Expression cloning of three Rhizobium leguminosarum lipopolysaccharide core galacturonosyltransferases

Abstract

The lipid A and core regions of the lipopolysaccharide in Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, are strikingly different from those of Escherichia coli. In R. leguminosarum lipopolysaccharide, the inner core is modified with three galacturonic acid (GalA) moieties, two on the distal 3-deoxy-D-manno-octulosonic acid (Kdo) unit and one on the mannose residue. Here we describe the expression cloning of three novel GalA transferases from a 22-kb R. leguminosarum genomic DNA insert-containing cosmid (pSGAT). Two of these enzymes modify the substrate, Kdo2-[4'-(32)P]lipid IV(A) and its 1-dephosphorylated derivative on the distal Kdo residue, as indicated by mild acid hydrolysis. The third enzyme modifies the mannose unit of the substrate mannosyl-Kdo2-1-dephospho-[4'-(32)P]lipid IV(A). Sequencing of a 7-kb subclone derived from pSGAT revealed three putative membrane-bound glycosyltransferases, now designated RgtA, RgtB, and RgtC. Transfer by tri-parental mating of these genes into Sinorhizobium meliloti 1021, a strain that lacks these particular GalA residues, results in the heterologous expression of the GalA transferase activities seen in membranes of cells expressing pSGAT. Reconstitution experiments with the individual genes demonstrated that the activity of RgtA precedes and is necessary for the subsequent activity of RgtB, which is followed by the activity of RgtC. Electrospray ionization-tandem mass spectrometry and gas-liquid chromatography of the product generated in vitro by RgtA confirmed the presence of a GalA moiety. No in vitro activity was detected when RgtA was expressed in Escherichia coli unless Rhizobiaceae membranes were also included.

FIGURE 1. Partial structures of the R. leguminosarum and E. coli core oligosaccharides

The two Kdo residues closest to lipid A and the α-(1–5) linkage of the heptose or mannose attached to the inner Kdo are conserved. Dashed lines represent partial substituents. The genes encoding the enzymes responsible for generating the relevant linkages are indicated. The galacturonic acid residues in R. leguminosarum are shown in italics. The abbreviations used are as follows: Glc, D-glucose; Gal, D-galactose; Hep; L-glycero-D-manno-heptose; GlcNAc, N-acetyl-D-glucosamine; Man, mannose.

FIGURE 2. Structure of the conserved intermediate Kdo₂-lipid IV_A and its enzymatic processing in extracts of R. leguminosarum versus E. coli

The seven enzymes leading to the intermediate Kdo₂-lipid IV_A are conserved in E. coli and R. leguminosarum. Key enzymes that have been identified to date as being required for the subsequent processing of Kdo₂-lipid IV_A are indicated.

FIGURE 3. Two hydrophilic products generated from Kdo₂-lipid IV_A by membranes of S. meliloti harboring pMKGE

Crude extracts of S. meliloti 1021/pMKGE were centrifuged at 100,000 × g to yield cytosolic and membrane fractions. The indicated fractions were then assayed at 0.25 mg/ml protein with 2.5 μM Kdo₂-[4′-³²P]lipid IV_A as the acceptor under standard assay conditions. Membranes from S. meliloti cells harboring the vector pRK404a were used as the control. NE, no enzyme.

FIGURE 4. Modification of Kdo₂-lipid IV_A or Kdo₂-1-dephospho-lipid IV_A by membranes of S. meliloti harboring pMKGE

The putative GalA transferase activities present in membranes of S. meliloti 1021/pMKGE were compared with wild type R. leguminosarum 3841, S. meliloti 1021, or S. meliloti 1021 harboring the vector pRK404a. Membranes were assayed at 0.25 mg/ml with 2.5 μM Kdo₂-[4′-³²P]lipid IV_A (panel A) or Kdo₂-1-dephospho-[4′-³²P]lipid IV_A (panel B). Their products were designated I, II, or I’,II’, respectively. The time course with Kdo₂-[4′-³²P]lipid IV_A (closed circles) versus the 1-dephosphorylated analogue (open circles) is shown in panel C. The percent conversion refers to the sum of both products.

FIGURE 5. Time course of mild acid hydrolysis of Kdo₂-[4′-³²P]lipid IV_A versus products I and II

Panel A, the Kdo₂-[4′-³²P]lipid IV_A control. Panel B, products I and II. The hydrolysis was carried out at 100 °C in 12.5 mM sodium acetate buffer, pH 4.5, in the presence of SDS (40). The two Kdo glycosidic linkages are similarly susceptible to cleavage under these conditions, allowing discrimination between modification of the outer versus the inner Kdo residues or lipid A.

FIGURE 6. Functions of enzymes encoded by genes in the rgt region of the R. leguminosarum chromosome

Panel A, the 7-kb DNA insert in pMKGE was sequenced and analyzed using the NCBI ORF finder program (42). The sequence shows four putative open reading frames that are transcribed in the indicated directions. The possible functions of the ORFs were evaluated using COGNITOR (46) and PSI-BLAST (64). The results are summarized in Table 3. Panel B, reactions were performed with Kdo₂-1-dephospho-[4′-³²P]lipid IV_A as substrate. Washed membranes of S. meliloti 1021 cells harboring the indicated plasmids were added at 0.25 mg/ml. The mixtures were incubated at 30 °C for the indicated times, and 4-μl portions were spotted onto a silica TLC plate to stop the reactions. The membranes of the constructs were assayed individually or in different combinations to determine the order of RgtA and RgtB function.

FIGURE 7. A third GalA transferase activity catalyzed by RgtC

S. meliloti 1021/pMKGE membranes were assayed with mannosyl-Kdo₂-1-dephospho-[4′-³²P]lipid IV_A under standard assay conditions without carrier substrate at 0.25 mg/ml membrane protein. With this substrate, a third product is observed at later times, designated product III’. The three products can also be generated by the sequential action of RgtA, RgtB, and RgtC expressed individually in S. meliloti 1021, as shown in the far right lane.

FIGURE 8. Negative ion ESI/MS of Kdo₂-1-dephospho-lipid IV_A and product I’

Product I’ was synthesized in vitro from Kdo₂-1-dephospho-lipid IV_A (see “Experimental Procedures”), and both the remaining substrate and product I’ were purified by DEAE-cellulose chromatography. The spectra in the m/z range 830–990 atomic mass units (amu) of the 240 mM fraction (panel A) and the 480 mM fraction (panel B) are shown. Both substrate (panel A) and product I’ (panel B) yield mainly [M — 2H]^2- ions with the expected masses and isotope distributions for Kdo₂-1-dephospho-lipid IV_A or GalA-Kdo₂-1-dephospho-lipid IV_A, respectively. Additional details are listed in Table 5. Unassigned peaks are because of lipid impurities carried over from the membranes that were used as the enzyme source.

FIGURE 9. MS/MS analysis of Kdo₂-1-dephospho-lipid IV_A and product I’

Panel A, MS/MS of the [M — 2H]^2- substrate ion at m/z 881.5 (see Fig. 8A). Panel B, MS/MS of the [M — 2H]^2- product I’ ion at m/z 969.5 (see Fig. 8B). Key peaks unique to the substrate and product are italicized. The mass shifts of the Kdo-containing ions are consistent with the addition of 1 HexA unit to the outer Kdo residue. amu, atomic mass units.

FIGURE 10. Identification of GalA in product I’ by GC/MS analysis

Trimethylsilyl ester derivatives of methyl glycosides, obtained by methanolic HCl hydrolysis of DEAE-cellulose-purified fractions (from Fig. 8), were separated by gas-liquid chromatography. Peaks were identified by the comparison of their retention times with those of standards prepared in parallel (see asterisks). The profile (selective ion monitoring at m/z 217) for product I’ is compared with that of the GalA and GlcA standards.

FIGURE 11. A Rhizobiaceae membrane component is required for RgtA activity in E. coli

Membranes of the indicated constructs in E. coli NovaBlue (DE3) were assayed at 0.25 mg/ml. Additional membranes from S. meliloti 1021 or R. leguminosarum 3841 were added at 0.25 mg/ml, as indicated. Product I’ formation upon reconstitution was also compared with the control membranes of S. meliloti 1021/pRgtA.

FIGURE 12. Order of possible enzymatic reactions for the attachment of GalA moieties to the R. leguminosarum LPS core

The enzymes responsible for the 1-dephosphorylation of lipid A and for the incorporation of the mannose have been reported elsewhere (15, 22). RgtA and RgtB are proposed to add the two GalA units to the outer Kdo. The locations and stereochemistry of the attachment sites as α-(1–4) and α-(1–5) are proposed based on the structures of the related oligosaccharides isolated from cells (17, 65). Our data do not establish which linkage is formed first by RgtA and whether or not the same linkages are generated in our in vitro system as occur in vivo (17). Larger quantities of each product will have to be synthesized to resolve these structural ambiguities. RgtC presumably attaches a third GalA moiety to the core mannose unit in α-(1–4) linkage (17, 65), given the substrate requirement for detecting RgtC activity as shown here.