Meier-Dieter U, Starman R, Barr K, Mayer H, Rick P D
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
J Biol Chem. 1990 Aug 15;265(23):13490-7.
Twelve independent Tn10 insertion mutants of Escherichia coli K12 were isolated that were defective in the synthesis of enterobacterial common antigen (ECA). The mutants were identified by screening a random pool of Tn10 insertion mutants for their ECA phenotype using a colony-immunoblot assay. All 12 of the Tn10 insertion mutants were found to be located in the chromosomal region of the rff-rfe genes. Four of the Tn10 insertions were in rfe genes while the remaining eight Tn10 insertions were in rff genes. All of the rfe::Tn10 insertion mutants were defective in the synthesis of GlcNAc-pyrophosphorylundecaprenol (C55-PP-GlcNAc, lipid I), the first lipid-linked intermediate involved in ECA synthesis. Biochemical characterization of the rff::Tn10 insertion mutants revealed that they were defective in various steps of ECA synthesis subsequent to the synthesis of lipid I. These defects included: (i) the inability to synthesize UDP-ManNAcA due to Tn10 insertions in the structural genes for UDP-GlcNAc-2-epimerase (rffE) and UDP-ManNAcA (N-acetyl-D-mannosaminuronic acid) dehydrogenase (rffD), (ii) defects in the synthesis of C55-GlcNAc-ManNAcA (lipid II) due to insertion of transposon Tn10 in the structural gene for the UDP-ManNAcA transferase (rffM), (iii) the inability to synthesize TDP-Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) due to Tn10 insertions in the structural gene for the transaminase that catalyzes the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-4-amino-4,6-dideoxy-D-galactose (rffA), and (iv) defects in steps subsequent to the synthesis of C55-GlcNAc-ManNAcA-Fuc4NAc (lipid III). In addition, a re-examination of a mutant possessing the rff-726 lesion revealed that it was defective in the synthesis of lipid III due to a defect in the structural gene for the Fuc4NAc transferase (rffT).
分离出了12个大肠杆菌K12的独立Tn10插入突变体，它们在肠杆菌共同抗原（ECA）的合成上存在缺陷。通过使用菌落免疫印迹分析，从Tn10插入突变体的随机库中筛选其ECA表型来鉴定这些突变体。发现所有12个Tn10插入突变体都位于rff - rfe基因的染色体区域。其中4个Tn10插入在rfe基因中，其余8个Tn10插入在rff基因中。所有rfe::Tn10插入突变体在GlcNAc - 焦磷酸化十一异戊烯醇（C55 - PP - GlcNAc，脂质I）的合成上存在缺陷，脂质I是参与ECA合成的第一个脂质连接中间体。rff::Tn10插入突变体的生化特性表明，它们在脂质I合成后的ECA合成的各个步骤中存在缺陷。这些缺陷包括：（i）由于在UDP - GlcNAc - 2 - 表异构酶（rffE）和UDP - ManNAcA（N - 乙酰 - D - 甘露糖胺糖醛酸）脱氢酶（rffD）的结构基因中存在Tn10插入，无法合成UDP - ManNAcA；（ii）由于转座子Tn10插入UDP - ManNAcA转移酶（rffM）的结构基因，在C55 - GlcNAc - ManNAcA（脂质II）的合成中存在缺陷；（iii）由于在催化TDP - 4 - 酮 - 6 - 脱氧 - D - 葡萄糖转化为TDP - 4 - 氨基 - 4,6 - 二脱氧 - D - 半乳糖的转氨酶（rffA）的结构基因中存在Tn10插入，无法合成TDP - Fuc4NAc（4 - 乙酰氨基 - 4,6 - 二脱氧 - D - 半乳糖）；以及（iv）在C55 - GlcNAc - ManNAcA - Fuc4NAc（脂质III）合成后的步骤中存在缺陷。此外，对具有rff - 726损伤的突变体进行重新检查发现，由于Fuc4NAc转移酶（rffT）的结构基因存在缺陷，它在脂质III的合成中存在缺陷。
