Rahman A, Barr K, Rick P D
Department of Microbiology, Uniformed Services University of Health Sciences, Bethesda, MD 20814-4799, USA.
J Bacteriol. 2001 Nov;183(22):6509-16. doi: 10.1128/JB.183.22.6509-6516.2001.
The polysaccharide chains of enterobacterial common antigen (ECA) are comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. Individual trisaccharide repeat units are assembled as undecaprenyl-linked intermediates in a sequence of reactions that culminate in the transfer of Fuc4NAc from TDP-Fuc4NAc to ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) to yield Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III), the donor of trisaccharide repeat units for ECA polysaccharide chain elongation. Most of the genes known to be involved in ECA assembly are located in the wec gene cluster located at ca. 85.4 min on the Escherichia coli chromosome. The available data suggest that the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase also resides in the wec gene cluster; however, the location of this gene has not been unequivocally defined. Previous characterization of the nucleotide sequence of the wec gene cluster in the region between o416 and wecG revealed that it contained three open reading frames: o74, o204, and o450. In contrast, the results of experiments described in the current investigation revealed that it contains only two open reading frames, o359 and o450. Mutants of E. coli possessing null mutations in o359 were unable to synthesize ECA, and they accumulated lipid II. In addition, the in vitro incorporation of [(3)H]FucNAc from TDP-[(3)H]Fuc4NAc into lipid II was not observed in reaction mixtures using cell extracts obtained from these mutants as a source of enzyme. The ECA-negative phenotype of these mutants was complemented by plasmid constructs containing the wild-type o359 allele, and Fuc4NAc transferase activity was demonstrated by using cell extracts obtained from the complemented mutants. Furthermore, partially purified o359 gene product, expressed as recombinant C-terminal His-tagged protein, was able to catalyze the in vitro transfer of [(3)H]Fuc4NAc from TDP-[(3)H]Fuc4NAc to lipid II. Our data support the conclusion that o359 of the wec gene cluster of E. coli is the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in the synthesis ECA trisaccharide repeat units.
肠杆菌共同抗原（ECA）的多糖链由三糖重复单元Fuc4NAc-ManNAcA-GlcNAc组成，其中Fuc4NAc是4-乙酰氨基-4,6-二脱氧-D-半乳糖，ManNAcA是N-乙酰-D-甘露糖胺糖醛酸，GlcNAc是N-乙酰-D-葡萄糖胺。单个三糖重复单元作为十一异戊二烯连接的中间体在一系列反应中组装，这些反应最终导致Fuc4NAc从TDP-Fuc4NAc转移到ManNAcA-GlcNAc-焦磷酸化十一异戊二烯醇（脂质II），生成Fuc4NAc-ManNAcA-GlcNAc-焦磷酸化十一异戊二烯醇（脂质III），这是ECA多糖链延伸的三糖重复单元供体。已知参与ECA组装的大多数基因位于大肠杆菌染色体上约85.4分钟处的wec基因簇中。现有数据表明，TDP-Fuc4NAc：脂质II Fuc4NAc转移酶的结构基因也位于wec基因簇中；然而，该基因的位置尚未明确确定。先前对o416和wecG之间区域的wec基因簇核苷酸序列的表征表明，它包含三个开放阅读框：o74、o204和o450。相比之下，当前研究中描述的实验结果表明，它仅包含两个开放阅读框，o359和o450。在o359中具有无效突变的大肠杆菌突变体无法合成ECA，并且它们积累了脂质II。此外，在使用从这些突变体获得的细胞提取物作为酶源的反应混合物中，未观察到[(3)H]FucNAc从TDP-[(3)H]Fuc4NAc体外掺入脂质II。这些突变体的ECA阴性表型被含有野生型o359等位基因的质粒构建体互补，并且通过使用从互补突变体获得的细胞提取物证明了Fuc4NAc转移酶活性。此外，部分纯化的o359基因产物，表达为重组C末端His标签蛋白，能够催化[(3)H]Fuc4NAc从TDP-[(3)H]Fuc4NAc体外转移到脂质II。我们的数据支持这样的结论，即大肠杆菌wec基因簇的o359是参与合成ECA三糖重复单元的TDP-Fuc4NAc：脂质II Fuc4NAc转移酶的结构基因。
