Deletion of flaA1 and flaA2 does not affect the synthesis of the sheath [ 20 ], whereas fcpA knockout mutants lack a sheath [ 19 , 26 ].
These results suggest that FcpA is a major sheath component and plays a central role in coiling via its interaction with the core filament. Recently, cryo-electron microscopy revealed that FcpB is a sheath protein that is localized along the outer curve of the PF, suggesting a contribution to PF coiling [ 22 , 23 ]. Spirochetes and externally flagellated species share fundamental motor parts for rotation, a rotor and a dozen stator units torque generators [ 24 ], but spirochetes flagellar motor has some spirochete-specific structures, resulting in a unique performance.
Motor torque is generated by interaction between the rotor and the stator [ 33 ]. Thus, the flagellar motor with a larger rotor ring allows more stators to surround the rotor. In addition to the geometrical advantage, the number of assembled stators of externally flagellated species is dynamically altered by changes in load against the motor and the input energy for rotation e. This knowledge predicts that the spirochetal motor can produce higher torque, which is supported by motility measurements showing that Leptospira spp.
In externally flagellated bacteria, when viewed from behind a swimming cell, a left-handed helical flagellum rotates counterclockwise CCW , which is balanced by the clockwise CW rotation of the cell body Figure 2 a [ 43 ]. In the case of spirochetes, the protoplasmic cylinder is believed to be rotated in the opposite direction of the PF rotation Figure 2 b [ 14 ].
Rotation of the PFs of Borrelia and Brachyspira drives wave propagation along the cell body, thus providing thrust for swimming [ 44 ]. In contrast, the swimming form of Leptospira is more complex. When viewing a swimming Leptospira cell from its posterior side, the PF transforms both ends of the cell body into a left-handed spiral or a hook shape and gyrates the bent ends in a CCW fashion; concurrently, the PF rotates the right-handed protoplasmic cylinder in a CW manner Figure 2 c [ 11 , 12 ].
The majority of thrust for Leptospira swimming is given by gyration of the spiral end and rolling of the protoplasmic cylinder [ 10 ]. However, correlative speed variation between the protoplasmic cylinder and the hook end was observed [ 14 ], suggesting that Leptospira swimming depends on mechanical communication among the three rotating parts.
Mechanical models for bacterial swimming. Torques of the cell body T Cell and flagellum T Flagellum are balanced, that is, their sum is zero. The protoplasmic cylinder PC is rotated by the counter torque of the periplasmic flagella PFs rotating at both ends of the cell body. Rotational directions are indicated by large arrows. The coupling ion used in torque generation by the flagellar motor depends on the type of stator units [ 45 ].
Such hybrid stator systems can exchange stator units in response to changes in environmental conditions, such as pH and viscosity [ 50 ]. Swimming of L. The flagellar motor rotates both CCW and CW, and a reversal of the direction of motor rotation results in a change in the swimming direction.
These motor reversal-based changes in swimming direction are related to bacterial chemotaxis, which may be stimulated by chemicals, temperature, light, and other trigger mechanisms [ 55 ]. In spirochetes, rotational directions of PFs are important for directed swimming [ 6 , 44 ]. According to the schematic structure shown in Figure 1 a, the flagellar motors residing at both cell ends have to rotate in opposite directions to each other; if they rotate in the same direction, the cell body will not be rotated due to the counterbalance of torques generated by the two motors or the inability to swim due to a twist of the cell body.
This mechanical model suggests that asymmetric rotation and synchronized motor reversal between PFs are required for the cells to swim smoothly and change swimming direction [ 44 ]. Coordinated rotation of E. CheY-P molecules generated in response to methylation of the methyl-accepting chemotaxis protein MCP bind to a rotor protein FliM and induce a conformational change of the rotor.
CheY is also involved in spirochete chemotaxis [ 57 , 58 , 59 , 60 ], but whether its diffusion can manage signal transduction between motors depends on the distance. CheY-P diffusion could be effective in E.
This estimation suggests that a CheY-independent mechanism could control the rapid swimming reversal observed in spirochetes. Furthermore, a chemotaxis-deficient B. FliG1 plays a central role for torque generation through interaction with stator units. FliG2 is essential for PF synthesis in B. Knockout of fliG1 does not affect PF synthesis, but subcellular localization studies on FliG1 tagged with green fluorescent protein GFP revealed that the localization of FliG1 is asymmetric [ 63 ].
This suggests the possibility that asymmetric PF rotation observed for B. Furthermore, a mathematical model predicted the importance of the interaction between PFs at the cell center.
In a borrelial model with a single PF, free swimming of the spirochete was reproduced by assuming that both ends of the PF are anchored to the cell body intimate interaction between PFs but not by assuming that only one end of the PF is anchored no interaction between PFs.
In the case of Leptospira with short PFs, given that the leptospiral cell body is stiffer than PFs [ 29 ], torque transmission from one end to the other may occur along the cell body instead of being mediated by direct contact between PFs.
Swimming speeds differ significantly among species Figure 3 a. In comparison with externally flagellated bacteria, the swimming speed of spirochetes in liquid media is much slower. The fastest swimmer is Leptospira spp. Swimming speeds are correlated with cell body rotation rates or wave frequencies Figure 3 b. B pilosicoli and L. Speeds of bacterial motility. Spirochete-derived data are enlarged in the inset. Refer to the following literature for the corresponding swimming measurements: E.
Although the swimming ability of spirochetes seems to be inferior to that of other flagellated bacteria Figure 3 , spirochete swimming is known to be improved by increased viscosity. Kaiser and Doetsch reported that the swimming speed of L. Similar phenomena have been observed in B. However, swimming motilities of these spirochetes cannot be improved by all types of viscous fluids but only by gel-like, heterogeneous polymer solutions, for example those containing methylcellulose, polyvinylpyrrolidone PVP , or mucin [ 8 , 69 , 83 , 84 ].
These linear polymers form a quasi-rigid network and are thus treated as viscoelastic fluids [ 85 ]. In contrast, the swimming speeds of B. Measurements in B. Although the mechanisms by which spirochete motilities are influenced by the differences in microscopic polymer structure are not fully understood, viscoelasticity is believed to be related to this unique phenomenon.
However, a recent motility study using Leptospira proposed another plausible model of taxis-like behavior, which was based on the result that a change in viscosity affects the reversal frequency in swimming direction [ 13 ]. When a leptospiral cell swims with the anterior spiral S end and the posterior hook H end SH form , the transformation into symmetric cell morphology SS or HH form interrupts swimming transiently, although the cell keeps rotating Figure 4 a.
Leptospiral swimming is restarted by transformation from symmetric to asymmetric forms, and the swimming direction after exhibiting symmetric morphologies is determined by the cell forming SH or HS. Takabe et al. The reversal movement returns the cell to its original position, indicating that there is no net migration.
Thus, viscosity-dependent impairment of net migration occurs due to the increment of the reversal event that results in trapping leptospires in areas with higher viscosity, which could assist the accumulation of spirochetes in the mucus layer in vivo Figure 4 d.
Effect of viscosity on Leptospira swimming. The spirochete can swim while displaying asymmetric morphologies SH or HS , with the front end pointing towards the swimming direction and usually displaying a spiral shape. Enhanced swimming reversal with elevated viscosity suppresses net migration of Leptospira cells, facilitating an accumulation of spirochetes in high viscosity areas. Early studies on chemotaxis using E. Notably, not all of the attractants and repellants are related to metabolism [ 87 , 88 ].
In spirochetes, S. Both pathogenic and saprophyte Leptospira spp. Chemotaxis to hemoglobin was observed in the pathogenic species L. Chemotaxis is closely related to the reversal of flagellar rotation, as described in Section 4. The swimming pattern of spirochetes involves back-and-forth motions, and attractants increase the persistency of their directed runs [ 91 ]. However, when swimming freely in liquid medium, the spirochetal back-and-forth movement cannot result in changes in direction as large as Vibrio , because the spirochete cell body is elastic but not too flexible to be buckled by mechanical stress.
A physical study on Leptospira showed that such a long and spiral body has a larger diffusion coefficient than a simple rod, suggesting that the exploration of spirochetes involves passive Brownian motion in addition to active swimming [ 94 ].
Pseudomonas aeruginosa not only swim with a polar flagellum but can also move on a solid surface using pili in a process called twitching motility [ 2 , 95 ]. To that effect, ambivalent motility of P. A major motility form of spirochetes is swimming, but Leptospira spp.
For moving while attached to surfaces, Mycoplasma mobile uses abundant leg-like protein complexes that are expressed on the cell surface; these legs successively catch and release sialylated oligosaccharides on surfaces, thereby propelling the cell [ 97 ].
Another gliding bacterium, Myxococcus xanthus , has a machinery that is composed of intracellular motor proteins and an external adhesive complex Agl-Glt [ 98 ]. Leptospiral swimming is a result of flagella-dependent motility, but a machinery specialized in crawling has yet to be identified.
Charon et al. A recent study by Tahara et al. Furthermore, it was revealed that modification of glass surfaces with anti-lipopolysaccharide LPS antibody affects the crawling speed and that anti-LPS antibody-coated microbeads move on the outer bacterial membrane. These results suggest that LPS is responsible for crawling, serving as one of the adhesins anchoring the cell to the surface Figure 5 b—d [ 73 ].
Electron microscopic observation of a hamster liver infected by pathogenic leptospires showed entry of leptospiral cells into the intercellular junction of hepatocytes [ ], implying that leptospiral pathogenicity could involve adherence of spirochetes to host cells, followed by crawling discussed in Section 7.
Crawling motility of Leptospira. Open bars indicate the fractions of cells adhered to the glass without crawling. Sequential frames of a movie were superimposed to show the bead trajectory.
Adhesive molecules red and purple symbols , such as LPS, anchor the cell to a surface, and PF-dependent rolling of the protoplasmic cylinder propels the cell. In general, bacterial flagella and motility are related to virulence, such as invasion, adhesion, and others [ , ]. Motility is an essential virulence factor for pathogenic spirochetes, and loss of motility due to a lack of flagellar genes attenuates infections with B.
Invasion of B. Motility analyses of B. The translocating state is similar to swimming in solutions, whereas the wriggling the entire cell body is fixed in place but keeps undulation and the lunging the cell body is partially fixed on the surface states are observed only in the dermis or the gelatin resembling the mouse dermis.
The translocation is essential for dissemination within the host, and transient adhesion by wriggling and lunging is thought to be involved in changing the moving direction and evading host immune system [ 70 ]. Although the details on the relationship between motility of Leptospira serovars and their host-dependent pathogenicity remain unknown, the crawling motility mediated by leptospiral LPS and other adhesion molecules is a potential key factor [ 73 , ].
Recently, we measured adhesivity and crawling of some leptospiral serovars on kidney cells derived from various mammalian hosts, including humans, showing close correlation of the measured parameters with the symptom severity of the host—serovar pairs; pairs causing more severe symptoms, such as hemorrhage, jaundice, and nephritis, show high adhesivity and persistent crawling of leptospires on the host cells [ ].
This knowledge is an important step toward understanding the host—pathogen relationship to develop novel antimicrobials for targeting pathogen dynamics. Members of the spirochetes share a basic cell structure, but their configurations, PF compositions, and motility forms are extremely diverse.
These are important clues to discuss high torque generation by the spirochetal flagellar motor. Motility measurements by optical microscopy showed improved efficiency of swimming motility in gel-like fluids and viscosity-dependent enhancement of swimming reversal, probably facilitating an accumulation of spirochetes in viscous milieus that exist abundantly within a host body.
A recent study showed the close relationship of the spirochetal movements over host cell surfaces and the severity of the symptoms caused, giving crucial insight into the practical role of bacterial motility as a virulence factor. Although the knowledge summarized in this review deepened the understanding of the mechanics of spirochete motility and its biological significance, there are still many issues remaining, such as the interaction between spirochetes and viscoelastic fluids, signal transduction for the coordinated rotation of PFs between both cell ends, and the molecular basis of crawling motility on the host cells.
The author thank K. Takabe, Md. Islam, J. Xu, A. Kawamoto, N. Koizumi, and S. Kudo for critical discussion related to research referred in this review. National Center for Biotechnology Information , U. Journal List Biomolecules v. Published online Apr 4. Shuichi Nakamura. Author information Article notes Copyright and License information Disclaimer.
Received Mar 11; Accepted Apr 3. This article has been cited by other articles in PMC. Abstract Spirochetes can be distinguished from other flagellated bacteria by their long, thin, spiral or wavy cell bodies and endoflagella that reside within the periplasmic space, designated as periplasmic flagella PFs.
Keywords: spirochetes, periplasmic flagella, motility, chemotaxis, molecular motor. Introduction Motility systems of living organisms are currently classified into 18 types [ 1 ]. Cell Structure A schematic of the basic structure shared among spirochete species is shown in Figure 1 a.
Open in a separate window. Figure 1. To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed.
Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella. They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins. Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function.
Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident. Future studies on spirochete motility should be exciting, as only recently have complete genome sequences and tools for allelic exchange mutagenesis become available.
A similar sub-tomogram averaging procedure as described above was followed for the fcpA - and fcpB - flagella tomograms. Specifically, IMOD was used for motion correction using the program alignframes and the tilt series were locally aligned through use of the fiduciary markers.
CTF correction was also performed in IMOD through phase flipping, followed by subtraction of the fiduciary markers from the tomograms. The final aligned tomograms were binned by 2, resulting in a pixel size of 4. Tomograms were then reconstructed in Tomo3D using weighted back projection, and 3dmod IMOD was used to select the filaments. A total of 24 tomograms were imported, corresponding to particles. The resultant fcpB - structure had a resolution of For fcpA - , a total of 25 tomograms were used, yielding particles; particles were imported into RELION and refined using similar parameters.
Nevertheless, the resulting fcpA - subunit x, y, z coordinates yielded continuous trajectories for many of the filaments. For the fcpB - dataset, the RELION-refined x, y, z coordinates and Euler angles of all 24 tomograms and particles were imported into emClarity, using the same in-house scripts as described above.
Due to reduced signal to noise ratio in the fcpB - data, the reference alignment procedure failed to unambiguously establish the polarity of many of the filaments, which were therefore discarded. After this analysis, particles corresponding to 14 filaments from 10 tomograms remained. As in the wild-type dataset, the flgCones and fscGoldSplitonTomos options were activated. Five cycles of averaging and alignment were used, in a manner similar to that described above, with each cycle using a binning of 2.
The rawAngleSearch range for the cycles, in order, were 0,0,7,1,0 , 5,1,0,0,0 , 0,0,5,1,0 , 4,1,0,0,0 , 0,0,3,0. After the fifth cycle, the half data sets were combined, giving an average FSC of Initial resolution estimates of final subtomogram average volumes were estimated using emClarity as described Himes and Zhang, ; Figure 2—figure supplement 2.
Additional masks used in combination with direction-dependent resolution calculations were defined as follows. To better match the filament cross-section, the intersection of three such masks, slightly offset from each other, was taken to produce a tighter mask with approximately elliptical cross-section.
Models of L. In the case of FcpB, we used our L. The highest-scoring fittings thus obtained gave alignments for two protofilaments of FcpA and one protofilament of FcpB within the outer convex sheath region. To identify additional docking locations within the sheath where resolution anisotropy due to the missing cone of Fourier data may have reduced the efficacy of the fitting procedure, we performed additional SITUS searches with the same parameters as before but with modified input maps and models.
Top-scoring hits from this second round of SITUS searching identified four additional protofilaments of FcpA, for a total of 6 protofilaments, and three additional protofilaments of FcpB for a total four protofilaments.
The best FcpB models, according to the Rosetta score, were used as input for an exhaustive full docking procedure with our FcpA X-ray model. The best docked configurations, according to the binding energy score, were rigidly fit into a segmented portion of the electron density map of the flagellar filament using SITUS colores and ranked according to the correlation coefficient.
To build a complete atomic model of the curved filament, an mer of modeled FlaB subunits co-assembled according to straight helical symmetry was first fit into the pseudo-helically symmetrized core volume. The mer was then deformed to follow a curved path that matched the measured curvature of the tomographically averaged wild-type filament, using UCSF Chimera Pettersen et al.
To refine this filament model and eliminate minor clashes, all the protein monomers protomers placed in density were subjected to iterative cycles of rigid body, side-chain and backbone minimization using positional and conformational constraints, restrained within the cryoET volume map using a customized protocol in Rosetta Das and Baker, Curvature was estimated for each reconstructed filament segment by comparing the 3D coordinates of neighboring filament subunits, and the resulting curvature values summed in histograms.
The curvature of the purified flagella was determined in the manner of Crenshaw et al. The final curvature value is then found by averaging the curvature at successive points, in the following manner:. These values were calculated for the 84 filaments of wild-type that were used in the emClarity analysis, the 14 filaments of the fcpB - sample that were used in the emClarity analysis, and 8 filaments of the fcpA - sample.
Due to the small size of the dataset and sample heterogeneity, a high-resolution structure of the fcpA - sample could not be determined.
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses. Thank you for submitting your article "An asymmetric sheath controls flagellar supercoiling and motility in the Leptospira spirochete" for consideration by eLife. Your article has been reviewed by four peer reviewers, including Edward H Egelman as the Reviewing Editor and Reviewer 1, and the evaluation has been overseen by John Kuriyan as the Senior Editor.
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This manuscript describes a cryo-EM tomography and X-Ray crystallography analysis of the flagella from Leptospira , with a focus on the location an arrangement of FcpA and FcpB. The authors show that these proteins localize to a sheath region in the along the outside curve of the flagellum, and that the core region is composed of FlaB, as has been observed in other spirochete flagella. Overall, this is a well-written manuscript that clearly describes the authors' findings. The localization of these proteins and the differences in flagellar conformation that arises in flagella lacking these proteins is informative about the functional role that these proteins play.
The main concern raised was that the interpretation is distorted by limited map resolution and ansotropic resolution of the cryo-ET reconstruction. While this does not necessarily reduce the impact of the story, the conclusions are occasionally pushed too far.
It would therefore be prudent to scale back some of the claims based on limited level of detail. These are enumerated below. Introduction: Why have the authors not mentioned the sheathed flagella of Vibrio species at all?
Discussion: Since the FcpA and FcpB complex binds to the convex side of the filament, the filament is responsible for determination of overall shape of the flagellum. Please discuss this point. Do the sheathed flagella show polymorphism under conditions tested in Salmonella?
If anything is known, please discuss the role of polymorphism. Subsection "3D reconstruction of flagellar filaments": "lasso-like supercoiling geometry". This term is incorrect. The noun lasso or lariat refers to a noose at the end of a rope — it does not have a supercoiled geometry.
Simply use "supercoiled". Subsection "Asymmetric sheath composition": "the finding of a break in the helical symmetry". Please do not over-interpret this feature — the authors should omit these far-reaching claims. The observed "break" may well be a result of the resolution anisotropy despite averaging of many subtomograms. As the authors point out themselves, the images suffer from a predominance of side views, resulting in insufficient sampling of data within the missing wedge, which is therefore present in all similar oriented subtomograms.
This is further illustrated in Figures 2F, G and Figure 3—figure supplement 5A, which clearly shows much lower resolution in the Z vertical direction of this cross section. This is compounded by the typical loss of resolution with increasing radius, as often observed in helical reconstructions. Thus, the apparent merging or absence of subunits in the outer sheath domain is not surprising under these circumstances and should not be explicitly claimed as a feature of the flagellum, but rather attributed to the reconstructed map.
The shifted features in the mutant reconstruction in Figure 3G, which has even lower overall resolution, are not convincing and may simply be a consequence of sampling different positions on the helical lattice. Furthermore, the rotational average presented in Figure 3—figure supplement 5C shows clearly, that the inner core FlgE is likely to share the fold symmetry of the Salmonella flagellum, which also argues that individual features of the asymmetric reconstruction cannot be trusted even at the level of subunit resolution.
The authors are aware of the problem, as they write in the figure legend of Figure 1—figure supplement 1C: " This restriction in the range of theta values prevents a meaningful 3D reconstruction from being obtained. I suggest simply to omit claims related to these map features across the manuscript. If the authors insist on this claim, they need to supply appropriate statistics to give credence that they can confidently resolve details at the level of one subunit or better at this location in the map.
This could be accomplished by providing a proper local resolution map e. Subsection "Asymmetric sheath composition": it is not clear what the "additional sheath features" are. While Figure 2C and 3A contain obvious differences, these differences may be artifacts of the reconstruction — see above. Simply say that the limited local resolution did not allow positioning of additional subunit models on the concave side and leave it at that.
Subsection "The sheath promotes filament curvature" paragraph two: This paragraph is a nice discussion how the sheath is not the structural reason of supercoiling itself, but that it contributes to stiffness and curvature of the filament. As such, it should better be moved to the Discussion section. Discussion end of paragraph one: Asymmetry in flagellar filaments has been addressed in recent papers — i.
Kato et al. Please modify the statement and cite these references. Discussion paragraph two: "unique hooks exhibiting covalent crosslinking between FlgE subunits" — this reference by Lynch et al. Remove this part of the sentence. Discussion paragraph two: "one function of the sheath elements This idea, although worthwhile mentioning, is not new — see work by Beeby et al.
Some references may be adequate. Relatedly, in the penultimate paragraph of the Discussion: "the asymmetric sheath composition While the former is supported by experiments in this paper, flagellar supercoiling cannot be caused by identical subunits or associated identical sheath proteins, since identical subunits would result in straight helical tubes.
Thus, they must be able to change their conformations dependent on their location on the supercoil. How does the unequal mechanical distortion arise spontaneously?
The evidence presented is insufficient to make the claim that the sheath proteins alone are responsible for supercoiling. Continued, in the final paragraph of the Discussion: Therefore, these claims about the asymmetric arrangement of the sheath components should be toned down or removed.
The authors also contradict themselves, since they write in the final paragraph of the Results section that filaments maintain supercoiling even in absence of these components. The final paragraph should be rewritten accordingly.
We thank the reviewers for pointing out this omission. We have added a mention of the Vibrio sheathed flagellum in the Introduction. We thank the reviewer for this insightful comment, which is closely connected to other points related to asymmetry, which we address below. We have added several paragraphs to the Discussion that address in more detail the role of filament supercoil shape in recruiting and being stabilized by the FcpA and FcpB sheath proteins.
In particular, the new Discussion now emphasizes evidence in our data that mutants lacking many or all sheath components still form supercoils, albeit with modified geometries.
We further discuss evidence for polymorphism in the Leptospira filaments, which although not investigated previously, is hinted at by our data. We thank the reviewer for noting this incongruity. We have deleted the term "lasso-like". We thank the reviewer for pointing out these weaknesses in our manuscript. We have carefully addressed these issues in detail.
First, we would like to emphasize that our conclusion of asymmetric composition in the sheath is supported by labeling studies, not just by our 3D structure data alone. Antibody labeling studies of both FcpA and FcpB, reported previously and cited in the text, showed specific localization of these sheath components to the filament outer curvature — directly supporting the asymmetric distribution of FcpA and FcpB indicated by our 3D structure analysis.
To further validate our claim of asymmetric organization in the Leptospira flagellar sheath, we have performed several additional analyses:. To this point, we have modified Figure 2 and added a new supplemental figure Figure 3—figure supplement 4 to better highlight the quality of map features throughout the filament cross section, including the sheath.
To further quantify the problem, we went beyond the originally reported estimates of the resolution anisotropy Figure 2—figure supplement 3 in the original submission , and used the 3D FSC tool to estimate resolution anisotropy not only for the entire map but also for isolated core and sheath regions see the new Figure 2—figure supplement 5. Again, observable features in our map are entirely consistent with the resolution estimates. The new Figure 3—figure supplement 4A includes an example of a "bad" viewing direction third panel from top , where features "smeared" in the horizontal direction due to the missing cone of Fourier data.
Even in this case, substructure in the sheath can be discriminated, and homology is evident with other, less blurred sheath views; this homology especially clear when comparing the right-hand sheath regions of panel 3 and panels all four of these sheath sites are predicted to have identical composition, with one molecule each of FcpA and FcpB.
These calculations reveal two categories of sites, precisely corresponding to our localizations of bound FcpA. Using resampled reference regions from the map itself rather than the PDB , we show a striking fall-off in reference cross-correlation values when comparing the sites we identify as occupied reference correlations ranged from 0. Together with our new local resolution estimates, this analysis strongly supports our interpretation that FcpA binding is absent or at the very least, severely perturbed on the filament inner curvature.
We have also modified the text to emphasize that our essential claim of asymmetry in the sheath composition does not rely on a positive identification of all features in the sheath which is not currently possible.
In our view, presence of the groove and other asymmetries in the sheath complements the above-mentioned antibody studies, providing robust evidence for compositional asymmetry in the sheath. We thank the reviewer for pointing out this shortcoming of our previous analysis. However, we also realize that our previous manuscript did not do a good job of representing the map quality in the sheath region.
We have modified Figure 2 and added a new supplemental figure Figure 3—figure supplement 4 to better highlight the quality of map features throughout the filament cross section, including the sheath. In particular, we note that subunit-level features even resolving ultrastructure within a single subunit are evident within the sheath region on both sides of the filament inner and outer curvatures. Moreover, as shown in Figure 3—figure supplement 4, panel 3, individual subunit features can even be resolved when the map is projected in the worst directions where features are smeared out in one direction due to the missing cone of Fourier data.
These observations are complemented by our additional resolution analysis above Figure 2—figure supplements 4 and 5 , which demonstrates that the local resolution in the sheath on the inner curvature side is not substantially worse than on the side where we located FcpA and FcpB. We further note that there is no room in our model of the outer curvature sheath to put any of the missing sheath elements FlaA1 and FlaA2 known to exist in the Leptospira filament.
Since these have to go somewhere, it seems clear they must bind to the inner curvature. We have added additional discussion to highlight these points, which in our view argue strongly that most of the inner-curvature sheath material is something other than FcpA and FcpB — most probably FlaA1 and FlaA2. We thank the reviewer for pointing this out. We have moved the paragraph to the Discussion and integrated it with additional discussion points. We thank the reviewer for pointing out this omission.
We have amended the Discussion to describe and cite these recent results. We thank the reviewer for pointing out the mismatch between this statement and the paper we mistakenly cited. We have replaced the citation with correct one that reports the discovery of crosslinking in FlgE hook subunits of the spirochete Treponema denticola.
We added additional citations to a pair of papers from Derosier and Macnab. We thank the reviewer for pointing out these gaps in our arguments. To address these points, we have added several paragraphs to the Discussion. In our view, the cartoon representation of our results in Figure 5F, G are now strongly supported by our new data and analysis — as noted above, the measured local resolutions and corresponding, visible density features in our maps give robust evidence for asymmetry at the subunit level of the sheath.
As for supercoiling, we agree with the reviewer that our data indicate that sheath-less or at least, mostly sheath-less fcpA — filaments still tend to supercoil and thus, are asymmetric. Our revised Discussion removes the implied suggestion that the sheath "introduces" asymmetry to the core.
We have added two citations to classic papers from Klug and Caspar that give allosteric mechanisms by which supercoiled filaments can be preferentially stabilized, through the introduction of a helical symmetry mismatch between inner and outer layers of a filament. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. We gratefully acknowledge Garrett Debs for help with the RELION subtomogram averaging pre-processing step, including his customizations of published python scripts for Relion sub-tomogram averaging.
We thank Prof. Felix Rey for providing insightful discussions about the manuscript. This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.
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