Lipid A biosynthesis in Rhizobium leguminosarum: role of a 2-keto-3-deoxyoctulosonate-activated 4' phosphatase.

Abstract

Lipid A from several strains of the N2-fixing bacterium Rhizobium leguminosarum displays significant structural differences from Escheriehia coli lipid A, one of which is the complete absence of phosphate groups. However, the first seven enzymes of E. coli lipid A biosynthesis, leading from UDP-GlcNAc to the phosphorylated intermediate, 2-keto-3-deoxyoctulosonate (Kdo2)-lipid IVA, are present in R. leguminosarum. We now describe a membrane-bound phosphatase in R leguminosarum extracts that removes the 4' phosphate of Kdo2-lipid IVA. The 4' phosphatase is selective for substrates containing the Kdo domain. It is present in extracts of R. keguminosarum biovars phaseoli, viciae, and trifolii but is not detectable in E. coli and Rhizobium meliloti. A nodulation-defective strain (24AR) of R. leguminosarum biovar trifolii, known to contain a 4' phosphate residue on its lipid A, also lacks measurable 4' phosphatase activity. The Kdo-dependent 4' phosphatase appears to be a key reaction in a pathway for generating phosphate-deficient lipid A.Endotoxins are lipopolysaccharides (LPSs) that comprise the outer leaflet of the outer membranes of Gram-negative bacteria (1-3). The biosynthesis of the lipid A portion of Escherichia coli LPS (Fig. 1A) is crucial for cell viability (1,5,6). Additionally, the lipid A moiety of LPS is responsible for the toxic effects observed when LPS is injected into animals, which include fever and shock (3,7,8). This toxicity is based on the potent stimulation by lipid A of the host's immune system, resulting in the production of lethal amounts of tumor necrosis factor and other cytokines (6, 9, 10). The presence of the phosphate groups, of the glucosamine disaccharide, and of certain fatty acyl chains is crucial for the biological activities of lipid A (3,11,12).There is considerable interest in lipid A analogs that can act as antagonists of the toxic lipid As found in human pathogens (1,(13)(14)(15) or that could function as partial agonists for use as adjuvants in vaccines (7). One of the most remarkable lipid As reported to date is that of R leguminosarum (Fig. 1B), a N2-fixing symbiont of certain legumes (4). The lipid A of R leguminosarum differs from that of E. coli in that it lacks phosphate groups (Fig. 1B) (4). It contains a galacturonic acid residue in place of the 4' phosphate and an aminogluconic acid moiety in place of glucosamine-1-phosphate (Fig. 1B) (4). It apparently has no acyloxyacyl substituents but, instead, bears some very long acyl chains (27-OH-C:28) (4). Despite the unique structural features of its lipid A, R. leguminosarum extracts contain all seven enzymes required for the synthesis of KdO2-lipid IVA ( Fig. 2A) (16), an important phosphorylated precursor of E. coli lipid A (18)(19)(20). The fact that R leguminosarum and E. coli make the same late intermediate ( Fig. 2A) (16) 7352The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 so...

Origin of the 2-Amino-2-deoxy-gluconate Unit inRhizobium leguminosarum Lipid A

“…The R. leguminosarum lpxE structural gene encoding the 1-phosphatase has recently 2 M. Karbarz and C. R. H. Raetz, manuscript in preparation. (43,44,(55)(56)(57). The only exceptions are the reactions that incorporate the 4Ј-galacturonic acid and the ␤-hydroxybutyrate residues.…”

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

“…Assay Conditions-For mannosyltransferase reactions, unless indicated, the standard reaction mixtures (10 -40 l) contained 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 10 M Kdo 2 -[4Ј- 32 (42,43). The mannosyltransferase, LpcC, is proposed to form the ␣,1-5 linkage between mannose and the inner Kdo.…”

“…2), are identical (42,43). In both systems, Kdo 2 -lipid IV A can be further acylated (2, 48 -50), but in R. leguminosarum, Kdo 2 -lipid IV Acan also be dephosphorylated at the 1-and 4Ј-positions to generate an unusual lipid A moiety lacking phosphate (23,43). In both systems, Kdo 2 -lipid IV A can also serve as an acceptor of several distinct core sugars, which are transferred one at a time from sugar nucleotide donors (18,19,23,44).…”

“…However, the plasmid pJK6 conferred upon E. coli membranes the ability to catalyze efficient GDP-mannose-dependent transfer of mannose to Kdo 2 -[4Ј- (Fig. 2), are identical (42,43). In both systems, Kdo 2 -lipid IV A can be further acylated (2, 48 -50), but in R. leguminosarum, Kdo 2 -lipid IV Acan also be dephosphorylated at the 1-and 4Ј-positions to generate an unusual lipid A moiety lacking phosphate (23,43).…”

The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359

“…The presence of this common lipid A precursor also implied that these rhizobial cells possess unique enzyme activities, which process their Kdo 2 lipid-IVa analog into the mature rhizobial lipid A structure. Several of these enzyme activities have been reported, including a membranebound phosphatase that removes the 4Ј-phosphate from Kdo 2 lipid-IVa and requires the presence of the Kdo residues for maximum activity (21), a second phosphatase that removes the 1-phosphate (22), and a unique acyl carrier protein required for the transfer of the 27-hydroxyoctacosanoyl residue to the lipid A (23). In addition, a rhizobial-specific mannosyl transferase activity has been identified that transfers mannose from GDPMan to Kdo 2 lipid-IVa (24).…”