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Tuning spherical cells into kinking helices in wall-less bacteria

In bacteria, cell shape is determined and maintained through a complex interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Spiroplasma species, members of the Mollicutes class, challenge this general understanding because they are characterized by a helical cell shape and motility without a cell wall. This specificity is thought to rely on five MreB isoforms and a specific fibril protein. In this study, combinations of these five MreBs and of the fibril from Spiroplasma citri were expressed in another Mollicutes, Mycoplasma capricolum. Mycoplasma cells that were initially pleomorphic, mostly spherical, turned into helices when MreBs and fibrils were expressed in this heterologous host. The fibril protein was essential neither for helicity nor for cell movements. The isoform MreB5 had a special role as it was sufficient to confer helicity and motility to the mycoplasma cells. Cryo-electron microscopy confirmed the association of MreBs and fibril-based cytoskeleton with the plasma membrane, suggesting a direct effect on the membrane curvature. Finally, the heterologous expression of these proteins, MreBs and fibril, made it possible to reproduce the kink-like motility of spiroplasmas without providing the ability of cell movement in liquid broth. We suggest that other Spiroplasma components, not yet identified, are required for swimming, a hypothesis that could be evaluated in future studies using the same model.


Introduction
Maintenance and dynamic reconfiguration of cell shape represent a selective value for bacteria for both primary and secondary cellular processes, in particular for nutrient acquisition, division, capacity to escape from predators, biofilm formation, and motility (Young, 2006). Therefore natural evolution has led most bacterial species to adopt one or a limited number of morphologies among many more or less complex possibilities, depending on their way of life and their ecological niche (Young, 2007). The main determinant of bacterial cell shape is the peptidoglycan layer surrounding the plasma membrane and forming the cell wall (Egan et al., 2020). The morphological transition of cells into spheres upon inhibition of cell wall synthesis in rod-shaped bacteria demonstrates the essential role of this structure in the maintenance of an elongated bacterial morphology (Claessen and Errington, 2019). In most rod-shaped bacteria, short internal filaments made up of actin-like proteins called MreBs guide the synthesis machinery of the cell wall to ensure cell elongation as deposition and crosslinking of new peptidoglycan units progress (Shi et al., 2018).
Along with cocci and rods, helical or corkscrew morphologies are major shapes adopted by phylogenetically distant bacteria including Helicobacter pylori, spirochetes and spiroplasmas.
In H. pylori, the helical shape of the cell body can significantly contribute to propulsive thrust, (Constantino et al., 2016), or to pathogenicity (Salama et al., 2013). In this species, the cell wall is differentially synthesized based on the curvature of the cell body, with two proteins MreB and CcmA defining the appropriate areas where synthesis is enhanced (Taylor et al., 2020). Helical or wave-like morphologies and motility in spirochetes are primarily determined by the periplasmic flagella, the cell wall and cytoplasmic MreB (Nakamura, 2020).
Spiroplasmas represent a group of helical bacteria apart. Indeed, spiroplasmas belong to the class Mollicutes, characterized by the lack of a peptidoglycan-based cell wall (Whitcomb, 1980). Most Spiroplasma species are pathogens or endosymbionts of arthropods and plants (Regassa, 2006). With the sole exception of the strain Spiroplasma citri ASP-1, all natural Spiroplasma isolates described to date are helical and motile (Harne et al., 2020b) suggesting a selective value of this specific shape and unique motility. Thus, spiroplasmas control their helicity without a peptidoglycan layer, the major determinant of bacterial shape in walled bacteria. In addition, these bacteria are motile in the absence of external appendages such as flagella or pili that allow the motility of the vast majority of bacteria (Nakamura and Minamino, 2019). Recently, studies aiming at elucidating the mechanisms of shape maintenance and motility in Spiroplasma are increasing. Indeed, Spiroplasma appears to be a particularly attractive model for the identification of shape and motility determining factors in a wall-less organism (Harne et al., 2020b).
Spiroplasma cells are polarized, with a tapered (also called tip) and a rounded end (Garnier et al., 1984). The discovery of an internal cytoskeleton composed of the protein fibril (Fib) unique to Spiroplasma (Williamson, 1974) came closely after the discovery of these bacteria (Davis et al., 1972).The main cytoskeleton structure corresponds to a monolayered, flattened ribbon positioned along the shortest helical path (Trachtenberg and Gilad, 2001).
Microscopic observations of cryofixed freeze-substituted preparations combined with tomographic reconstruction confirmed the overall organization and highlighted the membrane association of the internal protein ribbon made of both actin-like MreBs and fibril (Trachtenberg et al., 2008).The internal cytoskeleton also comprises a dumbbell-shaped structure at one cell pole (tapered-end) likely involved in cell polarization (Liu et al., 2017).
The motility of Spiroplasma is due to a helicity change, which is initiated at one of the two ends of the cell and introduces a "kink" in the cell whose propagation is responsible for the movement of the bacteria (Shaevitz et al., 2005). On the cytoplasmic side, the complex dumbbell-shaped structure could be responsible for the generation of the initial twist which is then propagated along the cell .
The presence of at least 5 mreB paralogs in the near-minimal genome of Spiroplasma species , raises some questions regarding the selective benefit provided by the different isoforms during evolution. In the non-helical strain S. citri ASP-1, the loss of helicity and motility is due to a nonsense mutation within the sequence encoding MreB5 that cannot be functionally compensated by any of the other 4 isoforms present in this species (Harne et al., 2020a).Thus MreB5 was identified as a major determinant of cell helicity in S. citri (Harne et al., 2020a).These in vivo results, coupled with differences in polymerization and depolymerization dynamics between isoforms (Masson et al., 2021;Pande et al., 2021;Takahashi et al., 2021), strengthen the hypothesis of functional differentiation between some MreB paralogs.
Despite the qualitative compositional information, structure and mechanisms of the Spiroplasma internal cytoskeleton remain unclear. Recently, based on the structure of the Fib filament, a molecular mechanism, in which fibril is responsible for cell helicity and for its shift has been proposed. Following this model, the length changes in MreB polymers would generate the helicity shift . The construction of Spiroplasma mutants expressing combinatorial sets of MreBs with or without fibril could help validating this motility model and identifying the different roles of MreBs. However, although different tools have been developed to modify Spiroplasma genome, it remains difficult to obtain conditional gene knockdowns, especially for paralogs (Harne et al., 2020b). To circumvent these limitations, one approach is to perform heterologous expression experiments of Spiroplasma cytoskeleton proteins within a phylogenetically-related Mycoplasma species also lacking a cell wall. The choice of Mycoplasma capricolum subsp. capricolum (Mcap) is justified as it belongs to the Spiroplasma phylogenetic group of the Mollicutes class, and it is amenable to genome engineering including using methods derived from synthetic biology (Labroussaa et al., 2016;Lartigue et al., 2009 (Davis et al., 1985;Rottem, 1980), an essential point when considering that cytoskeleton elements were found to be closely associated with the membrane (Trachtenberg et al., 2008).
Here, we investigated whether the reconstruction of Spiroplasma cell structure in Mcap was possible. We then took advantage of this model to obtain clues on the minimal requirements for helical shape and kinking motility in Mollicutes, by comparing the effect of the insertion of different combinations of S. citri genes in Mcap genome on morphology, motility and formation of internal cytoskeleton elements. they were mostly short-rod shaped and coccoid, a few elongated ones (up to 8 microns) but none with a helical morphology. In contrast, with the recombinants resulting from transformation with the transposon carrying the combination of mreB1-5 and fibril genes and named Mcap mreB1-5-fib , the cell morphology was heterogeneous and included helices, long, straight and soft filamentous cells, eventually branched (up to 80 microns), but also partially helical cells, and coccoid cells (Fig. 1A). Helical pitch of the cells, corresponding to the distance between two equivalent points separated by a single turn on the helix, measured parallel to the cell axis and determined from darkfield microscopy images (insert, Fig. 1B), was 1.83+/-1.55 µm in average, a value significantly different from 0.74+/-0.3 µm determined for S. citri (Fig. 1B). Helical cells exhibited bending and kinking movements, mimicking those seen with S. citri ( Fig.1C and Supplemental movie 1). However, kinks were initiated more irregularly than in S. citri and most of them did not spread down the entire length of the cell. Kinks locally travelled with a similar mean velocity V kink in S. citri (V kink =12.0 ± 2 µm/s) and in Mcap mreB1-5-fib (V kink =10.8 ± 2 µm/s). Motility with kink propagation was observed for helical pitches between 0.6 and 1.5 µm. Movements were also observed for non-helical filaments, with the propagation of a thrill along the cell body that induced bending of the elongated filaments. The kink-based cell movements did not provide a directional motility to helical Mcap mreB1-5-fib cells in liquid SP5. Since the medium viscosity minimizes Brownian motion and favors S. citri motility (Boudet et al., 2018), 0.2 to 1% methylcellulose was added to the medium but this additive did not improve the translational movement of the Mcap mreB1-5-fib recombinant (data not shown). Also, while S. citri grown on agar-containing plates form satellite colonies due to the migration of single cells away from the mother colony, no satellites were observed with the Mcap mreB1-5-fib recombinant after growth on nutrient medium containing 0.8% noble agar ( Fig. S2), suggesting that the propulsive force conferred by the addition of S. citri cytoskeleton proteins is not sufficient to efficiently move the cells in one direction under the conditions tested.

Expression of MreBs and Fib in
Mcap had not only a significant effect on cell morphology but also on cell division. Indeed, the presence of long filaments is likely the result of a defective septation during the cell division process. Elongation of the cells that were not correctly separated resulted in branching (Fig. 1A). Impairment in cell division was also correlated with a particular aspect of the colonies grown on SP4-agar medium: while the control transformants gave typical fried-egg colonies, Mcap mreB1-5-fib recombinant growth resulted in more brownish and granular colonies, some with irregular contours (Fig. S2). Such colony morphology could be associated with division arrest for some cells.

Fibril is essential neither for helicity nor for movements, but favors propagation of deformations at the membrane level along the cell body in Mcap
We took advantage of the Mcap heterologous system to assess whether or not helicity and motility could be conferred to the cells when fib only or mreB1-5 only were added to the genome of recombinants (Fig. S3). To avoid the possible impact of the localization of the added genes in the Mcap genome (Table S1), only features common to all clones tested are described below.
Mcap fib recombinants were characterized by a short-rod shape for most cells, but also by the presence of a few short helices having a helical pitch of 1.06+/-0.28 µm (Fig. 2). In the cell population of Mcap mreB1-5 recombinants, short and long helices were also observed, with a mean pitch of 1.72+/-0.67 µm, not significantly different from the pitch of Mcap mreB1-5+fib transformants (1.83+/-1.55 µm). Thus, MreBs were sufficient to confer helicity to Mcap cells.
It is noticeable that for both Mcap fib and Mcap mreB1-5 transformants, the range of helical pitch lengths overlaps the length of that of S. citri, indicating that the Spiroplasma shape can be found with either of the constructions. Defects in septation were observed for most Mcap mreB1-5 cells, with the formation of long, branched filaments. One possible reason is that one or several MreBs may interfere with the division process. Also, some cell bodies showed certain flexibility, while others were characterized by a significant stiffness, possibly due to differences in amounts of cytoskeleton proteins. These problems in division were associated with the formation of brownish, irregular colonies on agar plates smaller than those observed with the control cells, i.e. Mcap transformed with the empty vector (Fig. S2).  Taken together these results suggest that fibril is essential neither for helicity nor for motility, but likely increases the efficiency of propagation of the membrane deformation (kink) along the cell body. In addition, Fib can confer helicity and motility to short helices by itself.

A single MreB is sufficient to confer helicity and to initiate kinks
Proteomics analyses were performed to check the expression of MreBs in S. citri, in one clone of Mcap mreB1-5 and two clones of Mcap mreB1-5+fib . For one Mcap mreB1-5+fib clone, protein expression was also analyzed after 4 and 8 passages in axenic medium. Morphology of exponentially growing cells was checked before extraction of total proteins and LC-MS/MS analysis. MreB5 was the most expressed among MreBs in these transformants, as in S. citri (Table 1). Surprisingly, although all mreB genes were added in all clones, not all MreBs were detected by proteomics. Even more unexpected was the observation that the set of detected proteins was different from one clone to another. More generally, relative abundance of MreBs and Fib in Mcap mreB1-5+fib did not mimic those in S. citri. Fib was detected at a significantly lower level in Mcap mreB1-5+fib , and MreB5 amount after 8 passages in SP5 tet5 was more than 20 times higher than after 4 passages. All clones having shown to be able to form motile helices, different combinations of MreBs were then able to confer helicity and motility. MreB1+MreB5, MreB4+MreB5, MreB1+MreB4+MreB5, and all MreBs were among the MreB combinations generating motile helices (Table 1). It should however be stressed out that the apparently lacking MreBs may be expressed at a level too low to be detected.
Aiming at determining the minimal gene set required for helicity and/or motility, a transformant carrying only either mreB5 or mreB1 was constructed (Fig. S4).
While Mcap transformation efficiencies with all the constructions (except for pMT85-PStetMfib) were rather low (Table S2), the one with pMT85-PStetM-mreB5 was even lower.
Nevertheless, two clones could be recovered after three transformation assays. Mcap mreB5 population showed a large diversity of shapes from coccoid to filamentous ones, including helices with a helical pitch of 0.96+/-0.48 µm, which was not significantly different from those of S. citri helices. Short helices with a length inferior to 5 µm were also endowed with movements, associated with the propagation of membrane deformations along the cell body ( Fig. 3). Although the change in helicity could be visualized (Supplemental movie 2), the cellular stiffness was not sufficient enough to conserve the helicity on the whole length during movements, which led to the transient formation of unwound and untangled filaments.
Longer helices were not found to be motile. Subculturing of the Mcap mreB5 clones led to loss of helicity and motility.
All these observations show that addition of mreB5 is sufficient to confer helicity to Mcap and triggers kink-like membrane deformations for short helices. The morphology of Mcap mreB5 was then compared to those of Mcap mreB1 carrying mreB1 gene alone (Fig. S4). The latter shows helices with a larger pitch (1.81+/-1.08 µm) (Fig. 2). Twitching movements were observed on flexible, non-helical cells, but kink-based motility of helices was not recorded with Mcap mreB1 . Addition of fib gene together with mreB1 (Fig. S5) produced non-motile, stable helices with a mean pitch of 1.35+/-0.97 µm.

Expression of MreBs and Fib was associated with formation of cytoskeleton structures interacting with the plasma membrane
CryoEM was used to assess whether the different gene combinations led to formation of stable cytoskeleton filaments in Mcap. MreB5, and Fib was analyzed using cryoEM. Expression of these proteins induced the production of internal fibers positioned next to the membrane (Fig. 4C). The cytoskeleton could recover the whole inner side of the membrane in some cells, or appear only next to negatively curved membrane areas in others. The latter organization mimicked the Spiroplasma one, and differences observed between cells were likely due to differences from one cell to another in protein amounts. The internal cytoskeleton structure in Mcap mreB1-5-fib corresponded to a stack of filaments with a mean width ranging from 4 to 10 nm, and showing a regular striated pattern with a 5+/-1nm periodicity. Straight cells in which the cytoskeleton covers most of the membrane could correspond to cells showing a significant stiffness observed in cell suspensions as described above.
An internal structure was also observed in Mcap mreB1-5 , i.e. in the absence of fib (Fig. 4D).
The internal filament formed by one or several MreBs ran next to the membrane, in close interaction either with curved membrane areas, or with a larger membrane zone. The width of protofilaments ranged from 4 to 8 nm. The addition of fib together with at least one mreb triggered the formation of internal filaments associated with the membrane and showing the regular striated pattern described for Mcap mreB1-5-fib (Fig. 4C, F, H). showed also internal structures. Expression of MreB1 induced the formation of stable cytoskeleton fibers adjacent to the membrane (Fig. 4E). Filaments made of MreB1 (width ranging from 4 to 8 nm) conferred rigidity to cells when interacting with large membrane segments. These internal structures can recover the entire membrane inner side. In Mcap mreB5 , MreB5 filament was clearly observed only in a single cell (Fig. 4G). MreB5 filament, having a width of up to 27 nm, crossed the whole cell body with localized interactions with the plasma membrane. In the absence of any MreB, expression of Fib allowed the formation of an internal cytoskeleton made of a stack of approximately 10 nm wide filaments, in particular in some tubular protrusions observed for some cells (Fig 4J).

Interestingly, a large number of internalized membrane-bound structures were observed in
Mcap mreB1-5 , Mcap mreB1 and, even more frequently, in Mcap mreB5 (Fig 4I). This observation is in line with the great ability of MreB1 and MreB5 to induce membrane curvature at directed membrane areas, as an excessive curvature could lead to inverse membrane blebbing followed by detachment and internalization of a more or less spherical membrane-bound vesicle.

Identification of the minimal requirements for conferring helicity and motility to Mcap
The Spiroplasma cytoskeleton was reconstituted in a mycoplasma that was initially allowed the tightening of the helices, but the mean helical pitch remained higher than those of S. citri cells. On the contrary, MreB5 expression produced helices with a mean pitch similar to those found in S. citri. Hence each of these proteins represents a minimal requirement for helicity in a wall-less bacterium, and MreB5 the minimal one to mimic Spiroplasma helicity.
Regarding motility, the expression of MreB1 induced disorganized cellular movements, resembling tremors, but the observed membrane deformation did not propagate along the cell body, while MreB5 was able to confer kink-based motility to short helices. Therefore, a single MreB, such as MreB5, represents the minimal requirement to produce helical cells endowed with kink-like motility. However, the cytoskeleton resulting from the expression of a single MreB was not sufficient to allow the conservation of the cell length and helicity upon propagation of membrane deformation. Changes in cell length were likely responsible for loss of helix directionality. A similar phenotype was obtained when transforming Mcap with fib only. In Spiroplasma, fibril forms polymers whose length remains the same during motility , ensuring the efficient conservation of the helical shape when the kink travels along the cell. In Mcap, the sole addition of fib did not provide the required helix stiffness. As discussed above, the co-expression of Fib with MreB5 allowed the stabilization of long helices, suggesting that a MreB could help in stabilizing the Fib-based cytoskeleton. However, in this configuration, the energy transfer to the membrane coupled by MreB5 and by Fib was not sufficient to induce membrane deformations propagating along the entire cell length. This could be due to the limited efficacy of the Fib-MreB5 combination in deforming a cell, which had become more rigid thanks to the presence of a Fib filament as Spiroplasma, but also provides clues regarding the cytoskeleton organization. In Mcap, the width of MreB filaments could be as thin as 4 nm, similar to those of unidentified filaments previously observed in S. citri (Trachtenberg et al., 2003), which validates our heterologous system. In rod-shaped, walled bacteria, MreBs establish a direct interaction with the plasma membrane (Salje et al., 2011). The capacity of MreB5 to interact with lipid bilayers in vitro was previously demonstrated (Harne et al., 2020a). In Mcap, both MreB1 and MreB5 induced a plasma membrane curvature and formed filaments that were closely associated with the membrane. This ability to distort membranes is common to some other MreBs, as shown for TmMreB, an MreB from the thermophilic archaeon Thermotoga maritima (Salje et al., 2011) (Rottem, 1980), could occur and favor the interaction of Spiroplasma MreBs with membrane parts enriched in specific lipids. Of note, bundles of thick filaments were associated with rigid, straight cell morphology, indicating that helicity likely requires a thinner filament. Also, our cryoEM experiments indicate that MreB1 and B5 filaments could span the whole Mcap cell length over several micrometers. In most rod-shaped bacteria, the current prevalent hypothesis assumes that MreBs form short membrane-associated filaments (Shi et al., 2018) but their length is still debated (Errington, 2015). The length of MreB1 and B5 filaments in Mcap may differ from those of MreB filaments in Spiroplasma, as their expression level is probably not the same in the two species.

Role of MreBs in motility
The demonstration of a role of MreB in bacterial motility has so far only been obtained in gliding motility of Myxococcus xanthus (Treuner-Lange et al., 2015). A role for MreB5 in Spiroplasma swimming was also suggested since the complementation of the non-helical, non-motile S. citri ASP-1 with mreB5 from the helical and motile S. citri GII-3 restored not only helicity but also kinking motility (Harne et al., 2020a). Nonetheless, Spiroplasma kinking motility requires helicity. Therefore, motility restoration in mreB5-complemented ASP-1 could be due to helicity recovery and the role played by MreB5 in motility could then be indirect.

Differential polymerization kinetic parameters of MreB isoforms measured in vitro led
Sasajima and collaborators  to propose a theoretical model for Spiroplasma swimming mechanism in which two MreBs would generate a force similar to that of a bimetallic strip. This force would then be transmitted to fibril polymers and result in change in handedness of helical fibril filaments. This model would fit our data showing that expression of two distinct MreBs allowed both helicity and kinking motility. However, a single MreB, MreB5, was also able to endow Mcap cells with movements resulting from the spread of a membrane deformation along the cell body. In most species, MreB filaments are highly dynamic polymers that align along the greatest principal membrane curvature (Hussain et al., 2018) and show circumferential motion following a path mostly perpendicular to the long axis of the cell (Morgenstein et al., 2015). In rod-shaped, walled bacteria, this motion is

Role of fibril in Spiroplasma shape and motility
The present work also sheds light on the possible involvement of the fibril in the generation of helicity, its maintenance and motility. Fib filaments were observed in close interaction with S. citri plasma membrane in previous studies (Kürner et al., 2005;Trachtenberg et al., 2008). It was long thought that fibril was responsible for Spiroplasma motility by changing its length (Kürner et al., 2005). However recent studies indicated that the length of Fib polymers do not change during cell movement . The structure of Fib filaments has been studied by electronic microscopy: Fib filaments show an helicity with a pitch close to those of the Spiroplasma cell, leading to the conclusion that Fib was the determinant of helicity in Spiroplasma . Our results indicate that Fib is able to form membrane-interacting polymers in Mcap with a width similar to those observed in Fib isolated filaments (Trachtenberg et al., 2003;, and induce membrane curvature in the absence of any MreB. However, the helix constructed with Fib only is in a more relaxed form than those observed when MreB5 and Fib were co-expressed, suggesting that MreB5 could help positioning the Fib filament by interacting with both Fib and the membrane. This is in line with the ability of MreB5 to bind both fibril and liposomes in vitro (Harne et al., 2020a). Given its well-adapted helical structure and the rigidity it confers to cells, Fib could be a major determinant in helical shape maintenance. Regarding long helix generation, Fib had to be associated with MreB5 in Mcap, and probably in Spiroplasma also. Our work also provides some clues about the role played by Fib in motility. Fib allowed Mcap to form helices endowed with a motility very similar to those observed with MreB5. This is intriguing because Fib and MreB do not share any sequence similarity, and unlike MreBs, Fib lacks ATPase activity . In the most recently proposed model for Spiroplasma motility, MreBs transmit the force to the fibril filament which changes its handedness to generate the shift in the cell body helicity .
We propose that fibril is not only an essential structural component for transmission of the torque to the membrane, but also participates itself to the generation of the required force.
Interestingly the genome of a few motile Spiroplasma species including S. sabaudiense (Abalain-Colloc et al., 1987) lacks the fibril gene (Chang et al., 2014), but contains more than 5 mreB genes (Harne et al., 2020b;Ku et al., 2014). It is therefore tempting to suggest an evolutionary convergence between MreB and fibril to ensure motility.

Lack of an efficient swimming in Mcap recombinants
It is noteworthy that none of the Mcap recombinants exhibits translational motility in liquid medium. Among the hypotheses that can explain this observation is the lack of cell polarization in recombinants. It is reasonable to assume that a firm attachment of the internal cytoskeleton ribbon at the two poles of the cells is required to ensure translational movement. In Mcap recombinants, such attachment may be lacking. In addition, the Spiroplasma dumbbell-shaped core (Liu et al., 2017) was absent in Mcap transformants. We could show here that this structure was not required for kink generation and propagation, but it may be essential to generate an initial kink located at the extremity of the cell. Also, the tapered shape of the Spiroplasma tip may favor the penetration and propulsion in liquid media. Finally, the relative abundance of the proteins did not match those observed in S.
citri. More specifically, Fib was detected at a low level, whereas the level of expression of MreB5 was deregulated, as indicated by its increase during subculturing in Mcap mreB1-5+fib . A proper stoichiometry of these proteins is likely to be required for optimal cell stiffness, stability of the helical structure in Mcap recombinants during the propagation of the kink, and efficient swimming.

Conclusion
The present study demonstrates the importance of structure and organization of MreBs and fibril in determining helicity and motility in Spiroplasma. It provides some clues that leads to a structural model in which (i) the MreB5 filaments interact specifically with membrane areas enriched in yet unidentified lipids and induce membrane curvature, (ii) MreB5 determines the correct localization of MreB1 and fibril, which both participate to the membrane curvature, (iii) MreB1, MreB5 and Fib polymerize into filaments along the plasma membrane following the shortest path of the cell body, and the MreB/Fib association stabilize the copolymer structure. The fibril transmits the cumulative force generated by MreBs and itself to the membrane to form and propagate the kink along the cell helix. The role of the other MreBs (B2, B3 and B4) remains to be elucidated. As discussed above, it is plausible that MreB4 also constitutes the cytoskeleton ribbon, and that functions of MreB1 and MreB4 are overlapping. This study opens up new perspectives, in particular for understanding the swimming mechanism in Spiroplasma. Now that the involvement of MreBs in motility has been demonstrated, it appears essential to understand how the expression of MreBs is regulated during the cell cycle in order to distinguish their functions associated with division, from those linked to the maintenance of helicity and motility. Further studies will also be required to elucidate the mechanism of force generation by the cytoskeleton components and its transmission to the membrane, as well as to determine whether the tapered shape of the Spiroplasma end allows the propulsion of the cell in liquid medium.

Bacterial strains and culture conditions
Escherichia coli (NEB® 10-beta Electrocompetent E. coli or NEB® 5-alpha Competent E. coli High Efficiency) served as host strains for cloning experiments and plasmid propagation.
Plasmid-transformed E. coli cells were grown at 37°C in Luria-Bertani (LB) broth or on LB agar supplemented with ampicillin at 100µg/ml and tetracycline 5 µg/mL.
The restriction free M. capricolum subsp. capricolum strain California KidT (Mcap) was used in this study. This strain was obtained by inactivation of the CCATC-restriction enzyme in the wild-type strain (ATCC 27343) (Lartigue et al., 2009). Mcap as well as its derivatives were grown at 37°C in SP5 medium (Tully et al., 1977) supplemented with tetracycline 5 µg/mL.
One passage corresponds to the transfer of 10 μ L of mycoplasma culture (at pH~6.5) to 1 mL of mycoplasma medium (1/100 dilution) followed by an incubation period of ≥ 24h (depending on the constructions) at 37 °C. SP5 medium containing 0.8% noble agar was used to grow colonies of Mcap and its derivatives.
S. citri strain GII-3 was originally isolated from its leafhopper vector C. haematoceps captured in Morocco (Vignault et al., 1981). Spiroplasmas were cultivated at 32°C in SP4 medium. SP4 containing 0.8% noble agar was used to grow colonies of S. citri.

Plasmid construction
Seven plasmids were built during this study. All derive from the transposon based-plasmid Five plasmids were constructed using the NEBuilder® HiFi DNA Assembly Cloning Kit (Table S3 and Figures S1, S3, S4, S5). Depending on the assemblies, two to four overlapping DNA fragments were PCR amplified (Advantage HF 2 PCR Kit from Clontech) using primers described Table S4, purified and combined at 50°C according to the manufacturer's instructions. DNA cassettes were designed to contain ~40bp overlaps. One DNA cassette corresponds to the whole transposon based-plasmid pMT85-PStetM. Others correspond to different regions of S. citri GII-3 genome: the fibril gene (SPICI12_006), the mreB1 gene (SPICI13_009) and the locus composed of mreB2, mreB3, a hypothetical protein encoding gene (HP), mreB4 and mreB5 (SPICI01A_045 to SPICI01A_049). S. citri DNA cassettes were amplified so that native promoters were conserved.
All the oligonucleotides for plasmids constructions were supplied by Eurogentec and are described in Table S4.Prior to being used for transformation into Mcap, the purified plasmids were verified by restriction analyses and sequencing.

Mycoplasma transformation and screening
Mcap was grown in SOB+ medium at 30°C and transformed using the established 5% polyethylene glycol (PEG) mediated protocol (Labroussaa et al., 2016). Transformations were conducted using 5 to 10 µg of plasmids and transformants were grown on selective medium SP5 plus tetracycline 5 µg/mL (SP5 tet5) for 7 to 21 days depending on the construction.
Colonies obtained on selective plates were picked and transferred into 1 mL of SP5 tet5 liquid medium and incubated at 37 °C. After three passages, 200 µL were used for DNA extraction and PCR analysis and 800µL were stored at −80 °C.
Transformants genomic DNA was extracted with the NucleoSpin® Tissue kit (Macherey-Nagel, Düren, Germany) and further analyzed by (i) PCR to verify the presence of the tet(M) and S. citri genes (fibril, mreb1, mreb2-5) and (ii) direct sequencing to localize the transposon insertion site (see below).
Primers used for PCR, direct sequencing and localization of insertions into Mcap genomes are summarized Table S4.
The PCR amplification cycle was performed as previously described (Dordet Frisoni et al., 2013).
Transposon insertion sites were determined by Sanger sequencing of the PCR products with the nested primer MT85-1 (Table S4).

Dark-field microscopy, cell length measurements:
One volume of cultures of exponentially growing Mcap in SP5 tet5 and of S. citri in SP4 (pH 6.9) was diluted in two volumes of fresh medium. Bacterial suspensions were prepared between two microscope slides, with a liquid thickness of 15 µm. The morphology of Mcap transformants growing in SP5 media was observed using an Eclipse Ni (Nikon) microscope working in reflection and equipped with a dark field condenser. The Nikon oil immersion microscope objective was a 60X with a numerical aperture (N. A.) of 0.80. Pictures were taken with a camera Iris 9™ Scientific CMOS (2960 x 2960 pixels). Videos were recorded at the maximal frame rate, 10 to 30 frames per second (fps) using the software NIS-Elements Br (Nikon). Mycoplasma cell length and helicity parameters were measured from isolated frames using the same software. Helical pitch measurements excluded non-helical cells. The data are expressed as the mean ± standard deviation (SD). Statistical significance was estimated by the unpaired t test, two tailed. Time-lapse images were isolated from the video recordings.

Cryo Electron Microscopy
Lacey carbon formvar 300 mesh copper grids were used. They were first submitted to a standard glow discharged procedure (3 mbar, 3 mA for 40 s). Plunge freezing was realized using the EM-GP apparatus (Leica). Four microliters of the sample was deposited on the grid and immediately blotted for 2 s with a Whatmann paper grade 5 before plunging into a liquid ethane bath cooled with liquid nitrogen (−184 °C). The settings of the chamber were fixed at 80% humidity and 20 °C. Specimen were used non-diluted in culture medium. They were observed at −178 °C using a cryo holder (626, Gatan), with a ThermoFisher FEI Tecnai F20 electron microscope operating at 200 kV under low-dose conditions. Images were acquired with an Eagle 4k x 4k camera (ThermoFisher FEI) and processed in ImageJ.

Proteomics
Mycoplasma and Spiroplasma cells were harvested by centrifugation at 10,000 g, 20 min, before being washed twice with Dulbecco's phosphate-buffered saline (Eurobio) (mycoplasmas), or with a solution containing 8 mM Hepes and 280 mM sucrose (spiroplasmas). Protein concentration was determined using the DC Protein Assay (Bio-Rad). Fifteen micrograms of proteins were mixed with SDS loading buffer (75 mM Tris-HCl, pH 6.8, 50 mM DTT, 2% SDS, 0.02% bromophenol blue, 8.5% glycerol), then loaded onto a SDS-PAGE stacking gel (7%). A short electrophoresis was performed (10 mA, 45 min and 20mA, 2h) in order to concentrate proteins. After migration, gels were stained with Coomassie Blue and destained (50% ethanol, 10% acetic acid, 40% deionized water). The revealed protein band from each fraction was excised, washed with water, and then immersed in a reductive solution (5 mM DTT). Cysteines were irreversibly alkylated with 20 mM iodoacetamide in the dark. Following washing and drying steps, gel bands were submitted to protein digestion with trypsin added to a final protease to protein ratio of 1:25, for 3 hours at 37 °C, in ammonium bicarbonate buffer (10 mM and pH 8). Peptides were extracted with 50% CH 3 CN, followed by 0.1% TFA, and finally 100% CH 3 CN. The collected samples were then dried. For each clone, three technical replicates were performed.
Tandem Mass Spectrometry. Peptides were then analyzed by mass spectrometry. All experiments were performed on a LTQ-Orbitrap Elite (Thermo Scientific) coupled to an Easy-nLC II system (Thermo Scientific). One microliter of sample (200 ng) was injected onto an enrichment column (C18 Acclaim PepMap100, Thermo Scientific). The separation was performed with an analytical column needle (NTCC-360/internal diameter: 100 μ m; particle size: 5 μ m; length: 153 mm, NikkyoTechnos, Tokyo, Japan). The mobile phase consisted of H 2 O/0.1% formic acid (FA) (buffer A) and CH 3 CN/FA 0.1% (buffer B). Tryptic peptides were eluted at a flow rate of 300 nL/min using a three-step linear gradient: from 2 to 40% B over 76 min, from 40 to 100% B in 4 min and 10 min at 100% B. The mass spectrometer was operated in positive ionization mode with capillary voltage and source temperature set at 1.8 kV and 275 °C, respectively. The samples were analyzed using CID (collision induced dissociation) method. The first scan (MS spectra) was recorded in the Orbitrap analyzer (r = 60,000) with the mass range m/z 400-1800. Then, the 20 most intense ions were selected for tandem mass spectrometry (MS 2 ) experiments. Singly charged species were excluded for MS 2 experiments. Dynamic exclusion of already fragmented precursor ions was applied for 30 s, with a repeat count of 2, a repeat duration of 30 s and an exclusion mass width of ±5 ppm. Fragmentation occurred in the linear ion trap analyzer with collision energy of 35. All measurements in the Orbitrap analyzer were performed with on-the-fly internal recalibration (lock mass) at m/z 445.12002 (polydimethylcyclosiloxane). Resulting raw files were analyzed with Proteome Discoverer 1.4.1.14 (Thermo Scientific). Database search was performed with Mascot (version 2.8.0) algorithm against "GCF_000012765.1_ASM1276v1_protein" (801 entries) retrieved from NCBI and containing the protein sequences encoded in M. capricolum subsp. capricolum genome, and a multifasta containing S. citri GII-3 and plasmid-encoded protein sequences. The following search parameters were used: trypsin was specified as the enzyme allowing for one mis-cleavage; carbamidomethyl (C) and oxidation (M) were specified as variable modifications; precursor mass range was set between 350 and 5000 Da, with a precursor mass tolerance and a fragment ion tolerance of 5 ppm and 0.3 Da respectively. Peptide validation was performed using Percolator algorithm (Käll et al., 2007) and only « high confidence » peptides were retained corresponding to 1% false discovery rate. A minimum of 2 PSMs and 2 unique peptides were required to consider protein identification.