The emergence of microbes resistant to even the most powerful antibiotics represents a serious threat to global public health. The coordinated action of the bacterial machinery of peptidoglycan (PG) synthesis, a process essential for bacterial viability, represents an obvious target for the development of new antibiotics. In this proposed project, we aim to define the molecular interactions of the components of the PG degradation apparatus, consisting of enzymes involved in glycan chain hydrolysis or modification. Our previous studies in Neisseria meningitidis showed that targeting a hot spot on a single lytic transglycosylase (LgtA) also disables the function of the PG-modifying enzyme, Ape1, leading to a disruption of PG assembly, and results in an aberrant peptidoglycan composition, making this pathogen unable to survive in the host. Our studies will reveal, in molecular detail, how these peptidoglycan degrading enzymes work in concert to assemble the bacterial cell wall. Specifically, we will define, in a comprehensive way, how a network of lytic transglycosylases (LtgA, LtgD, LtgE) and their protein binding partners work to facilitate peptidoglycan degradation and the insertion of organelles into the bacterial cell envelope. In this project, we will utilize biochemical and biological approaches to probe protein-protein and enzyme-substate interactions of the various lytic transglycosylases, combined with determination of the molecular basis of activity of the multienzyme complexes in PG metabolism. Genetic modifications of the components of the PG biosynthetic nanomachine will be used to test the observations from our structural studies. Our approach, utilizing high resolution x-ray crystallographic tools along with cryo-EM single particle analysis, will allow visualization of the action of enzymes in PG assembly and degradation and should provide mechanistic insights into their orchestrated activity during the insertion of new PG during cell wall assembly and bacterial cell division. Our studies will lead the way towards the development of new therapies targeting multiple peptidoglycan metabolic enzymes.