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

Ovoid-shaped bacteria, such as Streptococcus 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. SIGNIFICANCEThis paper reports three types of single-molecule motions of PG synthesis proteins in the ovoid-shaped, pathogenic bacterium Streptococcus pneumoniae, not reported previously in other bacteria. The core elongasome exhibits zonal, circumferential motion in the absence of MreB filaments, independent of FtsZ treadmilling or the processive movement of the septal PG synthase. Class A aPBP1a also moves processively at midcell, but is not a persistent component of the core elongasome. These types of motions have implications for the functions of these PG synthases and indicate that processive motion in pneumococcus follows spatially separate tracks, possibly reflective of PG structure. In contrast, the MpgA muramidase displays a different kind of subdiffusive motion that is largely confined to midcell by an unknown mechanism.