A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum: Purification, substrate specificity, and expression in Salmonella waaC mutants

The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo₂-lipid IV_A. LpcC containing an N-terminal His₆ tag was assayed using GDP-mannose as the donor and Kdo₂-[4′-³²P]lipid IV_A as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo₂-[4′-³²P]lipid IV_A. A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IV_A or Kdo-lipid IV_A. The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the α-anomeric configuration) for the glycosylation of Kdo₂-[4′-³²P]lipid IV_A. Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner L-glycero-D-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo₂-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.