PROJECT SUMMARYBacteria are surrounded by a cell envelope that is essential for growth, integrity, and pathogenesis. Theenvelope and the biogenesis pathways that build it are also the target of many of our most effective antibioticand vaccine therapies. Because cell envelope biogenesis has been such a successful target, it has been anactive area of research for over half a century. Most of the genes responsible for the synthesis and remodelingof the different surface polymers have been identified and their biochemical activities characterized. However,our understanding of how these different assembly pathways are regulated and coordinated with each otherduring growth remains limited.This proposal focuses on two outstanding questions related to how bacteria coordinate envelope assembly,both principally focused on the cell wall peptidoglycan (PG). Cell growth requires PG synthesis but also theactivity of cell wall hydrolases to allow expansion of the PG meshwork. How these potentially lytic enzymes areregulated and coordinated with growth remains an unanswered question in all bacteria. The first two aims ofthis proposal focus on how the model gram-positive bacterium Bacillus subtilis regulates two functionallyredundant cell wall hydrolases and how it coordinates their activities with cell wall synthesis and envelopeexpansion. The third aim focuses on how cells sense and respond to perturbations to cell wall biogenesis. The?M-signaling pathway was identified over two decades ago as a stress-response pathway that is induced uponenvironmental stresses, including cell wall targeting antibiotics. This pathway is active at intermediate levelsduring unperturbed growth and functions in cell envelope homeostasis, monitoring envelope assembly andadjusting flux through the PG biogenesis pathway. What this pathway senses and how it transduces thisinformation across the membrane have remained mysterious. The results of the proposed studies will elucidatecritical regulatory pathways in envelope biogenesis and will inform the development of new treatments forinfections. The Specific Aims of this application are:Aim 1: Elucidate how cells sense and respond to the extent of PG crosslinking to ensure proper expansion ofthe cell wall.Aim 2 Investigate how cell wall hydrolysis is coordinated with cell wall synthesis during growth.Aim 3: Determine how cells sense and respond to perturbations to cell envelope biogenesis.