Assembly of the K40 antigen in Escherichia coli: identification of a novel enzyme responsible for addition of L-serine residues to the glycan backbone and its requirement for K40 polymerization.

摘要: Escherichia coli O8:K40 coexpresses two distinct lipopolysaccharide (LPS) structures on its surface. The O8 polysaccharide is a mannose homopolymer with trisaccharide repeat unit and synthesized by an ABC-2 transport-dependent pathway. K40LPS backbone structure composed of repeating N-acetylglucosamine (GlcNAc) glucuronic acid (GlcA) has uncommon substitution, L-serine moiety attached to acid. gene cluster responsible for synthesis the K40 previously been cloned sequenced was found contain six open reading frames (ORFs) (P. A. Amor C. Whitfield, Mol. Microbiol. 26:145-161, 1997). Here, we demonstrate that insertional inactivation orf1 results in accumulation semirough (SR)-K40LPS form which retains reactivity specific polyclonal serum Western immunoblots. Structural compositional analysis SR-K40LPS reveals it comprises single lipid A core. lack polymerization indicates encodes polymerase (Wzy) assembly occurs via Wzy-dependent pathway (in contrast polysaccharide). Inactivation orf3 also SR-LPS fails react Methylation linkage fast atom bombardment-mass spectrometry this biological GlcNAc-GlcA-GlcNAc. Additionally, lacks substitution GlcA. These show (i) enzyme addition residue (ii) individual repeats essential their recognition into Wzy.