CLONING AND OVEREXPRESSION OF GLYCOSYLTRANSFERASES THAT GENERATE THE LIPOPOLYSACCHARIDE CORE OF RHIZOBIUM-LEGUMINOSARUM

The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizo bium leguminosarum is more amenable to enzymatic study than that of Es cherichia coil because much of it is synthesized from readily availabl e sugar nucleotides. The inner portion of the R. leguminosarum core co ntains mannose, galactose, and three 3-deoxy-D-manno-octulosonate (Kdo ) residues, arranged in the order: lipid A-(Kdo)(2)-Man-Gal-Kdo-[O ant igen], A mannosyltransferase. ase that uses GDP-mannose and the conser ved precursor Kdo(2)-[4'-P-32]lipid IVA (Kadrmas, J. L., Brozek, H. A. , and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32119-32125) is propo sed to represent a key early enzyme in R. leguminosarum core assembly, Conditions for demonstrating efficient galactosyl- and distal Kdo-tra nsferase activities are now described using a coupled assay system tha t starts with GDP-mannose and Kdo(2)-[4'-P-32]lipid IVA. As predicted, mannose incorporation precedes galactose addition, which in turn prec edes distal Kdo transfer, LPS core mutants with Tn5 insertions in the genes encoding the putative galactosyltransferase (lpcA) and the dista l Kdo-transferase (lpcB) are shown to be defective in the correspondin g in vitro glycosylation of Kdo(2)-[4'-P-32]lipid IVA. We have also di scovered the new gene (lpcC) that encodes the mannosyltransferase, The gene is separated by several kilobase pairs from the lpcAB cluster, A ll three glycosyltransferases are carried on cosmid pIJ1848, which con tains at least 20 kilobase pairs of R. leguminosarum DNA, Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of al l three enzymes, which are not normally present in strain 1021, Expres sion of the lpc genes individually behind the T7 promoter results in t he production of each R. leguminosarum glycosyltransferase in E. coil membranes in a catalytically active form, demonstrating that lpcA, lpc B, and lpcC are structural genes.