PROJECT SUMMARY The cell envelope lies at the interface of bacterial pathogens and their host. It contains unique molecules that are recognized by the innate immune system, and it is where key virulence factors are embedded. The cell envelope and the biogenesis pathways that build it are also targets of many of our most effective antibiotic and vaccine therapies. Because cell envelope biogenesis has been such a successful target, it has been an active area of research for over half a century. Most of the genes responsible for the synthesis and remodeling of the different surface polymers have been identified and their biochemical activities characterized. However, a major gap in our knowledge is how these different pathways are coordinated with each other and how the cell monitors the envelope for defects and directs their repair. This proposal focuses on investigating the coordination between the synthesis of the distinct surface polymers and the repair of the envelope using the model gram-positive bacterium Bacillus subtilis. The gram-positive cell envelope is composed of distinct layers of surface polymers including lipid-linked anionic polymers called lipoteichoic acids (LTAs), the multi-layered cell wall peptidoglycan (PG) and the distinct anionic polymers called wall teichoic acids (WTAs) that are attached to it. Cell growth requires the coordinated synthesis of these envelope layers, but the mechanisms underlying this coordination remain poorly understood. A second outstanding question is how gram-positive bacteria monitor and repair defects in their envelope layers. In preliminary work, I have discovered that conserved enzymes that synthesize PG and transfer WTA onto it contain extracellular intrinsically disorder regions (IDRs). My data and previous studies in my host lab suggest that these regions direct the synthases to gaps or defects in the cell wall meshwork to fortify them. The goals of this proposal are (1) to establish how gram-positive bacteria monitor and repair their cell envelope layers and (2) use directed and unbiased approaches to investigate the interplay between the assembly of the PG and the teichoic acids. This project will be carried out in the laboratory of Dr. David Rudner in the Department of Microbiology at Harvard Medical School. I will be closely mentored by Dr. Rudner and will be embedded within a rich and collegial scientific community committed to scientific exchange and discovery. The formative training and connections that I make during my fellowship will be instrumental in launching my future career as an independent research investigator.