The peptidoglycan (PG) cell wall is a gigantic mesh-like molecule that determines bacterial size, shape, and chaining, required for survival in hosts and environmental niches. In Gram-(+) bacteria like Streptococcus pneumoniae, PG also acts as the scaffold for covalent attachment of other surface macromolecules. The regulation of PG synthesis is a fundamentally important spatial and temporal biological problem that involves interactions, assembly, and disassembly of a large ensemble of proteins and expression of these proteins at levels that are correct for normal growth and changed during stress. The long-term goal of this grant is to determine the protein interactions and circuits that regulate PG synthesis in the bacterial pathogen, S. pneumoniae (pneumococcus), which is used as a model for ovoid-shaped bacteria in these mechanistic, basic- science studies. This grant will answer the following important, interrelated questions about pneumococcal septal and peripheral (sidewall-like) PG synthesis, which both emanate from midcell FtsZ rings. Starting with FtsZ rings, how do new FtsZ rings find and assemble at equators of new daughter cells? What are the directional movements and chronology of interactions of proteins that assemble and stabilize the FtsZ ring at different stages of cell division? What roles do known and newly discovered regulatory proteins and their phosphorylation by a Ser/Thr kinase play in FtsZ ring assembly and stabilization and in PG synthesis? Moving to PG synthesis, what are the composition, directional movement, and coordination of the machines that carry out septal and peripheral synthesis during the cell cycle? Which interactions with regulatory proteins mediate the unidirectional movement of Class B penicillin-binding proteins (PBPs) detected along mature septal rings? What are the modalities and interactions of the Class A PBPs, SEDS transglycosylases, and regulatory proteins that balance septal and peripheral PG synthesis during the cell cycle? How do mutations that alter PG synthesis or its regulation affect PG composition and structure? On the related topic of PG remodeling, what is the mechanism by which FtsEX activates PcsB PG hydrolase activity? Which divisome proteins interact with FtsEX:PcsB to activate PG hydrolysis? What is the primary role of FtsEX:PcsB in cell separation? Finally, regarding setting protein amounts, how does the KhpAB RNA binding protein post-transcriptionally regulate FtsA amount, and does conserved KhpAB act as a general RNA chaperone? How does the second messenger cyclic-di-AMP regulate pneumococcal PG synthesis? How does alteration of the metabolite precursor pathway for PG synthesis suppress the requirement for essential PBPs? These questions will be answered by a systems approach that combines powerful genetic, physiological, cell biological (e.g., high-resolution 3D-SIM and TIRFm-SIM), and biochemical (e.g., UHPLC-MS/MS) methods to attack this multicomponent problem. This grant will fill in major gaps about the regulation of PG synthesis in a model ovoid-shaped bacterium, identify functions of reported virulence factors, and provide new targets and vulnerabilities for antibiotic development.