The structure of the lipid-A from Rhizobium species Sin-1, a nitrogen-fixing Gram-negative bacterial symbiont of Sesbania, was determined by composition, nuclear magnetic resonance spectroscopic, and mass spectrometric analyses. The lipid-A preparation consisted of a mixture of structures due to differences in fatty acylation and in the glycosyl backbone. There were two different disaccharide backbones. One disaccharide consisted of a distal glucosaminosyl residue ␤-linked to position 6 of a

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... ximal 2-aminoglucono-1,5-lactonosyl residue, and in the second disaccharide, the proximal residue was 2-amino-2,3-dideoxy-D-erythro-hex-2-enono-1,5-lactone. For both disaccharides, the distal glucosamine was acylated at C-2 primarily with ␤-hydroxypalmitate (␤-OHC16:0) which, in turn, was O-acylated with 27-hydroxyoctacosanoic acid. For some of the lipid-A molecules, the distal glucosaminosyl residue was also acylated at C-3 with ␤-hydroxymyristate (␤-OHC14: 0), whereas other molecules were devoid of this acyl substituent. Both the 2-aminoglucono-1,5-lactonosyl and 2-amino-2,3-dideoxy-D-erythro-hex-2-enono-1,5-lactonosyl residues were acylated at C-2, primarily with ␤-OHC16:0. Minor amounts of lipid-A molecules contained ␤-OHC14:0 at C-3 and/or ␤-hydroxystearate (␤-OHC18:0) or ␤-hydroxyoctadecenoate (␤-OHC18:1) as the C-2 and C-2 N-acyl substituents. Rhizobia refer collectively to the group of Gram-negative bacteria that belong to the Rhizobiaceae family and form nitrogen-fixing symbioses with legume plants. The major constituent of the Gram-negative bacterial cell wall is lipopolysaccharide (LPS). 1 The LPS molecule has three structural regions as follows: the O-chain polysaccharide, core oligosaccharide, and the hydrophobic lipid-A. The LPS has been shown to be important in the symbiotic infection process (1-4). Structural changes to both the O-chain polysaccharide and to the lipid-A appear to be important for symbiotic infection (5). These changes include methylation of the O-chain glycosyl residues and increased fatty acylation of the lipid-A with 27-hydroxyoctacosanoic acid (27-OHC28:0) (6), a long-chain fatty acyl component that is common to the lipid-A isolated from members of the Rhizobiaceae (7-9). As with other Gram-negative bacteria, the LPS of rhizobia most likely have important roles that enable these bacteria to adapt to different environments; in this case, the intracellular environment of the legume host cell. These roles probably include acting as a permeation barrier toward potential toxins (e.g. defense response molecules from the host) as well as other structural adaptations that allow survival within the host cell. Lipid-A is considered the least variable region in the LPS molecule. The lipid-A structure from enteric bacteria is largely conserved, consisting of a ␤-(136)-linked glucosamine disaccharide backbone with phosphate groups at C-1 and C-4Ј and ␤-hydroxy fatty acyl groups and acyloxyacyl residues at positions 2 and 3, and 2Ј and 3Ј, respectively (10, 11). Modifications to this structure that are thought to contribute to the virulence of enteric pathogens (e.g. Salmonella typhimurium) occur under certain physiological conditions. These modifications can include the addition of a palmitoyl residue, hydroxylation of a myristoyl substituent, and the addition of aminoarabinosyl and phosphoethanol amine moieties (12). The LPS from other bacterial species show some variability in the glycosyl backbone and fatty acylation patterns of their lipid-A structures. Some of these lipid-A structures have 2,3-diaminoglucosamine replacing one or both of the glucosaminosyl residues (13, 14) . Fatty acyl components can be present that have shorter chain lengths, sites of unsaturation, or keto functional groups (15). A structurally unusual lipid-A from the hyperthermophilic bacterium, Aquifex pyrophilus, has been reported recently (16) in which both the glycosidic and 4Ј-phosphate groups are replaced by galacturonosyl residues. Several reports indicate variation in the glycosyl components of lipid-A among Rhizobium species. The structure of Sinorhizobium meliloti lipid-A is similar to enteric bacterial lipid-A in that it contains an acylated and bis-phosphorylated glucosamine disaccharide (17). Other rhizobial lipid-As (from Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium lupini, and Mesorhizobium loti) have 2,3-diaminoglucose as a constituent of the lipid-A backbone (13, 14) . Perhaps one of the more unusual lipid-A structures is that reported for Rhizobium etli and Rhizobium leguminosarum (9, 18, 19) . Briefly, this unusual lipid-A contains a fatty acylated glycosyl backbone of ␤-glucosamine linked to C-6 of 2-aminogluconate in which