Cytoplasmic ATP Hydrolysis Powers Transport of Lipopolysaccharide Across the Periplasm in E. coli

Millions of molecules of lipopolysaccharide (LPS) must be assembled on the Escherichia coli cell surface each time the cell divides. The biogenesis of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane through the periplasm to the cell surface. However, no intermediate transport states have been observed. We developed methods to observe intermediate LPS molecules bound to Lpt proteins in the process of being transported in vivo. Movement of individual LPS molecules along these binding sites required multiple rounds of adenosine triphosphate (ATP) hydrolysis in vitro, which suggests that ATP is used to push a continuous stream of LPS through a transenvelope bridge in discrete steps against a concentration gradient. Theouter membrane of Gram-negative bacte-ria is an asymmetric bilayer, with the innerleaflet composed of phospholipids and the outer leaflet composed of lipopolysaccharide (LPS). The LPS layer allows Gram-negative bacteria to survive in harsh environments and in the presence of many antibiotics. However, phospholipids and LPS do not spontaneousl