Bulawa C E, Raetz C R
J Biol Chem. 1984 Apr 25;259(8):4846-51.
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.
在某些基因背景下，pgsB基因存在缺陷的大肠杆菌突变体缺乏磷脂酰甘油，并积累了新型的、源自葡萄糖胺的磷脂（西岛，M.，和雷茨，C.R.H.（1979年）《生物化学杂志》254，7837 - 7844）。这些化合物中最简单的是2,3 - 二酰基葡萄糖胺1 - 磷酸（2,3 - 二酰基 - GlcN - 1 - P）（大肠杆菌的“脂质X”），其中β - 羟基肉豆蔻酰基部分是唯一的脂肪酸取代基（高山，K.，库雷希，N.，马斯卡尼，P.，纳什德，M.A.，安德森，L.，和雷茨，C.R.H.（1983年）《生物化学杂志》258，7379 - 7385）。我们现在报告一种灵敏的放射化学方法，用于检测野生型大肠杆菌中的2,3 - 二酰基 - GlcN - 1 - P，并证明每个细胞中有大约4000个分子（占总氯仿可溶性磷的0.02%）。在携带pgsB1损伤的突变体中，其水平高100至300倍。此外，我们发现了一种新型的脂核苷酸，UDP - 2,3 - 二酰基 - GlcN，它也与pgsb1突变一起积累。这种物质占野生型磷脂的0.005%，在突变体中积累50至100倍。大肠杆菌中UDP - 2,3 - 二酰基 - GlcN的鉴定基于：1）在二维薄层色谱过程中，野生型和突变体细胞中一种少量的32P标记脂质与UDP - 2,3 - 二酰基 - GlCn标准品一起迁移；2）这种32P标记物质对脂核苷酸特异性焦磷酸酶切割的敏感性；3）色谱鉴定[32P]UMP和[32P]2,3 - 二酰基 - GlcN - 1 - P（脂质X）作为酶促降解的唯一产物。如随附文章所示，这种新型核苷酸对于大肠杆菌和鼠伤寒沙门氏菌提取物中脂质A二糖的生物合成至关重要。
