Molecular tug-of-war regulates Bacillus subtilis elongasome dynamics and bacterial cell shape

Abstract

The shape and size of a bacterial cell is determined its peptidoglycan cell wall. During growth in many rod-shaped bacteria, the elongasome, comprising peptidoglycan synthases and other shape-determining proteins, assembles upon an MreB filament. The elongasome moves processively around the cell circumference, inserting new material into the cell wall. We investigated how Rod complex dynamics are regulated to construct a uniform, rod-shaped sacculus In the Gram-positive model organism Bacillus subtilis I developed a novel method, single molecule Vertical Cell Imaging by Nanostructured Immobilisation (SM-VerCINI) that allows extended observation of MreB trajectories around the whole circumference of the cell. I found that MreB filaments are highly processive, travelling on average halfway around the cell circumference (1.6 µm) until pausing, reversing or unbinding. I found that the elongasome pauses and reverses far more frequent than previously thought, and the rate of elongasome pausing and reversal is set by cellular levels of elongasome synthase RodA. Further, RodA levels determine elongasome speed and processivity, which leads to subsequent changes in cell shape. These results are consistent with molecular motor tug-of-war, where synthesis complexes attach to opposite ends of the MreB filament, causing peptidoglycan synthesis termination and sudden reversal via tug-ofwar competition. My results show that elongasome synthase tug-of-war is a key regulator of MreB filament dynamics and likely also determines cell shape in B. subtilis.