Garrett T A, Que N L, Raetz C R
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1998 May 15;273(20):12457-65. doi: 10.1074/jbc.273.20.12457.
The lpxK gene has been proposed to encode the lipid A 4'-kinase in Escherichia coli (Garrett, T. A., Kadrmas, J. L., and Raetz, C. R. H. (1997) J. Biol. Chem. 272, 21855-21864). In cell extracts, the kinase phosphorylates the 4'-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipid A, but the enzyme has not yet been purified because of instability. lpxK is co-transcribed with an essential upstream gene, msbA, with strong homology to mammalian Mdr proteins and ABC transporters. msbA may be involved in the transport of newly made lipid A from the inner surface of the inner membrane to the outer membrane. Insertion of an Omega-chloramphenicol cassette into msbA also halts transcription of lpxK. We have now constructed a strain in which only the lpxK gene is inactivated by inserting a kanamycin cassette into the chromosomal copy of lpxK. This mutation is complemented at 30 degreesC by a hybrid plasmid with a temperature-sensitive origin of replication carrying lpxK+. When this strain (designated TG1/pTAG1) is grown at 44 degreesC, the plasmid bearing the lpxK+ is lost, and the phenotype of an lpxK knock-out mutation is unmasked. The growth of TG1/pTAG1 was inhibited after several hours at 44 degreesC, consistent with lpxK being an essential gene. Furthermore, 4'-kinase activity in extracts made from these cells was barely detectable. In accordance with the proposed biosynthetic pathway for lipid A, DS-1-P (the 4'-kinase substrate) accumulated in TG1/pTAG1 cells grown at 44 degreesC. The DS-1-P from TG1/pTAG1 was isolated, and its structure was verified by 1H NMR spectroscopy. DS-1-P had not been isolated previously from bacterial cells. Its accumulation in TG1/pTAG1 provides additional support for the pathway of lipid A biosynthesis in E. coli. Homologs of lpxK are present in the genomes of other Gram-negative bacteria.
有人提出lpxK基因在大肠杆菌中编码脂多糖A 4'-激酶（加勒特，T.A.，卡德马斯，J.L.，和雷茨，C.R.H.（1997年）《生物化学杂志》272，21855 - 21864）。在细胞提取物中，该激酶使脂多糖A的四酰基二糖1-磷酸前体（DS-1-P）的4'-位磷酸化，但由于该酶不稳定，尚未得到纯化。lpxK与一个必需的上游基因msbA共转录，msbA与哺乳动物的多药耐药蛋白和ABC转运蛋白有很强的同源性。msbA可能参与将新合成的脂多糖A从内膜的内表面转运到外膜。将一个ω-氯霉素盒插入msbA也会使lpxK的转录停止。我们现在构建了一个菌株，其中通过将卡那霉素盒插入lpxK的染色体拷贝，仅使lpxK基因失活。该突变在30℃时可被携带lpxK +且具有温度敏感型复制起点的杂交质粒互补。当该菌株（命名为TG1/pTAG1）在44℃生长时，携带lpxK +的质粒丢失，lpxK基因敲除突变的表型显现出来。TG1/pTAG1在44℃生长数小时后生长受到抑制，这与lpxK是一个必需基因一致。此外，从这些细胞制备的提取物中的4'-激酶活性几乎检测不到。根据提出的脂多糖A生物合成途径，DS-1-P（4'-激酶底物）在44℃生长的TG1/pTAG1细胞中积累。从TG1/pTAG1中分离出DS-1-P，并通过1H核磁共振光谱法验证了其结构。DS-1-P以前未从细菌细胞中分离出来。它在TG1/pTAG1中的积累为大肠杆菌中脂多糖A生物合成途径提供了额外的支持。其他革兰氏阴性细菌的基因组中存在lpxK的同源物。
