Spiroplasma are bacteria with a helical cell shape and a lack of 17 peptidoglycan. They belong to the phylum Tenericutes that evolved from 18 Firmicutes, which includes the bacterium Bacillus subtilis. Spiroplasma swimming 19 motility is unrelated to widespread bacterial motilities, such as flagellar motility, and 20 is caused by helicity switching with kinks traveling along the helical cell body. The 21 swimming force is likely generated by five classes of bacterial actin homolog 22 MreBs involved in the helical bone structure. We analyzed sequences of 23 Spiroplasma MreBs (SMreBs) to clarify their phylogeny and sequence features. 24 The maximum likelihood method based on around 5,000 MreB sequences showed 25 that the phylogenetic tree was divided into several radiations: MreBs of 26 conventional bacteria, MreBs of candidate phyla radiation, and radiations for 27 Firmicutes MreB isoforms. SMreBs formed a clade adjacent to the radiation of 28 MreBH, an MreB isoform of Firmicutes. Sequence comparisons of SMreBs and 29 Bacillus MreBs were also performed to clarify the features of SMreB. In SMreBs 2 30 and 4, amino acids involved in the interactions for inter-and intra-protofilament 31 were significantly different from those in Bacillus MreBs. Catalytic glutamic acid 32 and threonine were changed to aspartic acid and lysine, respectively, in SMreB3. 33 2 SMreB5 had a significantly longer C-terminal region than the other MreBs, which 34 possibly forms a protein-protein interaction unique to Spiroplasma. A membrane-35 binding region was not identified in most SMreBs 1 and 4, although it is conserved 36 in many walled bacterial MreBs. These features may support the functions 37 responsible for the unique mechanism of Spiroplasma swimming.38 39 IMPORTANCE Spiroplasma, a small bacterial group, is parasitic to plants and 40 arthropods. Spiroplasma species are globally widespread and swim via a "helicity 41 switching" mechanism. This mechanism is caused by fibril, a Spiroplasma-specific 42 protein, and five classes of MreBs, bacterial actin belonging to the same family as 43 major muscle protein. This study clarified the evolutional and structural features of 44 MreBs and provided possible clues to control the industrial damage caused by 45 Spiroplasma and develop manageable nano-devices to mimic helicity switching.46 47 48 50 The phylum Tenericutes consists of bacteria that evolved from the phylum 51 Firmicutes (1, 2). Currently, Tenericutes is comprised only of the class Mollicutes, 52 which includes the genera Spiroplasma and Mycoplasma. Species of these genera 53 are characterized by small genome size, low guanine and cytosine contents, being 54 mostly parasitic or commensal, and absence of peptidoglycan (3). In addition, they 55 cannot equip conventional machineries for bacterial motility, such as flagella or 56type IV pili, due to the lack of a peptidoglycan layer (4). Instead, they have 57 developed three unique motility systems: mobile type gliding, pneumoniae type 58 gliding, and Spiroplasma swimming (...