PROJECT SUMMARY The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and an aqueous compartment termed the periplasm that is located between them. A long-term goal of my lab has always been to understand the mechanisms of envelope biogenesis using Escherichia coli as a model system. This proposal concerns OM biogenesis and the stress responses that maintain cell envelope physiology. All of the components of the OM, phospholipids (PL), lipopolysaccharide (LPS), lipoproteins, and β-barrel proteins (OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified the essential proteins required to assemble LPS (LptABCDEFG) and OMPs (BamABCDE) in the OM and we have provided evidence of a diffusive mechanism of phospholipid transport between the IM and the OM. In the current funding period, we have shown that the conditional lethal phenotype of bamB bamE double mutants can be suppressed simply by deleting a surface-exposed lipoprotein, we revealed the existence of an alternate lipoprotein trafficking pathway, we uncovered a role for the cyclic form of Enterobacterial Common Antigen in maintaining the OM barrier, and we identified mutations that activate or prime the σE stress response that suppress a variety of OMP and Bam defects. In translational studies, we used our knowledge of OM biogenesis to discover a new class of antibiotics that work to inhibit BamA at the cell surface. We propose to use our large collection of mutations that alter the Bam components or various OMP substrates together with our collection of suppressors as tools to probe the OMP assembly process. In particular, we will probe the function of the non-essential BamBCE lipoproteins and test our hypothesis that BamD does not perform a truly essential mechanistic role, but rather functions as a regulator to control the activity of BamA. We will test the role of the chaperone Skp as a specific adaptor for the periplasmic protease DegP. We also posit that the trimeric nature of the major OMPs functions as a global organizer of OM architecture by providing multiple interacting faces to allow the protein-protein interactions necessary for the formation of protein islands. Our studies on LPS assembly will utilize a mutant O-antigen ligase and the enzyme sortase to attach peptides or proteins to LPS to challenge the capabilities of the LptDE translocon. We will also test our model that three essential IM proteins, YejM, YciM, and FtsH comprise a novel pathway that regulates LPS synthesis in response to the lipid status of the OM. The mlaA* mutation destabilizes the OM by increasing LPS levels. This causes membrane loss by OM vesiculation and IM PLs flow into the OM to replace the loss. We have identified a mutation that slows this lipid flow and we believe that continued study of this gene may provide insights into the poorly understood process of anterograde PL transport.