Biosynthesis of a Novel 3-Deoxy-D-manno-oct-2-ulosonic Acid-containing Outer Core Oligosaccharide in the Lipopolysaccharide of Klebsiella pneumoniae

Abstract

The core oligosaccharide region of Klebsiella pneumoniae lipopolysaccharide contains some novel features that distinguish it from the corresponding lipopolysaccharide region in other members of the Enterobacteriaceae family, such as Escherichia coli and Salmonella. The conserved Klebsiella outer core contains the unusual trisaccharide 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)-(2,6)-GlcN-(1,4)-GalUA. In general, Kdo residues are normally found in the inner core, but in K. pneumoniae, this Kdo residue provides the ligation site for O polysaccharide. The outer core Kdo residue can also be non-stoichiometrically substituted with an L-glycero-D-manno-heptopyranose (Hep) residue, another component more frequently found in the inner core. To understand the genetics and biosynthesis of core oligosaccharide synthesis in Klebsiella, the gene products involved in the addition of the outer core GlcN (WabH), Kdo (WabI), and Hep (WabJ) residues as well as the inner core HepIII residue (WaaQ) were identified. Non-polar mutations were created in each of the genes, and the resulting mutant lipopolysaccharide was analyzed by mass spectrometry. The in vitro glycosyltransferase activity of WabI and WabH was verified. WabI transferred a Kdo residue from CMP-Kdo onto the acceptor lipopolysaccharide. The activated precursor required for GlcN addition has not been identified. However, lysates overexpressing WabH were able to transfer a GlcNAc residue from UDP-GlcNAc onto the acceptor GalUA residue in the outer core.

KpsC and KpsS are retaining 3-deoxy- d - manno -oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules

“…The CMP-β-Kdo synthetase, KdsB, was expressed and purified as previously described (46 portions of the fusion proteins were equivalent to 4 μg. Reaction mixtures were incubated at 25°C, then stopped with the addition of an equal amount of 50% acetonitrile, 1% SDS, and 10 mM EDTA.…”

Three Enzymatic Steps Required for the Galactosamine Incorporation into Core Lipopolysaccharide

“…For quantitative analysis, the reactions were stopped by adding an equal volume of stop solution (27) and incubated on ice for 1 h. The LPS was recovered by filtration (0.45 m) and washed, and the amount of radioactivity incorporated into LPS was measured in a scintillation counter.…”

“…GlcNAc and GalNAc Transferase Assay-The transferase activity of His 6 -WabH R110 and His 6 -WabP 50/57 were determined essentially as described (27). Briefly, reactions were performed in a total 0.1-ml volume at a final concentration of 50 mM Tris-HCl (pH 8.0) containing 10 mM MgCl 2 and 1 mM dithiothreitol.…”

“…Both groups shared 60 and 89% levels of identity and similarity. An alignment between these proteins and those of K. pneumoniae strain C3 and 52145, where it has been shown that this protein transfers the GlcNAc from UDP-GlcNAc to core OS (27), was done to identify residues specific for the WabH-like 50/57 -WabH-like TG83 pair (Fig. 2).…”

“…WabH and WabP in Vitro Activity-In vitro enzymatic assays were performed (27) to test if the WabH R110 and WabP 50/57 are specific for the transfer of GlcNAc and GalNAc, respectively. This assay measured the amount of radiolabeled GlcNAc or GalNAc incorporated into acceptor LPS from UDP-[ 14 C]GlcNAc or UDP-[ 14 C]GalNAc.…”