Functional analysis of the galactosyltransferases required for biosynthesis of D-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae

D-Galactan I is an O-antigenic polymer with the repeat unit structure [-->3 )-beta -D-Galf-(1 -->3)-alpha -D-Galp-(1 -->], that is found in the lipopol ysaccharide of Klebsiella pneumoniae O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and as sembly of D-galactan I via an ATP-binding cassette (ABC) transporter-depend ent pathway. The galactosyltransferase activities that ape required for the processive polymerization of D-galactan I were identified by using in vitr o reactions. The activities were determined with endogenous lipid accepters in membrane preparations from Escherichia coli K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with spe cific lipid accepters. The D-galactan I polymer is built on a lipid accepto r, undecaprenyl pyrophosphoryl-G1cpNAc, a product of the WecA enzyme that p articipates in the biosynthesis of enterobacterial common antigen and O-ant igenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is di rected into D-galactan I biosynthesis by the bifunctional wbbO gene product , which sequentially adds one Galp and one Galf residue from the correspond ing UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltran sferase 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 resi due to form a lipid-linked tetrasaccharide. Chain extension of the D-galact an I polymer requires WbbM for Galp transferase, together with Galf transfe rase activity provided by WbbO. Comparison of the biosynthetic pathways for D-galactan I and the polymannose E. coli O9a antigen reveals some interest ing features that may reflect a common theme in ABC transporter-dependent O -PS assembly systems.