Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis.

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and 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 of Bacillus subtilis cells for minutes-long periods using single-molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently 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 associated with the MreB filament. Altogether these results demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

Evidencing New Roles for the Glycosyl-Transferase Cps1 in the Phytopathogenic Fungus Botrytis cinerea.

The fungal cell wall occupies a central place in the interaction between fungi and their environment. This study focuses on the role of the putative polysaccharide synthase Cps1 in the physiology, development and virulence of the grey mold-causing agent Botrytis cinerea. Deletion of the Bccps1 gene does not affect the germination of the conidia (asexual spores) or the early mycelial development, but it perturbs hyphal expansion after 24 h, revealing a two-phase hyphal development that has not been reported so far. It causes a severe reduction of mycelial growth in a solid medium and modifies hyphal aggregation into pellets in liquid cultures. It strongly impairs plant penetration, plant colonization and the formation of sclerotia (survival structures). Loss of the BcCps1 protein associates with a decrease in glucans and glycoproteins in the fungus cell wall and the up-accumulation of 132 proteins in the mutant's exoproteome, among which are fungal cell wall enzymes. This is accompanied by an increased fragility of the mutant mycelium, an increased sensitivity to some environmental stresses and a reduced adhesion to plant surface. Taken together, the results support a significant role of Cps1 in the cell wall biology of B. cinerea.