PMID:2180947
| Citation |
Galloway, SM and Raetz, CR (1990) A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J. Biol. Chem. 265:6394-402 |
|---|---|
| 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. |
| Links | |
| Keywords |
Acyltransferases/genetics; Acyltransferases/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Genes, Bacterial; Genotype; Kinetics; Lipid A/biosynthesis; Lipid A/isolation & purification; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mutation; Plasmids |
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