title: The biosynthesis of gram-negative endotoxin. Identification and function of UDP-2,3-diacylglucosamine in Escherichia coli.
abstract: Escherichia coli mutants defective in the pgsB gene are phosphatidylglycerol-deficient in certain genetic settings and accumulate novel, glucosamine-derived phospholipids (Nishijima, M., and Raetz, C. R. H. (1979) J. Biol. Chem. 254, 7837-7844). The simplest of these compounds is 2,3-diacylglucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) ("lipid X" of E. coli), in which beta-hydroxymyristoyl moieties are the sole fatty acid substituents (Takayama, K., Qureshi, N., Mascagni, P., Nashed, M. A., Anderson, L., and Raetz, C. R. H. (1983) J. Biol. Chem. 258, 7379-7385). We now report a sensitive radiochemical method for detection of 2,3-diacyl-GlcN-1-P in wild type E. coli and demonstrate that there are about 4000 molecules/cell (0.02% of the total CHCl3-soluble phosphorus). In mutants bearing the pgsB1 lesion, the levels are 100- to 300-fold higher. In addition, we have discovered a novel liponucleotide, UDP-2,3-diacyl-GlcN, that also accumulates in conjunction with the pgsb1 mutation. This material represents 0.005% of the wild type phospholipid and accumulates 50- to 100-fold in the mutant. The identification of UDP-2,3-diacyl-GlcN in E. coli is based on: 1) migration of a minor 32P-labeled lipid from wild type and mutant cells with a UDP-2,3-diacyl-GlCn standard during two-dimensional thin layer chromatography; 2) susceptibility of this 32P-labeled material to cleavage by a liponucleotide-specific pyrophosphatase; and 3) chromatographic identification of [32P]UMP and [32P]2,3-diacyl-GlcN-1-P (lipid X) as the sole products of the enzymatic degradation. As shown in the accompanying article, this novel nucleotide is crucial for biosynthesis of lipid A disaccharides in extracts of E. coli and Salmonella typhimurium.
mesh_terms: D004731:Endotoxins / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism*; D008050:Lipid A / Q000302:isolation & purification; D013058:Mass Spectrometry; D009154:Mutation; D010743:Phospholipids / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D013045:Species Specificity; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
chemical_list: D004731:Endotoxins; D006017:Glycolipids; D008050:Lipid A; D010743:Phospholipids; D010761:Phosphorus Radioisotopes; D014539:Uridine Diphosphate Sugars; D014537:Uridine Diphosphate N-Acetylglucosamine; C041030:UDP-N(2),3-di(3-hydroxymyristoyl)glucosamine