PROJECT ABSTRACT The bacterial outer membrane is a lipid bilayer that plays a key role in resistance to antibiotics, detergents, and other external stresses. Despite decades of research on the bacterial envelope, it is unknown how phospholipids are trafficked between the bacterial inner and outer membranes, through the intervening hydrophilic space of the periplasm. We recently discovered that members of the mammalian cell entry (MCE) protein family form structurally diverse hexameric rings and barrels, and that some of these proteins may form “bridges” or “pipes” between the inner and outer membrane to facilitate lipid transport (Ekiert, et al. Cell 2017). In the future, we will work to understand how MCE proteins help to create a periplasm-spanning transport system that links the inner and outer membranes. We will use cryo-EM and X-ray crystallography to unravel how the structure of the individual components supports their biological functions, and how these components assemble into larger inner membrane, outer membrane, and even transenvelope complexes. We will also employ complementary genetic and biochemical approaches to test hypotheses and probe the mechanism of trafficking by MCE systems, including the direction of transport, how transport activity is regulated, how lipids are are extracted from and inserted into the inner and outer membranes, and how lipids are transported across the periplasm. This work will advance our understanding of a fundamental yet poorly understood aspect of bacterial cell biology, and may open up avenues to the development of new antibiotics that target the essential process of outer membrane biogenesis. In addition, the presence of MCE proteins in some double- membraned organelles, such as chloroplasts, suggests that understanding E. coli MCE systems will also have direct implications for lipid trafficking in other bacterial-derived organelles.