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

Abstract

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. However, the mechanism is unknown. Here, to outline this mechanism, we analyzed MreB behaviors optically. We tried MreB4 fluorescence labeling by protein fusion. The labeling was not achieved, because the fusion of fluorescent protein or peptide at 16 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 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 by photobleaching and photoactivation, suggesting 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. These results suggest that the movement is caused by conformational change of MreB5 filament induced by MreB4 without obvious replacements of MreB5 subunits.

Caption of Graphical Abstract

MreB4 switches right-handed cell to left-handed with no obvious replacement of MreB5 molecules, which is responsible for the cell helicity (Orange dotted line). Addition of polymerization inhibitor A22 causes helix deformation, movement stall, and diffusion of MreB5 molecules when the cell is moving (Blue dotted line).