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

The polysaccharide chains of enterobacterial common antigen (ECA) are compr ised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminur onic acid, and GlcNAc is N-acetyl-D-glucosamine. Individual trisaccharide r epeat units are assembled as undecaprenyl-linked intermediates in a sequenc e of reactions that culminate in the transfer of Fuc4NAc from TDP-Fuc4NAc t o ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid H) to yield Fuc4NAc-ManN Ar-A-GlcNAc-pyrophosphorylundecaprenol (lipid III), the donor of trisacchar ide 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 l ocated at ca. 85.4 min on the Escherichia coli chromosome. The available da ta suggest that the structural gene for the TDP-Fuc4NAc:lipid H Fuc4NAc tra nsferase also resides in the wec gene cluster; however, the location of thi s 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 inve stigation revealed that it contains only two open reading frames, o359 and o450. Mutants of E. coli possessing null mutations in o359 were unable to s ynthesize ECA, and they accumulated lipid IL In addition, the in vitro inco rporation of [H-3]FucNAc from TDP-[H-3]Fuc4NAe into lipid H was not observe d in reaction mixtures using cell extracts obtained from these mutants as a source of enzyme. The ECA-negative phenotype of these mutants was compleme nted by plasmid constructs containing the wild-type o359 allele, and Fuc4NA c transferase activity was demonstrated by using cell extracts obtained fro m the complemented mutants. Furthermore, partially purified o359 gene produ ct, expressed as recombinant C-terminal His-tagged protein, was able to cat alyze the in vitro transfer of [H-3]Fuc4NAe from TDP- [H-3]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 H Fuc4NAc transfera se involved in the synthesis ECA trisaccharide repeat units.