The outer leaflet of the outer membrane of Gram-negative bacteria is composed almost entirely of lipopolysaccharide (LPS) and acts as an efficient permeability barrier against antibiotics. Lipid A, also known as endotoxin, is the hydrophobic anchor of LPS and is the cause of Gram negative septic shock. While the enzymes involved in lipid A biosynthesis are now known, understanding the molecular details of LPS transport and secretion from its place of synthesis in the cytoplasmic face of the inner membrane to the outer face of the outer membrane remains one of the most intriguing, unsolved problems of LPS biochemistry. Indeed, LPS transport is a potential target for antibacterial agents. There is evidence which suggests that the essential, ABC family transporter msbA is involved in the translocation of lipid A across the inner membrane in E.coli. This proposal describes a biochemical and genetic approach to understand the function of msbA in LPS biosynthesis. Specifically, this proposal focuses on (I) the isolation and characterization of conditional, temperature-sensitive mutants of msbA, (II) Purification and functional reconstitution of msbA, and (III) further genetic studies of spontaneous revertants and suppressors of msbA mutants to identify other genes involved in LPS secretion.
