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, Kdo 2 -[4- 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.

Characterization of Galacturonosyl Transferase Genes rgtA, rgtB, rgtC, rgtD, and rgtE Responsible for Lipopolysaccharide Synthesis in Nitrogen-fixing Endosymbiont Rhizobium leguminosarum

“…Colonies were picked and streaked on TY/gentamicin plates. Mutant colonies were isolated using a PCR screen and given the strain names EL202 (rgtE ) mutants were, respectively, complemented with the previously described plasmids pMKGE, pRK-RgtA, pRK-RgtB, and pRK-RgtC by triparental mating (3,4).…”

“…The rgtE gene, previously referred to as orf3, was predicted to encode the GalAT that synthesizes Dod-P-GalA by KanjilalKolar et al (3,4); however, the gene function was not determined. The predicted amino acid sequence of RgtE is similar to a class of glycosyl transferases that has been demonstrated experimentally to synthesize polyprenyl-phosphate glycosyl lipid donors necessary for the glycosylation of lipid A (supplemental Fig.…”

“…In Vitro Reconstitution of GalAT Activity-The activity assay methods were adapted from Kanjilal-Kolar et al (3,4). Briefly, the standard reaction mixture contained 50 mM MES, pH 6.5, 0.05% Nonidet P-40, 2 mM MgCl 2 , and 2.5 M Kdo 2 -1-dephospho-[4Ј-32 P]lipid IV A (10 6 cpm/nmol).…”

“…The cell pellets were resuspended in prechilled 50 mM HEPES, pH 7.5. Membranes were prepared as previously described (4).…”

Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol

“…DEAE Fractionation of Total Lipid Extracts-Total lipid extracts derived from UE54 and MG1655 whole cells were fractionated on DE-52 cellulose (Whatman) as described previously (18).…”