Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non-legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod-factors which are N-acylated chitin pentamers carrying a variety of substituents. The terminal non-reducing glucosamine is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyl groups. The reducing N-acetyl-glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated or sulphated or non-substituted. All three internal residues are N-acetylated. At pico- to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non-sulphated lipo-oligosaccharide signals.

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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...

Previous work in our laboratory showed that L-sorbose utilization in Candida albicans is subject to a novel form of regulation which involves a reversible increase or decrease in the copy number of chromosome 5. Furthermore, the structural gene SOU1 is required for L-sorbose utilization and encodes a member of the short chain dehydrogenase family. However, the precise function of SOU1 was not known and neither was the pathway for L-sorbose utilization. We have now expressed SOU1 at a high level from a replicative plasmid having a constitutive ADH1 promoter and purified a version of Sou1p tagged with the FLAG epitope at the N-terminus. Sou1FLAGNp has a sorbose reductase activity which utilizes NADPH as a co-factor and converts L-sorbose to D-sorbitol. It can also less efficiently utilize fructose as a substrate with NADPH as a co-factor, converting fructose to mannitol. In agreement with prediction, the purified enzyme has a subunit molecular weight of 31 kDa and a pI of about 4.8. It probably consists of four identical subunits and has a pH optimum of 6.2. The L-sorbose utilization pathway in C. albicans probably converts L-sorbose to fructose-6-phosphate via D-sorbitol as an intermediate. The first step is catalysed by Sou1p. We also found that C. albicans extracts have a D-sorbitol-6-phosphate dehydrogenase activity, not encoded by SOU1, which utilizes NADP as a co-factor. This activity has not been described previously in yeasts and may be involved in the conversion of phosphorylated D-sorbitol to fructose-6-phosphate or glucose-6-phosphate.

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