Peptidoglycan (PG) is the mesh-like scaffolding that determines the shape and size of bacterial cells and protects them from osmotic shock. In Gram-positive bacteria, thick PG provides the outer cellular layer to which wall- teichoic acids, capsule, and extracellular proteins are covalently attached. The human respiratory pathogen Streptococcus pneumoniae (pneumococcus) has emerged as a leading model for PG synthesis and its regulation in ovoid-shaped Gram-positive bacteria. Pneumococcal PG synthesis shows fundamental differences from PG synthesis in rod-shaped and spherical bacteria. Most notably, PG synthesis is zonal and carried out by two separable PG synthase nanomachines confined to the midcell of dividing pneumococcal cells. The septal PG synthase (bPBP2x:FtsW) locates to the leading edge of the closing septal annulus, while the elongasome PG synthase (bPBP2b:RodA with RodZ, MreCD) locates to the outer rim of the septal annulus and pushes peripheral PG outward. Both the septal and elongasome PG synthases move circumferentially at midcell driven by PG synthesis, but not by FtsZ treadmilling. This project builds on previous findings to address the most important current questions about PG synthesis in S. pneumoniae. One set of questions centers on the functions and regulation of the three Class A PBP PG synthases (aPBP1a, aPBP1b, aPBP2a) in exponentially growing and stressed pneumococcal cells, about which relatively little is known. By using a comprehensive approach that includes innovative assays of septal and elongation PG synthesis rates in live cells, single-molecule (sm) motion dynamics, and high-resolution microscopy, we will determine the contributions of the aPBPs to PG synthesis and how their dynamics and localization are altered by mutational changes and cell-wall stress. Unbiased and directed approaches will be used to identify aPBP interactors at different stages of division and how these interactions regulate aPBP functions. A second set of questions center on the mechanisms that organize and regulate the septal and elongasome PG synthase nanomachines. We will determine mechanisms that organize circumferential motion and nodal distributions of PG synthases at midcell. We will also determine the assembly pathway of the septal divisome in early-divisional cells and the mechanisms by which the septal and elongasome PG synthesis machines separate and are regulated later in division. We will continue studies of newly discovered mechanisms that link PG synthase functions to the availability of PG precursor metabolites and to second messengers. A third set of questions concerns the roles, interactions, and regulation of the FtsEX:PcsB hydrolase in PG remodeling and the MpgB and MpgA muramidases in PG-chain release in septal and elongasome PG synthesis, respectively. Altogether, this project will fill in major knowledge gaps about the dynamics, functions, and regulation of aPBPs, about the organization and regulation of septal and elon...