The bacterial cell envelope is a remarkable and complex structure that guards bacteria from their surrounding environment. A defining feature of Gram-negative bacteria is the presence of an outer membrane (OM) that encapsulates the peptidoglycan layer of these organisms. While the inner membrane (IM) is composed of glycerophospholipids (GPLs), the OM is a bilayer with extreme lipid asymmetry with GPL confined to the inner leaflet and lipopolysaccharide (LPS) localized to the outer leaflet. This unique membrane organization affords Gram-negative bacteria protection from large polar molecules, as well as lipophilic compounds, serving as an essential innate barrier to a variety of antibiotics and toxic compounds. Remarkably, the high-priority Gram-negative pathogen Acinetobacter baumannii can completely inactivate LPS biosynthesis as an alternative mechanism of resistance to the “last-resort” antibiotics called polymyxins. The primary objective of this application is to investigate the mechanisms required for maintenance of the cell envelope of A. baumannii, regardless of LPS status. While the benefit of an asymmetric OM relative to a GPL bilayer is apparent due to the impermeable barrier it provides, the lack of LPS essentiality in A. baumannii can be used a tool to explore novel mechanisms of OM stability in both the presence or absence of LPS. In Aim 1, we will investigate changes to the bacterium during its transition from an LPS-deficient to a LPS-positive cell, including how GPL transport influences LPS structure. For Aim 2 our focus will be the identification of genes that support LPS-deficiency, including the role of lipoproteins and how they are transported to the cell surface regardless of LPS status. Finally, in Aim 3, we will characterize novel gene products necessary for OM stability in LPS-positive A. baumannii uncovered by a genetic and chemical synthetic lethality screen. Given the current literature, the application is built on a strong scientific premise addressing major gaps in our understanding of the A. baumannii cell envelope and other Gram-negative pathogens. Furthermore, the Aims focus on highly conserved pathways that impact membrane biogenesis, bacterial pathogenesis, and antimicrobial development.