Rick P D, Hubbard G L, Barr K
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
J Bacteriol. 1994 May;176(10):2877-84. doi: 10.1128/jb.176.10.2877-2884.1994.
The Escherichia coli O8 antigen is a mannan composed of the trisaccharide repeat unit -->3)-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1--> (K. Reske and K. Jann, Eur. J. Biochem. 67:53-56, 1972), and synthesis of the O8 antigen is rfe dependent (G. Schmidt, H. Mayer, and P. H. Mäkelä, J. Bacteriol. 127:755-762, 1976). The rfe gene has recently been identified as encoding a tunicamycin-sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase (U. Meier-Dieter, K. Barr, R. Starman, L. Hatch, and P. D. Rick, J. Biol. Chem. 267:746-753, 1992). However, the role of rfe in O8 side chain synthesis is not understood. Thus, the role of the rfe gene in the synthesis of the O8 antigen was investigated in an rfbO8+ (rfb genes encoding O8 antigen) derivative of E. coli K-12 mutant possessing a defective phosphoglucose isomerase (pgi). The in vivo synthesis of O8 side chains was inhibited by the antibiotic tunicamycin. In addition, putative lipid carrier-linked O8 side chains accumulated in vivo when lipopolysaccharide outer core synthesis was precluded by growing cells in the absence of exogenously supplied glucose. The lipid carrier-linked O8 antigen was extracted from cells and treated with mild acid in order to release free O8 side chains. The water-soluble O8 side chains were then purified by affinity chromatography using Sepharose-bound concanavalin A. Characterization of the affinity-purified O8 side chains revealed the occurrence of glucosamine in the reducing terminal position of the polysaccharide chains. The data presented suggest that GlcNAc-pyrophosphorylundecaprenol functions as the acceptor of mannose residues for the in vivo synthesis of O8 side chains in E. coli K-12.
大肠杆菌O8抗原是一种由三糖重复单元组成的甘露聚糖，即→3)-α-甘露糖-(1→2)-α-甘露糖-(1→2)-α-甘露糖-(1→（K. Reske和K. Jann，《欧洲生物化学杂志》67:53 - 56，1972年），并且O8抗原的合成依赖于rfe（G. Schmidt、H. Mayer和P. H. Mäkelä，《细菌学杂志》127:755 - 762，1976年）。rfe基因最近被鉴定为编码一种对衣霉素敏感的UDP - GlcNAc：十一异戊烯磷酸GlcNAc - 1 - 磷酸转移酶（U. Meier - Dieter、K. Barr、R. Starman、L. Hatch和P. D. Rick，《生物化学杂志》267:746 - 753，1992年）。然而，rfe在O8侧链合成中的作用尚不清楚。因此，在具有缺陷磷酸葡萄糖异构酶（pgi）的大肠杆菌K - 12突变体的rfbO8 +（编码O8抗原的rfb基因）衍生物中研究了rfe基因在O8抗原合成中的作用。O8侧链的体内合成受到抗生素衣霉素的抑制。此外，当在没有外源供应葡萄糖的情况下培养细胞从而阻止脂多糖外核心合成时，假定的脂质载体连接的O8侧链在体内积累。从细胞中提取脂质载体连接的O8抗原并用弱酸处理以释放游离的O8侧链。然后使用结合了伴刀豆球蛋白A的琼脂糖通过亲和色谱法纯化水溶性O8侧链。对亲和纯化的O8侧链的表征揭示了在多糖链的还原末端位置存在葡糖胺。所呈现的数据表明，GlcNAc - 焦磷酸化十一异戊烯醇在大肠杆菌K - 12中作为甘露糖残基的受体用于O8侧链的体内合成。
