Identification of the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in synthesis of enterobacterial common antigen in Escherichia coli K-12.
Identification of the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in synthesis of enterobacterial common antigen in Escherichia coli K-12.
复制标题
大肠杆菌 K-12 中参与肠杆菌共同抗原合成的 TDP-Fuc4NAc:lipid II Fuc4NAc 转移酶结构基因的鉴定。
DOI：
10.1128/jb.183.22.6509-6516.2001
复制
发表时间：
2001
影响因子：
3.2
通讯作者：
Rick,PD
中科院分区：
文献类型：
--
作者：
Rahman,A;Barr,K;Rick,PD
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 isN-acetyl-d-mannosaminuronic acid, and GlcNAc isN-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 thewecgene cluster located at ca. 85.4 min on theEscherichia colichromosome. The available data suggest that the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase also resides in thewecgene cluster; however, the location of this gene has not been unequivocally defined. Previous characterization of the nucleotide sequence of thewecgene cluster in the region betweeno416andwecGrevealed that it contained three open reading frames:o74,o204, ando450. In contrast, the results of experiments described in the current investigation revealed that it contains only two open reading frames,o359ando450. Mutants ofE. colipossessing null mutations ino359were unable to synthesize ECA, and they accumulated lipid II. In addition, the in vitro incorporation of [3H]FucNAc from TDP-[3H]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-typeo359allele, and Fuc4NAc transferase activity was demonstrated by using cell extracts obtained from the complemented mutants. Furthermore, partially purifiedo359gene product, expressed as recombinant C-terminal His-tagged protein, was able to catalyze the in vitro transfer of [3H]Fuc4NAc from TDP-[3H]Fuc4NAc to lipid II. Our data support the conclusion thato359of thewecgene cluster ofE. coliis the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in the synthesis ECA trisaccharide repeat units.
