Isolation and structure of the fibril protein, a major component of the internal ribbon for Spiroplasma swimming

Yuya Sasajima; Takayuki Kato; Tomoko Miyata; Akihiro Kawamoto; Keiichi Namba; Makoto Miyata

doi:10.3389/fmicb.2022.1004601

Isolation and structure of the fibril protein, a major component of the internal ribbon for Spiroplasma swimming

Front Microbiol. 2022 Oct 6:13:1004601. doi: 10.3389/fmicb.2022.1004601. eCollection 2022.

Authors

Yuya Sasajima¹, Takayuki Kato², Tomoko Miyata³, Akihiro Kawamoto², Keiichi Namba^{3

4

5}, Makoto Miyata^{1

6}

Affiliations

¹ Graduate School of Science, Osaka Metropolitan University, Osaka, Japan.
² Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
³ Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
⁴ RIKEN Center for Biosystems Dynamics Research and Spring-8 Center, Suita, Osaka, Japan.
⁵ JEOL YOKOGUSHI Research Alliance Laboratories, Osaka University, Suita, Osaka, Japan.
⁶ The OCU Advanced Research Institute for Natural Science and Technology (OCARINA), Osaka Metropolitan University, Osaka, Japan.

Abstract

Spiroplasma, which are known pathogens and commensals of arthropods and plants, are helical-shaped bacteria that lack a peptidoglycan layer. Spiroplasma swim by alternating between left- and right-handed helicity. Of note, this system is not related to flagellar motility, which is widespread in bacteria. A helical ribbon running along the inner side of the helical cell should be responsible for cell helicity and comprises the bacterial actin homolog, MreB, and a protein specific to Spiroplasma, fibril. Here, we isolated the ribbon and its major component, fibril filament, for electron microscopy (EM) analysis. Single-particle analysis of the fibril filaments using the negative-staining EM revealed a three-dimensional chain structure composed of rings with a size of 11 nm wide and 6 nm long, connected by a backbone cylinder with an 8.7 nm interval with a twist along the filament axis. This structure was verified through EM tomography of quick-freeze deep-etch replica sample, with a focus on its handedness. The handedness and pitch of the helix for the isolated ribbon and fibril filament agreed with those of the cell in the resting state. Structures corresponding to the alternative state were not identified. These results suggest that the helical cell structure is supported by fibril filaments; however, the helical switch is caused by the force generated by the MreB proteins. The isolation and structural outline of the fibril filaments provide crucial information for an in-depth clarification of the unique swimming mechanism of Spiroplasma.

Keywords: cytoskeleton; electron tomography; filament; helical shape; motility; quick freeze replica electron microscopy; single particle analysis.