Elucidation of fibril helix structure responsible for swimming in 2 Spiroplasma using electron microscopy 3 4 5

30 Spiroplasma, known pathogens and commensals of arthropods and plants, are helical-shaped 31 bacteria lacking the peptidoglycan layer. They swim by alternating between leftand right-handed 32 cell helicity, which is driven by an internal structure called the ribbon. This system is unrelated to 33 flagellar motility that is widespread in bacteria. The ribbon comprises the bacterial actin homolog 34 MreB and fibril, the protein specific to Spiroplasma. Here, we isolated the ribbon and its core, the 35 fibril filament, and using electron microscopy, found that the helicity of the ribbon and the cell is 36 linked to the helicity of the fibril. Single particle analysis using the negative-staining method 37 revealed that the three-dimensional structures of the fibril filament comprise a repeated ring 38 structure twisting along the filament axis. The handedness of these structures were verified by the 39


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Mollicutes, which are parasitic or commensal bacteria, have evolved from the phylum Firmicutes 59 that includes Bacillus and Clostridium by reducing their genome sizes (1-4). In the course of 60 evolution, the cells have become softer and smaller owing to the loss of the peptidoglycan layer. and right-handed cell helicity (Fig. 1A). This swimming is driven by an intracellular structure called 75 the "ribbon" which localizes along the innermost line of the helical cell structure, and structural 76 changes in the ribbon may switch the cell helicity (15, 16). Therefore, the detailed structure of the 77 ribbon should be elucidated to determine this swimming mechanism.

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In previous studies using electron microscopy, two types of filaments in the ribbon were 79 visualized (15, 16). One comprises a protein "fibril," specific to Spiroplasma. The other is possibly 80 MreB, the bacterial actin homolog (14-16). Interestingly, all Spiroplasma species have as much 81 as five MreB classes (9, 17-19). As Spiroplasma MreBs are distantly located from other MreBs in 82 the phylogenetic tree, here, we use the term SMreB (17, 18). Fibril protein has been studied as a 83 linear motor protein which is thought to be responsible for the helicity-switching through 84 contraction and extension (16,20,21). The fibril filament is considered to function as a chain of 85 elliptical rings. However, the structure and function of the fibril protein remains unclear.

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In the present study, we clarified the role of the fibril filament as the determinant of cell helicity, 87 using optical and electron microscopy (EM), and image analyses. Then, we proposed a scheme 88 for the helicity-switching swimming of Spiroplasma.

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Cell helicity is derived from the internal ribbon structure

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To clarify which structure forms the helical cell morphology of Spiroplasma, we first measured the 94 helical pitches of the swimming cells using optical microscopy. The helical shape of the cells can 95 be observed as a series of density segments in the defocused image plane, relative to the cell 96 axis under phase contrast microscopy (Fig. 1B). The helical pitches between the left-and right-97 handed segments along the cell axis were 696 ± 32 (n = 159) and 697 ± 37 nm (n = 146), 98 respectively. Next, we performed EM to analyze the internal ribbon structure to compare the 99 helical pitches of the cells and the ribbons. The cells were bound to EM grids non-specifically, 100 chemically fixed by glutaraldehyde, and then stained with uranyl acetate. Negative-staining EM 101 showed the images of helical-shaped cells with a narrow tip at one side (Fig. 1C). Next, we 102 exposed the internal ribbon structure by treating the cells with 0.1% Triton X-100 on the grid (Fig.   103 1D). The ribbon showed a "spiral" flat structure comprising protofilaments. However, generally, in 104 negative-staining EM, the specimens are placed in vacuum and dried and can result in 105 distortions, which is disadvantageous for helix observation. Therefore, we applied quick-freeze, 106 deep-etch (QFDE) EM to visualize the structure as close to the original as possible. In QFDE, a 107 sample is frozen in milliseconds, exposed by fracturing and etching, and then a platinum replica is 108 made by shadowing. The observation of the replica by transmission EM gives images with high 109 contrast and resolution much better than conventional scanning electron microscopy (SEM) (22, 110 23). The cells were non-specifically bound to mica flakes and fixed by quick freezing in a liquid 111 condition. Then, we prepared replicas by fracturing and platinum coating. QFDE-EM showed cell 112 morphology consistent with the results from negative-staining EM (Fig. 1E). Using QFDE-EM, we 113 also observed the ribbon exposed with 0.1% Triton X-100 treatment (Fig. 1F). The ribbon showed 114 the "helicoid" structure in which the twisted positions were aligned in a line. When the cells were 115 starved in phosphate-buffered saline (PBS) without glucose for 30 min, they all showed a left 116 helix with the same pitch. Therefore, we assumed that this structure is the default state of the cell.

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We recovered and observed the fraction under EM and found that the ribbon comprised 134 protofilaments with a width of 66 ± 12 nm and length longer than 2 μm (Fig. 2C a). The ribbons 135 were twisted with a pitch of 350 ± 17 nm (n = 47) (Fig. 2D a) consistent with the helical pitches of 136 the cells and the ribbons prepared on the grid (Fig. 1, Table 1) (P = 0.7 > 0.01). To analyze the 137 number and width of the protofilaments involved in the isolated ribbon, we traced a sectional 138 image profile of the ribbon (Fig. 2D b). Six to nine protofilaments were detected with widths 139 ranging between 4-16 nm (Fig. 2D c, d and Fig. S1). SDS-PAGE and peptide mass fingerprinting 140 analyses of this fraction showed five protein bands including six proteins ( Table 2). The band (v) 141 was shown to contain SMreBs 2 and 4 (Table. S1). The whole ribbon fraction mainly comprised 142 fibril protein (band iii) and the protein mixture of SMreBs 2 and 4 (band v) with an intensity ratio of 143 47% and 37%, respectively (Fig. 2E).

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Next, we examined the effects of A22 on the ribbon. We kept cells in 1 mM A22 for 2.5 h at 30 °C   To detect the conformational changes in the fibril three dimensionally, we performed single 184 particle analysis based on negative-staining EM. The double-stranded fibril filament was not 185 suitable for image averaging owing to the positional variety in the binding of the two filaments 186 ( Fig. 3 and Fig. S2). Therefore, we sonicated the purified fibril fraction to increase the proportion 187 of single-stranded forms and succeeded in the acquisition of single-stranded images (Fig. 4A).

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From the selected 11 867 particles with good quality, the 2D-averaged images were summarized 189 into three types (i), (ii), and (iii) (Fig. 4A b). Then, the initial three-dimensional (3D) model was

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Then, we calculated the structures of fibril filaments as C2 structures.

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Two dimensional re-projections from these three structures corresponded well to the 2D class  single-stranded filament (Fig. 3E), which was consistent with the images obtained using the other 219 methods.

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Although we found variations in curvatures and twists among the three classes, no variations 221 were detected in the lengths of periodic structures comprising rings and cylinders (Fig. 4A).

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The 3D images reconstituted from negative-staining EM have common features, although they 224 showed variations in curvature and twist. They have rings and cylinders rising to the right along 225 the filament axis when they are viewed from front and back sides, respectively (Fig. 4A d),

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showing that the three classes have the same handedness. As the images from negative-staining 227 EM are transparent, the structures reconstituted here may be mirror images of the real structures.

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Then, we intended to verify the handedness of the reconstituted structures by tomography of 229 replica from QFDE EM (Fig. 5), because tomogram cannot be a mirror image (27, 28).

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We made QFDE replicas from the fraction containing single-stranded fibril filaments, acquired 231 images every 1.5 degree to 50 degrees for both sides, reconstituted a tomogram (Movie_S4) 232 (Fig. 5A), and then a structure was obtained by averaging 60 subtomograms (Fig. 5B). The Although the interconnected ring structure in the fibril filament has been observed (14, 20), the 3D 244 reconstruction of the fibril has not been achieved to date. In the present study, we clarified the 3D 245 structure of the fibril filament for the first time. The sonication in the isolation process was 246 effective in isolating the single stranded filament, whose uniform structure was advantageous for 247 image averaging (Fig. 4). The structure determined here showed a width of 10.5 nm, and was in 248 good agreement with the corresponding filament structure obtained through electron 249 cryotomography (16), suggesting that the filaments isolated here retained the original structure.

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The left-handed conformation accounted for 73% of the fibril filament, suggesting that this 251 conformation is more stable than others, as an intrinsic character of fibril protein. This observation 252 may explain the fact that both the cells in a default state and the isolated ribbons were mostly left-253 handed (29, 30). The fibril structure is likely more stable in the left-handed conformation than in 254 the right-handed one. The fibril filament did not show any polarity along the filament axis,

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although Spiroplasma cells swim in a directed manner (Fig. 1) (9, 14). This directionality could be 256 caused by structures other than the fibril, for example, SMreB proteins and the dumbbell formed cylinder did not show significant variations (Fig. 4), suggesting that the helicity shift is not resulted 264 from the conformational changes of fibril filaments.

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Based on these results, we can now suggest the core part of the helicity-switching mechanism 267 (Fig. 6). The ribbon comprises 6-9 fibril filaments connected laterally and oriented along the 268 innermost part of the helical cell (Fig. 6A). The fibril should support the cell membrane through 269 their ring structures, because the fibril filament has a positive curvature toward the backbone (Fig.   270   6B a upper). Thus, the fibril twist forms the twist of the ribbon and the cell with the same 271 handedness, because the fibril filament binds to the adjacent fibril filaments through their fixed 272 positions (Fig. 6B a lower). If the fibril filaments in the ribbon have strong cooperativity along the 273 ribbon axis and transmit the twist to the next levels, the helicity shift travels along the ribbon axis, 274 with accumulation of the rotational angle (Fig. 6B b) (29). If we assume that such a ribbon is fixed   Generally, the energy for motility is supplied from ATP hydrolysis or membrane potential (2).

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Class Mollicutes lack the respiration pathway to generate membrane potential and produce ATP 288 through metabolism such as glycolysis and arginine fermentation (33). In Mollicutes, the 289 membrane potential is generated from ATP hydrolysis and not the primary energy source.

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Moreover, in the present study, we showed that SMreBs have roles to bundle fibril filaments (

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To isolate the internal structure, 10 mL of cell suspension in PBS was treated with 1% Triton X-