PROJECT SUMMARY Bacteria interact with the outside world through their cell envelope. The cell envelope defines cell boundaries and provides structural integrity and cell shape to bacteria. Therefore, properly building their multi-structure envelope is critical to ensure growth and survival. In Gram-negative bacteria, the cell envelope is delimited by two lipid bilayers, the inner membrane and the outer membrane. These membranes are separated by an aqueous compartment known as the periplasm, which also contains a thin peptidoglycan cell wall. The structure and composition of the Gram-negative outer membrane is unusual mainly because it is not a phospholipid bilayer. Its inner leaflet contains glycerophospholipids, but its outer leaflet is composed of lipopolysaccharides (LPS). By densely packing their cell surface with LPS molecules, Gram-negative bacteria create a permeability barrier against small hydrophobic molecules that otherwise could diffuse across phospholipid bilayers. Consequently, these bacteria are naturally resistant to many antibiotics and detergents. The long-term plan of our research program is to investigate how Gram-negative bacteria build their cell envelope. This project focuses on understanding outer membrane biogenesis by studying the mechanism of function of lipid transporters that build this bilayer. Outer-membrane lipids are synthesized in the inner membrane, so they must travel through the aqueous periplasmic compartment and be asymmetrically delivered to the outer membrane. The proposed research plan will leverage our expertise in applying genetic and biochemical approaches to study the cell envelope in order to investigate two highly conserved systems that are essential for outer-membrane biogenesis and growth of the Gram-negative bacterium Escherichia coli. We will investigate how the Lpt system extracts newly synthesized LPS molecules from the inner membrane and transports them across the cell envelope through a protein bridge so that they can be assembled at the cell surface. Our studies will focus on how LPS extraction and transport are powered by the LptB2FGC components, which constitute an ATP-binding cassette (ABC) transporter at the inner membrane. ABC transporters are ATP-driven machines that all cells use to move substrates across cellular compartments. We will also investigate transport of phospholipids from the inner to the outer membrane by newly discovered bridge-like proteins belonging to the AsmA-like protein family. These proteins have recently been recognized as the bacterial ancestors of lipid-transfer proteins that transport phospholipids at contact sites between membranes of different organelles in eukaryotic cells. Our research will therefore provide fundamental knowledge about bacterial physiology and growth that is also applicable to homologous systems conserved across many organisms. In addition, since the barrier imposed by the outer membrane is the main reason why very few novel antibiotics ...