Functional Analysis of the Galactosyltransferases Required for Biosynthesis of d-Galactan I, a Component of the Lipopolysaccharide O1 Antigen of Klebsiella pneumoniae

AUTOR(ES)

Guan, Shukui

FONTE

American Society for Microbiology

RESUMO

d-Galactan I is an O-antigenic polymer with the repeat unit structure [→3)-β-d-Galf-(1→3)-α-d-Galp-(1→], that is found in the lipopolysaccharide of Klebsiella pneumoniae O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly of d-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization of d-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from Escherichia coli K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. The d-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-GlcpNAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed into d-galactan I biosynthesis by the bifunctional wbbO gene product, which sequentially adds one Galp and one Galf residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Galf precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of d-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Galp residue to form a lipid-linked tetrasaccharide. Chain extension of the d-galactan I polymer requires WbbM for Galp transferase, together with Galf transferase activity provided by WbbO. Comparison of the biosynthetic pathways for d-galactan I and the polymannose E. coli O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.

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