following final concentrations: chloramphenicol (6.25 µg/ml for \u003cem\u003eV. alginolyticus\u003c/em\u003e, 12.5 µg/ml for \u003cem\u003eV. cholerae\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e), ampicillin (50\u0026nbsp;µg/ml), and streptomycin (100\u0026nbsp;µg/ml). Bacterial growth was monitored by measuring optical density at 600\u0026nbsp;nm (OD\u003csub\u003e600\u003c/sub\u003e) using a spectrophotometer (Amersham Bioscience).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic Manipulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromosomal mutations were introduced into the genomes of \u003cem\u003eV. alginolyticus\u003c/em\u003e and \u003cem\u003eV. cholerae\u003c/em\u003e using the suicide plasmid pRE112, which carries the \u003cem\u003esacB\u003c/em\u003e gene for sucrose-based counterselection\u003csup\u003e32\u003c/sup\u003e. Approximately 600 bp of DNA up- and downstream of the target gene were amplified and assembled into pRE112 using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, catalogue number: E2621L), then transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α λ-pir⁺ or \u003cem\u003eE. coli\u003c/em\u003e CC118 λ-pir⁺ and after confirmation of correct assembly into \u003cem\u003eE. coli\u003c/em\u003e SM10 λ-pir⁺. Conjugation was performed between donor \u003cem\u003eE. coli\u003c/em\u003e SM10 λ-pir⁺ and recipient \u003cem\u003eVibrio\u003c/em\u003e strains. Recombinants resulting from single crossover events were selected on LB/LBS plates containing chloramphenicol, supplemented with ampicillin (\u003cem\u003eV. alginolyticus\u003c/em\u003e) or streptomycin (\u003cem\u003eV. cholerae\u003c/em\u003e). To select for the second crossover and excision of the plasmid backbone, cultures were grown for 4\u0026nbsp;h in LB/LBS, and plated on no-salt LB agar supplemented with 10% sucrose and streptomycin (\u003cem\u003eV. cholerae\u003c/em\u003e) or on tryptic soy agar (TSA) supplemented with 10% sucrose and ampicillin (\u003cem\u003eV. alginolyticus\u003c/em\u003e). Colonies were screened for loss of chloramphenicol resistance to confirm plasmid excision, and the desired chromosomal modifications were verified by PCR and Sanger sequencing. Lists of plasmids and oligonucleotides used in strain construction are provided in \u003cstrong\u003eTable S4\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eTable S5\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of flagellar filaments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA single colony of wild-type \u003cem\u003eV. alginolyticus\u003c/em\u003e was inoculated into 5 ml LB medium and cultured overnight at 30 °C. The next day, 250 mL LB medium was inoculated 1:100 with the overnight culture and grown to OD 0.5-0.8. Cells were harvested at 4,000 × g at 4 °C for 10 min. The cell pellet was resuspended in 40 mL ice-cold sucrose buffer (20mM Tris-HCl, 20% w/v sucrose, pH 8.0) on ice. Flagellar filaments were sheared off by passing the cell-suspension through a 23ga, 0.7 x 50 mm needle using a 20 mL syringe 10 times. To separate the cells from the sheared filaments, the cell suspension was centrifuged at 20,000 × g at 4 °C for 30 min, and the supernatant containing the sheared filaments was collected and centrifuged at 100,000 × g 4 °C for 1h. The filaments in the pellet were resuspended in 200 µL ice-cold sucrose solution (20mM Tris-HCl, 20% w/v sucrose, pH 8.0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-particle cryo-EM sample preparation, data acquisition, and processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFilament resuspension (3 μL) was applied to glow-discharged holey carbon grids (Quantifoil R2/2, 300 mesh). Samples were incubated for 30 s at 4 °C and 88% humidity before being blotted by Leica EM GP1 and then rapidly plunged into liquid ethane. Grids that were blotted for 5, 6, 7, 8 s were screened on 200 kV Glacios microscope (Thermo Fisher). The grids with good ice thickness were deposited to 300 kV Krios G3i microscope with a Gatan K3 direct electron detector (Thermo Fisher). Dataset was collected using a physical pixel size of 1.078 Å at the magnification of 81,000 ×. Finally, 21,747 movies were collected with a total dose rate of 40 e-/Å\u003csup\u003e2\u003c/sup\u003e, with 40 frame fractionations. All movies were collected over 40 frames with a defocus range of -0.9 μm to -2.7 μm.\u003c/p\u003e\n\u003cp\u003eFor all movies, motion-correction and CTF estimation were processed in CryoSPARC v4.7\u003csup\u003e33\u003c/sup\u003e using patch motion correction and patch CTF estimation respectively. A total of 21,634 movies were used. Filament tracer was performed for particle picking, with a filament diameter of 200 Å and a separation distance of 0.265-fold diameters. 3,059,499 particles were extracted with a box size of 500×500 pixels and subjected to 2D classification. Two rounds of 2D classification were performed, to remove junk particles and to separate sheathed and unsheathed filament, respectively. For the sheathed filament, a subset of 76,818 particles were subjected to a round of helical refinement, followed by a round of local CTF refinement. Two rounds of 3D classification were performed to remove particles that present abnormal sheaths. Finally, a local refinement with a tight mask was performed with 72,779 particles and a map with a resolution of 3.6 Å was obtained. For the unsheathed filament, a subset of 443,577 particles were subjected to a round of helical refinement, followed by local CTF refinement. A round of 3D classification was performed to remove junk particles. Finally, a local refinement with a tight mask was performed with 439,378 particles and a map with a resolution of 3.16 Å was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building and refinement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAtomic models for all \u003cem\u003eV. alginolyticus\u003c/em\u003e NCTC 10675 flagellin homologs, namely FlaD1, FlaD2, FlaD3, FlaD4, FlaD5, were obtained from the AlphaFold database\u003csup\u003e24\u003c/sup\u003e, and fitted in the cryo-EM maps. Based on the density, we identified FlaD2 as the main flagellin in the structure (See results). Therefore, we used FlaD2 to build the atomic models for both sheathed and unsheathed filaments. 33 flagellin monomers (sheathed filament) or 55 flagellin monomers (unsheathed) were manually fitted into the reconstructed maps, and the complex was flexibly refined by ISOLDE\u003csup\u003e34\u003c/sup\u003e with secondary structure restraints gained from the AlphaFold model in UCSF ChimeraX\u003csup\u003e35\u003c/sup\u003e. Real-space refinement in PHENIX\u003csup\u003e36\u003c/sup\u003e with secondary structure, rotamer and Ramachandran restraints but without NCS restraints was next employed. Coot\u003csup\u003e37\u003c/sup\u003e was then used to correct rotamer outliers, side-chain clashes, and unattributed density. The final model was validated using the validation program in PHENIX\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe atomic model of the bacterial OM was generated with the membrane builder in CHARMM-GUI\u003csup\u003e21\u003c/sup\u003e, using a 1:1:1 ratio of Phosphatidylethanolamine:Phosphatidylglycerol: Cardiolipin for the inner leaflet, and LPS for the outer leaflet.\u003c/p\u003e\n\u003cp\u003eAtomic models of the \u003cem\u003eV. alginolyticus\u003c/em\u003e, \u003cem\u003eV. cholerae\u003c/em\u003e and \u003cem\u003eH. pylori\u003c/em\u003e FliD orthologues were obtained from the AlphaFold Database. A model of the \u003cem\u003eV. alginolyticus\u003c/em\u003e cap-filament complex was generated by aligning five copies of the \u003cem\u003eV. alginolyticus\u003c/em\u003e FliD model, and the structure of the \u003cem\u003eV. alginolyticus\u003c/em\u003e filament (this study), onto the structure of the \u003cem\u003eS. enterica\u0026nbsp;\u003c/em\u003efilament-cap complex; this model was fitted in the tomogram of filament tips using ChimeraX\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-ET sample preparation, data collection and processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures of the respective strains were diluted 1:100 in 10 mL LBS and incubated for 1.5 h. Cells were centrifuged at 2,500 × g for 10 min and resuspended in\u0026nbsp;ice-cold sucrose buffer (20mM Tris-HCl, 20% w/v sucrose, pH 8.0) in a gentle manner. The pipette tip was cut off to reduce flagella to be sheared off. 4 μL cell suspension was applied to glow-discharged lacey carbon grids (Agar scientific, 200 mesh). Samples were incubated for 30 s at 5 °C and 95% humidity before being blotted by Vitrobot Mark IV and then rapidly plunged into liquid ethane. Grids that were blotted for 8, 12, 16, 20 s were screened on 200 kV Glacios microscope (Thermo Fisher). \u0026nbsp;Cryo-EM tomography data were collected using a Titan Krios TEM (Thermo Fisher) operated at 300 kV and equipped with a K3 direct camera. Tomograms were collected using the TOMO5 software (Thermo Fisher) with a pixel size of 2.1 Å at 46,000 x. 21 tilt series ranging from -50 to +50 degrees with an increment of 5 degree and a fixed defocus of –3.5 μm were collect for each position\u0026nbsp;in dose-symmetric tilt scheme. A dose of 1.5 ~ 2.0 e\u003csup\u003e−\u003c/sup\u003e/Å\u003csup\u003e−2\u003c/sup\u003e per tilt was set, resulting in a total dose of 30~40 e\u003csup\u003e−\u003c/sup\u003e/Å\u003csup\u003e−2\u003c/sup\u003e. Relion 5\u003csup\u003e38\u003c/sup\u003e\u0026nbsp; was used for Motion correction, CTF correction, tilt series alignment\u0026nbsp;(AreTomo3), tomogram reconstruction, and tomogram denoising\u0026nbsp;(cryoCARE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMotility assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSwimming motility was studied using tryptone broth-based soft agar swim plates containing 0.3% Bacto agar, supplemented with 2% NaCl for \u003cem\u003eV. alginolyticus\u003c/em\u003e. Motility plates were inoculated with 2 µL of overnight culture and incubated at 37 °C for 6-8 h. Images were acquired by scanning the plates and the diameters of the swimming halos were measured using Fiji\u003csup\u003e39\u003c/sup\u003e. The swimming diameters of the mutant strains were normalized to those of the respective WT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOuter membrane and peptidoglycan staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures were diluted 1:100 into 5\u0026nbsp;mL M9 minimal medium supplemented with 0.4% glucose, 2\u0026nbsp;mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.1\u0026nbsp;mM CaCl\u003csub\u003e2\u003c/sub\u003e and 2% NaCl. For each strain, 500 µL aliquots were transferred into a new tube, while the remaining 4.5 mL culture was used to monitor growth at 37 °C, 180 rpm. To perform dual labelling, HADA (Tocris BioScience, catalogue number: 6647/5) was added to the culture to a final concentration of 500 µM. Cells were incubated for 4-5 h at 37 °C in the dark with shaking at 650 rpm. Thirty minutes before harvesting, FM1-43 (Invitrogen, catalogue number: T3163) was added to a final concentration of 15 µg/mL. After staining, 1 mL of fresh M9 medium was added to dilute the stains. Cells were pelleted by centrifugation at 2500 × g for 5 min, and the supernatant was discarded. Pellets were washed twice: first resuspended in 1 mL of fresh medium, pelleted again at 2500 × g for 2 min, and then resuspended in 500 µL of the same medium. The samples were applied to home-made flow cells prepared with poly-L-lysine (PLL)-coated coverslips that were prepared as described previously\u003csup\u003e40\u003c/sup\u003e. Briefly, coverslips were incubated with 0.1% PLL for 10 min, then air-dried, and subsequently fixed to an objective slide via two layers of pre-heated parafilm to create a chamber. The side of the coverslip incubated with PLL faced the objective slide. Cells were allowed to adhere for 10 min in the dark in an inverted position. Non-adherent cells were gently washed off by rinsing twice with 40 µl of M9 medium. Mounting medium (Fluoroshield, Sigma Aldrich, catalogue number: F6182) was added before imaging. Fluorescence microscopy was performed using a Ti-2 Nikon inverted microscope equipped with a CFI Plan Apochromat DM 60× Lambda oil Ph3/1.40 (Nikon) oil objective, an Orca Fusion BT camera (Hamamatsu), and a SPECTRA III LED light source (Lumencor). Z-stack images were acquired every 0.4\u0026nbsp;µm across a 1.6\u0026nbsp;µm range (5 slices). FM1-43 was excited with a 488\u0026nbsp;nm laser at 20% power and 100\u0026nbsp;ms exposure. HADA was excited with a 365 nm laser at 20% power and 100 ms exposure. Emission was collected using the GFP emission filter (499–530\u0026nbsp;nm; FF01-515/30, Em2) and DAPI emission filter (414–450\u0026nbsp;nm; FF01-432/36, Em1) from the LED-DA/FI/TR/Cy5/ Cy7-A Full Multiband Penta filter (Semrock, IDEX). Images were analysed using Fiji\u003csup\u003e39\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism 10 (GraphPad Software, Inc., San Diego, CA), and values of P \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular modeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA homology model of the \u003cem\u003eV. alginolyticus\u003c/em\u003e FliD pentamer was built with SwissModel, with the \u003cem\u003eS. enterica\u003c/em\u003e structure (PDB ID: 9GNZ) as a template. The structure of the \u003cem\u003eV. alginolyticus\u003c/em\u003e FliD D3-D4 region was modeled using the AlpfaFold3 server\u003csup\u003e24\u003c/sup\u003e, and this model was added to each FliD chain, aligned on the D3 domain.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the full filament-cap model, the FliD pentamer model was aligned to the \u003cem\u003eS. enterica\u003c/em\u003e FliD-filament structure (PDB ID: 9GNZ), and the \u003cem\u003eV. alginolyticus\u003c/em\u003e filament structure (See above) was aligned to the \u003cem\u003eS. enterica\u003c/em\u003e filament in the same structure.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe coordinates and/or EM maps that are displayed in this paper have been deposited in PDB and/or EMDB databases with the following accession code: sheathed filament, PDB: 9RCD, EMDB: EMD-53917; unsheathed filament, PDB: 9RCB, EMDB: EMD-53912; tomogram of multiple flagella tips in \u003cem\u003e\u0026Delta;flhG\u003c/em\u003e: EMD-53992; tomogram of multiple short flagella in\u003cem\u003e\u0026nbsp;\u0026Delta;flhG \u0026Delta;fliS\u003c/em\u003e: EMD-53993. The raw micrographs for the purified flagella dataset has been deposited to the EMPIAR database, with the accession code EMPIAR-47487467.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.Q. is supported by a PhD studentship from the China Scholarship Council. \u0026nbsp;M.E. acknowledges funding from the European Research Council (ERC) under the European Union\u0026rsquo;s Horizon 2020 research and innovation program (grant agreement n\u0026nbsp;864971) and from the Max Planck Society as Max Planck Fellow. J.R.C.B. acknowledges funding from the BBSRC (BB/R009759/2) and HFSP program (RGY0080/2021). We thank members of the Erhardt, Bergeron labs for helpful discussions. We thank Christian Goosmann (Max Planck Institute for Infection Biology) for TEM grid preparations and observation of \u003cem\u003eVibrio\u003c/em\u003e cells. Cryo-EM grids were screened at the Imperial College London cryo-EM facility (funded by BBSRC grant BB/V019732/1), and data was collected at the LonCEM facility; we acknowledge Paul Simpson, and Nora Cronin, respectively, for support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.R.C.B., M.E. and KQ conceptualized the research project and J.R.C.B. and M.E. ensured funding. R.E. generated chromosomal \u003cem\u003eV. cholerae and V. alginolyticus\u003c/em\u003e mutants, performed and analysed motility assays\u0026nbsp;and fluorescent microscopy experiments on mutants with the help of C.K.. K.Q. prepared negative-staining grids and collected TEM images for \u003cem\u003eV. cholerae.\u0026nbsp;\u003c/em\u003eK.Q.\u0026nbsp;isolated the\u0026nbsp;flagella of \u003cem\u003eV. alginolyticus,\u0026nbsp;\u003c/em\u003eprepared the EM grids, collected and processed the EM data. K.Q. reconstructed and refined the EM map of the\u0026nbsp;sheathed and unsheathed filament, and built and refined their atomic models. For tomography on \u003cem\u003eVibrio\u003c/em\u003e flagella, K.Q. collected the tomography data, and W.Z. processed the data, with support from J.A.. J.R.C.B.,\u0026nbsp;K.Q. and R.E. wrote the first draft of the manuscript, with comments from all authors. K.Q. and R.E. prepared figures and K.Q. prepared movies. M.E. and J.R.C.B. reviewed and edited the manuscript. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBerg, H. C. \u0026amp; Anderson, R. A. 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Biol.\u003c/em\u003e\u003cstrong\u003e1593\u003c/strong\u003e, 37-46 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6983709/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6983709/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn several pathogenic bacteria, including \u003cem\u003eVibrio\u003c/em\u003especies, the filament of the bacterial flagellum is encased by a membranous sheath, an extension of the bacterial outer membrane. It has been proposed that having sheathed flagella permit bacteria to evade an immune response against flagellar components, suggesting a role in virulence. However, the molecular details of the interaction between sheath and filament, and how it impacts filament rotation and assembly, have remained largely uncharacterized.\u003c/p\u003e\n\u003cp\u003eHere, we combine single-particle cryo-electron microscopy, cryo-electron tomography, and genetic analyses to resolve the molecular architecture and biogenesis of the sheathed flagellum in \u003cem\u003eVibrio alginolyticus\u003c/em\u003e. We show that the flagellar filament forms a canonical 11-stranded supercoil made of the flagellin FlaD2 and enveloped by a bilayered sheath. We observed that the filament surface is highly electronegative, suggesting that electrostatic repulsion between filament and sheath may reduce friction and supports high-speed flagellar rotation. We also show that the filament cap protein FliD possesses a unique domain in sheathed flagella, that may coordinate sheath assembly with filament elongation. Collectively, this structural insight into the structure of the \u003cem\u003eVibrio alginolyticus\u003c/em\u003e flagellum suggests a molecular mechanism for the rotation of sheathed flagella.\u003c/p\u003e","manuscriptTitle":"The structure of the Vibrio alginolyticus flagellar filament suggests molecular mechanism for the rotation of sheathed flagella","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 13:16:57","doi":"10.21203/rs.3.rs-6983709/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"aeaa4b03-ca53-4b34-84d3-a9904e28eeba","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59690252,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":59690253,"name":"Biological sciences/Microbiology/Bacteriology"}],"tags":[],"updatedAt":"2026-04-24T07:11:44+00:00","versionOfRecord":{"articleIdentity":"rs-6983709","link":"https://doi.org/10.1038/s41467-026-71203-7","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-04-23 04:00:00","publishedOnDateReadable":"April 23rd, 2026"},"versionCreatedAt":"2025-12-16 13:16:57","video":"","vorDoi":"10.1038/s41467-026-71203-7","vorDoiUrl":"https://doi.org/10.1038/s41467-026-71203-7","workflowStages":[]},"version":"v1","identity":"rs-6983709","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6983709","identity":"rs-6983709","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}