Journal of Biological Chemistry
Volume 273, Issue 41, 9 October 1998, Pages 26432-26440
Journal home page for Journal of Biological Chemistry

CARBOHYDRATES, LIPIDS, AND OTHER NATURAL PRODUCTS
Cloning and Overexpression of Glycosyltransferases That Generate the Lipopolysaccharide Core of Rhizobium leguminosarum *

https://doi.org/10.1074/jbc.273.41.26432Get rights and content
Under a Creative Commons license
open access

The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia colibecause much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three 3-deoxy-d-manno-octulosonate (Kdo) residues, arranged in the order: lipid A-(Kdo)2-Man-Gal-Kdo–[O antigen]. A mannosyltransferase that uses GDP-mannose and the conserved precursor Kdo2-[4′-32P]lipid IVA (Kadrmas, J. L., Brozek, K. A., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32119–32125) is proposed to represent a key early enzyme in R. leguminosarum core assembly. Conditions for demonstrating efficient galactosyl- and distal Kdo-transferase activities are now described using a coupled assay system that starts with GDP-mannose and Kdo2-[4′-32P]lipid IVA. As predicted, mannose incorporation precedes galactose addition, which in turn precedes distal Kdo transfer. LPS core mutants with Tn5 insertions in the genes encoding the putative galactosyltransferase (lpcA) and the distal Kdo-transferase (lpcB) are shown to be defective in the corresponding in vitroglycosylation of Kdo2-[4′-32P]lipid IVA. 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. leguminosarumDNA. 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 eachR. leguminosarum glycosyltransferase in E. colimembranes in a catalytically active form, demonstrating thatlpcA, lpcB, and lpcC are structural genes.

Cited by (0)

*

This work was supported in part by National Institutes of Health Grant GM-51796 (to C. R. H. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by National Institutes of Health Pharmacology Training Program 5T32GM07105 at Duke University. Present address: Dept. of Chemistry, University of Utah, Salt Lake City, UT 84112.