Cloning and Overexpression of Glycosyltransferases That Generate the Lipopolysaccharide Core of Rhizobium leguminosarum

Abstract

The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia coli because much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three We have also discovered the new gene (lpcC) that encodes the mannosyltransferase. The gene is separated by several kilobase pairs from the lpcAB cluster. All three glycosyltransferases are carried on cosmid pIJ1848, which contains at least 20 kilobase pairs of R. leguminosarum DNA. Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of all three enzymes, which are not normally present in strain 1021. Expression of the lpc genes individually behind the T7 promoter results in the production of each R. leguminosarum glycosyltransferase in E. coli membranes in a catalytically active form, demonstrating that lpcA, lpcB, and lpcC are structural genes.

Effect of Cold Shock on Lipid A Biosynthesis inEscherichia coli

“…Isolation and Preparation of Lipid Substrates-Lipid IV A and (Kdo) 2 -lipid IV A were isolated and purified as described (14,24,25 (25)(26)(27), except that membranes of the 4Ј-kinase overproducing strain BLR(DE3)pLysS/pJK2 (28) were used to increase the yield of [4Ј-32 P]lipid IV A . Lauroyl, myristoyl, palmitoyl, R-3-hydroxymyristoyl, and palmitoleoyl-ACP were synthesized from the corresponding fatty acids and commercial acyl carrier protein, using solubilized membranes from the acyl-ACP synthase overproducing strain E. coli LCH109/ pLCH5/pGP1-2, as described previously (10,29).…”

Expression Cloning of Three Rhizobium leguminosarum Lipopolysaccharide Core Galacturonosyltransferases

“…3). There was no dependence of activity on the addition of cytosolic components (data not shown), in contrast to the behavior of the R. leguminosarum core glycosyltransferases LpcA, LpcB, and LpcC, which are membranebound enzymes requiring cytosolic sugar nucleotide donors (15).…”

“…5). Unlike the membrane-bound R. leguminosarum core glycosyltransferases LpcA, LpcB, and LpcC, which require a cytosolic sugar nucleotide donor (15), the proposed GalA transferases require only membrane components (Fig. 3).…”

“…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 supplementary components added for mannosylation were 50 mM HEPES, pH 7.5, 0.1% Triton X-100, 1 mM GDP-mannose, and 0.3-0.5 mg/ml pIJ1848/S. meliloti 1021 membranes (15,40) in a total volume of 20 l, making the final reaction volume 120 l. The reaction was allowed to proceed at 30°C for 30 min. This was followed by the addition of another 0.3-0.5 mg/ml membranes with 30 min of incubation at 30°C.…”