Structure of the O-antigen of the lipopolysaccharide isolated from Pantoea ananatis AEP17, a rhizobacterium associated with rice.

The lipopolysaccharide of a Gram-negative bacterium having a putative plant-growth promoting activity (Pantoea ananatis AEP17) has been isolated and subjected to partial hydrolysis. The O-antigen has been studied by mass spectrometry and NMR experiments. On the basis of these experiments it is concluded that the following repeating unit is present in the polysaccharide: →3)-ß-d-GlcpNAc-(1→3)[α-d-GalpAN-(1→2)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→ The occurrence of d-galacturonamide (GalAN) is unusual in bacterial O-polysaccharides. It has only been reported in Escherichia coli O65 [Perry, M. B.; MacLean, L. L. Carbohydr. Res.1999, 322, 57-66].

Structure of the O-antigen of the main lipopolysaccharide isolated from Sinorhizobium fredii SMH12.

The lipopolysaccharide of Sinorhizobium fredii SMH12, a wide-range host bacterium isolated from nodulated soybean plants growing in Vietnam, has been studied. Isolation of lipopolysaccharide by the phenol-water method leads to a mixture of two polysaccharides; polyacrylamide gel electrophoresis indicates that both are possibly lipopolysaccharides. The structures of the O-antigen of the main lipopolysaccharide and its deacetylated form are determined by sugar and methylation analysis, partial hydrolysis, lithium degradation, ESI-MS/MS, and NMR studies. Here we show that the fast-growing S. fredii SMH12 produces a lipopolysaccharide whose O-antigen has a repeating unit consisting of the trisaccharide -->4)-alpha-D-Gal pA-(1-->3)-2-O-Ac-alpha-L-Rha p-(1-->3)-2-O-Ac-alpha-D-Man p-(1-->. The position O-6 of the mannose residue in the repeating unit is unsubstituted, acetylated, or methylated in an approximate ratio 1:1:2. The tandem mass spectrometry studies rule out both an alternating and a random distribution of methyl groups and suggest the existence of zones in the polysaccharide rich in methyl groups interspersed with zones without methyl groups.

Signal molecules in the peanut-bradyrhizobia interaction.

Main nodulation signal molecules in the peanut-bradyrhizobia interaction were examined. Flavonoids exuded by Arachis hypogaea L. cultivar Tegua were genistein, daidzein and chrysin, the latest being released in lower quantities. Thin layer chromatography analysis from genistein-induced bacterial cultures of three peanut bradyrhizobia resulted in an identical Nod factor pattern, suggesting low variability in genes involved in the synthesis of these molecules. Structural study of Nod factor by mass spectrometry and NMR analysis revealed that it shares a variety of substituents with the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium spp. Nodulation assays in legumes nodulated by these rhizobia demonstrated differences between them and the three peanut bradyrhizobia. The three isolates were classified as Bradyrhizobium sp. Their fixation gene nifD and the common nodulation genes nodD and nodA were also analyzed.

Structural determination of the Nod factors produced by Rhizobium gallicum bv. gallicum R602.

Rhizobium gallicum is a fast-growing bacterium found in European, Australian and African soils; it was first isolated in France. It is a microsymbiont which is able to nodulate plants of the genus Phaseolus. Rhizobium gallicum bv. gallicum R602 produces four extracellular signal molecules consisting of a linear backbone of N-acetyl glucosamine, bearing on the nonreducing terminal residue an N-methyl group and different N-acyl substituents. The four acyloligosaccharides terminate with a sulfated N-acetylglucosaminitol. This unit may be also acetylated. These structures were determined using carbohydrate and methylation analysis, mass spectrometric analysis and one-dimensional- and two-dimensional-nuclear magnetic resonance experiments. This work establishes the common structure that a lipochito-oligosaccharide must have so that the Rhizobium that produces and excretes it is able to nodulate plants of Phaseolus vulgaris. The substituents common to all the molecules are an N-methyl group and a C(18:1) fatty acid on the nonreducing terminal residue.

Structural analysis of the capsular polysaccharide from Sinorhizobium fredii HWG35.

We have determined the structure of a capsular polysaccharide from Sinorhizobium fredii HWG35. This polysaccharide was isolated following the standard protocols applied for lipopolysaccharide isolation. On the basis of monosaccharide analysis, methylation analysis, mass spectrometric analysis, one-dimensional (1)H and (13)C NMR, and two-dimensional NMR experiments, the structure was shown to consist of a polymer having the following disaccharide repeating unit: -->6)-2,4-di-O-methyl-alpha-d-Galp-(1-->4)-beta-d-GlcpA-(1-->. Strain HWG35 produces a capsular polysaccharide that does not show the structural motif (sugar-Kdx) observed in those S. fredii strains that, while effective with Asiatic soybean cultivars, are unable to form nitrogen-fixing nodules with American soybean cultivars. Instead, the structure of the capsular polysaccharide of S. fredii HWG35 is in line with those produced by strains HH303 (rhamnose and galacturonic acid) and B33 (4-O-methylglucose-3-O-methylglucuronic acid), two S. fredii strains that form nitrogen-fixing nodules with both groups of soybean cultivars. Hence, in these three strains that effectively nodulate American soybean cultivars, the repeating unit of the capsular polysaccharide is composed of two hexoses, one neutral (methylgalactose, rhamnose, or methylglucose) and the other acidic (glucuronic, galacturonic, or methylglucuronic acid).

A purL mutant of Sinorhizobium fredii HH103 is symbiotically defective and altered in its lipopolysaccharide.

The pleiotropic phenotype of an auxotrophic purL mutant (SVQ295) of Sinorhizobium fredii HH103 has been investigated. SVQ295 forms colonies that are translucent, produce more slime and absorb less Congo red than those of wild-type strain HH103. SVQ295 did not grow in minimal medium unless the culture was supplemented with thiamin and adenine or with thiamin and AICA-riboside (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), an intermediate of purine biosynthesis. Bacterial cultures supplemented with AICA-riboside or adenine reached the same culture density, although the doubling time of SVQ295 cultures containing AICA-riboside was clearly longer. S. fredii SVQ295 induced pseudonodules on Glycine max and failed to nodulate six different legumes. On Glycyrrhiza uralensis, however, nodules showing nitrogenase activity and containing infected plant cells were formed. SVQ295 showed auto-agglutination when grown in liquid TY medium and its lipopolysaccharide (LPS) electrophoretic profile differed from that of its parental strain HH103-1. In addition, four monoclonal antibodies that recognize the LPS of S. fredii HH103 failed to recognize the LPS produced by SVQ295. In contrast, (1)H-NMR spectra of K-antigen capsular polysaccharides (KPS) produced by SVQ295 and the wild-type strain HH103 were similar. Co-inoculation of soybean plants with SVQ295 and SVQ116 (a nodA mutant derivative of HH103) produced nitrogen-fixing nodules that were only occupied by SVQ116.

Structural determination of the lipo-chitin oligosaccharide nodulation signals produced by Rhizobium giardinii bv. giardinii H152.

Rhizobium giardinii bv. giardinii is a microsymbiont of plants of the genus Phaseolus and produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis. We report here the structures of seven lipochitooligosaccharide (LCO) signal molecules secreted by R. giardinii bv. giardinii H152. Six of them are pentamers of GlcNAc carrying C 16:0, C 18:0, C 20:0 and C 18:1 fatty acyl chains on the non-reducing terminal residue. Four are sulfated at C-6 of the reducing terminal residue and one is acetylated in the same position. Six of them are N-methylated on the non-reducing GlcN residue and all the nodulation factors are carbamoylated on C-6 of the non-reducing terminal residue. The structures were determined using monosaccharide composition and methylation analyses, 1D- and 2D-NMR experiments and a range of mass spectrometric techniques. The position of the carbamoyl substituent on the non-reducing glucosamine residue was determined using a CID-MSMS experiment and an HMBC experiment.