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Abstract
Gram-negative bacteria are able to survive in various conditions and environments due in part to their intricate cell envelope. They have an asymmetrical outer membrane, where the surface layer is composed primarily of lipopolysaccharide (LPS) and the inner leaflet is composed of glycerophospholipids (GPLs), while the inner membrane is a symmetric layer of GPLs. The lipid anchor of LPS, lipid A, is highly negatively charged, but can be modified to alter the chemical properties of the outer membrane. Critical for antimicrobial resistance, antibiotics like polymyxins take advantage of the negative charge of lipid A to engage with the outer membrane leading to disruption of the cell envelope and eventual cell death. However, Gram-negative organisms have evolved enzymatic machinery to covalently modify their lipid A structure and alter the negative charge of LPS. The addition of phosphoethanolamine by the phospho-form transferase, EptA, is one such modification. EptA modification requires the major GPL phosphatidylethanolamine as its substrate donor. The reaction results in the production of the neutral lipid, diacylglycerol that is recycled back into GPL synthesis. This dissertation explores the impact that diacylglycerol recycling has on lipid A modifications and antibiotic resistance. We demonstrate that loss of diacylglycerol recycling reduced phosphoethanolamine lipid A modification by inhibiting EptA to prevent further build-up of diacylglycerol, a dead-end lipid intermediate. Loss of diacylglycerol recycling could thus revert a normally polymyxin resistant strain back to a susceptible state. To further determine if there was a mechanism of antibiotic resistance independent of lipid A modification, we used strains lacking diacylglycerol recycling to select for polymyxin resistance. The isolated suppressors revealed a link between a carbon catabolite repression regulatory complex and lipid A modifications, as disrupting carbon catabolite repression restored polymyxin resistance. This unique connection between seemingly separate networks in the cell reveals new considerations for antibiotic resistance mechanisms, such as how changes in carbon source and metabolism can impact the composition of the Gram-negative outer membrane.