Structure and functional analysis of glycosyltransferases which are involved in the biosynthesis of bacterial polysaccharides

本研究在过去的三年中取得了以下结果：1.对亚克隆到质粒中的大肠杆菌染色体rfe-rff区进行了分析。我们发现rfe和orf 2基因编码的蛋白是膜蛋白。2.克隆了克雷伯氏菌K2-cps基因的全序列，该基因编码K2荚膜多糖生物合成相关酶。在<56>cps区鉴定出一个sigma-1启动子和19个开放阅读框，并通过同源性检索推测了其中几个开放阅读框的功能。3.我们还测定了大肠杆菌O 9-rfb区的全序列，该区域参与O 9 LPS的O多糖生物合成。4.从大肠杆菌K12的rfa区域中克隆了rfaG、rfaB、rfaI和rfaJ四种糖基转移酶基因，并在大肠杆菌中进行了表达。我们还构建了这些基因的缺陷突变体，分别与基因打靶方法。利用这些突变体，检测了上述糖基转移酶的表达和酶活性。目前正在进行定点突变基因的构建以鉴定活性位点。

Following results were obtained in the past three years.1.The rfe-rff region of Escherichia coli chromosome subcloned into a plasmid was analyzed. We found that the proteins encoded by rfe and orf2 genes were membrane proteins. We also analyzed the topology of these proteins with TnphoA method.2.We determined the entire nucleotide sequence of the K2-cps region of Klebsiella which encoded enzymes involved in the biosynthesis of K2 capsular polysaccharide. A sigma^<56> promoter and 19 ORFs were identified in the cps region and the functions of several ORFs were speculated from the results of homology search with the data base.3.We also determined the entire nucleotide sequence of the O9-rfb region of E.coli which was involved in the biosynthesis of the O polysaccharide of O9 LPS.GDPmannose synthetase, and three mannosyltransferases were identified in the region.4.We subloned 4 kinds of glycosyltransferase such as rfaG,rfaB,rfaI and rfaJ from the rfa region of E.coli K12 into a expression vector and expressed these enzymes. We also constructed deficient mutants of these genes, respectively, with a gene targeting method. By using these mutants, the expression and enzymatic activities of above glycosyltransferases were examined. Construction of site-directed mutant genes were currently proceeded for the identification of active sites.