A 2-0-methylfucose moiety is present in the lipo-oligosaccharide ABSTRACTBradyrhizobiumjaponicum is a soil bacterium that forms nitrogen-fixing nodules on the roots of the agronomically important legume soybean. Microscopic observation of plant roots showed that butanol extract of B. japonicum strain USDA110 cultures induced for nod gene expression elicited root hair deformation, an early event in the modulation process. The metabolite produced by B. japonicum responsible for root hair deformation activity was purified. Chemical analysis of the compound revealed it to be a pentasaccharide of N-acetylglucosamine modified by a Ci8:, fatty acyl chain at the
Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane, and for Rhizobium spp. has been shown to play a critical role in the establishment of an effective nitrogen-fixing symbiosis with a legume host. Many genes required for O-chain polysaccharide synthesis are in the lps alpha region of the CE3 genome; this region may also carry lps genes required for core oligosaccharide synthesis. The LPSs from several strains mutated in the alpha region were isolated, and their mild acid released oligosaccharides, purified by high performance anion-exchange chromatography, were characterized by electrospray- and fast atom bombardment-mass spectrometry, NMR, and methylation analysis. The LPSs from several mutants contained truncated O-chains, and the core region consisted of a (3-deoxy-D-manno-2-octulosomic acid) (Kdo)-(2-->6)-alpha-Galp-(1-->6)-[alpha-GalpA-(1-->4)]-alpha-Ma np-(1-->5)- Kdop (3-deoxy-D-manno-2-octulosomic acid) (Kdo)pentasaccharide and a alpha-GalpA-(1-->4)-[alpha-GalpA-(1-->5)]-Kdop trisaccharide. The pentasaccharide was altered in two mutants in that it was missing either the terminal Kdo or the GalA residue. These results indicate that the lps alpha region, in addition to having the genes for O-chain synthesis, contains genes required for the transfer of these 2 residues to the core region. Also, the results show that an LPS with a complete core but lacking an O-chain polysaccharide is not sufficient for an effective symbiosis.
Lipopolysaccharides (LPSs) isolated from several strains of Rhizobium, Bradyrhizobium, Agrobacterium, and Azorhizobium were screened for the presence of 27-hydroxyoctacosanoic acid. The LPSs from all strains, with the exception of Azorhizobium caulinodans, contained various amounts of this long-chain hydroxy fatty acid in the lipid A fractions. Analysis of the lipid A sugars revealed three types of backbones: those containing glucosamine (as found in Rhizobium meliloti and RhizobiumfrediO), those containing glucosamine and galacturonic acid (as found in Rhizobium leguminosarum bv. phaseoli, trifolii, and viciae), and those containing 2,3-diamino-2,3-dideoxyglucose either alone or in combination with glucosamine (as found in Bradyrhizobium japonicum and Bradyrhizobium sp.[Lupinus] strain DSM 30140). The distribution of 27-hydroxyoctacosanoic acid as well as analysis of lipid A backbone sugars revealed the taxonomic relatedness of various strains of the Rhizobiaceae.Bacteria belonging to the family Rhizobiaceae are gram negative and are able to form nitrogen-fixing symbiotic relationships with legume plants. There are three distinct genera: the symbiotic nitrogen-fixing Rhizobium and Bradyrhizobium spp. and the plant pathogenic Agrobacterium spp. Quite recently a new genus, so far comprising only the stem-nodulating nitrogen-fixing species Azorhizobium caulinodans, was defined (13). Of these genera, the species of Rhizobium are taxonomically closely related and show genetic similarities to the genus Agrobacterium as evidenced by 16S rRNA homology studies (12). On the other hand, the slowly growing species of Bradyrhizobium are rather distantly related to the other two genera as revealed by their low SAB values determined by DNA-rRNA hybridization studies (1). In addition to the nucleotide sequence homology studies, differentiation of various members of the Rhizobiaceae has been attempted by several chemotaxonomic approaches such as cellular fatty acid analysis (21, 31), polyacrylamide gel electrophoresis of cellular proteins (19), and composition analysis of extracellular gum (26). However, results of these studies were not sufficient to adequately distinguish between members of the Rhizobiaceae. More recently, the backbone sugar composition of lipid A fractions of lipopolysaccharide (LPS) has been used as a taxonomic marker for recognition and relatedness of various nonsulfur bacteria (23). Therefore, in this study, the lipid A fractions from rhizobial LPSs were examined to see whether they represented a marker for determining the relatedness of these bacteria.The surface polysaccharides, including the LPS, of strains of Rhizobium have been hypothesized to be involved in the molecular mechanisms of symbiotic infection (5). In an attempt to elucidate the structure of LPS from rhizobial strains, an unusual very-long-chain hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27-OH-28:0), was discovered to be the major fatty acid constituent of the lipid A region (15). More recently, we have also identified this long-cha...
The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and -elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrixassisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and 1 H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization The Rhizobium are Gram-negative bacteria capable of forming a nitrogen-fixing symbiosis with legumes in a host-specific manner. The initial stages of this process have been fairly well studied and involve an exchange of signal molecules (e.g. flavonoids and nod factors) (1-4) between the bacterium and plant, leading to the induction of specific bacterial and plant genes. Relatively little is known about the molecular events occurring during the later stages of infection, including the subsequent development of the infection threads and bacteroids. Morphological studies have shown that the rhizobial cell surface is in contact with the plant cell surface throughout these stages, suggesting a prominent role for cell surface macromolecules in these events.Rhizobium cells cultured as free-living bacteria express a variety of glycoconjugates on the cell surface, including the lipopolysaccharides (LPS), 1 capsular polysaccharides (K-antigens), and the extracellular polysaccharides (5-7). The LPS are major structural components of the rhizobial outer membrane and the dominant antigens of the rhizobial cell surface; antibodies raised to whole rhizobial cells are directed most strongly to the LPS (5, 8 -10). Studies with monoclonal antibodies directed to the O-chain moiety of various rhizobia have revealed that different types of LPS can be expressed by a single Rhizobium strain and that this expression depends on the in planta or physiological environment (11)(12)(13)(14)(15)(16). The majority of these antigenic changes appear to occur in the O-chain portion of the LPS and are associated with the different stages of symbiotic infection (11, 12, 14 -18). In addition, recent studies have shown that certain LPS structural changes can be induced by plant flavonoids, indicating that LPS expression can be modulated by nod/nol gene products (19 -21). In one example, an Rhizobium etli LPS O-chain epitope was suppressed by adding bean root or seed exudate to the growth medium (22), and the active compound was identified as an anthocyanin (21). Other studies have examined rhizobial LPS mutants and their symbiotic phenotypes and have shown that mutants that lack the O-antigen portion of their LPS or that contain truncated O-chains are unable to form normal infection threads (10,23,24) and/or are unable to invade the root nodule cells (25)(...
Lipopolysaccharide (LPS) was isolated from free-living Rhizobium leguminosarum bv. phaseoli CE3 cells grown at pH 4.8 (antigenically similar to bacteroid LPS) and compared with that from cells grown at pH 7.2 (free-living bacteria). Composition analysis revealed that pH 7.2 LPS differs from pH 4.8 LPS in that 2,3,4-tri-O-methylfucose is replaced by 2,3-di-0-methylfucose. The amount of 2-O-methylrhamnose is greater in the pH 4.8 LPS than in the pH 7.2 LPS. Analysis of the structural components of LPS (0-chain polysaccharide, core oligosaccharides, and the lipid A) revealed that all the composition differences in the various LPSs occur in the 0-chain polysaccharide. These structural variations between pH 4.8 and pH 7.2 LPSs provide a chemical basis for the observed lack of cross-reactivity with pH 4.8 LPS of two monoclonal antibodies, JIM28 and JIM29, raised against free-living bacteria grown at pH 7.2. An LPS preparation isolated from bacteroids contained both 2,3,4-tri-O-and 2,3-di-O-methylfucose residues. This result is consistent with the finding that the two monoclonal antibodies react weakly with bacteroid LPS. It is concluded that methylation changes occur on the LPS 0-chain of R. leguminosarum bv. phaseoli when the bacteria are grown at low pH and during nodule development.Rhizobia are gram-negative soil bacteria that form nitrogen-fixing symbiotic associations with leguminous plants. In terms of host selection, the bacteria exhibit a great deal of specificity. The surface carbohydrates of rhizobia, which include the lipopolysaccharide (LPS), extracellular polysaccharide, and capsular polysaccharide, have all been hypothesized to play a role in symbiosis (9,11,18,20,22,23). The LPSs of Rhizobium species resemble their enterobacterial counterparts in having structurally distinct regions; the 0-chain polysaccharide, the core oligosaccharide, and the hydrophobic lipid A (10, 13-17); however, there are many differences in the structural details of Rhizobium LPSs compared with enterobacterial LPSs (1-3, 12, 25-27).Earlier reports on the role of LPSs in symbiosis were directed toward the possibility that LPSs were involved in the specific attachment of the symbiont to the host plant (8,9,30). Recent studies indicate involvement of LPSs at a later stage in the nodule development. Mutants which do not have the 0-chain region either fail to form normal infection threads or are not released properly from the infection thread into the host root cells (7,13,19,33,34,37).Using monoclonal antibodies to Rhizobium leguminosarum bv. viciae bacteroids, it has been shown that there are changes in LPS epitopes which occur during differentiation of bacteria into bacteroids (6,35,39,42). Some of these changes can be produced ex planta, by growing the bacteria at low pH or low 02 tension, conditions which mimic, in part, the microenvironment of the root nodule (28). Similar results have been obtained for another strain of R. leguminosarum bv. viciae and for R leguminosarum bv. phaseoli (21, 38). These epitope changes occur only ...
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