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

Spiroplasma swim by switching their helical body into right- and left-handed. Helicity formation and switching can be reconstituted in an immotile minimal synthetic bacterium, JCVI-syn3B by introducing the pair of bacterial actin, MreB4 and MreB5. Here, we analyzed MreB behaviors optically, to investigate this unknown mechanism. We tried MreB4 fluorescence labeling by protein fusion. The labeling was not achieved, because the fusion of fluorescent protein or peptide to 16 positions resulted in immotile constructs. These results may suggest that MreB4 has many interaction interfaces with other proteins. Induced expression of MreB4 to cells with constitutive MreB5 expression resulted in earlier and higher frequency of motile cells, distinct from the results of constitutive MreB4 and inducible MreB5. Next, the behavior of labeled MreB5 was analyzed. Photobleaching and photoactivation suggested static behavior of MreB5 during cell movements. Cell treatment by A22, a 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. SignificanceSpiroplasma MreB is a unique bacterial actin that causes motility with a different mechanism from other actins. Here, we investigated the helicity switching involved in the motility. Fluorescently traced MreB behaviors suggested that the motility mechanism is not coupled with its replacement in the internal structure.