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

Abstract

ABSTRACT d -Galactan I is an O-antigenic polymer with the repeat unit structure [→3)-β- d -Gal f -(1→3)-α- d -Gal p -(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-Glc p NAc, 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 Gal p and one Gal f residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Gal f 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 Gal p residue to form a lipid-linked tetrasaccharide. Chain extension of the d -galactan I polymer requires WbbM for Gal p transferase, together with Gal f 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.