The most defining and distinguishing feature of Gram-negative bacteria, such as Escherichia coli, is the presence of an asymmetric outer membrane, which is essential for their viability. This asymmetry is due to the presence of lipopolysaccharide (LPS) in the outer leaflet of OM and phospholipids facing the inner leaflet. LPS is the major virulence factors and is the causative agent of bacterial sepsis. Globally, sepsis is one of the leading causes of mortality, with more than 1500 deaths per day. For balanced bacterial growth, a tight balance exists between phospholipids and LPS as they share a common metabolic precursor. Any imbalance in the ratio of LPS vs phospholipids causes bacterial lethality. This balance is achieved by the regulation of the amounts of LpxC, which catalyzes the first committed step in LPS biosynthesis. Over the past several years, we have shown that FtsH and LapB mediate LpxC proteolysis and the newly discovered essential LapC protein inhibits LapB activity to maintain a balance. Since it is unknown how the activity of LapB and LapC is regulated in response to LPS demand, and which amino acid residues in LapB and LapC mediate interaction with LPS, we investigate these aspects in the project. Our recent studies revealed that when bacteria synthesize underacylated LPS, or when LapD or LapC are dysfunctional they require cardiolipin for their viability. However, the molecular basis of this lethality is not understood. To address these gaps, several approaches are taken that include: mutagenesis of LapB and LapC to identify amino acid residues that are required for LPS binding and protein-protein interactions, which will be established by measuring binding affinities. To understand the requirement of cardiolipins, we identified a new protein TesD with a predicted thioesterase activity. Based on initial biochemical studies, TesD was found to interact with several enzymes involved in LPS and phospholipid synthesis, including the central cofactor acyl carrier protein. Thus, we will quantify these interactions as well as implore the impact on LpxC stability and LPS/fatty acid content, when either TesD is absent or overproduced, measure its thioesterase activity and examine if TesD has selectivity towards specific acyl chain length of fatty acids.