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.
