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
A. Rahman et al., 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, J BACT, 183(22), 2001, pp. 6509-6516
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.