Assembly of the k40 antigen in Escherichia coli: Identification of a novelenzyme responsible for addition of L-serine residues to the glycan backbone and its requirement for K40 polymerization

Escherichia coli O8:K4O coexpresses two distinct lipopolysaccharide (LPS) s tructures on its surface. The O8 polysaccharide is a mannose homopolymer wi th a trisaccharide repeat unit and is synthesized by an ABC-2 transport-dep endent pathway. The K40(LPS) backbone structure is composed of a trisacchar ide repeating unit of N-acetylglucosamine (GlcNAc) and glucuronic acid (Glc A) and has an uncommon substitution, an L-serine moiety attached to glucuro nic acid. The gene cluster responsible for synthesis of the K40 polysacchar ide has previously been cloned and sequenced and was found to contain six o pen reading frames (ORFs) (P. A. Amor and C. Whitfield, Mol. Microbiol, 26: 145-161, 1997). Here, we demonstrate that insertional inactivation of orf1 results in the accumulation of a semirough (SR)-K40(LPS) form which retains reactivity with specific polyclonal serum in Western immunoblots, Structur al and compositional analysis of the SR-K40(LPS) reveals that it comprises a single K40 repeat unit attached to lipid A core, The lack of polymerizati on of the K40 polysaccharide indicates that orf1 encodes the K40 polymerase (Wzy) and that assembly of the K40 polysaccharide occurs via a Wzy-depende nt pathway (in contrast to that of the O8 polysaccharide). Inactivation of orf3 also results in the accumulation of an SR-LPS form which fails to reac t with specific polyclonal K40 serum in Western immunoblots. Methylation li nkage analysis and fast atom bombardment-mass spectrometry of this SR-LPS r eveals that the biological repeat unit of the K40 polysaccharide is GlcNAc- GlcA-GlcNAc. Additionally, this structure lacks the L-serine substitution o f GlcA. These results show that (i) orf3 encodes the enzyme responsible for the addition of the L-serine residue to the K40 backbone and (ii) substitu tion of individual K40 repents with L-serine is essential for their recogni tion and polymerization into the K40 polysaccharide by Wzy.