Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell ofStreptococcus pneumoniae

Abstract

ABSTRACTOvoid-shaped bacteria, such asStreptococcus pneumoniae(pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed non-processive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.SIGNIFICANCEThese results demonstrate that unlike in rod-shaped bacteria, the core elongasome ofS. pneumoniaeexhibits zonal, circumferential motion. This motion is independent of FtsZ treadmilling or the presence of MreB filaments and is separate from the circumferential motion of the septal PG synthase that closes the septal annulus. Also unlike in rod-shaped bacteria, a Class A PBP moves processively at midcell, distinctly from components of the core PG elongasome or septal PG synthase. Thus, processive, circumferential motion in pneumococcal cells follows spatially separate linear tracks that may reflect a common ordered structure in the existing peptidoglycan itself. In contrast, the MpgA muramidase displays a different kind of subdiffusive motion that is largely confined to midcell by an unknown mechanism.