Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics inBacillus subtilis

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is primarily performed by a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference ofBacillus subtiliscells for minutes-long periods using single molecule fluorescence microscopy. We found that theB. subtiliselongasome is highly processive and that processive synthesis events are frequent terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes bound to the MreB filament. Our data, thus, demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics inB. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.