title: A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis.
abstract: Using localized mutagenesis of whole cells, we have isolated a temperature-sensitive UDP-N-acetylglucosamine acyltransferase mutant of Escherichia coli that loses all detectable acyltransferase activity and quickly dies after a shift from 30 to 42 degrees C. Acyltransferase activity and temperature resistance are restored by transforming the mutant with a hybrid plasmid containing the E. coli gene for UDP-GlcNAc acyltransferase (lpxA). In addition, a new assay has been developed for quantitating the amount of lipid A (the active component of endotoxin) in E. coli and related Gram-negative strains. Cells are labeled with 32Pi and extracted with chloroform/methanol/water (1:2:0.8, v/v) to remove glycerophospholipids. The residue is then hydrolyzed with 0.2 M HCl to liberate the "monophosphoryl" lipid A degradation products (Qureshi, N., Cotter, R. J. and Takayama, K. (1986) J. Microbiol. Methods 5, 65-77), each of which bears a single phosphate residue at position 4'. The amount of lipid A is normalized to the total amount of labeled glycerophospholipid present in the cells. The steady state ratio of lipid A to glycerophospholipid in wild-type cells is approximately 0.12. The lipid A content of the acyltransferase mutant is reduced 2-3-fold, and the rate of lipid A synthesis is reduced 10-fold when compared to wild-type after 60 min at 42 degrees C. These results provide physiological evidence that UDP-N-acetylglucosamine acyltransferase is the major committed step for lipid A biosynthesis in E. coli and that lipid A is an essential molecule.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison 53706.
authors.forename: S M
authors.initials: SM
authors.lastname: Galloway
authors.forename: C R
authors.initials: CR
authors.lastname: Raetz
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial; D005838:Genotype; D007700:Kinetics; D008050:Lipid A / Q000096:biosynthesis* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009154:Mutation; D010957:Plasmids
chemical_list: D008050:Lipid A; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase