Kajimura Junko, Rahman Arifur, Rick Paul D
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
J Bacteriol. 2005 Oct;187(20):6917-27. doi: 10.1128/JB.187.20.6917-6927.2005.
We describe here the purification and quantification of a water-soluble cyclic form of enterobacterial common antigen (ECA(CYC)) from Escherichia coli K-12 as well as information regarding its subcellular location and the genetic loci involved in its assembly. Structural characterization of purified ECA(CYC) molecules obtained from E. coli K-12 revealed that they uniformly contained four trisaccharide repeat units, and they were substituted with from zero to four O-acetyl groups. Cells from overnight cultures contained approximately 2 microg ECA(CYC) per milligram (dry weight), and cell fractionation studies revealed that these molecules were localized exclusively in the periplasm. The synthesis and assembly of ECA(CYC) were found to require the wzxE and wzyE genes of the wec gene cluster. These genes encode proteins involved in the transmembrane translocation of undecaprenylpyrophosphate-linked ECA trisaccharide repeat units and the polymerization of trisaccharide repeat units, respectively. Surprisingly, synthesis of ECA(CYC) was dependent on the wzzE gene, which is required for the modulation of the polysaccharide chain lengths of phosphoglyceride-linked ECA (ECA(PG)). The presence of ECA(CYC) in extracts of several other gram-negative enteric organisms was also demonstrated; however, it was not detected in cell extracts of Pseudomonas aeruginosa. These data suggest that in addition to ECA(PG), ECA(CYC) may be synthesized in many, if not all, members of the Enterobacteriaceae.
我们在此描述了从大肠杆菌K-12中纯化和定量水溶性环状形式的肠杆菌共同抗原（ECA(CYC)），以及关于其亚细胞定位和参与其组装的基因位点的信息。从大肠杆菌K-12获得的纯化ECA(CYC)分子的结构表征显示，它们均一性地含有四个三糖重复单元，并且被零至四个O-乙酰基取代。过夜培养物中的细胞每毫克（干重）含有约2微克ECA(CYC)，细胞分级分离研究表明这些分子仅定位在周质中。发现ECA(CYC)的合成和组装需要wec基因簇的wzxE和wzyE基因。这些基因分别编码参与十一碳烯基焦磷酸连接的ECA三糖重复单元的跨膜转运和三糖重复单元聚合的蛋白质。令人惊讶的是，ECA(CYC)的合成依赖于wzzE基因，该基因是调节磷酸甘油酯连接的ECA（ECA(PG)）多糖链长度所必需的。还证明了几种其他革兰氏阴性肠道菌的提取物中存在ECA(CYC)；然而，在铜绿假单胞菌的细胞提取物中未检测到。这些数据表明，除了ECA(PG)之外，ECA(CYC)可能在许多（如果不是全部）肠杆菌科成员中合成。
