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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32AI010613-01
Application #
6293629
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
2000-11-01
Project End
Budget Start
2000-11-01
Budget End
2001-10-31
Support Year
1
Fiscal Year
2000
Total Cost
$32,416
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Thompkins, Kandi; Chattopadhyay, Ballari; Xiao, Ying et al. (2008) Temperature sensitivity and cell division defects in an Escherichia coli strain with mutations in yghB and yqjA, encoding related and conserved inner membrane proteins. J Bacteriol 190:4489-500
Doerrler, William T (2007) Density gradient enrichment of Escherichia coli conditional msbA mutants. Appl Environ Microbiol 73:7992-6
Doerrler, William T; Raetz, Christian R H (2005) Loss of outer membrane proteins without inhibition of lipid export in an Escherichia coli YaeT mutant. J Biol Chem 280:27679-87
Doerrler, William T; Gibbons, Henry S; Raetz, Christian R H (2004) MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli. J Biol Chem 279:45102-9
Doerrler, William T; Raetz, Christian R H (2002) ATPase activity of the MsbA lipid flippase of Escherichia coli. J Biol Chem 277:36697-705
Doerrler, W T; Reedy, M C; Raetz, C R (2001) An Escherichia coli mutant defective in lipid export. J Biol Chem 276:11461-4