Segmented filamentous bacteria undergo a structural transition at their adhesive tip during unicellular to filament development

Domain: biology
Published: 2025-12-17
Editorial outcome: minor_revision

Abstract
Segmented filamentous bacteria (SFB) are intestinal commensals that promote immune system development and pathogen protection through intimate attachment to the ileal epithelium. Attachment occurs via the tip of unicellular teardrop-shaped SFB, called intracellular offsprings (IOs), before outgrowth into filaments. To characterize this critical stage of the SFB lifecycle, we imaged SFB using cryo-electron microscopy and tomography. IOs were surrounded by a repetitive surface (S)-layer that became replaced by disordered hair-like structures specifically at the IO tip. During outgrowth into filaments, the S-layer was exchanged for a morphologically distinct repetitive hair-like layer. The bacterial structures and morphological transition were conserved across SFB from mouse and rat origin, while growth of mouse-SFB under non-attachment conditions in a heterologous host affected the SFB tip length and relative proportion of the tip stages. Moreover, a major Th17 and B cell antigen, while being a ubiquitous cell wall-associated protein, was immunologically accessible only at the filament tip, underscoring the unique properties of the tip structure. This study identifies an IO-specific S-layer and reveals a conserved developmental transition of the SFB tip surface including the transient appearance of structures consistent in location and timing with being involved in host cell attachment.

Introduction
The intimate interactions between the intestinal microbiota and its host play a crucial role in shaping host physiology including the maturation of the immune system1. Segmented Filamentous Bacteria (SFB) are ubiquitous commensals in vertebrates but most extensively studied in mice where they strongly stimulate both innate and adaptive immunity2–7 and provide protection against intestinal and respiratory pathogens3,8,9. However, these Clostridium-related monoderm bacteria can also be harmful by exacerbating symptoms severity in a number of disease models10. Despite their immunomodulatory roles, SFB remain poorly studied due to technical constraints such as limited in vitro culturing11 and a lack of genetic tools.

Unlike most intestinal commensals, SFB are part of the more restricted mucosa-associated microbiota1. To establish their niche, teardrop-shaped unicellular SFB, called intracellular offsprings (IOs), attach to ileal epithelial cells and cells overlaying Peyer’s patches12. IOs attach using their tip, an unusual hook-like structure, and elongate to form segmented filaments that can reach over 100µm in length11,12. Filament differentiation leads to the formation of two new IOs per bacterial segment and eventual IO release at the distal end12,13.

The host-specific interaction14,15 mediated by the SFB tip leads to actin re-organization at the attachment site without evidence of SFB penetration beyond the terminal web or apparent plasma membrane disruption16–18. Endocytic vesicle formation near the attachment site18 results in the uptake of a major SFB antigen of the B cell19 and T helper 17 (Th17) response2,3,7,18, a hallmark host response to SFB colonization2,3,7,18. However, genome analyses of mouse- and rat-SFB have provided little insights regarding the composition of the SFB surface20–23. The typical cell wall binding motifs and domains present in most Clostridia are largely absent20–23. This includes surface layer homology (SLH) domains characteristic of some, but not all, surface layer (S-layer) proteins from monoderm bacteria24. Instead, likely surface-anchoring domains in SFB include a domain of unknown function (DUF4214) identified in multiple predicted surface proteins20–23. Some genes potentially involved in polysaccharide capsule formation were also found20–23 but scanning and transmission electron microscopy have not identified bacterial supramolecular structures such as an S-layer or capsule in SFB17,25.

To obtain a more detailed view of the SFB surface, we imaged purified IOs and filaments from mouse- and rat-SFB in a hydrated state using a combination of cryogenic electron microscopy (cryo-EM) and tomography (cryo-ET). We identified a number of intracellular structures but mainly focused on the SFB surface at the bacterial tip for which we describe a morphological transition during the outgrowth of unicellular IOs into filaments. This transition includes the replacement of an IO-specific S-layer by a repetitive hair-like layer, the appearance of tip structures, and a unique accessibility of a major Th17 and B cell antigen at the filament tip.

Results
Unicellular SFB are surrounded by an S-layer
We aimed to visualize the cellular structures that compose SFB in their native state and assess their conservation in SFB from different hosts. For this, we used cryo-EM and cryo-ET to image whole cells of SFB isolated from monocolonized mice (mouse-SFB) and rats (rat-SFB)26,27. We mainly focused on the SFB tip structure (Fig. 1a), which has a diameter of approximately 168.8±28.9nm (n=10, Supplementary Data 1), and therefore can be studied in toto. A total of 130 mouse-SFB (85 IOs and 45 filaments) and 68 rat-SFB (48 IOs and 20 filaments) were imaged (Supplementary Data 1). The SFB surface was characterized at both the bacterium’s tip and distal end (Fig. 1a–c). Bacterial tip imaging included part of the SFB cell body that, together with the separate imaging of the IO distal end, was considered to represent the overall bacterial surface beyond the tip.

Exterior to the SFB membrane, a region of low electron density was followed by a more electron-dense region although this region was not always well defined (Fig. 1d–g). These two layers likely constitute the inner wall (IW) and outer wall (OW) zones of the cell wall (CW), as previously described for other monoderm bacteria such as Bacillus subtilis28.

Exterior to the cell wall, mouse-SFB IOs (Fig. 1d, e) and rat-SFB IOs (Fig. 1f, g) exhibited another electron-dense layer at both the tip (Fig. 1d/f(ii), h, j) and the distal end (Fig. 1e/g(ii), i, j). In cross-section views, this outer layer appeared to be composed of repetitive crescent-like elements and regularly-spaced globular densities (Fig. 1d–g(ii), j) that showed resemblance to the S-layer of Caulobacter crescentus, Clostridioides difficile, and Clostridium thermocellum29–31. These data are consistent with the coverage of the entire IO surface by an S-layer, similarly to what is described for other bacteria32.

The distance between the repetitive globular subunits was measured manually, by Fourier spectra analysis, and by plot profile tracing on the side view of tomograms from the IO tip (Fig. 1d/f(iii–vi), k). The three methods consistently estimated the globular subunit spacing at around 8nm. Spacing was similar for mouse-SFB (8±1nm, n=50) and rat-SFB (8±1nm, n=50) (p=0.4109, Fig. 1k) and was within the range of the typical center-to-center S-layer spacings (2.5–35nm) of other bacteria33.

Top views of tomograms from the IO tip suggest that the electron dense subunits are arranged in rows (Fig. 1l and Supplementary Fig. 1a–d). Additionally, an apparent hexagonal organization was detected (Supplementary Fig. 1e, f) and most clearly seen in a tomogram of a rat-SFB IO with a broken tip phenotype whereby the S-layer showed abrupt discontinuity and was absent at the tip end (Supplementary Fig. 1f). The lack of a clear symmetry seen in the top view of SFB tomograms may be explained by the need to accommodate differences in SFB tip curvature. In the top views of intact SFB tips, the likely arrangement of S-layer subunits revealed a twisted appearance at the tip (Supplementary Fig. 1a).

Identification of the S-layer symmetry will require a more detailed analysis beyond what whole SFB cryo-ET can provide. In comparison, in the relatively closely-related species C. difficile, a p2-symmetric S-layer was observed30, while the morphologically more similar C. crescentus is covered by a p6-symmetrical S-layer29. Together, these data reveal the presence of a repetitive outermost layer with the typical S-layer center-to-center spacings that surrounds IOs and is conserved in SFB from various hosts.

Identification of intracellular and extracellular SFB features
During the SFB surface characterization, several intracellular and extracellular structures were identified (Fig. 2a–f and Supplementary movies 1–4). These structures were characterized at the SFB tip and proximal cell body (Fig. 2a–f and Supplementary Fig. 2a–e) although some of these features were also found at the SFB distal end (Supplementary Fig. 3a, b and Supplementary Data 1).

The presence of flagella at the concave side of SFB IOs, as previously described34, was confirmed for both mouse- and rat-SFB (Fig. 2a, e/f(i) and Supplementary Fig. 2a−d(i)). A structure typical of a chemosensory array, predicted to be present by genome analysis20,21, was found localized at the convex side of the cell body, opposite to where flagella are anchored, in IOs from both hosts (Fig. 2b, e/f(iii), and Supplementary Fig. 2a−d(ii)).

Vesicles with a median diameter of 51nm for mouse-SFB (average diameter of 54±15nm, n=15) and 41nm for rat-SFB (average diameter of 62±53nm, n=6) (Supplementary Table 1) were identified inside the tip of IOs and filaments (Fig. 2a, e/f(ii), Supplementary Fig. 2a(v), b(iii), c/d(vi), and Supplementary Data 1).

We also identified electron-dense lines parallel to the cell wall at the SFB tip, here designated as tracks (Fig. 2a and Supplementary Fig. 2a/b(v), c(vi), d(vii)). Tracks were commonly identified at the mouse-SFB tip but rarely at the rat-SFB tip (Supplementary Data 1 and Supplementary Fig. 2d(vii)), possibly due to the shorter tip length of rat-SFB compared to that of mouse-SFB (Supplementary Fig. 4). Given that our cryo-ET dataset mainly focused on the tip structure, these intracellular features were only characterized in this region even though they could also be found at the distal end (Supplementary Fig. 3a and Supplementary Data 1).

Tracks were designated as short (Fig. 2a and Supplementary Fig. 2a/b(v), c(vi), d(vii)) when their distance could be measured and as long when we could not fully visualize them in the field of view imaged (Supplementary Fig. 5a). While these features were commonly present on both sides of the tip, we also observed them only on one side (Supplementary Fig. 5b). Short and long tracks could be found in the same bacterium (Supplementary Fig. 5c). Tracks were only identified in SFB that showed evidence of cytoplasmic membrane retraction (Supplementary Fig. 5d(i)) and were present in the majority of SFB also harboring intracellular vesicles (61%, n=18) (Supplementary Fig. 5d(ii)). The length and thickness of short tracks was on average 137±56nm (n=15) and 58±30nm (n=10), respectively (Supplementary Fig. 5e(i/ii)). The length and thickness could not be estimated for long tracks due to the limited region imaged (Supplementary Fig. 5e(i/ii)). Nevertheless, given their similar location and width (Supplementary Fig. 5e(iii)), we hypothesize that both short and long tracks may play a role in maintaining the SFB shape or in anchoring the cytoplasmic membrane within the elongated tip and beyond.

Two types of intracellular filament-like structures were also identified in both mouse- and rat-SFB IOs (Supplementary Data 1). One type consisted of multiple filaments clustered together with a thickness of approximately only 26±12nm (n=23), which made it challenging to follow them in every tomographic slice (Fig. 2c, e/f(iv)), while the other filaments resembled more plate-like structures with a thickness of approximately 63±20nm (n=6) (Fig. 2d, e/f(v)). The difference between the estimated thickness of these two intracellular features was statistically significant (Supplementary Fig. 3c(i), p=0.0110). The width of individual clustered filaments and plate-like structures was also significantly different (Supplementary Fig. 3c(ii), p=0.0055). Clustered filaments had a width of approximately 9nm (mouse-SFB, 9±1nm, n=23; rat-SFB, 9±1nm, n=9) while the width of plate-like structures was 4/6nm (mouse-SFB, 4±1nm, n=6; rat-SFB, 6±1nm, n=4) (Fig. 2c, d and Supplementary Fig. 2a(ii, iii), b−d(iii, iv), and Supplementary Table 1). Clustered filaments and plate-like structures were at least 50 and 100nm in length, respectively. Due to the technical limitations stemming from sample thickness and from the limited field of view, their full length could not always be determined.

Clustered filaments were found both in the center of the cell body or parallel to the membrane in the concave side of IOs along the curvature of the cell (Supplementary Fig. 2a/b(iii)). These structures showed a potential resemblance to the cytoskeletal filaments formed in C. crescentus by the CTP synthase35, an enzyme also encoded in the genome of mouse- and rat-SFB20–23. In C. crescentus, these filaments are ~500nm in length, play a role in the regulation of bacterial curvature and, in initial developmental stages, they are found in the center of the cell body but their position changes to the cell periphery parallel to the inner membrane in later developmental stages35. Conversely, plate-like structures were less common (Supplementary Data 1) and were found in the center of the IOs cell body, but also at the SFB distal end (Supplementary Fig. 3b), either isolated or in groups of 2–3 plate-like structures, showing a maximum of 200nm in length when their full length could be measured (Supplementary Fig. 2a(ii), b(iv)). To our knowledge, these plate-like structures do not resemble any previously characterized structures.

Lastly, two types of extracellular vesicles (eVs) were identified: undecorated (Supplementary Fig. 2e) and decorated (Supplementary Fig. 2a(iv), b(iii), c/d(v)). Morphologically similar undecorated eVs have been identified for the monoderm gut commensal Bifidobacterium longum36 and decorated vesicles are commonly found in diderm bacteria37. In our samples, eVs were found in close proximity (Supplementary Fig. 2a(iv), b(iii)) and in direct contact with the SFB tip (Supplementary Fig. 2e(iii)). Additionally, both undecorated and decorated vesicles were identified near SFB that possess both intact and broken tip phenotypes (Supplementary Fig. 2a(iv), b(iii) and Supplementary Data 1). Overall, the average diameter of eVs was 85±40nm (n=15) in mouse-SFB and 103±54nm (n=26) in rat-SFB samples (Supplementary Table 1), placing them within the range of Gram-positive eVs38. While there is some evidence that similar vesicles can also be found in vivo, on SFB IOs attached to the epithelium of cows39, we are currently unable to determine if the eVs are SFB-derived since their molecular composition remains unknown. Overall, whole-cell cryo-ET thereby enabled the identification of expected, predicted, as well as unexpected cellular features of mouse- and rat-SFB.

The SFB tip undergoes a morphological developmental transition
We next refocused on the surface characterization of the SFB tip (Fig. 3a–n and Supplementary movies 5–8) as this structure mediates adhesion to the host epithelium and is therefore the primary location for potential attachment-related structures. SFB were grouped into stages based on the surface structures identified at the tip. In mouse-SFB IOs of less than 3.2µm in length (n=55), 24% were fully covered by an S-layer (stage 1 IOs, Fig. 3a, f). In the remaining IOs below 3.2µm, the tip showed varying degrees of hair-like structures with a disordered appearance seen from both side (Fig. 3b, c, f and Supplementary Fig. 6a) and top views (Supplementary Fig. 7a). These structures were found only at the tip edge in 20% of IOs (stage 2 IOs, Fig. 3b, f), while they were consistently present further down the tip in 18% of IOs (stage 3 IOs, Fig. 3c, f and Supplementary Fig. 6a).

In 20% of the IOs below 3.2µm, discontinuities were observed in the bacterial S-layer without disordered hair-like structures, leading to the broken tip phenotype and preventing us from identifying the tip stage (Unidentified: UD, Fig. 3f and Supplementary Fig. 8a). This broken phenotype was only found at the tip, suggesting increased fragility of this region, and was predominantly found in IOs (Supplementary Fig. 8b, c), potentially due to the transition from an S-layer to disordered hair-like structures.

The distinctly repetitive S-layer subunits could no longer be seen in the regions occupied by disordered hair-like structures (Fig. 3c and Supplementary Fig. 6a). However, the S-layer remained present in stages 2 and 3 IOs further down the tip towards the cell body (Fig. 3b(iii), g) as well as in the distal end, where disordered hair-like structures were never observed (Fig. 3j, and Supplementary Fig. 9a, b). Through tracking of the disordered hair-like structures in tomograms, these structures were found to be in contact, or close proximity, to the electron dense line corresponding to the outermost region of the cell wall (Fig. 3c, g and Supplementary Fig. 6a) and to be 34±6nm in length (n=250) (Fig. 3n) and 1.3±0.4nm in width (n=250) (Source Data). Together, these results reveal a structural transition from an S-layer to disordered hair-like structures specifically at the tip of IOs.

As IOs transitioned to filaments, the bacterial surface became covered by a morphologically distinct outer layer, here designated as hair-like layer (HLL). This outer layer was found in 18% of IOs below 3.2µm (Fig. 3f and Supplementary Fig. 6b, d) and in 100% of bacteria above this length (Fig. 3d, e, h, i, l, m, and Supplementary Fig. 8b, c). It is composed of an array of ordered hair-like structures whose arrangement is challenging to identify from the tip’s top view (Supplementary Fig. 7b). The HLL was found at the SFB tip end (stage 5, Fig. 3e and Supplementary Fig. 6d(i−ii)), tip side (stage 4 and 5, Fig. 3h, i and Supplementary Fig. 6b−d(iii)) and distal end (stage 4 and 5, Fig. 3k, Supplementary Fig. 9c, d). In stage 4 SFB, the end of the tip possessed disordered hair-like structures (Fig. 3d and Supplementary Fig. 6b, c), which, however, were significantly smaller (p<0.0001) than those of stage 3 (28±5nm, n=250, Fig. 3n). The cell body of stage 4 SFB was fully covered by the HLL. We only identified one mouse-SFB IO (3.3µm in length) out of the 14 stage 4 SFB imaged (7%) that showed all three features: HLL, disordered hair-like structures and the likely S-layer, which has neither HLL nor disordered hair-like structures (Supplementary Fig. 10a). This IO may therefore represent a rare transitional stage between stages 3 and 4.

In stage 5 SFB, the disordered hair-like structures were no longer present at the tip (Fig. 3e, i, k, and Supplementary Fig. 6d). Conservation of the HLL at the SFB distal end was confirmed in stages 4 and 5 SFB of variable lengths (Supplementary Data 1), including filaments of over 30µm (Supplementary Fig. 9d). As stages 4 and 5 SFB were significantly longer than the SFB stages 1–3 (p values from p=0.0040 to p<0.0001, Fig. 3l, m), stages 4 and 5 correspond to more advanced growth stages.

The filament-associated HLL was exterior to the cell wall and could be divided into an inner and outer part, separated by an electron dense layer. This electron dense layer was in a similar location to where the crescent-like elements of the IO-associated S-layer were in stages 1–3 SFB (Fig. 3g−i). Through manual tracing, individual electron dense hair-like subunits, comprising inner and outer parts of the HLL, were found to be 1.4±0.4nm (n=250) in width (Source Data) and, at 28±4nm (n=250) in length, to be significantly shorter than the disordered hair-like structures of stage 3 (p<0.0001, Fig. 3n). Together, these results reveal a morphological transition of the SFB surface from an S-layer, surrounding small IOs, to a filament-associated hair-like layer characterized by ordered elongated subunits with the transient appearance of longer disordered hair-like structures occurring only at the SFB tip. Given the location of the disordered hair-like structures and their presence in unicellular SFB, we hypothesize that these structures may play a role in SFB attachment to the host.

We have also explored a potential association between the different mouse-SFB stages and the intracellular and extracellular features identified. All flagellated IOs belonged to an early developmental stage (stages 1–3), and the proportion of early- and late-stages (stages 4 and 5) in flagellated and non-flagellated IOs was significantly different (Supplementary Fig. 11a, b). Furthermore, stage 1 mouse-SFB comprised 37% of the flagellated IOs (n=11) and only 12% of the non-flagellated IOs (n=74). However, this change in stage proportions was not statistically significant (Supplementary Fig. 11a, b). Similarly, the chemosensory array was only found in SFB IOs and may be characteristic of early SFB stages since it was identified in five S-layer-covered IOs and in only one HLL-covered IO (Supplementary Data 1). However, the limited field of view of our tomograms prevented us from properly assessing a potential stage-association not only for the chemosensory array but also for other cytosolic features, including filaments and plate-like structures, which, however, were also only found in SFB IOs (Supplementary Data 1).

Conversely, SFB tracks were found in both IOs and filaments. Long tracks were associated with late-stage SFB (Supplementary Fig. 5f) and tracks were found in significantly longer SFB when either any tracks or only long tracks were analyzed (Supplementary Fig. 5g). Overall, these results provide preliminary evidence for potential associations between intracellular and surface features and the here identified developmental stages.

The developmental transition is conserved in SFB from different hosts
We next aimed to determine whether the morphological transition of the SFB outer layer was conserved across SFB from a different host by characterizing the outer layer of rat-SFB during the outgrowth of IOs into filaments. As observed for the mouse-SFB tip, features of the early development stages (stages 1 and 3), including the S-layer and the replacement of this layer by disordered hair-like structures at the tip, were only found in IOs (Fig. 4a, b). In addition, the IO-associated S-layer and the absence of disordered hair-like structures were conserved in rat-SFB at the distal end of stages 1 and 3 IOs (Fig. 4e and Supplementary Fig. 9e, f). Similarly, the later developmental stages (stages 4 and 5), characteristic also of longer rat-SFB (p values from p=0.0023 to p<0.0001, Fig. 4g and Supplementary Fig. 8d), possessed the filament-associated HLL at the tip (Fig. 4c, d) and at the distal end (Fig. 4f and Supplementary Fig. 9g). As noted for mouse-SFB, it was challenging to identify a clear arrangement of the HLL subunits from the top view of the rat-SFB tip. An exception was a rare rat-SFB IO showing a potential transitional stage between stages 3 and 4 (Supplementary Fig. 10b and Supplementary movie 9), where arrangements resembling rows could be seen (Supplementary Fig. 7c). Nevertheless, some differences were noted. There was a significant increase (p<0.0001) in the proportion of IOs (74% of rat-SFB IOs vs 14% of mouse-SFB IOs) that could not be assigned to a tip stage (UD, Supplementary Fig. 8e−g). Of these, 31% also showed considerable S-layer discontinuities at the distal end (Supplementary Fig. 9h and Supplementary Data 1). As the purification conditions were the same for both mouse and rat-SFB, this may therefore be due to an increased fragility of the rat-SFB S-layer. Overall, our results confirm the conservation of the main developmental transitions at the tip and the distal end of rat-SFB, including the S-layer, the disordered hair-like structures at the tip, and the HLL surrounding SFB as they grow out into filaments.

The filament-associated hair-like layer is composed of repetitive subunits
We next aimed to obtain a more precise characterization of the hair-like layer by focussing on the inner (I-HLL) and outer (O-HLL) parts of this filament-associated layer. The I-HLL from stages 4 and 5 SFB was in a similar location to the IO-associated S-layer globular subunits from stages 1 to 3 IOs (Figs. 3g–i, and 5a, b), whereas the O-HLL extended beyond the IO-associated crescent-like elements of the S-layer (Fig. 5c–e). While the outer and inner subunits appeared to be aligned, and may therefore be connected, an electron dense layer separated these subunits. The I-HLL and O-HLL were often challenging to identify in the same Z plane (Fig. 5a–d). However, we could simultaneously observe hair-like structures both in the exterior and in the interior of the HLL electron dense line for both mouse-SFB (Fig. 3h, i) and rat-SFB (Fig. 4f).

The distance between the individual subunits of the inner and outer HLL was estimated by Fourier spectra analysis and plot profile tracing on the side view of tomograms of the SFB filament tips and combined with manual measurements (Fig. 5a−d). The subunits in both the inner and outer layer were found to be repetitive and approximately 5nm apart (n=50), both in mouse and rat-SFB (Fig. 5a−d, f). This distance between subunits was significantly smaller (p<0.0001) than the approximately 8nm separating the globular subunits of the S-layer (Fig. 5f). These results support the replacement of the IO-associated S-layer with a morphologically distinct hair-like layer composed of more tightly arranged repetitive units as SFB transition from IOs to filaments.

The major Th17 antigen is accessible at the tip of SFB filaments
The hair-like layer described is, to our knowledge, morphologically distinct from any surface structure previously identified by cryo-EM/ET and its molecular composition remains unknown. The major Th17 antigen (here referred to as Th17Ag)7, which was recently shown to also be a major B cell antigen, eliciting IgA and IgG responses in the blood and IgA responses in the intestinal lumen19, was previously found to surround attached SFB of the mouse-SFB-NYU strain18. This makes the Th17Ag a potential candidate for a component of the hair-like structures constituting the HLL. To assess the localization of this SFB factor in more detail, we recombinantly expressed and purified the mouse-SFB-NL homolog (AID45212) (Supplementary Fig. 12a, b) and generated a Th17Ag-specific nanobody (VHH). The Th17Ag was localized at the mouse-SFB-NL surface both by immunogold labeling followed by cryo-EM and by immunofluorescence followed by confocal microscopy (Fig. 6a−h, Supplementary Fig. 13a, b and Supplementary Fig. 14a−g).

Labeling with the Th17Ag-specific VHH fused to the Fc region of human IgG1 (VHH-Fc) (Supplementary Fig. 12c) revealed distinct labeling patterns in SFB filaments and IOs. For filaments, Th17Ag labeling was seen in 82% of the filaments imaged (n=17) and, for 93% of these, was strikingly restricted to the filament tip (n=13) (Fig. 6a, f and Supplementary Data 2). The Th17Ag labeling was seen in regions containing disordered hair-like structures (Fig. 6e(i)) but not the HLL (Fig. 6e(ii)). For IOs, Th17Ag labeling was observed in only 30% of the IO imaged (n=27) (Fig. 6f) and, in 75% of those, occurred at the tip but also on the remaining cell body, including at the distal end (Fig. 6c). In agreement, IOs showed labeling with the VHH-Fc targeting the Th17Ag using immunofluorescence (Fig. 6g). In contrast with the pattern observed in filaments, labeling of IOs was, albeit weakly, observed in regions containing the HLL (Supplementary Fig. 15a, b) but was not always present in regions including disordered hair-like structures (Supplementary Fig 15c). Additionally, as the Th17Ag was detected in the IOs distal end (Fig. 6c and Supplementary Fig. 14c) and in other regions where an S-layer was present (Supplementary Fig. 15c), it is unlikely that the Th17Ag is a component of the disordered hair-like structures.

While labeling was detected across the different IO stages (Supplementary Data 2), late-stage IOs (stages 4/5, Supplementary Fig. 15a, b) were present at a significantly higher proportion in the labeled population (36%, n=11) compared to the unlabeled population (0%, n=17) (Supplementary Fig. 16a(i-iii)). The same trend was identified for stage 3 IOs (Supplementary Fig. 14c and Supplementary Fig. 15c) among early SFB stages (stages 1 to 3) with 71% of labeled IOs (n=7) vs 18% of unlabeled IOs (n=17) (Supplementary Fig. 16a(iv)).

The tip-restricted labeling of SFB filaments, which could also be seen by immunofluorescence (Fig. 6a, f, g and Supplementary Fig. 14a, e, f), contrasts with earlier reports showing labeling of the Th17Ag surrounding SFB in histological sections18. As histological processing involves sample permeabilization, we treated PFA-fixed SFB with 0.5% triton X-100 at 95°C for 5min, which disrupts the outermost layer without compromising cell shape integrity. This treatment largely prevented all labeling with the VHH-Fc (Fig. 6f and Supplementary Fig. 14b) but using the rabbit polyclonal antibody raised against the Th17Ag7 recapitulated the published findings of extensive labeling surrounding 100% of the filaments (n=7), including the tip structure (Fig. 6b, f, h). In addition, the rabbit polyclonal antibodies showed strong staining of the bacterial surface in 86% of IOs imaged (n=7) (Fig. 6d, f and Supplementary Data 2). Under non-denaturing conditions, the rabbit polyclonal antibody displayed similar staining patterns as the VHH-Fc (Supplementary Fig. 14a, c, e, f). Epitope mapping of the VHH, following assessment of binding affinity (Supplementary Fig. 17a and Supplementary Table 2), identified three target regions of the Th17Ag towards the protein C-terminus (Fig. 6i). The nearly non-existent immunolabelling with the VHH-Fc under denaturing conditions (Fig. 6f and Supplementary Fig. 14b, g) is therefore likely due to the disruption of the conformational epitope recognized by VHH (Fig. 6i and Supplementary Fig. 17b, c). These results identify the Th17Ag as an abundant component of the SFB surface for both IOs and filaments whose accessibility is largely restricted to the tip in filaments and moderately accessible over the full surface in a subset of IOs. The Th17Ag, which contains a domain of unknown function also found in SFB proteins annotated as peptidoglycan hydrolases20–23, is therefore likely a cell wall-associated protein whose accessibility in filaments is prevented by the HLL. For IOs, as only a small subset, enriched in late-stage IOs, was labeled, we hypothesize that there may be intermediate accessibility stages (Supplementary Fig. 15a, b) prior to the impermeability of the HLL seen in filaments. Overall, our results point towards an increased exposure of the cell wall specifically at the SFB tip.

SFB growth in a heterologous host affects adhesive tip morphology
We next evaluated the tip stages of mouse-SFB in a context where adhesion was not supported. Attachment, but not growth, of SFB has been shown to be host-specific for rat-SFB under monocolonization conditions in mice14. Similarly, mouse-SFB did not attach to the ileal epithelium in rats, while mouse-SFB could still grow robustly in the rat intestinal lumen (Supplementary Fig. 18). When mouse-SFB were grown in rats, all five developmental stages were identified as well as the association of stages 4 and 5 with the outgrowth into filaments (Fig. 7a−f). However, we observed a decrease in tip length for mouse-SFB, both in IOs (575.6±146.8nm in mice, n=24, vs 467.4±95.0nm in rats, n=36, p=0.0010) and filaments (625.0±138.4nm in mice, n=39, vs 439.2±193.4nm in rats, n=10, p=0.0087) (Fig. 7g, h, k, l) when SFB of equivalent lengths (IOs, p=0.1717; filaments, p=0.2939) were compared (Fig. 7i, j). Additionally, stage 2 IOs were present at a significantly higher proportion when mouse-SFB were grown in rats compared to when grown in mice (61% vs. 32%, p=0.0147) (Fig. 7m–n). These findings support a role of attachment in the elongation of the tip and in the potential transition of SFB from stage 2 to stage 3, the stage where the disordered hair-like structures are most prominent.

Methods
SFB monocolonization
Germ-free C57BL/6J mice were bred and housed either at the animal facility of Institut Pasteur (IP) (agreement number 75-15-01) or at the animal facility of SFR Necker (agreement number A75-15-34), in conditions that answer to regulatory requirements (i.e., approved by the French Ministry of Agriculture). The light/dark cycle was 10/14, the temperature was controlled between 20 and 24°C, and the relative humidity around 50%. Mice were selected based on availability and regardless of their sex. Germ-free male F344 rats between 5 and 9 weeks-old were obtained from the Plateforme Anaxem de l’Institut Micalis (INRAE, Jouy-en-Josas) and housed at the IP animal facility. Animal sex was not considered in the study design since SFB colonization was the only parameter of interest and no other host parameter was recorded. Monocolonization with mouse-SFB-NL21,26,27 was established for 7-13-week-old germ-free mice. Monocolonization with mouse-SFB-NL or rat-SFB-Yit20,48 was established for 7-17-week-old germ-free rats. To achieve SFB monocolonization, all rodents were kept under axenic conditions and gavaged with previously frozen fecal pellets of rodents monocolonized with the SFB strain of interest resuspended in water. Monocolonization was verified by Gram staining (Sigma Aldrich) and culture-based techniques. All rodents were sacrificed between 7- and 18-days post gavage for further SFB purification.

SFB purification from rodent intestinal content
SFB were purified from the intestinal content of monocolonized mice and rats (one or two rodents per biological replicate) as previously described34 with some modifications. Briefly, all liquid reagents were pre-equilibrated overnight in a hypoxic chamber at 2% oxygen. Intestines were dissected in hypoxic conditions and contents homogenized in 20mL of PBS 1X by gentle pipetting. The intestinal suspension was layered in one volume of 50% nycodenz (Proteogenix) and spun down at 4000xg for 15min in a swinging bucket at room temperature. The interphase was collected, homogenized, layered in ½ volume of 30% nycodenz and spun down in the conditions above. Pellets were discarded and the remaining sample was washed in PBS 1× and spun down at 100xg for 5min. Pellets were again discarded, and supernatants spun down at 4000xg for 20min. When necessary, samples were passed through a 5μm filter (Cytiva) to obtain an IO-only fraction and concentrated at 4000xg for 30min. Reverse filtration was used to recover the filament-enriched fraction. The pellet was resuspended in PBS 1× and the suspension layered on 1 volume of 15% nycodenz and spun at 4000xg for 15min. The pellet was recovered, washed in PBS 1×, and the suspension was spun down as described above. Pellets corresponding to SFB fractions were resuspended in approximately 200µL of PBS 1× and diluted from 1:2 to 1:10 depending on the purified SFB density verified by Gram staining. SFB suspensions were kept at 4°C for a maximum of 24h prior to preparation of cryo-EM samples.

SFB preparation for Cryo-electron microscopy and tomography
Gold particles of 5 or 10nm (Protein A-gold, Aurion) were added to SFB samples in a 1:20 or 1:40 dilution, respectively, and gently homogenized. Four microliters of this suspension were deposited onto Lacey S166-3 mesh 300 copper grids (Agar Scientific) or Quantifoil R2/1 or R2/2 200 mesh copper grids previously glow-discharged at 2mA for 1min using an ELMO glow discharge system (Cordouan Technologies). 2µL of PBS 1× were deposited on the grid and back blotting was performed for 2–6s using an automatic plunge freezer (EMGP, Leica). During blotting, the temperature and humidity of the chamber were set to 10°C and 98%, respectively. The samples were then cryofixed at −180°C in liquid ethane using an automatic plunge freezer (EMGP, Leica) and stored in liquid nitrogen until imaging. Grids containing frozen-hydrated SFB were subsequently clipped into autogrids (Thermo Fisher Scientific: TFS) whenever subsequent imaging was performed using a 300kV Titan Krios electron microscope.

Cryo-electron microscopy and tomography
Initial screening of the samples was performed using a 200kV Tecnai F20 (TFS) equipped with a direct detector Falcon II (TFS) at the IP Ultrastructural BioImaging Platform. Projection images of the tip and distal end of SFB were acquired using the software EPU (TFS) at a pixel size of 2.01 or 4.14 Å and are listed in Supplementary Data 1. Representative images of the identified phenotypes (Supplementary Data 1) were acquired with a 300kV TITAN Krios electron microscope equipped with a Falcon 4i direct electron detector and a Selectris X energy filter (10eV slit width), here designated as TITAN Krios IP, unless otherwise stated. Imaging was performed at the IP NanoImaging Core facility. Projection images of the tip and distal end of SFB were acquired with −10µm defocus at a calibrated pixel size of 1.59 or 2.01Å. Data acquisition was controlled by TOMO 5 (version 5.14, 5.17 or 5.22, TFS). The magnification used to obtain a full SFB in the field of view to determine SFB length varied according to the bacterial size (from less than 2µm to more than 65µm in length). Projection images were used to classify tip stages and to measure SFB length, tip length and vesicle diameter.

To obtain a 3D view and enable the measurement of the SFB structures observed, tilt series were acquired mainly at the SFB tip at high magnifications. We used either the TITAN Krios IP or a Titan Krios microscope (TFS) operated at 300kV equipped with a post-column energy filter (20eV slit width, Gatan) and a K2 direct electron detector (Gatan) at the University of Zurich, here designated as TITAN Krios UZH. For the latter microscope, data acquisition was controlled by SerialEM 3.849. The tilt series acquisition parameters used for the TITAN Krios IP (a) and the TITAN Krios UZH (b) were set as follows. Tilt series were acquired at a calibrated pixel size of 1.59 Å (a), 1.75 Å (b), 1.9 Å (a) or 2.21 Å (b) (Supplementary Data 1). Two types of tilt-series acquisition schemes were used: a dose-symmetric scheme50 starting at 0° from −51° to +51° with a 3° increment (a) and a bidirectional scheme starting at −30° from −60° to +60° with a 3° increment (b). The projection dose was calibrated for each sample to reach a total dose of approximately 120 to 140 e−/A2 and the defocus used was between −2 and −6µm.

Cryo-EM data processing and visualization
For each tilt series acquired, frame alignment was performed using either MotionCor2 1.6.4 or the Alignframes program from the IMOD software package version 4.11 (RRID:SCR_003297)51. IMOD was also used for tilt alignment using fiducial markers and tomogram reconstruction at a binning factor of 2 or 4 with an antialiasing filter using dose weighting and a SIRT-like filter with 10-20 iterations. For tomograms selected as representative (binned by a factor of 4, Supplementary Data 1), fiducials were erased with the IMOD findbeads3d tool. These tomograms were further subjected to Topaz denoising52 using a default 10Å/px pre-trained model, for visualization purposes. A maximum of 10 tomographic slices were summed using the IMOD slicer tool to increase contrast and shown as representative tomographic slices of the tomograms containing features of interest. Tomographic slices may have been rotated along the x and y axis for presentation and comparison purposes.

For representative tomograms showing tip stages, intracellular and extracellular features, IMOD models were manually prepared for the membrane, cell wall and outer layer and for the features to be highlighted in each model. Linear interpolation was performed for all models except for vesicles (in which spherical interpolation was performed), flagella, filament-like structures and tracks (for which no interpolation was performed). IMOD meshes were prepared for all features except tracks. Upon volume meshing, the cap and tube tool were used for filament-like structures and vesicles and for flagella, respectively, to ensure the features were represented as closed models. IMOD models for the three layers (membrane, cell wall and outer layer), vesicle membranes, intracellular filaments and flagella were used as segmentation masks in UCSF ChimeraX version 1.7 (RRID:SCR_015872)53 to prepare volume masks using the slab tool with the width measured for each feature. The outer layer corresponded to the electron dense layer seen in the S-layer and in the hair-like layer. The pad tool was used to represent the tomogram densities from the cytosol, the interior of extracellular vesicles, and the chemosensory array while the coating of extracellular vesicles was defined using the slab tool. Tracks and hair-like structures were represented as IMOD contours, and their thickness adjusted using the atom and stickRadius commands. Final segmentation models were clipped, rotated and colored to show the features of interest.

Projection images selected as representative were binned 4 times and opened in FIJI version 2.14.0 (RRID:SCR_002285)54. Contrast was adjusted and a Gaussian low-pass filter with 0.5 to 1px radius was applied using Fiji’s unsharped mask tool, for visualization purposes. No image treatment was applied to images acquired using the 200kV Tecnai F20 (TFS). For two representative images of morphological stages, IMOD models were prepared for the membrane, cell wall, outer layer and hair-like structures. IMOD models were opened as contours in ChimeraX and their thickness adjusted using the atom and stickRadius commands. The cytoplasm of each original projection image was isolated using the imodmop command and included in the segmentation model prepared. A cytoplasm mask was defined and the segmentation models colored to show the features of interest.

All schematics were prepared using PowerPoint Version 2501.

Cryo-EM data analysis
To measure SFB length, projection images containing a full SFB in the field of view were opened in IMOD or in FIJI and a contour/line was traced from the SFB distal end to the edge of the tip. Tip length was measured by tracing a contour/line from the base to the tip edge, as shown in Fig. 7k-l. To measure vesicle diameters, projection images or tomograms (binned 4 times) acquired in a 300kV electron microscope were open in IMOD, a circle shape was used to establish the contour of each vesicle at its maximum diameter and its area was measured using the imodinfo tool. Vesicle diameter was then deduced from the circle area formula (πr²).

Measurements of intracellular filaments, hair-like structures length and width and distance between hair-like structures and S-layer subunits from SFB tip tomograms (binned 4 times) were performed manually in IMOD by defining individual contours and using the imodinfo tool. SFB tip diameter was measured as described above in a tomographic slice from the center of the volume. At least five tomograms were selected for the measurements of each feature/stage with the exception of plate-like structures since these were only present in three tomograms for mouse-SFB-NL and two tomograms for rat-SFB-Yit. Ten measurements were performed to estimate the width of tracks, intracellular filaments, and plate-like structures identified in each of the tomograms selected. The average of the measurements performed for each tomogram was used for statistical analysis. Tip diameter and length and vesicle diameter was measured once per tomogram/feature. Ten measurements were performed for each filament-like structure and for the distance between S-layer and hair-like layer subunits. Ten measurements in five different tomographic slices were performed to estimate hair-like structures length and width. Individual measurement values and their corresponding mean were plotted while mean and standard deviation are indicated in the text. The distance between S-layer and hair-like layer subunits was also confirmed by selecting regions from tomograms binned by a factor of 2 that contained clear S-layer or HLL repeats and by using a Fast Fourier transform in Fiji to assess repetitiveness. Plot profiles of the selected regions were obtained and the distances between two peaks were measured.

Histological analysis
Tissue from the terminal ileum of rodents monocolonized with SFB was fixed in 4% paraformaldehyde (Electron Microscopy Sciences) and frozen in OCT (TFS). Sections of 10µm thickness were cut using a cryostat (Leica), fixed in 4% paraformaldehyde, stained for 10min using Giemsa staining (Sigma Aldrich), and mounted using Eukitt Quick-hardening mounting medium (Sigma Aldrich). Imaging was performed at room temperature using an Olympus BX53 microscope equipped with an Olympus cellSens Standard 4.2.1 software. The Olympus UPLFLN 40× objective (0.75 NA) and the UPLFLN 100X objective (1.3 NA) were used.

Protein expression and purification of the Th17Ag
The Th17Ag gene from mouse-SFB-NL, without the signal peptide sequence, was synthesized with Escherichia coli codon optimization and cloned in the plasmid pET151/D-TOPO (GeneArt, TFS). Protein expression of the Th17Ag in the BL21 (DE3) Star E. coli strain was performed at 16°C in Luria-Bertani broth with an overnight induction using 1mM Isopropyl β-d-1-thiogalactopyranoside (IPTG) (Sigma Aldrich). The recombinantly expressed Th17Ag was produced with a poly-histidine and V5 epitope-containing N-terminal tag. The tagged protein was purified by affinity chromatography followed by size-exclusion using a Cobalt 5mL HiTrap TALON crude column (Cytiva) and a Superdex 200 Increase 10/300 GL column (Cytiva), respectively. Protein purification was performed using an AKTA-FPLC purification system equipped with a UNICORN 5.31 following the instructions of the manufacturer. On-tube digestion using a GST-tagged Tobacco Etch Virus (TEV) protease (PXP1108, Proteogenix) was performed to cleave the tag from purified Th17Ag using 1 TEV unit per 6µg of purified protein. Removal of the remaining tags and TEV was achieved through re-purification by affinity chromatography using a Cobalt 5mL HiTrap TALON crude column (Cytiva) and a 5mL GSTrap HP column (Cytiva). Protein-containing fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot using a mouse monoclonal anti-polyHistidine antibody (Sigma Aldrich, 1:5000, catalog number: A7058, RRID:AB_258326) and a horse anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Cell Signaling, 1:20000, catalog number: #7076). Images of the Coomassie-stained gels and of the protein blots were acquired using a ChemiDoc (BioRad). The tagged protein was used for subsequent experiments unless otherwise stated.

ELISA with polyclonal antibody against the Th17Ag
Purified Th17Ag was coated onto Nunc MaxiSorp flat-bottom plates (TFS) at a concentration of 10µg/mL at 4°C overnight. Coated plates were washed four times with 0.05% tween 20 (Sigma Aldrich) in PBS 1X (TFS) and incubated for 1h at room temperature in blocking buffer (0.05% tween 20 in PBS 1× with 1% bovine serum albumin, BSA, Sigma Aldrich). Rabbit polyclonal antibodies produced against the Th17Ag from mouse-SFB-NYU (1:100 of a 1:1 mix containing two rabbit anti-3340 antibodies)7 and a goat anti-rabbit IgG (whole molecule) conjugated with alkaline phosphatase (Sigma Aldrich, 1:10000, catalog number: A3687, RRID:AB_258103) were consecutively added to each well in blocking buffer for 2h and 1h, respectively. Four washing steps were performed after each incubation. A solution of 4.5M NaCl (Sigma Aldrich), 0.1M Tris-HCl (TFS) and 1mg/mL 4-nitrophenyl phosphate disodium salt (Sigma Aldrich) was used to reveal binding. The absorbance was measured at 415nm using an iMark Microplate Absorbance Reader (BioRad) equipped with an MPM6 software (Version 6.3, BioRad). The polyclonal antibody anti-Th17Ag used was kindly provided by Dr. Dan R Littman.

Nanobody library and phage selection
An alpaca (Lama pacos) named Picchu was immunized three times by subcutaneous injection with SFB purified from the intestinal content of monocolonized mice (1×109 to 4×1010 SFB genomes) and fixed with 4% paraformaldehyde (PFA). Two extra booster immunizations were performed 3 months later with SFB purified from the SFB-TC7 cell co-culture (3×107 to 2×109 SFB genomes) described by Schnupf et al.11 and fixed with 4% PFA. SFB were mixed with complete Freund’s adjuvant for the first immunization and with incomplete Freund’s adjuvant for subsequent injections. Approximately 250mL of alpaca blood were collected 6 days after the last immunization. Peripheral blood lymphocytes were isolated by Ficoll (Sigma Aldrich) density gradient centrifugation and stored at −80°C until further use. Total RNA was extracted from the lymphocytes and converted to cDNA as described by Lafaye et al.55. DNA sequences encoding the nanobodies, naturally produced by the immunized alpaca (VHH, variable domain of heavy chain), were amplified by PCR from cDNA using 8 pairs of primers (Supplementary Table 4), and cloned into the pHEN6 phagemid vector. The pHEN6 vector features a pUC backbone for ampicillin selection and high-copy replication in E. coli and includes the f1 phage origin for single-stranded DNA production. The ligation mixture was then electroporated into E. coli TG1 to obtain a VHH library of approximately 108 different clones. Phage-nanobodies from the VHH library were produced using the helper phage M13K7 (New England Biolabs) and Th17Ag-binding VHH were selected by phage display56. Briefly, 1011 Phage-VHH were incubated with purified Th17Ag previously coated on Nunc-Immunotubes (TFS) for 1h at room temperature. Three rounds of panning were performed with increasingly lower concentrations of coated Th17Ag, starting at 10µg/mL, and increasingly higher numbers of extensive washes. Th17Ag-binding phage-VHH were eluted in 100mM triethylamine (TEA, Sigma Aldrich) and used to infect E. coli TG1. Individual colonies were used to produce phage-VHH in 96-wells plates, which were then tested by phage-ELISA for binding to the Th17Ag previously coated onto 96-wells Nunc MaxiSorp flat-bottom plates (TFS) using an anti-M13 bacteriophage mouse monoclonal antibody conjugated with HRP (Abcam, 1:2500, catalog number: ab305291). The sequence of positive clones was determined by Sanger sequencing using the M13 rev-29 primer (5’ CAGGAAACAGCTATGACC 3’) (Eurofins).

Nanobody expression and selection
Positive VHH were expressed from pHEN6 vectors with an N-terminal pelB signal sequence and a C-terminal c-Myc and poly-histidine-containing tag. Periplasmic expression was performed in E. coli TG1 after overnight induction at 30°C with 1mM IPTG (Euromedex). Bacterial pellets were resuspended in PBS 1× containing 300mM NaCl, 1mg/mL polymyxin B sulfate (Sigma Aldrich) and Complete, EDTA-free Protease Inhibitor Cocktail (Roche) and incubated at 4°C for 1h with vigorous shaking (300rpm). Periplasmic extracts obtained by centrifugation and used to purify the produced nanobodies by affinity chromatography using a Cobalt 5mL HiTrap TALON crude column (Cytiva) followed by size exclusion chromatography on a Superdex 75pg column (Cytiva). An AKTA-Start purification system equipped with a UNICORN start 3.1 was used according to the manufacturer instructions. Purified VHH were concentrated and stored in PBS 1× at −20°C until further use.

Binding of purified VHH (10µg/mL) to the Th17Ag was assessed by ELISA using a mouse monoclonal antibody anti-c-Myc (Bio-Techne, 1:500, catalog number: #NB600-302) and a goat polyclonal anti-mouse IgG (whole molecule) conjugated with alkaline phosphatase (Sigma Aldrich, 1:10000, catalog number: A9316, RRID:AB_258446).

To enable large scale VHH production, a positive VHH, with c-Myc and 6xHis tag followed by a stop codon, was subcloned into the vector pFuse, which was transfected into Expi293 cells using the Expi293 Expression System Kit (TFS, catalog number: A14527), according to the manufacturer’s instructions. Expi293 cells were grown at 37°C for 5 days and VHH-Fc were purified from their supernatants by affinity chromatography as described above. Purified VHH were concentrated and stored in PBS 1× at −20°C until further use.

Expression of nanobodies fused to Fc region
The coding region of the selected VHH was subcloned into the vector pFuse57 in order to allow the expression of dimeric VHH-Fc fusion proteins. VHH-Fc were expressed in Expi293 cells as described above, and purified using a HiTrap protein A HP (Cytiva) and an AKTA-Start purification system, according to the manufacturer instructions. Purified VHH were concentrated and stored in PBS 1× at −20°C until further use.

Binding of purified VHH-Fc (10µg/mL) to the Th17Ag was assessed by ELISA using a mouse monoclonal antibody anti-human IgG1 Fc conjugated with HRP (TFS, 1:1000, catalog number: A-10648, RRID:AB_2534051) and Nunc MaxiSorp flat-bottom plates (TFS) coated with 10µg/mL of purified protein. 3,3′,5,5′-Tetramethylbenzidine (TMB, Sigma Aldrich) was used to reveal binding following the manufacturer’s instructions. The absorbance was measured at 450nm.

VHH-Fc was biotinylated using EZ-Link Sulfo NHS-Biotin (TFS) according to the manufacturer guidelines for subsequent use in immunofluorescence.

Immunofluorescence and immunogold labeling of the Th17Ag
Purified SFB from monocolonized mice (IOs only and filament-enriched fractions) were fixed in 4% PFA at room temperature for 30min and kept in 1% PFA at 4°C overnight. SFB samples were washed with PBS 1x and either left untreated (fixed SFB) or incubated in PBS 1X (TFS) with 0.5% triton X-100 (Sigma Aldrich) at 95°C for 5min followed by a PBS 1× wash (fixed and denatured SFB). Immunolabelling was performed in 1.5mL tubes using PBS 1× with 1% BSA (Sigma Aldrich) as blocking buffer and PBS 1× as washing buffer. After 1h in blocking buffer, SFB were incubated either with rabbit polyclonal (1:100) or with VHH-Fc (10µg/mL) in blocking buffer for 2h at room temperature. For immunogold labeling, Protein A coupled to 10nm gold particles (Aurion, 1:20) was added for 1h in blocking buffer. For immunofluorescence, streptavidin coupled to Alexa 568 (1:200, TFS, catalog number S11226, RRID:AB_2315774) and a goat polyclonal anti-rabbit IgG (H+L) conjugated with Alexa Fluor 568 (1:500, TFS, catalog number: A-11036, RRID: AB_10563566) was used for detection of labeling with the biotinylated VHH-Fc and the polyclonal antibody, respectively. DAPI (4µg/mL, TFS) was added together with the fluorescently coupled antibody/streptavidin to make the identification of SFB easier. Two washing steps were performed after each incubation.

For immunogold labeling, samples were diluted from 1:4 to 1:2 and cryo-EM grids were prepared immediately after staining, as described above, and imaged using a 200kV Tecnai F20 (TFS) equipped with a direct detector Falcon II (TFS).

For immunofluorescence, samples were diluted from 1:10 to 1:20, dried onto glass slides and preserved in ProLong Gold antifade medium (TFS) at 4°C until imaging by confocal microscopy. Imaging was performed at room temperature using an SP8 confocal microscope (Leica) equipped with both PMTs and HyD detection systems and a LAS X acquisition software (version 3.5.7). Slides were imaged at room temperature using a HC PL APO 63x (1.40 NA) objective and z-stacks were acquired at 0.3µm intervals with scanning at 1024×1024 pixels and a zoom factor of 2.5 or at 2048×2048 pixels and a zoom factor of 1.25. DAPI and the fluorescent dye Alexa Fluor 568 were excited using 405nm and 561nm lasers, respectively.

Quantification of immunogold labeling
Representative projection images acquired using a 200kV Tecnai F20 (TFS) were selected for each immunogold labeling condition and the ccderaser function of imod was used to erase the black pixel located at the center of the original images. Counting of gold particles co-localized with SFB cells was performed manually in IMOD. Images with over 10 gold particles in the background were not considered for analysis. Additionally, to avoid considering poorly labeled SFB, SFB were only considered labeled if co-localization with at least 20 gold particles was observed, or at least 10 gold particles if labeling was restricted to the SFB tip.

VHH binding kinetics
The binding kinetics of the anti-Th17Ag VHH were obtained by Biolayer Interferometry (BLI) using an Octet HTX system (Sartorius) equipped with an Octet Data Acquisition software version 13.0 (Sartorius) at 30°C. The His-tagged VHH was immobilized onto Ni-NTA sensor tips at 5μg/mL for 180s following a baseline in PBS 1× supplemented with tween 20 at 0.1% (PBS/T) for 120s. The VHH-coated tips were then dipped into different concentrations of the Th17Ag (2264, 905, 226.4, 90.5 and 0nM) for 600s to allow association. Subsequently, biosensors were placed in PBS/T for 15min to initiate dissociation. Raw data were processed using Octet Data Analysis Studio (version 13.0) and the data were fitted to a 1:1 Langmuir binding model.

Identification of VHH binding epitopes on the Th17Ag
A summary of the Hydrogen/Deuterium eXchange-Mass Spectrometry (HDX-MS) data is provided in Supplementary Table 358. The quality of the Th17Ag was assessed by intact mass measurement (measured mass: 113 762.00 Da; expected mass: 113 760.7381 Da; Δm=1.26 Da 11.1ppm). The Th17Ag alone (7.1µM in PBS 1×) or in complex with a 1.5× molar excess VHH was equilibrated for 30min at room temperature. Continuous labeling was initiated by adding 107µL of deuterated buffer (PBS 1×, pD 7.45) to 13µL of equilibrated protein solution, resulting in a final D2O/H2O ratio of 90/10. The experimental KD value was used to adjust the protein concentrations so that more than 90% of complex remained during labeling. The exchange reaction was quenched after 10 seconds, 1, 10, 30, 60 and 120min labeling at room temperature by mixing 20µL of the labeling reaction with 40µL of an ice-cold solution of 0.15% formic acid and 6M guanidinium chloride to reduce the pH to 2.5. A similar procedure was used for the preparation of undeuterated control samples. Quenched samples were snap-frozen in liquid nitrogen and stored at −80°C until further use. Triplicate labeling experiments were performed for each time point and condition (1 biological and 2 technical replicates).

Quenched samples were injected onto a nanoACQUITY UPLC system (Waters Corporation) equipped with an HDX manager maintained at 0°C. Approximately 11pmol of labeled Th17Ag either alone or with 16.5pmol of VHH were digested using an in-house packed cartridge (2.0×20mm, 63µL bed volume) of immobilized pepsin beads (TFS) for 2min at 20°C. Peptides were directly trapped and desalted onto a C18 trap column (VanGuard BEH 1.7mm, 2.1×5mm, Waters Corporation) at a flow rate of 100µL/min (0.15% formic acid, pH 2.5) and separated by an 8min linear gradient of acetonitrile from 5 to 30%, followed by a 2min increase from 30 to 40%, at 40µL/min using an ACQUITY UPLC BEH C18 analytical column (1.7µm, 1.0mm×100mm, Waters Corporation). After each run, the pepsin column was manually cleaned with two consecutive washes of 1.0% formic acid, 5% acetonitrile, 1.5M guanidinium chloride. Blank injections were performed after each sample to confirm the absence of carry-over.

Mass spectra were acquired in resolution and positive ion mode (m/z 50-1950) on a Synapt G2-Si HDMS mass spectrometer (Waters Corporation) equipped with a standard ESI source and lock-mass correction. Peptides were identified in undeuterated samples by a combination of data independent acquisition (MSE) and exact mass measurement (below 5.0ppm mass error) using the same chromatographic conditions as for the deuterated samples. To maximize sequence coverage, the fragmentation of Th17Ag peptide ions was conducted using four distinct collision energy ramps in the trap region of the instrument: Low: 10–30V; Med: 15–35V; High: 20–45V; Mix: 10–45V.

The initial peptide map of the Th17Ag was generated by database searching in ProteinLynX Global server 3.0 (Waters corporation) using the following processing and workflow parameters: low and elevated intensity thresholds set to 250.0 and 100.0 counts; intensity threshold=750.0 counts; automatic peptide and fragment tolerance; non-specific primary digest reagent; false discovery rate=4%. Each fragmentation spectrum was manually inspected for assignment confirmation. The peptide map was refined in DynamX 3.0 (Waters corporation) using the following import PLGS results filters: minimum intensity=10,000; minimum products per amino acid value=0.3; minimum PLGS score=7.0; maximum MH+ error=5.0ppm. A total of 154 peptides covering 90.6% of the Th17Ag protein sequence were selected and analyzed by HDX-MS.

DynamX 3.0 was used to extract the centroid masses of all peptides selected for HDX-MS analyses. Results are reported as relative deuterium exchange levels (no back exchange correction) expressed in either mass unit or as fractional exchange. Fractional exchange values were calculated by dividing the experimental uptake value by the theoretically maximum number of exchangeable backbone amide hydrogens that could be replaced in each peptide, considering the final excess of deuterium present in the labeling mixture.

To facilitate HDX-MS data interpretation, models of the Th17Ag were generated with Alphafold59 using the default parameters without energy minimization.

Statistics and Reproducibility
Data was plotted and statistical analysis was performed using GraphPad Prism 9.10 (GraphPad Software Inc, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001). Statistical significance was assessed for individual measurements following a normal distribution using an unpaired t-test or a one-way ANOVA in cases where only two or multiple groups were compared, respectively. Statistical significance was assessed for individual measurements that did not pass a normality test using a Mann-Whitney U test or a Kruskal–Wallis test followed by a Dunn’s test in cases where only two or multiple groups were compared, respectively. To compare proportions between two groups, a Fisher’s exact test was used. The MEMHDX software (version 0.01) was used to statistically validate HDX-MS datasets (Wald test, false discovery rate of 5%)60. Supplementary Data 1 and Supplementary Data 2 detail the number of SFB displaying each phenotype and feature described, and the corresponding number of biological replicates (“Experiment no.” column) for the cryo-EM/ET and the immunogold labeling datasets, respectively. The immunofluorescence and histological staining experiments were repeated for two biological replicates showing similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "SFB have major immunostimulatory properties that are dependent on adhesion to host cells and SFB surface structures are\nlikely crucial in host interaction. This study is important because it identifies a putatively novel S-layer and reveals a\ndevelopmental transition of the SFB tip surface including the transient appearance of structures (disordered hair-like\nstructures) consistent in location and timing with being involved in host cell attachment.\nAlthough there is no molecular characterisation of the identified surface (or intracellular) structures, I fully endorse the\npublication of this manuscript because the cryo-ET images are first-class despite SFB being an extremely challenging study\nsystem (use of monocolonised mice is necessary for bacterial propagation, a lack of availability of large quantities of\nintracellular offsprings, lack of genetic tools, etc). I should, however, point out that cryogenic electro-tomography lies outside\nmy field of expertise.\nI congratulate the authors for stepping outside the realm of classic (model) organisms, the solid cryogenic-ET and I only\nhave edits to hopefully improve the readability and the clarity of the manuscript:\nline 22: specifically localised instead of uniquely.\nline 26: Maybe better: d Moreover, although the major Th17 antigen is ubiquitous cell wall protein, this was only expose on\nthe filament tip, underscoring its unique properties.\nline 60: please clarify what you. mean by near native conditions.\nline 64: in line 54 and 55 the authors write that typical surface layer proteins are absent but then here, in line 64, the authors\nare sure that what they see is an S layer: maybe better \"putative S layer\"? Or can the authors be 100% sure that it is an S\nlayer?\nline 69: There are so many abbreviations that sometimes the reading is super hard: maybe avoid abbreviations in the titles?\nAnd replace IO with intracellular offspring in this line?\nline 77: it is probably going to be hard to change this but \"back\" sounds really strange to me! I had preferred to call proximal\ntip the tip and distal tip the back. I am curious what the other reviewers will say!\nline 113: please clarify \"mainly\", if possible.\nlines 121-123: was it always the case that when vesicles were pesent at the tip or when the cytoplasmatic membrane did not\nfully extend (by the way, extend rather than extended) into the tip track were visible? Or how often was this the case?\nFigure 2 legend: maybe specify that Fig.2 deals with the SFB tip in the title of the legend?\nline 131: what do you mean by challenging to track?\nline 170: images instead of imaged.\nline 177: I guess you can delete \"the presence of\".\nline 286: sorry if I missed it: the small subset which was labelled, which stage was it?\nline 306 and elsewhere: I would refrain from abbreviating disordered hair like structures. As said, there are already too many\nabbreviations.\nline 326: please clarify/rephrase \"bacteria with similar cell ends\".\nline 327: why polar tip? I would delete polar.\nline 330: I believe the acronym EA1 has not been used before, if so, please explain.\nlines 333 - 342: I would refrain from using the abbreviation ordHLS.\nline 345: better: decrease of iOS with fully developed (double negation is hard to read).\nline 371: better: only a quarter of non-permeabilised IOs showed labelling.\nline 374: delete generally.", "reviewer_2": "Significance:\n1. The novelty of this study is the observations of the S-layer, hair-like, and other structural features and their associated\ndevelopmental phases in the tip and back regions of the SFB from both mouse and rat species. The authors have done a\nthorough morphological analysis and surface/functional characterization to provide insights into the SFB life cycle. While the\npresented work offers some critical insights into the unknown structural land of SFB, more analyses and further work will be\nneeded to support the main claim that the morphological changes including the appearance and disappearance of the S-\nlayer and disordered and ordered hair-like structures, are critical for the conserved developmental transition of the SFB tip\nand involvement in host cell attachment.\n2. The observation of IO-specific S-layer and its transition is one of the main points throughout the manuscript. The claim that\nthe replacement of the S-layer surface by disordered hair-like structures, is primarily supported by morphological\nobservations. The S-layer assumes a hexagonal-like, seemingly 6-fold symmetry, which is very interesting. The sample\navailability and enrichment have been a bottleneck for more detailed structural analysis, as stated by the authors as well.\nWhile I fully understand the challenge, it would still be very valuable to further characterize the data by doing some\nsubtomogram averaging and FFT lattice characterization. The existing datasets were collected at pixel sizes of 1.5~2 Å\nrange. I wonder if the authors tried to analyze the pattern via STA? Is it possible to have enough particles using the existing\ntomograms from one of those pixel sizes for STA? The particle picking for regular arranged or lattice-like structures could be\ndone via the geometric picking solution as shown in Qu et al. 2018 PMID: 30478053, Burt et al. 2020", "reviewer_labeling-like": "ffect. It is important to provide a control image to show\nthe gold immunolabeling specificity.\n7. The authors hypothesize that the presence of S-layer and HLS are involved in cell wall integrity. While the eV is seen\nnear the broken phenotype, as stated above, it is unclear where the eV comes from. To prove this hypothesis, cell wall\nintegrity needs to be checked for the various developmental stages and see if there is a difference between stage 1 and\nstage 3 and stage 5. While Th17Ag, a cell wall protein, has been explored in the paper, did the author check other\nlandmarks that prove the cell integrity of the SFB after the purification?\n8. Cryo-ET data have been collected at multiple pixel sizes with various defocus and processing. While the\nmethod section is comprehensive, it will be good to provide a table that states which pixel size data sets\nare used for what data analysis e.g. S-layer structure characterizations in Fig. xxx, etc.\nMinor:\nIn Fig. 3 d iii, it is challenging to see the presence of both ordered HLS and disHLS in the current tomogram slice view. Fig 3.\nB is a 2D projection instead of a tomogram slice.\nIn Fig 3.f, it is hard to pinpoint each stage and their percentages due to the similar shades of grey. It might be better to use\nother more distinct colors to represent in the pie chart.\nLine 320, the HLL may itself constitute an S-layer since it is not repetitive but also appears to prevent the\naccess of the protein. There is no direct evidence in the manuscript to suppport this claim.\nLine 735, please state the original pixel size and binned pixel size after the bin4 has been performed.", "reviewer_3": "The manuscript by Gruz and colleagues presents a high-resolution study of segmented filamentous bacteria - an important\ngut commensal microbe known for its potent immunostimulatory effects using state-of-the-art cryo-electron microscopy and\ncryo-electron tomography (cryo-EM/ET). The authors capture high-resolution images of SFB at different developmental\nstages, from short unicellular intracellular offsprings, IO, to long filamentous cells. The authors discover a progression of\nsurface structures, an S-layer transforming into an entirely new, “hair-like” layer, identify novel “hair-like structures”\npotentially involved in host-cell attachment, and demonstrate differential surface accessibility of the major Th17 antigen at\nthe tip of filamentous forms.\nThe cryo-EM/ET data is of high quality, the tomograms look excellent, and are analyzed rigorously with respect to the\ndevelopmental stages. The manuscript is well-written and clear. It provides novel insights into the development and provides\nfood for thought for future research.\nThe major drawback of the manuscript is the unknown molecular identity of the hair-like structures. This is a very important\nlimitation of the study, and as it is, the manuscript is very descriptive. It may be possible to identify the molecules by\npulldowns of Th17, proteomics, bioinformatics, subvolume averaging, or their combination.\nFurthermore, it would be interesting to describe the mutual arrangement of the hair-like molecules throughout the\ndevelopmental process, at least where they are ordered. This could be another important step for the identification of the\nmolecules. Even though the cryo-ET dataset is of moderate size, there should be enough particles for subvolume averaging,\nand even at moderate resolution, it can provide useful insights."}, "author_response": "Point-by-point response to reviewers’ comments for the manuscript NCOMMS-25-16988-T\nText in black – reviewer comments\nText in orange – author response\nText in green – changes in the manuscript\nWe thank the reviewers for their careful review of our work and for the constructive feedback, which has\nsubstantially improved this manuscript. Our point-by-point response is below.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nSFB have major immunostimulatory properties that are dependent on adhesion to host cells and SFB\nsurface structures are likely crucial in host interaction. This study is important because it identifies a\nputatively novel S-layer and reveals a developmental transition of the SFB tip surface including the transient\nappearance of structures (disordered hair-like structures) consistent in location and timing with being\ninvolved in host cell attachment.\nAlthough there is no molecular characterisation of the identified surface (or intracellular) structures, I fully\nendorse the publication of this manuscript because the cryo-ET images are first-class despite SFB being\nan extremely challenging study system (use of monocolonised mice is necessary for bacterial propagation,\na lack of availability of large quantities of intracellular offsprings, lack of genetic tools, etc). I should,\nhowever, point out that cryogenic electro-tomography lies outside my field of expertise.\nI congratulate the authors for stepping outside the realm of classic (model) organisms, the solid cryogenic-\nET and I only have edits to hopefully improve the readability and the clarity of the manuscript:\nWe thank the reviewer for the encouraging comments and for their acknowledgement of the challenges of\nworking with Segmented Filamentous Bacteria (SFB).\nline 22: specifically localised instead of uniquely.\nHas been changed accordingly.\nLine 23: IOs were surrounded by a repetitive surface (S)-layer that became replaced by disordered hair-\nlike structures specifically at the IO tip.\nline 26: Maybe better: d Moreover, although the major Th17 antigen is ubiquitous cell wall protein, this was\nonly expose on the filament tip, underscoring the unique properties of the tip structure.\nHas been changed to:\nLine 28: Moreover, the major Th17 antigen, which is a ubiquitous cell wall protein, was only exposed on the\nfilament tip, underscoring the unique properties of the tip structure.\nline 60: please clarify what you. mean by near native conditions.\nHas been changed to:\nLine 63: To obtain a more detailed view of the SFB surface, we imaged purified IOs and filaments from\nmouse- and rat-SFB in a hydrated state using a combination of cryogenic electron microscopy (cryo-EM)\nand tomography (cryo-ET).\nline 64: in line 54 and 55 the authors write that typical surface layer proteins are absent but then here, in\nline 64, the authors are sure that what they see is an S layer: maybe better \"putative S layer\"? Or can the\nauthors be 100% sure that it is an S layer?\nWhile typical surface layer proteins and cell wall anchoring domains are not encoded in the SFB genome,\nthe absence of typical S-layer homology (SLH) and other typical S-layer protein (SLP) cell-wall anchoring\ndomains is not unique to SFB and has been reported for SLP from other monoderm bacteria such as\nLactobacillus acidophilus1 and Lactobacillus brevis2. Instead, a domain of unknown function (DUF4214) is\npresent in 33 proteins of mouse-SFB-NL including the cell-wall protein Th17Ag and other predicted surface\nproteins and it has been hypothesized to play a role in cell wall anchoring. The introduction has been\nmodified as follows to clarify this point:\nLine 55: The typical cell wall binding motifs and domains present in most Clostridia are largely absent19–22.\nThis includes surface (S-) layer homology (SLH) domains characteristic of some, but not all, surface layer\n(S-layer) proteins from monoderm bacteria23. Instead, likely surface-anchoring domains in SFB include a\ndomain of unknown function (DUF4214) identified in multiple predicted surface proteins19–22.\nRegarding the reviewer’s suggestion to use the nomenclature “putative S-layer” instead of “S-layer”, while\nit is true that the molecular identity of its building blocks remains unknown, the surface layer we identified\nin SFB IOs resembles other bacterial S-layers and it possesses all the remaining characteristics of bacterial\nS-layers. It is a repetitive outermost layer exterior to the cell wall, possess the typical center-to-center S-\nlayer spacings, has the electron density compatible with a proteinaceous structure and is morphologically\nsimilar to other bacterial S-layers. Thus, we would prefer to maintain the S-layer nomenclature but clarify\nwhy we are referring to this layer as an S-layer. The manuscript has been modified as follows:\nLine 113: Together, these data reveal the presence of a repetitive outermost layer with the typical S-layer\ncenter-to-center spacings that surrounds IOs and is conserved in SFB from various hosts.\nline 69: There are so many abbreviations that sometimes the reading is super hard: maybe avoid\nabbreviations in the titles? And replace IO with intracellular offspring in this line?\nWe thank the reviewer for this suggestion to improve the manuscript readability. We removed abbreviations\nfrom the titles (with the exception of SFB) and have reduced the abbreviations for hair-like structures (HLS).\nLine 71: Unicellular SFB are surrounded by an S-layer\nLine 306: The major Th17 antigen is accessible at the tip of SFB filaments\nline 77: it is probably going to be hard to change this but \"back\" sounds really strange to me! I had preferred\nto call proximal tip the tip and distal tip the back. I am curious what the other reviewers will say!\nWe acknowledge the reviewer’s comment on the terminology used for the two SFB poles. Given that “tip”\nhas already been used in the literature to designate the structure of the teardrop-shaped SFB that attach\nto the intestinal epithelium3, we propose to alter the designation of “back” to “distal end”. This has been\nchanged throughout the text and manuscript figures.\nline 113: please clarify \"mainly\", if possible.\nThe characterization of most intracellular features identified required the use of tomograms and our Cryo-\nET dataset mainly targeted the SFB tip and not the distal end, due to the sample thickness limits inherent\nof cryo-ET (i.e. while we have 66 tomograms that allow us to visualize the SFB tip, we only have 3\ntomograms for the distal end of mouse-SFB grown in mice in our dataset). Thus, we focused our analysis\non the intracellular features identified at the SFB tip and proximal cell body region.\nNevertheless, we have also screened both tomograms and projection images of the distal end of mouse-\nSFB for the presence of tracks. The same screening was performed for both plate-like structures and\nfilaments using tomograms and the data was included in new columns in Supplementary Table 1.\nExamples of the tracks and plate-like structures identified at the SFB distal end are shown in\nSupplementary Fig. 3a/b.\nThe manuscript has been changed to:\nLine 118: These structures were characterized at the SFB tip and proximal cell body (Fig. 2a-f,\nSupplementary Fig. 2a-e) although some of these features were also found at the SFB distal end\n(Supplementary Fig. 3a/b, Supplementary Table 1).\nLine 135: Given that our cryo-ET dataset mainly focused on the tip structure, these intracellular features\nwere only characterized in this region even though they could also be found at the distal end\n(Supplementary Fig. 3a, Supplementary Table 1).\nLine 173: Conversely, plate-like structures were less common (Supplementary Table 1) and were found in\nthe centre of the IOs cell body, but also at the SFB distal end (Supplementary Fig. 3b), either isolated or in\ngroups of 2-3 plate-like structures, showing a maximum of 200 nm in length when their full length could be\nmeasured (Supplementary Fig. 2a(ii),b(iv)).\nlines 121-123: was it always the case that when vesicles were pesent at the tip or when the cytoplasmatic\nmembrane did not fully extend (by the way, extend rather than extended) into the tip track were visible? Or\nhow often was this the case?\nTo answer to the reviewer’s question, we inspected again our dataset and performed a more detailed\nanalysis of the tracks identified at the SFB tip. We now show a significant association between the presence\nof tracks and both membrane retraction and intracellular vesicles. This analysis is shown in Supplementary\nFig. 5d and the corresponding information was added to new columns in Supplementary Table 1. We\nhave also included examples of the potentially different types of tracks that we see at the filament tip\n(Supplementary Fig. 5a-c) and show that the presence of these features does not seem to be restricted\nto the SFB tip (Supplementary Fig. 3a, Supplementary Table 1).\nThe text altered in the manuscript is below.\nLine 131: We also identified electron-dense lines parallel to the cell wall at the SFB tip, here designated as\ntracks (Fig. 2a, Supplementary Fig. 2a/b(v),c(vi) and d(vii)). Tracks were commonly identified at the mouse-\nSFB tip but rarely at the rat-SFB tip (Supplementary Table 1, Supplementary Fig. 2d(vii)), possibly due to\nthe shorter tip length of rat-SFB compared to that of mouse-SFB (Supplementary Fig. 4). Given that our\ncryo-ET dataset mainly focused on the tip structure, these intracellular features were only characterized in\nthis region even though they could also be found at the distal end (Supplementary Fig. 3a, Supplementary\nTable 1).\nTracks were designated as ‘short’ (Fig. 2a, Supplementary Fig. 2a/b(v),c(vi) and d(vii)) when their distance\ncould be measured and as ‘long’ when we could not fully visualize them in the field of view imaged\n(Supplementary Fig. 5a). While these features were commonly present on both sides of the tip, we also\nobserved them only on one side (Supplementary Fig. 5b). Short and long tracks could be found in the same\nbacterium (Supplementary Fig. 5c). Tracks were only identified in SFB that showed evidence of cytoplasmic\nmembrane retraction (Supplementary Fig. 5d(i)) and were present in the majority of SFB also harboring\nintracellular vesicles (61%, n = 18) (Supplementary Fig. 5d(ii)). The length and thickness of short tracks\nwas on average 126 ± 61 nm (n = 17) and 55 ± 30 nm (n = 11), respectively (Supplementary Fig. 5e(i/ii)).\nThe length and thickness could not be estimated for long tracks due to the limited region imaged\n(Supplementary Fig. 5e(i/ii)). Nevertheless, given their similar location and width (Supplementary Fig.\n5e(iii)), we hypothesize that both short and long tracks may play a role in maintaining the SFB shape or in\nanchoring the cytoplasmic membrane within the elongated tip and beyond.\nFigure 2 legend: maybe specify that Fig.2 deals with the SFB tip in the title of the legend?\nThe Fig. 2 and Supplementary Fig. 2 legend titles have been changed to:\nFig. 2. Description of intracellular and surface SFB features identified at the SFB tip and proximal region of\nthe cell body.\nSupplementary Fig. 2. Additional examples of the intracellular and extracellular features identified at or near\nthe SFB tip and proximal region of the cell body of SFB; Fig. 2.\nline 131: what do you mean by challenging to track?\nWe clarified this sentence in the manuscript. Has been changed to:\nLine 153: One type consisted of multiple filaments clustered together with a thickness of approximately only\n26 ± 12 nm (n = 23), which made it challenging to follow them in every tomographic slice (Fig. 2c,e/f(iv)),\nwhile the other filaments resembled more plate-like structures with a thickness of approximately 63 ± 20\nnm (n = 6) (Fig. 2d,e/f(v)).\nline 170: images instead of imaged.\nHas been changed to:\nLine 195: SFB were grouped into stages based on the surface structures identified at the tip.\nline 177: I guess you can delete \"the presence of\".\nHas been deleted:\nLine 203: In 20% of the IOs below 3.2 µm, discontinuities were observed in the bacterial S-layer without\ndisordered hair-like structures, leading to the 'broken tip’ phenotype and preventing us from identifying the\ntip stage (Undefined: UD, Fig. 3f, Supplementary Fig. 8a).\nline 286: sorry if I missed it: the small subset which was labelled, which stage was it?\nWe thank the reviewer for raising this important point, it was not previously mentioned in the manuscript. A\nnew Supplementary Table (Supplementary Table 3) was prepared, which includes the information on the\ntip stage of the SFB that were or not considered labelled after immunogold labelling. Additionally, we have\nalso attempted to determine if a particular IO stage was more enriched in the small subset of IOs labelled.\nThe results of this analysis have been added to the manuscript and are included below:\nLine 323: While labelling was detected across the different IO stages (Supplementary Table 3), late-stage\nIOs (stages 4 and 5) were present at a significantly higher proportion in the labelled population (23%, n =\n13) compared to the unlabeled population (0%, n = 26) (Supplementary Fig. 15a(i-iii)). The same trend was\nidentified for stage 3 IOs among early SFB stages (stages 1 to 3), although this difference was not\nsignificant (60% of labelled IOs n = 5 vs 15% of unlabeled IOs n = 13) (Supplementary Fig. 15a(iv)).\nLine 349: For IOs, as only a small subset, enriched in late-stage IOs, was labelled, we hypothesize that the\nincreased accessibility may be related to the transition of the S-layer to the HLL rather than a general\ngreater permeability of the S-layer.\nline 306 and elsewhere: I would refrain from abbreviating disordered hair like structures. As said, there are\nalready too many abbreviations.\nWe removed abbreviations of disHLS and ordHLS throughout the text. We have only kept these\nabbreviations in the figures and supplementary movies due to space restraints. The abbreviations for HLS\nwere kept in the discussion for readability although we redefine these terms at the beginning of this section.\nline 326: please clarify/rephrase \"bacteria with similar cell ends\".\nHas been changed to:\nLine 389: S-layers can play a role in host adhesion in bacteria with symmetric poles31.\nline 327: why polar tip? I would delete polar.\nHas been deleted:\nLine 389: However, since in SFB it is the tip that mediates epithelial cell attachment, it is unlikely that the\nS-layer or the HLL are involved in adhesion but rather may function in bacterial protection and/or\nmaintenance of cell wall integrity.\nline 330: I believe the acronym EA1 has not been used before, if so, please explain.\nHas been clarified:\nLine 384: The potential replacement of one surface layer with another is reminiscent of the transition\npreviously described for Bacillus anthracis39. In B. anthracis, an exponential phase-associated S-layer\ncomposed of the surface array protein (Sap) is replaced by a stationary phase-associated S-layer\ncomposed of the extracellular antigen 1 (EA1).\nlines 333 - 342: I would refrain from using the abbreviation ordHLS.\nWe removed abbreviations of disHLS and ordHLS throughout the text. We have only kept these\nabbreviations in the figures and supplementary movies due to space limitations.\nline 345: better: decrease of iOS with fully developed (double negation is hard to read).\nWe agree with the reviewer’s comment regarding the readability of this sentence. Even though there is a\ndecrease in the proportion of IOs with fully developed hair-like structures (30% of Stage 3 mouse-SFB\ngrown in mice in comparison to 14% of Stage 3 mouse-SFB grown in rats, Fig. 7m), this increase is not\nstatistically significant. However, the increase of Stage 2 mouse-SFB from 32% in mice to 61% in rats is\nstatistically significant. To avoid commenting on non-significant differences we propose to keep the\nsentence below in the manuscript:\nLine 416: Furthermore, their emergence prior to bacterial elongation and the increase in the proportion of\nIOs without fully developed disordered HLS in a heterologous host, where attachment is not supported,\nfurther support this hypothesis.\nline 371: better: only a quarter of non-permeabilised IOs showed labelling.\nHas been changed to:\nLine 397: However, only approximately a quarter of non-permeabilized IOs showed labelling and labelling\nwas not restricted to a specific region of the bacterial surface.\nline 374: delete generally.\nHas been deleted:\nLine 399: These results suggest that the HLL in SFB filaments prevents access to this potential cell wall-\nanchored protein while the S-layer appears to have a similar function for IOs.\nReviewer #2 (Remarks to the Author):\nSignificance:\n1. The novelty of this study is the observations of the S-layer, hair-like, and other structural features and\ntheir associated developmental phases in the tip and back regions of the SFB from both mouse and rat\nspecies. The authors have done a thorough morphological analysis and surface/functional characterization\nto provide insights into the SFB life cycle. While the presented work offers some critical insights into the\nunknown structural land of SFB, more analyses and further work will be needed to support the main claim\nthat the morphological changes including the appearance and disappearance of the S-layer and disordered\nand ordered hair-like structures, are critical for the conserved developmental transition of the SFB tip and\ninvolvement in host cell attachment.\nWe thank the reviewer for acknowledging the novelty of our morphological characterization of the SFB\nsurface. While we describe the presence of morphologically-distinct features at the surface of SFB from\ndistinct developmental stages, we had no intention to claim these changes are critical for the SFB\ndevelopmental transition and host cell attachment.\nTo address this, we would have to identify the molecular composition of the identified structures, to perform\ngenomic deletions of the corresponding genes and to assess the ability of SFB mutants to undergo the\ncharacteristic unicellular to filament transition and attach to host cells. Given the lack of available tools to\nperform these experiments, for the moment we can only provide a descriptive analysis and claim that the\nS-layer we identified was only observed in unicellular SFB while the HLL was characteristic of longer SFB\nand, thus, associated with later developmental stages.\nSimilarly, we cannot currently demonstrate the potential role of disordered hair-like structures in SFB\nattachment. We hypothesize that these structures may be involved in this close interaction due to the\nfollowing observations:\n(1) disordered hair-like structures are localized specifically at the SFB adhesive tip;\n(2) disordered hair-like structures are found in unicellular bacteria, appearing prior to bacterial elongation\nand, thus, prior to bacterial outgrowth;\n(3) the proportion of unicellular bacteria without fully developed hair-like structures (Stage 2 SFB) is\nstatistically higher when mouse-SFB is grown in rats (a setting in which attachment does not occur) in\ncomparison to when these bacteria are grown in mice (a setting in which attachment occurs), suggesting a\npotential delay in the development of these features in the absence of attachment.\nTo clarify these points in the manuscript we performed the following modifications:\nLine 65: We identified a number of intracellular structures but mainly focused on the SFB surface at the\nbacterial tip for which we describe a morphological transition during the outgrowth of unicellular IOs into\nfilaments.\nLine 67: This transition includes the replacement of an IO-specific S-layer by a repetitive hair-like layer, the\nappearance of tip structures, and a unique accessibility of the major Th17 antigen, a ubiquitous cell wall\nprotein, at the filament tip.\nLine 247: Given the location of the disordered hair-like structures and their presence in unicellular SFB, we\nhypothesize that these structures may play a role in SFB attachment to the host.\n2. The observation of IO-specific S-layer and its transition is one of the main points throughout the\nmanuscript. The claim that the replacement of the S-layer surface by disordered hair-like structures, is\nprimarily supported by morphological observations. The S-layer assumes a hexagonal-like, seemingly 6-\nfold symmetry, which is very interesting. The sample availability and enrichment have been a bottleneck for\nmore detailed structural analysis, as stated by the authors as well. While I fully understand the challenge,\nit would still be very valuable to further characterize the data by doing some subtomogram averaging and\nFFT lattice characterization. The existing datasets were collected at pixel sizes of 1.5~2 Å range. I wonder\nif the authors tried to analyze the pattern via STA? Is it possible to have enough particles using the existing\ntomograms from one of those pixel sizes for STA? The particle picking for regular arranged or lattice-like\nstructures could be done via the geometric picking solution as shown in Qu et al. 2018 PMID: 30478053,\nBurt et al. 2020"}, {"round": 2, "reviewer_comments": {"reviewer_2": "I appreciate the authors took time to address the questions brought up, particularly analyzing the OI tip with\nSTA. I commend the authors for the time and work that went into it. I have the following questions and hope\nto further improve the manuscript.\nWe thank the reviewer for their kind comments and his/her further contribution to improve the manuscript.\n1) Line 108: Here, the symmetry observation is being described and discussed. I agree that the claim would\nbe \"potential\" 6-fold symmetry. If the author would like to mention the performance of STA, it would be\nnecessary/important to include the STA results as part of the Supplemental/Extended Fig. I think in this\ncase, the lack of a clear definition of 6-fold symmetry organization could result from the missing wedge\nartifact despite the missing wedge compensation in PEET or Dynamo. I wonder if the author has tried to\nlimit the angular search range and translational search range as well after all subtomogram were aligned\nperpendicular to the beam direction. I have seen lattice structures suffer from the missing wedge effect\n(despite correction) during the alignment. As a result, the wrong alignment led to misleading 6-fold\nsymmetry. Two approaches could be helpful. One would be to align the subtomograms towards a fixed\ndirection, e.g. Revision Fig. 1e, where the membrane is centered and perpendicular to the Y. Then apply a\nlimited search angle/translation so that the aligned/guided positions are not lost during the subsequent\nalignment in PEET or Dynamo. The other one would be to provide some guidance for the angular search\nor predetermined orientation in Relion 3D refinement.\nThe subsequent analysis could require more time. I would suggest removing the sentence \"nevertheless,\nxxxx (data not shown)\".\nWe thank the reviewer for their guidance on alternative approaches that can be applied to avoid missing\nwedge artifacts that may prevent the identification of a clear 6-fold symmetry. We have tried both\napproaches suggested.\nDuring picking, the orientation of each particle relative to the picking surface was determined using the\nDynamo workflow. The angular information was converted to Relion convention using the dynamo2m\npackage (https://github.com/alisterburt/dynamo2m) and used in Relion as a starting point for classification\nor refinement. We tried giving this information both as the particle orientation (using the rlnAngle header),\nor as a constraint on the angle using the rlnAnglePrior header. The latter is not overwritten during\nrefinement, and provides a soft guidance for the alignment. Subsequently, we used the —sigma_ang flag\nto limit the angular search in Relion 3D Refinement to orientations close to those pre-orientations\ndetermined during picking.\nNeither of these approaches helped us converge on a global S-layer symmetry. In our opinion, the high\ncurvature of the SFB tip provides a strong feature, which enforces alignment of the individual particles in\nthe beam direction, and thus with aligned missing wedges. We tried to mask only the central density during\nalignment to overcome the curvature of the tip. However, due to the small repeat distance, the masked\nvolume was too small and provided too little density for STA to be successful.\nGiven the current limitations towards understanding the putative S-layer structure, we accepted the\nreviewer’s suggestion and removed the following sentence from the manuscript: “Nevertheless,\nsubtomogram averaging of the S-layer globular densities did not allow de novo identification of the lattice\nsymmetry (data not shown).”.\nLine 107: Identification of the S-layer symmetry will require a more detailed analysis beyond what whole\nSFB cryo-ET can provide.\nAdditionally, we no longer mention “potential six-fold symmetry” in the manuscript given the limitations faced\nwith the current structural analysis. The text was modified as follows:\nLine 100: Additionally, an apparent hexagonal organization was detected (Supplementary Fig. 1e,f) and\nmost clearly seen in a tomogram of a rat-SFB IO with a ‘broken tip’ phenotype whereby the S-layer showed\nabrupt discontinuity and was absent at the tip end (Supplementary Fig. 1f).\n2) Line 108, \"a more relaxed S-layer\". Is there any evidence or measurement, like curvature measurements,\nto prove the claim?\nSince in IOs with a ’broken tip’ phenotype the restraint to maintain the tip shape would be lost, we\nhypothesized that this may lead to a more relaxed S-layer lattice. However, the reviewer is correct, we do\nnot have direct measurements that allow us to prove our hypothesis that the potential 6-fold symmetry seen\nin the rat IO with a ‘broken tip’ phenotype can be visualized due to a more relaxed S-layer.\nGiven that this potential 6-fold symmetry was only seen in one bacterium with a ’broken tip’ phenotype,\ncurvature measurements would not allow us to confirm this hypothesis. We have removed the sentence “A\npotential 6-fold symmetry could be seen in the rat IO with a ‘broken tip’ phenotype, possibly due to a more\nrelaxed S-layer (Supplementary Fig. 1f).” from the manuscript.\nLine 107: Identification of the S-layer symmetry will require a more detailed analysis beyond what whole\nSFB cryo-ET can provide.\n3) Fig. 6, the HLS is too small to see in z (white arrowhead), the HLS will need to be zoomed in/enlarged.\nIn f, the merged and Alexa-658 are mislabeled/backwards. h, the residue font size is too small to read.\nThe projection images included in Fig. 6(a-d) were acquired using a 200 kV Tecnai F20 electron\nmicroscope, making it more challenging to prepare high resolution close ups to see the hair-like structures.\nThus, we decided to image other grids from this experiment, in which fixed SFB were labelled with a VHH-\nFc, using a 300 kV Titan Krios electron microscope. This allowed us to add a new panel to Fig. 6 (panel e)\nto better show the hair-like structures labelled at the SFB filaments tip (Fig. 6e(i)) and unlabeled at the SFB\nfilaments distal end (Fig. 6e(ii)).\nThe text in the manuscript was adjusted accordingly.\nLine 322: The Th17Ag labelling was seen in regions containing disordered hair-like structures (Fig. 6e(i))\nbut not the HLL (Fig. 6e(ii)).\nWe have also placed the panels of Fig. 6f (now Fig. 6g) in the correct position and increased the font size\nof the amino acid residues numbers of Fig. 6h (now Fig. 6i).\n4) Could the authors comment on why Th17Ag shows the labeling is more predominant in the late-stage\nIOs compared to the early stage, where the S-layer is present? Also, is Th17Ag present in both ordered\nand disordered HLS?\nTo better reply to these questions, we imaged the fixed SFB labelled with a VHH-Fc and imaged using a\n300 kV Titan Krios electron microscope, as mentioned above, since it allowed us to visualize S-layer\nsubunits and differentiate between Stage 4 and Stage 5 filaments. The additional SFB imaged were added\nto Supplementary Table 3 and the analyses shown in Fig. 6f and Supplementary Fig. 16 were updated with\nthe new data collected. The manuscript was also updated accordingly.\nAlso, is Th17Ag present in both ordered and disordered HLS?\nIn fixed filaments, the Th17Ag is predominantly inaccessible in regions containing ordered HLS as shown\nby the lack of labelling at the filaments distal end (Fig. 6a/e and Supplementary Fig. 14a). Additionally, the\ntip-restricted labelling in SFB filaments imaged using the 300 kV Titan Krios electron microscope occurs in\nStage 4 filaments, in regions containing the disordered hair-like structures (Fig. 6e, Supplementary Table\n3).\nHowever, in fixed IOs a different pattern was observed. Th17Ag labelling can be seen in regions containing\nordered HLS (Supplementary Fig. 15 a/b) but is not always present in regions containing disordered HLS\n(Supplementary Fig. 15c). In addition, it was also found in S-layer-containing regions (Supplementary Fig.\n15c).\nThe manuscript has been modified to clearly present these observations.\nLine 319: For filaments, Th17Ag labeling was seen in 82% of the filaments imaged (n = 17) and, for 93%\nof these, was strikingly restricted to the filament tip (n = 13) (Fig. 6a/f, Supplementary Table 3). The Th17Ag\nlabelling was seen in regions containing disordered hair-like structures (Fig. 6e(i)) but not the HLL (Fig.\n6e(ii)). For IOs, Th17Ag labelling was observed in only 30% of the IOs imaged (n = 27) (Fig. 6f) and, in 75%\nof those, occurred at the tip but also on the remaining cell body, including at the distal end (Fig. 6c). In\nagreement, IOs showed labelling with the VHH-Fc targeting the Th17Ag using immunofluorescence (Fig.\n6g). In contrast with the pattern observed in filaments, labelling of IOs was, albeit weekly, observed in\nregions containing the HLL (Supplementary Fig. 15a/b) but was not always present in regions including\ndisordered hair-like structures (Supplementary Fig 15c). Additionally, as the Th17Ag was detected in the\nIOs distal end (Fig. 6c, Supplementary Fig. 14c) and in other regions where an S-layer was present\n(Supplementary Fig. 15c), it is unlikely that the Th17Ag is a component of the disordered hair-like structures.\nCould the authors comment on why Th17Ag shows the labeling is more predominant in the late-stage\nIOs compared to the early stage, where the S-layer is present?\nWe hypothesize that late-stage IOs may be more predominantly labelled due to the transition between the\nS-layer to the HLL, which may facilitate access to the cell wall localized Th17Ag. Late-stage IOs may\nrepresent transitional stages of Th17Ag accessibility due to the formation of the HLL that, in filaments,\nblocks the access to this cell wall protein. We modified the manuscript to better present this hypothesis.\nLine 359: For IOs, as only a small subset, enriched in late-stage IOs, was labelled, we hypothesize that\nthere may be intermediate accessibility stages (Supplementary Fig. 15a/b) prior to the impermeability of\nthe HLL seen in filaments.\n5) Line 439, it might be good to specify the pixel size for the lower mag because too small a mag (big pixel\nsize) could lead to loss of important structural details that are required for further analysis. A bigger pixel\nsize (4~5 A/pixel) or montage cryoET (Reference) could help overcome the field of view limitation.\nWe agree with the reviewer, it would be better to increase the field of view without compromising the\nresolution of our tomogram. We modified this sentence in the discussion to suggest the use of montage\ncryo-ET schemes to overcome the field of view limitation instead of using a lower magnification.\nLine 448: Future studies using cryo-FIB45 and montage cryo-ET schemes46 to increase the field of view\nwithout compromising tomogram resolution could overcome this limitation.\n6) Line 448, I would suggest removing \"the extraction and purification of the S-layer... may overcome these\nlimitations\". Do the authors mean purifying the S-layer stems followed by reconstitution, or just purifying\nand putting them under the microscope? Purification and extraction may break the samples. To further\nstudy the organization of the S-layer stems below, a native, in-situ approach, as done here still remains the\nbest. I would suggest removing this comment.\nOur previous suggestion was to extract the S-layer directly from SFB followed by imaging using cryo-ET,\nsimilarly to what was performed for Caulobacter crescentus1. As discussed above, we have already\nperformed multiple attempts to identify the S-layer in situ. Thus, we believe that it may be necessary to try\na different strategy.\nWe proposed this approach since we hypothesize that extracting the S-layer from SFB may eliminate the\npotential restraints necessary to maintain the tip shape and the tip curvature limitation leading to a more\nrelaxed S-layer lattice and to the isolation of lattice sheets that could potentially facilitate the symmetry\nidentification.\nHowever, we agree with the reviewer, this approach does not guarantee the maintenance of the S-layer\nlattice structure. As suggested by the reviewer, we removed the following sentences: “The extraction and\npurification of the S-layer from IOs may overcome these limitations. Yet, given the challenges related to the\nstudy of SFB, including the use of monocolonized mice for bacterial propagation, a lack of availability of\nlarge quantities of IOs, and the lack of knowledge on the proteins that compose the IO-associated S-layer,\nobtaining S-layer sheets in solution will be challenging.”\n7) Line 444, no evidence for \"twisted lattice\" was provided here. I would think the tip curvature and the\nsample size would be the limiting factors for higher resolution STA analysis.\nWhen analyzing the top view of mouse-SFB IOs tips, the arrangement of the S-layer subunits led to the\nvisual appearance of a “twisted lattice” or “twisted tip”. This can be seen in Fig. 1l and Supplementary Fig.\n1a. We modified the results section to clarify that this designation was based on the visual appearance of\nthe S-layer subunits arrangement from the top view.\nLine 104: In the top views of intact SFB tips, the arrangement of S-layer subunits revealed a twisted\nappearance at the tip (Supplementary Fig. 1a).\nAdditionally, we took the reviewers suggestion and removed this descriptive designation from the\ndiscussion.\nLine 452: However, the strong SFB tip curvature, and smaller center-to-center spacing of the SFB S-layer\n(~8 nm) in comparison to the S-layer from C. crescentus (20-22 nm28,47) may have contributed to the\ndifficulties encountered in the identification of the lattice symmetry for SFB.\n8) In Fig5 a-d, most of the I or O-HLL don't seem to go together. e.g. in all of them, I barely see a uniform\ncoating of I-HLL. Is it because they appear on different tomographic slices? Could summing a few slices in\nSlicer/IMOD help to display them both at the same time? They seem to share a very similar spacing. The\nschematic view (c) shows O and I-HLL appear at the same time with almost identical spacing. If the\norganization of I/O-HLL in the slice view is not due to the different Z planes, then c needs to be modified.\nWe understand the reviewer’s concern and acknowledge that it is difficult to see a uniform coating for the\nI-HLL. As the reviewer pointed out, this occurs since the subunits of the HLL present in the inner (I-HLL)\nand outer (O-HLL) region of the layer cannot always be seen in the same tomographic slice, as shown in\nrevision Fig. 1a/b. Nevertheless, a more electron dense region (“shade”) that includes the inner part of the\nHLL subunits can be seen in the different tomographic slices (Revision Fig. 1c).\nThe projection images included in the manuscript (Fig. 5a-d(i)) and in the Revision Fig. 1 were prepared\nusing the Slicer tool in IMOD and 10 tomographic slices were summed. Thus, unfortunately this strategy\nwas not enough to clearly display both I-HLL and O-HLL at the same time for large regions of the bacterial\nsurface.\nIt is also noteworthy to mention that the projection images included in Fig. 5a-d are from tomograms\nsubjected to binning 2 (pixel size: 4.42 Å or 4.02 Å) since these were used for the FFT analysis presented.\nUsing tomograms subjected to binning 4 and filtered using topaz denoising2 helps to better display both I-\nHLL and O-HLL in the same tomographic slice. This can be seen in the tomographic slices included in Fig.\n3h/i (mouse-SFB, pixel size: 8.84 and 7.00, respectively). A projection image where both I-HLL and O-HLL\ncan clearly be seen at the same time is also included in Fig. 4f (rat-SFB). To clarify that I-HLL and O-HLL\nare challenging to display in the same tomographic slice and point towards other examples in which they\ncan be seen more clearly, we included the following sentence in the manuscript:\nLine 294: The I-HLL and O-HLL were often challenging to identify in the same Z plane (Fig. 5a-d). However,\nwe could simultaneously observe hair-like structures both in the exterior and in the interior of the HLL\nelectron dense line for both mouse-SFB (Fig. 3h/i) and rat-SFB (Fig. 4f).\nRevision Fig. 1. Visualization of HLL subunits in different tomographic slices. a-c, Representative\ntomographic slices from reconstructed tomogram EMD-52677 at (a/c) z=639 and at (b) z=621. (c)\nIdentification of the different HLL-related layers shown in (c(i)) a representative tomographic slice and in\n(c(ii)) a schematic. HLL subunits viewed from the outer hair-like layer (O-HLL) and the inner hair-like layer\n(I-HLL) are shown by red and white arrows, respectively. Scale bars: a-c: 20 nm.\nReviewer #3 (Remarks to the Author):\nI thank the authors for their thorough and detailed response to the initial reviews. The manuscript has been\nimproved by the revisions, and the additional analyses and clarifications strengthened the work. The\nauthors have addressed my previous comments satisfactorily. They have acknowledged the limitations\nregarding the molecular identity of the newly described hair-like structures and have convincingly argued\nin their rebuttal that the identification of these components is fraught with significant technical challenges\ninherent to the SFB system. I appreciate the addition of new supplementary figures showing the top-down\nviews of the disordered and ordered hair-like structures, which support their assertion that subtomogram\naveraging is not currently feasible due to the flexibility and lack of a clear, repetitive lattice.\nDespite these improvements, the central and significant gap of the study remains the unknown molecular\nidentity of the S-layer, the disordered hair-like structures, and the hair-like layer. This lack of molecular\ninformation necessarily renders the manuscript highly descriptive in nature. While the morphological\ncharacterization is of very high quality and provides novel insights into the SFB life cycle, the absence of\nmolecular data is a major limitation. The manuscript does not provide the molecular link, but it is correct\nand provides a foundation for future molecular studies.\nWe thank the reviewer for acknowledging our work towards the improvement of the manuscript, the\ntechnical challenges associated with the study of SFB, and the quality of our morphological\ncharacterization. We also appreciate the reviewer’s interest in the molecular identity of the novel structures\nhere described. We will continue our work in this direction and hope to be able to provide molecular insights\ninto SFB’s life cycle in the future.\nReferences\n1. Bharat, T. A. M., Kureisaite-Ciziene, D., Hardy, G. G., Yu, E. W., Devant, J. M., Hagen, W. J. H.,\nBrun, Y. V., Briggs, J. A. G. & Löwe, J. Structure of the hexagonal surface layer on Caulobacter\ncrescentus cells. Nat Microbiol 2, 1–6 (2017).\n2. Bepler, T., Kelley, K., Noble, A. J. & Berger, B. Topaz-Denoise: general deep denoising models for\ncryoEM and cryoET. Nat Commun 11, 5208 (2020)."}, "author_response": ""}]

Decision letter