The lipopolysaccharide of Rhizobium leguminosarum differs from that of other Gram-negative organisms. R. leguminosarum lipid A lacks phosphate groups, but it contains a galacturonic acid residue at the 4-position and an aminogluconate moiety in place of the usual glucosamine 1-phosphate unit. R. leguminosarum lipid A is esterified with a peculiar long chain fatty acid, 27-hydroxyoctacosanoate, not found in enteric Gramnegative bacteria, and the inner core of R. leguminosarum contains mannose and galactose in place of heptose. Despite these differences, the biosynthesis of R. leguminosarum In addition, at least two hydrophobic metabolites were generated from (Kdo) 2 -[4-32 P]-lipid IV A in a manner that was dependent upon both membranes and a cytosolic factor from R. leguminosarum. These compounds are attributed to novel acylations of (Kdo) 2 -[4-32 P]-lipid IV A . E. coli membranes and cytosol did not catalyze any of the unique reactions detected in R. leguminosarum extracts. Our findings establish the conservation and versatility of (Kdo) 2 -lipid IV A as a lipid A precursor in bacteria.

Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a ␤1-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo) 2 -lipid IV A , a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo) 2 -[4-32 P]-lipid IV A as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf * contains the consensus sequence, DSLD, for attachment of 4-phosphopantetheine. When the entire orf * gene was sequenced, it was found to encode a protein of 92 amino acids. Orf * is a new kind of acyl carrier protein because it is only ϳ25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf * confirmed the presence of 4-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf * acylation with hydroxylated 20, 22, 24, and 26 carbon fatty acids were also observed. Given the specialized function of Orf * in lipid A acylation, we suggest the new designation AcpXL.

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...