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Rhizobium tropici strain CIAT 899 possesses outstanding agronomic properties as it displays tolerance to environmental stresses, a broad host range and high effectiveness in fixing nitrogen with the common bean (Phaseolus vulgaris L.); in addition, it carries intriguing features such as five copies of the regulatory nodD gene, and the capacity to synthesize a variety of nodulation factors (NFs), even in a flavonoid-independent manner, when submitted to abiotic stresses. However, the roles of several nod genes of the repertoire of CIAT 899 remain to be determined. In this study, we obtained mutants for the hsnT, nodF and nodE genes of CIAT 899 and investigated their expression, NF structures and symbiotic properties. Either in the presence of the flavonoid apigenin, or of salt the expression of hsnT, nodF and nodE in wild-type CIAT 899 was highly up-regulated in comparison to the mutants of all five copies of nodD, indicating the roles that regulatory nodD genes play in the activation of hsnT, nodF and nodE; however, NodD1 was recognized as the main inducer. In total, 29 different NF structures were synthesized by wild-type CIAT 899 induced by apigenin, and 36 when induced by salt, being drastically reduced by mutations in hsnT, nodF and nodE, especially under osmotic stress, with specific changes related to each gene, indicating that the three genes participate in the synthesis of NFs. Mutations in hsnT, nodF and nodE affected differently symbiotic performance (nodule number and shoot dry weight), according to the host plant. Our results indicate that the expression of hsnT, nodF and nodE genes of CIAT 899 is mediated by nodD genes, and although these three genes do not belong to the main set of genes controlling nodulation, they contribute to the synthesis of NFs that will impact symbiotic performance and host specificity.

Aeromonas sp. AMG272 is a Gram-negative bacterium that has been isolated from agricultural soil and studied for its plant growth-promoting activities. Structures of the O-specific polysaccharide chain of the AMG272 lipopolysaccharide and its capsular polysaccharide were elucidated using GLC-MS and NMR spectroscopy. The structure of the O-specific polysaccharide, →4)-α -l- Rhap-(1 → 3)-β -d- GlcpNAc-(1→, has been found in other Aeromonas strains and related bacteria, whereas the structure of the capsular polysaccharide has not been reported before: →6)[β- d -Fucp3NAc4Ac-(1 → 3)]-α -d- GlcpNAc-(1 → 4)-α -d- Galp-(1 → 3)-α -d- GalpNAc-(1 → 4)-α -d- Galp-(1 → .

Seed germination and early seedling development have been studied in the recalcitrant species Quercus ilex using targeted transcriptional, hormonal, and sugar analysis. Embryos and seedlings were collected at eight morphologically defined developmental stages, S0–S7. A typical triphasic water uptake curve was observed throughout development, accompanied by a decrease in sucrose and an increase in glucose and fructose. Low levels of abscisic acid (ABA) and high levels of gibberellins (GAs) were observed in mature seeds. Post-germination, indole-3-acetic acid (IAA), increased, whereas GA remained high, a pattern commonly observed during growth and development. The abundance of transcripts from ABA-related genes was positively correlated with the changes in the content of the phytohormone. Transcripts of the drought-related genes Dhn3 and GolS were more abundant at S0, then decreased in parallel with increasing water content. Transcripts for Gapdh, and Nadh6 were abundant at S0, supporting the occurrence of an active metabolism in recalcitrant seeds at the time of shedding. The importance of ROS during germination is manifest in the high transcript levels for Sod and Gst, found in mature seeds. The results presented herein help distinguish recalcitrant (e.g., Q. ilex) seeds from their orthodox counterparts. Our results indicate that recalcitrance is established during seed development but not manifest until germination (S1–S3). Post-germination the patterns are quite similar for both orthodox and recalcitrant seeds. España Ministerio de Ciencia e Innovación AGL2009-12243-C02-02

The symbiosis between rhizobia and legumes is characterized by a complex molecular dialogue in which the bacterial NodD protein plays a major role due to its capacity to activate the expression of the nodulation genes in the presence of appropiate flavonoids. These genes are involved in the synthesis of molecules, the nodulation factors (NF), responsible for launching the nodulation process. Rhizobium tropici CIAT 899, a rhizobial strain that nodulates Phaseolus vulgaris, is characterized by its tolerance to multiple environmental stresses such as high temperatures, acidity or elevated osmolarity. This strain produces nodulation factors under saline stress and the same set of CIAT 899 nodulation genes activated by inducing flavonoids are also up-regulated in a process controlled by the NodD2 protein. In this paper, we have studied the effect of osmotic stress (high mannitol concentrations) on the R. tropici CIAT 899 transcriptomic response. In the same manner as with saline stress, the osmotic stress mediated NF production and export was controlled directly by NodD2. In contrast to previous reports, the nodA2FE operon and the nodA3 and nodD1 genes were up-regulated with mannitol, which correlated with an increase in the production of biologically active NF. Interestingly, in these conditions, this regulatory protein controlled not only the expression of nodulation genes but also the expression of other genes involved in protein folding and synthesis, motility, synthesis of polysaccharides and, surprinsingly, nitrogen fixation. Moreover, the non-metabolizable sugar dulcitol was also able to induce the NF production and the activation of nod genes in CIAT 899. España, MINECO AGL2016-77163-R

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)-β- 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].

Root exudates affect the concentration of nutrients in the plant rhizosphere and indirectly enhance microbial activity. Flavonoids are polyphenolic compounds that play an important role as signal molecules in the early stages of the symbiosis between legumes and rhizobia. Flavonoids from root exudates of Phaseolus vulgaris cv. Bush Blue Lake obtained under control conditions, salt stress (50 mM), and/or the presence of the plant growth-promoting rhizobacterium (PGPR) Chryseobacterium balustinum Aur9 were analyzed by HPLC coupled to ESI-MS/MS. Six flavonoids were identified in the root exudates of P. vulgaris cv. BBL: naringenin, isoliquiritigenin, quercetin umbelliferone, 7′,4-dihydroxyflavone, and hesperetin. The latter three flavonoids have not previously been reported in bean root exudates. The presence of C. balustinum , but not salt stress, modified the pattern of flavonoids exuded by the bean roots. The capacity of flavonoids from root exudates to induce the expression of the nod genes and the production of lipochitooligosaccharides (LCOs) was investigated in two bean-nodulating rhizobia, Rhizobium tropici CIAT899 and Rhizobium etli ISP42. The R. etli ISP42 LCOs profile changed when it was induced by the exudates collected in the presence of the PGPR with and without salt. In the case of R. tropici CIAT899, changes in the LCOs profile were detected when the bacterium was induced with the different bean root exudates in comparison with the LCOs synthesized upon induction with apigenin.

Legume-nodulating rhizobia use N-acyl homoserine lactones (AHLs) to regulate several physiological traits related to the symbiotic plant–microbe interaction. In this work, we show that Sinorhizobium fredii SMH12, Rhizobium etli ISP42 and Rhizobium sullae IS123, three rhizobial strains with different nodulation ranges, produced a similar pattern of AHL molecules, sharing, in all cases, production of N-octanoyl homoserine lactone and its 3-oxo and/or 3-hydroxy derivatives. Interestingly, production of AHLs was enhanced when these three rhizobia were grown in the presence of their respective nod-gene-inducing flavonoid, while a new molecule, C14-HSL, was produced by S. fredii SMH12 upon genistein induction. In addition, expression of AHL synthesis genes traI from S. fredii SMH12 and cinI and raiI from R. etli ISP42 increased when induced with flavonoids, as demonstrated by qRT-PCR analysis.

In this work we studied how biotic and abiotic stresses can alter the pattern of flavonoids exuded by Osumi soybean roots. A routine method was developed for the detection and characterization of the flavonoids present in soybean root exudates using HPLC-MS/MS. Then, a systematic screening of the flavonoids exuded under biotic stress, the presence of a plant growth promoting rhizobacterium, and salt stress was carried out. Results obtained indicate that the presence of Chryseobacterium balustinum Aur9 or 50 mM NaCl changes qualitatively the pattern of flavonoids exuded when compared to control conditions. Thus, in the presence of C. balustinum Aur9, soybean roots did not exude quercetin and naringenin and, under salt stress, flavonoids daidzein and naringenin could not be detected. Soybean root exudates obtained under saline conditions showed a diminished capacity to induce the expression of the nodA gene in comparison to the exudates obtained in the absence of salt. Moreover, lipochitooligosaccharides (LCOs) were not detected or weakly detected when Sinorhizobium fredii SMH12 was grown in the exudates obtained under salt stress conditions or under salt stress in the presence of C. balustinum Au9, respectively.

The structure and conformational behaviour of the core region of the lipopolysaccharide isolated from Sinorhizobium fredii SMH12, a wide-ranging host bacterium isolated from nodulated soybean plants growing in Vietnam, have been studied. A mixture of oligosaccharides was isolated from the lipopolysaccharide by mild hydrolysis followed by size-exclusion chromatography. The structures were determined by sugar analysis, ESI-MS/MS and NMR studies. Conformational analysis was performed by molecular dynamics calculations and compared with the NMR spectroscopic data. The results indicate that the core oligosaccharide contains a disaccharide made up of α-D-glucopyranuronic acid (1→4)-linked to D-Kdo. In solution, this disaccharide exists as an equilibrium of three different structures. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

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.

The conformational behavior of the capsular polysaccharide obtained from a fast‐growing soybean‐nodulating rhizobia (strain B33) isolated from Xinjiang Autonomous Region (Eastern China) has been analyzed by NMR and molecular mechanics simulations. This polysaccharide has the repeating unit →6)‐4‐O‐Me‐α‐d‐Glcp‐(1→4)‐3‐O‐Me‐β‐d‐GlcpA‐(1→. The NMR results indicate that the α‐(1→4) linkage may adopt a variety of conformations, and that at least two of the resulting minima must exist in solution. NOE data agree with an 85:10:5 ratio for the lowest‐energy conformations. In the case of the β‐(1→6) linkage, NMR indicates that the rotamer gg is highly populated. Experimental and calculated NOE intensities match well when the global energy minimum conformation for this linkage has exclusively the gg orientation. The influence of the adjacent methyl group on the glycosyloxymethyl population has been evaluated by simulation of a disaccharide without this group. A relative destabilization of gt rotamer has been found....

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.

The structure of a hemicellulose B fraction (B-1) isolated from grape skins (Vitis vinifera) of the Palomino variety has been studied by methylation analysis, (1)H NMR and (13)C NMR spectroscopy, and partial acid hydrolysis. Hemicellulose B-1 appeared to be homogeneous by gel filtration with a weight-average molecular weight of 22 600. This polysaccharide is a linear xyloglucan chain composed of xylopyranosyl and glucopyranosyl residues linked by beta-(1--4) glycosidic bonds. Attached to this backbone, 4-O-methyl-D-glucuronopyranosyl acid, D-glucopyranosyl, and L-fucopyranosyl residues occur at position 2 in a ratio of one residue for every five units of xylose in the main chain, with D-xylopyranosyl residues attached at position 6 of glucose units in a ratio of one residue for every two glucose-derived moieties in the main chain.

The conformational behavior of the homopolysaccharide isolated from Sinorhizobium fredii HH103 and its monosaccharide repeating unit (5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)- l -glycero- l -manno-nonulosonic acid) was analyzed by nuclear magnetic resonance (NMR) spectroscopy and extensive molecular dynamics simulations (MD). The results indicate that the glycosidic linkages and lateral chains may adopt a variety of conformations. MD simulations using the generalized Born solvent-accessible surface area (GB/SA) continuum solvent model for water and the MM3∗ force field provide a population distribution of conformers that satisfactorily agrees with the experimental NMR data for the torsional degrees of freedom of the molecule.

The structure of a polysaccharide from Sinorhizobium fredii HH103 has been determined. This polysaccharide was isolated by following the protocol for lipopolysaccharide extraction. On the basis of monosaccharide analysis, methylation analysis, fast atom bombardment MS, matrix-assisted laser desorption ionization MS, electron-impact high-resolution MS, one-dimensional 1H-NMR and 13C-NMR and two-dimensional NMR experiments, the structure was shown to consist of a homopolymer of a 3:1 mixture of 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)- and (S)-3-hydroxybutyramido]-L-glycero-L-manno-nonulosonic acid. The sugar residues are attached via a glycosidic linkage to the OH group of the 3-hydroxybutyramido substituent and thus the monomers are linked via both glycosidic and amidic linkages. In contrast with the Sinorhizobium K-antigens previously reported, which are composed of a disaccharide repeating unit, the K-antigen polysacharide of S. fredii HH103 is a homopolysaccharide.

The conformational behaviour of the major trisaccharide repeating unit (α-D-Galp-(1→2)-β-D-Ribf-(1→9)-α-5-O-Me-Kdnp-) of the polysaccharide from Sinorhizobium fredii SVQ293, a mutant derivative has been analysed by NMR spectroscopy and extensive molecular dynamics simulations. The results obtained indicate that the five-membered ring adopts an almost unique conformation as do the pyranose rings. The Ribf-(1→9)-α-5-O-Me-Kdnp linkage may adopt a variety of conformations while the α-D-Galp-(1→2)-β-D-Ribf- also populates an extended surface of the Φ/Ψ map. Two 10 ns MD simulations using the GB/SA continuum solvent model for water and the MM3* force field provides a population distribution of conformers which satisfactorily agrees with the experimental NMR data for both the glycosidic linkages and the hydroxymethyl groups.

The structure of one of the hemicellulose B fractions (HB-1) extracted from the seeds of the grape variety Palomino (Vitis vinifera cv. Palomino) has been studied by means of methylation analysis, 1H NMR, 13C NMR, and partial acid hydrolysis. This hemicellulose seems to be a homogeneous polysaccharide with an apparent molecular weight of 35 000. Its structure is that of an acidic arabinoxylan, a linear chain of beta-D-xylopyranosyl units, bonded together by (1-->4) glycosidic links, containing a single L-arabinofuranosyl, alpha-D-xylopyranosyl and 4-O-methyl-alpha-D-glucopyranuronosyl residues joined by glycosidic links to position 2 of the xylose units of the main chain, in proportions of one branch to every seven units of xylose.

The exopolysaccharide produced by a ropy strain of Lactobacillus spp. G-77 in a semi-defined medium, was found to be a mixture of two homopolymers composed of D-Glc. The two poly-saccharides were separated and, on the basis of monosaccharide and methylation analyses, 1H, 13C, 1D and 2D NMR experiments, one of the polysaccharides was shown to be a 2-substituted-(1-3)-beta-D-glucan, identical to that described for the EPS from Pediococcus damnosus 2.6 (M.T. Dueñas-Chasco, M.A. Rodríguez-Carvajal, P. Tejero-Mateo, G. Franco-Rodríguez, J. L. Espartero, A. Irastorza-Iribar, and A.M. Gil-Serrano, Carbohydr. Res., 303 (1997) 453-458), and the other polysaccharide was shown to consist of repeating units with the following structure [formula: see text]

The structure of one hemicellulose A fraction (A-1) isolated from grape skins ( Vitis vinifera ) of the Palomino variety has been studied by methylation analysis, NMR spectroscopy and partial acid hydrolysis. Hemicellulose A-1 appeared to be homogeneous with a weight-average M r of 35 544. It was shown to be a linear acidic xyloglucan constituted by a linear chain of xylopyranosyl and glucopyranosyl residues linked by β-(1 → 4) glycosidic bonds containing 4- O -methylglucuronopyranosy acid, l -arabinofuranosyl and xylopyranosyl residues attached at position 2, in a ratio of one residue for every 10 units of xylose in the main chain, and l -arabinofuranosyl and glucopyranosyl residues attached at position 6 of the units of glucose, in a ratio of one residue for every four glucoses in the main chain.

The exopolysaccharide produced by a ropy strain of Pediococcus damnosus (2.6) in a semi-defined medium was found to be an homopolymer composed of D-glucose. On the basis of monosaccharide and methylation analysis, 1H, 13C, 1D and 2D NMR experiments the polysaccharide was shown to consist of repeating units with the following structure. [sequence: see text]

Rhizobium fredii HH103 produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis of soybean. This strain produces a large variety of nodulation factors, consisting of a linear backbone of GlcNAc with different degrees of polymerization, bearing on the non-reducing residue various different N-acyl residues. The reducing terminal residue is 2-O-methylfucosylated at position 6. Several analogous molecules substituted with fucose were also detected.

The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors. Fil: Guasch Vidal, B.. Universidad de Sevilla; España Fil: Estévez, J.. Universidad de Sevilla; España Fil: Dardanelli, Marta Susana. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soria Díaz, M. E.. Universidad de Sevilla; España Fil: Fernández de Córdoba, F.. Universidad de Sevilla; España Fil: Balog, C. I. A.. Leiden University; Países Bajos Fil: Manyani, H.. Universidad de Sevilla; España Fil: Gil Serrano, A.. Universidad de Sevilla; España Fil: Thomas Oates, J.. University of York; Reino Unido Fil: Hensbergen, P. J.. Leiden University; Países Bajos Fil: Deelder, A. M.. Leiden University; Países Bajos Fil: Megías, M.. Universidad de Sevilla; España Fil: Brussel, A. A. N. van. Leiden University; Países Bajos

The structure of the O-antigen chain of the lipopolysaccharide isolated from Rhizobium tropici CIAT899, by the phenol-water procedure, and recovered from the phenol layer, has been investigated by hydrolysis, methylation analysis and 1D and 2D 1H and 13C NMR spectroscopy of the complete polysaccharide and of oligosaccharides obtained by partial hydrolysis. The O-antigen has the repeating unit [formula: see text]

Margaret, Isabel et al., The Sinorhizobium fredii HH103 rkp-3 region has been isolated and sequenced. Based on the similarities between the S. fredii HH103 rkpL, rkpM, rkpN, rkpO, rkpP, and rkpQ genes and their corresponding orthologues in Helicobacter pylori, we propose a possible pathway for the biosynthesis of the S. fredii HH103 K-antigen polysaccharide (KPS) repeating unit. Three rkp-3 genes (rkpM, rkpP, and rkpQ) involved in the biosynthesis of the HH103 KPS repeating unit (a derivative of the pseudaminic acid) have been mutated and analyzed. All the rkp-3 mutants failed to produce KPS and their lipopolysaccharide (LPS) profiles were altered. These mutants showed reduced motility and auto-agglutinated when early-stationary cultures were further incubated under static conditions. Glycine max, Vigna unguiculata (determinate nodule¿forming legumes), and Cajanus cajan (indeterminate nodules) plants inoculated with mutants in rkpM, rkpQ, or rkpP only formed pseudonodules that did not fix nitrogen and were devoid of bacteria. In contrast, another indeterminate nodule¿forming legume, Glycyrrhiza uralensis, was still able to form some nitrogen-fixing nodules with the three S. fredii HH103 rifampicin-resistant rkp-3 mutants tested. Our results suggest that the severe symbiotic impairment of the S. fredii rkp-3 mutants with soybean, V. unguiculata, and C. cajan is mainly due to the LPS alterations rather than to the incapacity to produce KPS., This work was supported by grants from the Spanish Ministry of Sci- ence and Innovation (BIO2008-05736-C02-02 and BIO2011-30229-C02- 01) and the Andalusia Government (P07-CVI-02506).

The induction of root nodules by the majority of rhizobia has a strict requirement for the secretion of symbiosis-specific lipochitooligosaccharides (nodulation factors [NFs]). The nature of the chemical substitution on the NFs depends on the particular rhizobium and contributes to the host specificity imparted by the NFs. We present here a description of the genetic organization of the nod gene cluster and the characterization of the chemical structure of the NFs associated with the broad-host-range Rhizobium sp. strain LPU83, a bacterium capable of nodulating at least alfalfa, bean, and Leucena leucocephala. The nod gene cluster was located on the plasmid pLPU83b. The organization of the cluster showed synteny with those of the alfalfanodulating rhizobia, Sinorhizobium meliloti and Sinorhizobium medicae. Interestingly, the strongest sequence similarity observed was between the partial nod sequences of Rhizobium mongolense USDA 1844 and the corresponding LPU83 nod genes sequences. The phylogenetic analysis of the intergenic region nodEG positions strain LPU83 and the type strain R. mongolense 1844 in the same branch, which indicates that Rhizobium sp. strain LPU83 might represent an early alfalfa-nodulating genotype. The NF chemical structures obtained for the wild-type strain consist of a trimeric, tetrameric, and pentameric chitin backbone that shares some substitutions with both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83 most of the NFs were sulfated in their reducing terminal residue, none of the NFs isolated from the nodH mutant LPU83-H were sulfated. The evidence obtained supports the notion that the sulfate decoration of NFs in LPU83 is not necessary for alfalfa nodulation., Instituto de Biotecnologia y Biologia Molecular

The structure of the extracellular cyclic (1 → 2)-β- d -glucan secreted by Rhizobium tropici CIAT 899 has been studied by methylation analysis, 1 D and 2 D NMR experiments, HPLC and FAB-MS. Molecular mechanics (MM2) and theoretical 3 J HH coupling constants calculations were performed.

A novel flavonol glycoside, rhamnazin-3- O - β -glucopyranosyl-(1→5)- α -arabinofuranoside, was isolated from the aerial parts of Retama sphaerocarpa (Fabaceae) and its structure determined by mp, UV, IR, FAB-MS, GC–MS and NMR data.

5 páginas, 1 esquema -- PAGS nros. 3469-3473, The conformational and dynamic behavior of the O-antigen of the lipopolysaccharide of Sinorhizobium fredii SMH12, a wide-range host bacterium isolated from nodulated soybean plants growing in Vietnam, was studied. The O-antigenhas a repeating unit consisting of the trisaccharide →4)-α-D-GalpA-(1→3)-2-O-Ac-α-L-Rhap-(1→3)-2-O-Ac-α-D-Manp-(1→, where the O-6 position of the mannose residue in the repeating unit is unsubstituted, acetylated, or methylated in an approximate ratio 1:1:2. For the analysis of the conformational and dynamic behavior, the deacetylated polysaccharide was employed. A combination of NMR spectroscopic methods assisted by molecular mechanics and dynamic simulations was employed to deduce its major conformational and dynamic features, The work was supported by the Ministerio de Ciencia y Tecnología (CTQ2006-10874-C02-01, AGL2005-07923-C05, and AGL2006-13758-C05) and the Junta de Andalucía (Grupo CVI0135). F. J. F. de C. was supported by a fellowship from Ministerio de Ciencia y Tecnología

The strain Lactobacillus pentosus LPS26 produces a capsular polymer composed of a high- (2.0 × 10 6 Da) (EPS A) and a low-molecular mass (2.4 × 10 4 Da) (EPS B) polysaccharide when grown on semi-defined medium containing glucose as the carbon source. The structure of EPS A and its deacetylated form has been determined by monosaccharide and methylation analysis as well as by 1D/2D NMR studies ( 1 H and 13 C). We conclude that EPS A is a charged heteropolymer, with a composition of d -glucose, d -glucuronic acid and l -rhamnose in a molar ratio 1:2:2. The repeating unit is a pentasaccharide with two O- acetyl groups at O-4 of the 3-substituted α- d -glucuronic acid and at O-2 of the 3-substituted β- l -rhamnose, respectively. →4)-α- d -Glc p -(1→3)-α- d -Glc p A4Ac-(1→3)-α- l -Rha p -(1→4)-α- d -Glc p A-(1→3)-β- l -Rha p 2Ac-(1→ This unbranched structure is not common in EPSs produced by Lactobacilli. Moreover, the presence of acetyl groups in the structure is an unusual feature which has only been reported in L. sake 0–1 [Robijn et al. Carbohydr. Res. , 1995 , 276 , 117–136].

Aims: To study the influence of medium constituents on growth, and exopolysaccharide (EPS) production by a strain of Oenococcus oeni. The structure of one of the EPSs has also been characterized. Methods and Results: EPS concentration was estimated by the phenol/sulfuric acid method. After purification and fractionation of crude EPSs, the sugar composition was determined by GLC-MS of the TMS methyl glycosides. The major polysaccharide is 2-substituted-(1–3)-β-d-glucan. This structure was determined by methylation analysis and conventional 1H- and 13C-nuclear magnetic resonance spectroscopy. In addition, O. oeni synthesized two heteropolysaccharides, although a lesser proportion, constituted by galactose and glucose, and one of them also showed rhamnose. The sugar source has a clear influence on growth and EPS synthesis, and EPS production was not enhanced by adding ethanol or increasing the nitrogen source. EPS biosynthesis starts in the exponential growth phase, and continued during the stationary growth phase. Conclusions: Higher EPS yields were obtained on cultures grown on glucose + fructose. O. oeni produces a β-glucan, as the predominant EPS, and it is also able to produce two heteropolysaccharides. Significance and Impact of the Study: This work provides a better understanding of EPS synthesis by O. oeni and shows the first EPS structure described for this species.

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 C18:1 fatty acid on the nonreducing terminal residue.

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).

The exopolysaccharide (EPS) and capsular polysaccharide (CPS) composition of four Azospirillum brasilense strains differing in their aggregation capacity was analyzed by high performance anion exchange chromatography. When growing the different strains in an aggregation inducing medium containing a high carbon:nitrogen (C:N) ratio, both EPS and CPS showed a positive correlation between aggregation and the relative amount of arabinose. Arabinose was not detected in polysaccharides from Sp72002, a pleiotrophic Tn5 mutant strain impaired in aggregation. Arabinose was also not detected in extracellular polysaccharides of bacteria grown in a low C:N ratio, non-inducing aggregation medium, with exception for a relatively small amount found in the CPS of FAJ0204, a super-aggregating mutant strain. The only monosaccharides able to significantly inhibit aggregation at low sugar concentration when tested in a bioassay were arabinose (at a higher extent) and galactose. The possibility that residues of arabinose present in the extracellular polysaccharides are involved in the aggregation of A. brasilense is discussed.

The solution conformation of two lipooligosaccharides related to Nod factors or lipochitoligosaccharides have been analysed by 1D and 2D 1H and 13C NMR spectroscopy, molecular mechanics and dynamics calculations. The obtained data indicate that the glycosidic torsion angles have restricted fluctuations, but may adopt a variety of shapes. Remarkably, the relative orientation of the fatty acid chain towards the oligosaccharide backbone is solvent dependent. In water solution, the acyl residue and the oligosaccharide adopt a quasi-parallel orientation for a significant amount of time.

The structure of a polysaccharide from Sinorhizobium fredii SVQ293, a thiamine auxotrophic mutant of S. fredii HH103, has been determined. This polysaccharide was isolated following the protocol for lipopolysaccharide extraction. On the basis of monosaccharide analysis, methylation analysis, fast atom bombardment MS, collision-induced dissociation tandem MS, one-dimensional 1H and 13C NMR and two-dimensional NMR experiments, the structure was shown to consist of the following trisaccharide repeating unit-->2)-alpha-d-Galp-(1-->2)-beta-d-Ribf-(1-->9)-alpha-5-O-Me-++ +Kdnp- (2-->, in which Kdn stands for deaminated neuraminic acid; 25% of the Kdn residues are not methylated. The structure of this polysaccharide is novel and this is the first report of the presence of Kdn in a rhizobial polysaccharide, as well as being the first structure described containing 5-O-Me-Kdn. This Kdn-containing polysaccharide is not present in the wild-type strain HH103, which produces a 3-deoxy-d-manno-2-octulosonic acid (Kdo)-rich polysaccharide. We conclude that it is likely that the appearance of this new Kdn-containing polysaccharide is a consequence of the mutation.

We have purified and characterized the nodulation factors produced by Rhizobium tropici CIAT899. This strain produces a large variety of nodulation factors, these being a mixture of sulfated or nonsulfated penta- or tetra-chito-oligosaccharides to which any of six different fatty acyl moieties may be attached to nitrogen of the nonreducing terminal residue. In this mixture we have also found methylated or nonmethylated lipo-chitin oligosaccharides. Here we describe a novel lipo-chitin-oligosaccharide consisting of a linear backbone of 4 N-acetylglucosamine residues and one mannose that is the reducing-terminal residue and bearing a C18:1 fatty acyl moiety on the nonreducing terminal residue. In addition, we have identified, cloned, and sequenced R. tropici nodH and nodPQ genes, generated mutations in the nodH and nodQ genes, and tested the mutant strains for nodulation in Phaseolus and Leucaena plants. Our results indicate that the sulfate group present in wild-type Nod factors plays a major role in nodulation of Leucaena plants by strain CIAT899 of R. tropici.

The structure of the extracellular polysaccharide secreted by Rhizobium leguminosarum var. phaseoli CIAT 899 has been studied by methylation analysis, 1H-n.m.r. spectroscopy, and partial acid hydrolysis. The repeating unit is an octasaccharide made up of d -glucose, d -galactose, pyruvic acid, and acetic acid in the molar ratios 6:2:1.5:1.5. Half of the terminal Gal groups are 4,6-substituted by pyruvic acid acetal groups and the other half by O-acetyl groups at position 3. Also, one of the 3-linked glucosyl residues carries a pyruvic acid 4,6-acetal group and one of the 4-linked glucosyl residues is acetylated at position 6.

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.

A purified hemicellulose A, isolated from olive pulp (‘hojiblanca’ variety) contained xylose and 4- O -methylglucuronic acid in the molar ratio 24:1. The molecular weight was estimated to be 9500 by GPC. From the results of partial hydrolysis, enzymatic hydrolysis, methylation analysis and identification of an aldobiuronic acid, it was concluded that hemicellulose A is a polymer of β(1 → 4)-linked xylopyranosyl residues, having branches of 4- O -methylglucuronic acid groups on the O-2 atoms of the main chain.