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

Abstract

(Fig. 1B). D-Galactan II provides the epitope or epitopes that define the O1 antigen (55). The presence of D-galactan II is required for the resistance of the bacteria to complement-mediated killing in the host, and therefore Klebsiella pneumoniae mutants that produce only D-galactan I are serum sensitive (33).There are two major pathways for the biosynthesis of LPS O-PS. These are designated Wzy dependent and ATP-binding cassette (ABC) transporter dependent (reviewed in references 52 and 53). Both pathways are widely distributed among different genera and are involved in the synthesis of diverse O-PS structures. A third pathway (synthase dependent) is currently confined to a single O-PS (17). In the Wzy-dependent mechanism, undecaprenyl pyrophosphoryl (und-PP)-linked oligosaccharide repeat units are assembled at the cytoplasmic face of the inner membrane. These intermediates are then exported to the periplasmic face, where they provide the direct substrates for polymerization. Export and polymerization require the activity of members of the Wzx and Wzy protein families, respectively. In the ABC transporter-dependent mechanism, the polymer is built by processive transfer of glycosyl residues to an initiating und-PP-linked lipid intermediate at the cytoplasmic face of the inner membrane. After the lipid-linked polymer is synthesized at the cytoplasmic face of the inner membrane, it is exported to the periplasmic face by an ABC transporter (5, 45, 58). The various O-PS biosynthesis pathways are believed to converge with the presence of und-PPlinked polymer at the periplasmic face of the inner membrane, at which point the nascent O-PS is ligated to preformed lipid A core. The completed LPS molecule is then translocated to the outer leaflet of the outer membrane by a process that is still undetermined.In members of the family Enterobacteriaceae, initiation of ABC transporter-dependent

A Membrane-located Glycosyltransferase Complex Required for Biosynthesis of the d-Galactan I Lipopolysaccharide O Antigen in Klebsiella pneumoniae

“…The predicted N-terminal domain (WbbO N ) could not complement the ⌬wbbO mutation as expected (data not shown). Therefore, when expressed from a plasmid, WbbO does not require the N terminus for galactosyltransferase function and the reported ␣-galactopyranosyl-and ␤-galactofuranosyltransferase activity (14,20) of the enzyme is confined to the C-terminal domain of WbbO.…”

“…Localization and Domain Analysis of WbbM-WbbM is classified in GT family 8 and in vitro data shows that it adds galactose residues to elongate the product of WbbO (20). However, the precise structure of the residue(s) added has not been established.…”

“…From both in vivo and in vitro experiments, WbbO is proposed (11). The adaptor region consists of two galactose residues believed to be added to und-PP-GlcNAc by WbbO (14,20). The precise glycosyltransferase activities required for extension of the D-galactan I domain remain undetermined.…”

“…to add the first two sugars (Galp-Galf) to the und-PP-GlcNAc acceptor (14,20), forming the adaptor region of the O-PS (Fig. 1).…”

“…The goal of this study was to examine potential proteinprotein interactions among enzymes required for D-galactan I biosynthesis. Bioinformatic analysis indicates that the K. pneumoniae D-galactan I biosynthesis gene cluster encodes three predicted galactosyltransferases, designated WbbM, WbbN, and WbbO, with confirmatory experimental evidence being available for WbbO and WbbM (20). The cluster also encodes the UDP-galactopyranose mutase (Glf) enzyme responsible for production of the UDP-Galf precursor (21) (see Fig.…”