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 still unclear how phospholipids are trafficked between the bacterial inner and outer membranes. In addition, many other kinds of hydrophobic molecules must be imported or exported from the cell, and dedicated transport systems are required to move many of these molecules across the aqueous periplasm and outer membrane. Research in my lab focuses on studying transport mechanisms in the bacterial cell envelope. We have shown 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 tunnels between the inner and outer membrane to facilitate lipid transport. Very recently, several studies of the Mla pathway have been published by our lab and others, leading to mechanistic insights into how this pathway may transport lipids across the cell envelope. Several other MCE systems remain largely uncharacterized, and our initial work suggests that these function by fundamentally different mechanisms relative to the Mla pathway. In the future, we will work to understand how these unexplored MCE transport systems drive the transport of a range of hydrophobic substrates across the cell envelope. 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 potentially 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 identification of transporter substrates, how transport activity is regulated, how lipids/substrates are are extracted from and inserted into the inner and outer membranes, and how lipids/substrates 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 important cellular processes. 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.