Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

Abstract

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on f,-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being f3-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.

Composition des lipopolysaccharides de la souche sauvage P678 et de la souche mutante PM61 de Escherichia coli K12

“…Plusieurs types de lipopolysaccharides ont 6t6 caract6ris6s chez Escherichia coli. Le groupe de E. coli K12 a 6t6 6tudi6 avec les souches CR34, D21, W300 et avec des mutants obtenus ~ partir de ces souches [1][2][3][4][5][6]. On distingue plusieurs types s6rologiques l'int6rieur du groupe K12.…”

relA gene control of the synthesis of lipid A fatty acyl moieties

“…RESULTS Stringent control of fi-HM synthesis. ,3-HM, the major lipid component of lipid A, is the only hydroxy fatty acid found in E. coli K-12 (18,19). This acid is found only in lipid A; no 38-HM is found in the cellular phospholipids.…”

“…We now report that the synthesis of f3-hydroxymyristic acid (,8-HM), a fatty acid not 1 Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139. found in phospholipids but rather in the lipid A component of the cell walls (4,10,18,19), is under stringent control. This result strengthens the previous finding of Nunn and Cronan (14) in that it demonstrates relA control of fatty acid synthesis in strains that are wild type with respect to lipid metabolism.…”

“…The liberated fatty acids were extracted into petroleum ether-ethyl ether (1:1). Bound f3-HM and bound nonhydroxy fatty acids (the total fatty acid content of lipid A [4,10,18,19]) were obtained by acid hydrolysis of the residue remaining after free fatty acids and phospholipids had been removed by several cycles of extraction in the solvent of Bligh and Dyer (2). Total j3-HM, bound f8-HM, and nonhydroxy fatty acids were conveniently isolated by thin-layer chromatography on silica gel plates in petroleum ether-ethyl ether-acetic acid (70:30:2).…”

“…This acid is found only in lipid A; no 38-HM is found in the cellular phospholipids. /3-HM can be released from lipid A by acid hydrolysis (18) and is readily separated (due to the hydroxyl group) from the phospholipid fatty acyl moieties by thin-layer chromatography. Figure 1 shows the effect of amino acid starvation on the rate of synthesis of,8-HM in rel+ and relA strains.…”