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

Abstract

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, Kdo(2)-lipid IV(A). LpcC containing an N-terminal His(6) tag was assayed using GDP-mannose as the donor and Kdo(2)-[4'-(32)P]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 Kdo(2)-[4'-(32)P]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 alpha-anomeric configuration) for the glycosylation of Kdo(2)-[4'-(32)P]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 Kdo(2)-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.

Fig. 1. Structures of R. leguminosarum and Escherichia coli lipid A and of the inner core oligosaccharides

The closely related structures of L-glycero-D-manno-heptose and D-mannose are highlighted for comparison. There is, however, no sequence similarity between the enzyme that attaches the innermost heptose in E. coli (WaaC) and the enzyme that attaches the mannose residue in R. leguminosarium (LpcC) (14, 19). Only the major molecular species are indicated, which, in the case of R. leguminosarum, is designated component D-1 (2, 8, 9, 12).

Fig. 2. Proposed reaction catalyzed by the R. leguminosarum mannosyl transferase LpcC

The actual linkage generated in vitro by the cloned mannosyl transferase has not yet been confirmed but is presumed to be the same as that seen in the core domain of the LPS isolated from cells (11, 12).

Fig. 3. SDS Gel electrophoresis of fractions generated during the purification of LpcC

Lanes 1 and 2 contain 10 μg of protein from membranes and solubilized membranes, respectively, of BLR(DE3)/pLysS/pMKHN. Lane 3 contains 1.0 μg of LpcC after the nickel affinity column. Much of the overexpressed LpcC protein is not solubilized by Triton X-100, most likely because it is trapped in small inclusion bodies. However, about half of the mannosyl transferase activity present in whole membranes is recovered in the Triton X-100-solubilized fraction (see Table I).

Fig. 4. Sugar nucleotide specificity of purified LpcC

Reactions were carried out under standard conditions at pH 7.5 using a 1 mM concentration of each of the sugar nucleotides, as indicated. Purified enzyme was used at 5 μg/ml. The incubation was carried out at 30 °C for 30 min. Under conditions where product evolution is linear with time (data not shown), the very slow reactions with UDP-glucose and UDP-galactose are not detectable.

Fig. 5. Lipid acceptor specificity of purified LpcC

Reactions were carried out under standard conditions at pH 7.5 using either 10 μM Kdo₂-[4′-³²P]lipid IV_A, Kdo-[4′-³²P]lipid IV_A, or [4′-³²P]lipid IV_A in the presence of 2.5 μg/ml protein.

Fig. 6. Time course of hydrolysis at 100 °C of Kdo₂-[4′-³²P]lipid IV_A versus mannosyl-Kdo₂-[4′-³²P]lipid IV_A

A, the Kdo₂-[4′-³²P]lipid IV_A control. B, mannosyl-Kdo₂-[4′-³²P]lipid IV_A. The hydrolysis is carried out in sodium acetate buffer at pH 4.5 in the presence of SDS (19). The two Kdo glycosidic linkages are about equally susceptible to cleavage under these conditions, allowing discrimination between mannose addition to the outer versus the inner Kdo (19). LpcC modifies the inner Kdo as shown by the absence of unmodified Kdo-[4′-³²P]lipid IV_A during the time course of the hydrolysis of the mannosyl-Kdo₂- [4′-³²P]lipid IV_A.

Fig. 7. SDS gel electrophoresis of LPS from Salmonella waaC(rfaC) mutants complemented with R. leguminosarum lpcC

LPS preparations were separated on a 12% SDS gel and silver-stained by the method of Hitchcock and Brown (43). Lane 1, SL3770 (waa⁺); lane 2, SL1377 (waaC630); lane 3, SL1377 with pBluescript KS⁺; lane 4, SL1377 with pMKCA.

Fig. 8. MALDI-TOF mass spectra of LPS from E. coli WBB06 (ΔwaaC-waaF) containing pBluescript KS⁺ or pMKCA

Spectra were acquired in the negative mode. A, LPS isolated from WBB06 cells containing the vector pBluescript⁺. B, LPS isolated from WBB06 cells containing pMKCA (lpcC⁺). C, the proposed structure of the mannosyl-Kdo₂-lipid A.

Fig. 9. Two-dimensional ¹H NMR spectra at 600 MHz in the sugar region of ADP-mannose

A, COSY in D₂O at 25 °C showing through-bond connectivities. Only the ¹H resonances of the mannose residue are numbered. B, NOESY in D₂O at 25 °C showing through-space connectivities. The mannose sugar ¹H resonances and cross-peaks are as indicated. X indicates the residual HOD peak after reduction with presaturation and software processing. The NOESY was obtained with a 450-ms mixing time. No cross-peaks are seen between mannose H-1″ and H-3″ or between H-1″ and H-5″ (expected locations designated by the thin arrows), demonstrating that the anomeric configuration of the mannose residue must be α.