大肠杆菌44277(O2:K1)中rmlB基因功能

Function of rmlB in the pathogenic Escherichia coli 44277(O2:k1)

【目的】确定rmlB基因在大肠杆菌(O2:K1)L-型鼠李糖合成中的作用。【方法】将基因rmlB进行原核表达并测定酶活;用同源重组的方法将rmlB基因敲除,分析表型变化,并运用质谱,以及核磁共振等手段分析脂多糖O侧链的结构,以确定rmlB在O抗原合成中的作用。【结果】成功对rmlB基因进行了表达并测定了重组蛋白的酶活,确定蛋白RmlB具有dTDP-D-glucose 4,6-dehydratase活性。成功构建了rmlB基因缺失突变株,对突变株进行表型分析发现突变株的表型与野生株相比无变化。对突变株分析发现突变株中的O抗原仍含有L-型鼠李糖,说明在该菌株中可能存在RmlB的同功能酶或者存在其它的L-型鼠李糖合成途径。【结论】rmlB基因编码的蛋白具有dTDP-D-glucose 4,6-dehydratase活性但此基因对于L-型鼠李糖的合成不是必需的。

[Objective] To identify the role of rmlB in synthesizing L-rhamnose in the pathogenic Escherichia coli 44277（O2：K1：H4）.[Methods] The rmlB gene was expressed and the activity of the recombinant protein was assayed by measuring the quantity of reaction product.The rmlB gene was deleted by homologous recombination,then phenotypic changes of the △rmlB mutant was analyzed by Electron Microscope,Tricine SDS-PAGE and immunological methods.Further,various methods including MALDI-TOF-MS/MS,GC-MS and NMR was used to investigate the O antigen structure of the △rmlB mutant.[Results] RmlB was confirmed to be a protein harboring the activity of dTDP-D-glucose 4,6-dehydratase through enzyme assay.The △rmlB mutant was successfully constructed and no phenotypic change was observed after compared with the wild type strain.L-rhamnose still existed in the △rmlB mutant,indicating that there may be isoenzyme of RmlB presenting in the mutant or there was a novel way synthesizing L-rhamnose in the mutant.[Conclusion] RmlB has the activity of dTDP-D-glucose 4,6-dehydratase but it is not essential for the synthesis of L-rhamnose.