Live imaging of bacterial actin MreBs from Spiroplasma causing helicity switching of a minimal synthetic cell

Spiroplasma swim by switching the handedness of their helical bodies between right- and left-handed. Helicity formation and switching can be reconstituted in an immotile minimal synthetic bacterium, JCVI-syn3B by introducing a pair of bacterial actins, MreB4 and MreB5 from Spiroplasma eriocheiris. However, the mechanism is unknown. Here, to elucidate this mechanism, we analyzed MreB behaviors optically. We tried MreB4 fluorescence labeling by protein fusion. Labeling was unsuccessful because the fusion of fluorescent proteins or peptides at 16 different positions resulted in immotile cells. These results may suggest that MreB4 has many interfaces interacting with other proteins. To obtain suggestions for roles of MreB4 and MreB5, we tried induction of individual MreBs. Induced expression of MreB4 in cells with constitutive MreB5 expression resulted in earlier onset and higher frequency of motile cells, distinct from the results of constitutive MreB4 and inducible MreB5. Next, the behavior of labeled MreB5 was analyzed by photobleaching and photoactivation, suggesting static behavior of MreB5 during cell movements. Cell treatment with A22, an MreB polymerization inhibitor caused helix deformation, movement stall, and diffusion of MreB5 fluorescence, suggesting that A22 sensitive MreB5 interaction should be involved in helix formation and motility. These results suggest that the movement is caused by conformational change of MreB5 filament induced by MreB4 without obvious replacements of MreB5 subunits.