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...

Endotoxin (lipopolysaccharide (LPS))

At least 18 lipopolysaccharide (LPS) extraction methods are available, and no single method is universally applicable. Here, the LPSs from four R.etli, one R.leguminosarum bv. trifolii mutant, 24AR, and the R.etli parent strain, CE3, were isolated by hot phenol/water (phi;/W), and phenol/EDTA/triethylamine (phi/EDTA/TEA) extraction. The LPS in various preparations was quantified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE), and by immunoblotting. These rhizobia normally have two prominent LPS forms: LPS I, which has O-polysaccharide, and LPS II, which has none. The LPS forms obtained depend on the method of extraction and vary depending on the mutant that is extracted. Both methods extract LPS I and LPS II from CE3. The phi/EDTA/TEA, but not the phi/W, method extracts LPS I from mutants CE358 and CE359. Conversely, the phi;/W but not the phi;/EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide. phi/EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while phi/W extraction gives very small amounts of LPS I. The LPS yield from all the strains using phi/EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi/W extraction are highly variable (850-fold range). The phi/EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the phi/W method partitions LPS II exclusively into the phenol phase, making its recovery difficult. Overall, phi/EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to phi/W extraction. Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method.

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 proximal 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...

Bradyrhizobium japonicum 532C nodulates soybean effectively under cool Canadian spring conditions and is used in Canadian commercial inoculants. The major lipo-chitooligosaccharide (LCO), bacteria-to-plant signal was characterized by HPLC, FAB-mass spectroscopy MALDI-TOF mass spectroscopy and revealed to be LCO Nod Bj-V (C18:1, MeFuc). This LCO is produced by type I strains of B. japonicum and is therefore unlikely to account for this strains superior ability to nodulate soybean under Canadian conditions. We also found that use of yeast extract mannitol medium gave similar results to that of Bergerson minimal medium.

A 2-kb region that complements the Tn5-derived lipopolysaccharide (LPS) rough mutant Rhizobium leguminosarum RU301 was sequenced. Two open reading frames (ORFs) were identified. The first ORF (lpcA) is homologous to a family of bacterial sugar transferases involved in LPS core tetrasaccharide biosynthesis. ORF2 (lpcB), in which Tn5 transposed, has no significant homology to any DNA in the GenBank-EMBL databases. Chemical characterization of LPS produced by strain RU301 demonstrated that the 3-deoxy-Dmanno-2-octulosonic acid (Kdo) residue which normally attaches the core tetrasaccharide to the O chain was missing, suggesting that lpcB may encode a CMP-Kdo:LPS Kdo transferase.The lipopolysaccharides (LPSs) of gram-negative bacteria are a major chemical constituent of the outer membrane and are implicated as having a role in infection of the legume symbiont by rhizobia (3, 10, 11). In determinate bean nodules, LPS rough mutants of Rhizobium leguminosarum bv. phaseoli accumulate in the infection thread without forming bacteroids, and nitrogen fixation does not occur (11). Development may proceed further in indeterminate nodules, and low rates of nitrogen fixation can sometimes be detected (3, 5). Two forms of LPS exist: LPS I, consisting of lipid A, core tri-and tetrasaccharides, and the O-chain polysaccharide, and LPS II, which lacks the O-chain polysaccharide (10, 16). R. leguminosarum bv. viciae RU301 has been isolated and characterized as a Tn5 mutant that contains only LPS II (12). Complementing clones were obtained from both R. leguminosarum bv. phaseoli and R. leguminosarum bv. viciae gene libraries, and a common clustering of LPS and dct genes was demonstrated (12).Noel (10) defined five regions involved in LPS biosynthesis in Rhizobium etli CE3: the ␣-, ␤-, and ␥-lps regions, exoBC, and a poorly defined region which contains the lps-166 mutation. Strain RU301 is not complemented by cosmids corresponding to the ␣-, ␤-, or ␥-lps or the exoB region (12). A 3.4-kb EcoRI-HindIII fragment from the cosmid pIJ1848 (9), which is derived from R. leguminosarum bv. phaseoli, complemented strain RU301 (Fig. 1) and was used as a probe to determine if there was any homology to cosmid DNA encoding the ␣-, ␤-, or ␥-lps region. Hybridization analysis supported the view that the complementing region constituted a region separate from those previously described (12). The 3.4-kb fragment also complements the mutant strain VF-39-86 (14), which has been characterized as having a truncated core tetrasaccharide component containing the disaccharide mannose (1-5) 3-deoxy-Dmanno-2-octulosonic acid (Kdo) (16). A smaller PstI-EcoRI subclone, pRU74 (2.4 kb), complemented strain RU301 but not VF-39-86, indicating that at least two genes involved in LPS core tetrasaccharide synthesis may be present in this previously undefined region (12).Subclones derived from pRU74 were cycle sequenced [ Exo FIG. 1. Map of the LPS region of R. leguminosarum bv. phaseoli. pIJ1848 complements the LPS mutant strains RU301 and VF-39-86. The position of Tn...

SUMMARYWe established in previous studies that the binding of Salmonella lipopolysaccharide (LPS) to constitutive receptors of low af®nity triggers the expression of the inducible LPS-binding molecule CD14 in bone marrow cells (BMC) of C3H/HeOU mice, but not in BMC from C3H/HeJ mice. We show in this study that BMC from C3H/HeJ and C57BL/10ScCr mice do not express CD14 after exposure to LPSs from Salmonella enterica and Bordetella pertussis, but do express this marker when treated with several LPSs from Rhizobiaceae, or their lipid A fragments. This shows that the constitutive LPS receptor in BMC from C3H/HeJ and C57BL/10ScCr mice is fully able to trigger a complete signalling cascade. Results of cross-inhibition of the binding of radiolabelled LPS indicated that active LPSs (from R. species Sin-1 and R. galegae) and inactive LPSs (from S. enterica and B. pertussis) bind to the same site of the constitutive LPS receptor of C3H/HeJ cells. Furthermore, binding of R. species Sin-1 LPS, and signalling induced by this LPS, were both inhibited by pre-exposure of C3H/HeJ cells to B. pertussis lipid A. This correlation between binding and signalling suggests that in C3H/HeJ cells, the constitutive receptor, which recognizes a large panel of LPSs from different origins, appears selectively unable to be activated by some particular LPSs, such as those of Enterobacteria and Bordetella.

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