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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.

The symbiosis between rhizobia and their host legumes is initiated by a complex molecular dialogue in which the activation of bacterial NodD proteins by appropriate plant flavonoids triggers the expression of the bacterial nodulation (nod) genes. These genes are involved in the synthesis and export of the Nodulation factors (NF), which are signal molecules that, when recognized by plant receptors, launch the symbiotic process. The core of NF is synthesized by proteins encoded by the nodABC genes, whereas the rest of the nod genes protein products are involved in the decoration of NF with different chemical substituents and their export to the environment. Rhizobium tropici CIAT 899, the microsymbiont of common bean (Phaseolus vulgaris), is characterized for tolerating multiple physical stresses and for synthesizing a large variety of NF not only in the presence of inducing flavonoids but also when high concentrations of salt are present. In addition, another interesting feature of the R. tropici CIAT 899 genome is the presence of three different nodA genes on the symbiotic plasmid, although their exact roles remain to be elucidated. In this work, we characterize the role of the three NodA proteins of R. tropici CIAT 899 in symbiosis. We have analyzed by quantitative PCR the expression of the nodA1, nodA2 and nodA3 genes of CIAT 899, showing that only nodA1 and nodA2 are inducible by flavonoids. We have also constructed single, double and triple non-polar mutants in these genes in order to study their importance in NF production and in nodulation of four different host legumes of CIAT 899: P. vulgaris, Leucaena leucocephala, Lotus japonicus, and L. burttii. The nodA1 and nodA2 genes of CIAT 899 (both inducible by flavonoids) are more related between them than with nodA3 (non-flavonoid inducible). Interestingly, the presence of NodA1 or NodA3 is crucial for nodulation with L. leucocephala and L. japonicus, since both NodA1 or NodA3 guarantee Nod factor production upon apigenin induction. Interestingly, osmotic-stressing conditions increase the quantity and diversity of the Nod factors synthesized by NodA2, being these molecules able to induce the formation of nodule primordia on P. vulgaris. We concluded that R. tropici CIAT 899 requires at least the flavonoid-induced nodA1 or the flavonoid-independent nodA3 genes for ensuring symbiotic success in the four assayed host-legumes. The inducible nodA2 gene is sufficient to induce nodulation on P. vulgaris and L. burttii but not in L. leucocephala and L. japonicus. To our knowledge, in this work we report for the first time that a non-flavonoid inducible copy of nodA is enough to induce nodulation on legumes.

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 → .

Sinorhizobium fredii HH103 Rif R , a broad-host-range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen-fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 Rif R nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen-fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod-factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 Rif R . Complementation of the nodD2 and nolR mutants provoked a decrease in Nod-factor production, the incapacity to form nitrogen-fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 Rif R nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod-factor overproduction, although other bacterial symbiotic signals could also be involved.

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].

In the symbiotic associations between rhizobia and legumes, NodD promotes the expression of the nodulation genes in the presence of appropriate flavonoids. This set of genes is implied in the synthesis of Nodulation factors, which are responsible for launching the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. This strain produces Nodulation factors under abiotic stress such as acidity or high concentration of salt. Genome sequencing of CIAT 899 allowed the identification of five nodD genes. Whereas NodD1 is essential to nodulate Leucaena leucocephala, Lotus japonicus and Macroptilium atropurpureum, symbiosis with P. vulgaris and Lotus burtii decreased the nodule number but did not abolish the symbiotic process when NodD1 is absent. Nodulation factor synthesis under salt stress is not regulated by NodD1. Here we confirmed that NodD2 is responsible for the activation of the CIAT 899 symbiotic genes under salt stress. We have demonstrated that NodD1 and NodD2 control the synthesis of the Nod factor necessary for a successful symbiosis with P. vulgaris and L. burtii. This is the first time that NodD is directly implied in the activation of the symbiotic genes under an abiotic stress.

Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rifr surface-polysaccharide mutants gained effective nodulation with L. japonicus.

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.

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.

Background Nodulation and symbiotic nitrogen fixation are mediated by several genes, both of the host legume and of the bacterium. The rhizobial regulatory nodD gene plays a critical role, orchestrating the transcription of the other nodulation genes. Rhizobium tropici strain CIAT 899 is an effective symbiont of several legumes—with an emphasis on common bean (Phaseolus vulgaris)—and is unusual in carrying multiple copies of nodD, the roles of which remain to be elucidated. Results Phenotypes, Nod factors and gene expression of nodD1 and nodD2 mutants of CIAT 899 were compared with those of the wild type strain, both in the presence and in the absence of the nod-gene-inducing molecules apigenin and salt (NaCl). Differences between the wild type and mutants were observed in swimming motility and IAA (indole acetic acid) synthesis. In the presence of both apigenin and salt, large numbers of Nod factors were detected in CIAT 899, with fewer detected in the mutants. nodC expression was lower in both mutants; differences in nodD1 and nodD2 expression were observed between the wild type and the mutants, with variation according to the inducing molecule, and with a major role of apigenin with nodD1 and of salt with nodD2. In the nodD1 mutant, nodulation was markedly reduced in common bean and abolished in leucaena (Leucaena leucocephala) and siratro (Macroptilium atropurpureum), whereas a mutation in nodD2 reduced nodulation in common bean, but not in the other two legumes. Conclusion Our proposed model considers that full nodulation of common bean by R. tropici requires both nodD1 and nodD2, whereas, in other legume species that might represent the original host, nodD1 plays the major role. In general, nodD2 is an activator of nod-gene transcription, but, in specific conditions, it can slightly repress nodD1. nodD1 and nodD2 play other roles beyond nodulation, such as swimming motility and IAA synthesis. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1458-8) contains supplementary material, which is available to authorized users.

We examined the bean rhizobia community other than the predominant species Rhizobium etli present in soils of a region that is part of the range occupied by the host in Northwest Argentina, which showed Rep and 16S rDNA RFLP polymorphism. Two populations represented by isolates T29N3L and T44N22P were found to be distinct chromosomal genotypes and closely related to species Rhizobium tropici and Agrobacterium rhizogenes. Their symbiotic genes were analyzed and found to cluster with those from R. tropici as well as with rhizobia isolated from leguminous trees. Three nodulation metabolites produced by T44N22P were detected which are tetra- and pentameric chitocompounds, N-methylated, O-carbamoylated, and N-substituted either by a C18:0 or C18:1 acyl chain at their non-reducing end, and all them sulphated at the reducing end. Isolates T29N3L and T44N22P exhibited broad host range but unlike T29N3L, only T44N22P was able to efficiently nodulate Medicago truncatula. Fil: Aguilar, Orlando Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Lopez, Maria Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Donato, Mariano Humberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Laboratorio de Sistemática y Biología Evolutiva; Argentina Fil: Morón, Belén. Universidad de Sevilla; España Fil: Soria Diaz, M. Eugenia. Universidad de Sevilla; España Fil: Mateos, Clemente. Universidad de Sevilla; España Fil: Gil Serrano, Antonio. Universidad de Sevilla; España Fil: Sousa, Carolina. Universidad de Sevilla; España Fil: Megías, Manuel. Universidad de Sevilla; España

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rifr mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rifr. S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.

SummaryRhizobium tropici CIAT899 has been cataloged as a nodulator of bean, a plant often growing in areas characterized by highly acidic soils. The purpose of this work was to explore the effects of acidity on the production of Nod factors by this strain and their impact on the establishment of effective symbioses. We report that acidity increases rhizobial Nod factors production, and we exhaustively study the nodulation factor structures produced under abiotic stress. Significant differences were observed between the structures produced at acid and neutral pH: 52 different molecules were produced at acid pH, 29 at neutral pH, and only 15 are common to bacteria grown at pH 7.0 or 4.5. The results indicate that R. tropici CIAT899 has successfully adapted to life in acidic soils and is a good inoculant for the bean under these conditions.

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.

We have determined the structure of a polysaccharide from strain B33, a fast-growing bacterium that forms nitrogen-fixing nodules with Asiatic and American soya bean cultivars. On the basis of monosaccharide analysis, methylation analysis, one-dimensional 1H- and 13C-NMR and two-dimensional NMR experiments, the structure was shown to consist of a polymer having the repeating unit → 6)-4-O-methyl-α-d-Glcp-(1 → 4)-3-O-methyl-β-d-GlcpA-(1 → (where GlcpA is glucopyranuronic acid and Glcp is glucopyranose). Strain B33 produces a K-antigen polysaccharide repeating unit that does not have the structural motif sugar-Kdx [where Kdx is 3-deoxy-d-manno-2-octulosonic acid (Kdo) or a Kdo-related acid] proposed for different Sinorhizobium fredii strains, all of them being effective with Asiatic soya bean cultivars but unable to form nitrogen-fixing nodules with American soya bean cultivars. Instead, it resembles the K-antigen of S. fredii strain HH303 (rhamnose, galacturonic acid)n, which is also effective with both groups of soya bean cultivars. Only the capsular polysaccharide from strains B33 and HH303have monosaccharide components that are also present in the surface polysaccharide of Bradyrhizobium elkanii strains, which consists of a 4-O-methyl-d-glucurono-l-rhamnan.

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 establishment of a symbiotic interaction involves a signal exchange between the host legume (flavonoids) and the nitrogen-fixing rhizobia (nodulation factors (NFs)). Likewise, abiotic stress conditions, such as salinity and drought, strongly reduce the nodulation process, possibly affecting also the signal exchange. In this work we characterized the structure and biological activity of NFs produced by Bradyrhizobium japonicum USDA 138 under control, salt, and osmotic stress conditions. This strain is the most widely used in Argentine soybean culture; under control conditions, it produces a mixture of four types of NFs (V(C16:0,MeFuc), V(C18:1,MeFuc), IV(C18:1), and V(C18:1,Ac,MeFuc)). Interestingly, under stress conditions, this strain produces new types of NFs, one common for both stress conditions (V(C16:1,MeFuc)) and another one only present under salt stress (IV(C18:1,MeFuc)). All mixtures of NFs, extracted from control, salt, and osmotic stress conditions, showed biological activity in soybean plants, such as root hair deformation, and the radical application of purified NFs induced systemic differences in dry matter accumulation. The inoculation of soybean with genistein-induced bacteria cultured under both control and stress conditions had a positive effect on the number of nodules formed and in some cases on dry matter accumulation. These responses are not related to changes in chlorophyll fluorescence or greenness index. Fil: Muñoz, Nacira Belén. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soria Díaz, Maria Eugenia. Universidad de Sevilla; España Fil: Manyani, Hamid. Universidad de Sevilla; España Fil: Contreras Sánchez Matamoros, Rocío. Universidad de Sevilla; España Fil: Gil Serrano, Antonio. Universidad de Sevilla; España Fil: Megías, Manuel. Universidad de Sevilla; España Fil: Lascano, Hernan Ramiro. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

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.

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharide (KPS) biosynthesis, is constituted by the rkpU, rkpAGHIJ, and kpsF3 genes. Two mutants in this region affecting the rkpA (SVQ536) and rkpI (SVQ538) genes were constructed. Polyacrylamide gel electrophoresis and (1)H-NMR analyses did not detect KPS in these mutants. RT-PCR experiments indicated that, most probably, the rkpAGHI genes are cotranscribed. Glycine max cultivars (cvs.) Williams and Peking inoculated with mutants SVQ536 and SVQ538 showed reduced nodulation and symptoms of nitrogen starvation. Many pseudonodules were also formed on the American cv. Williams but not on the Asiatic cv. Peking, suggesting that in the determinate nodule-forming S. fredii-soybean symbiosis, bacterial KPS might be involved in determining cultivar-strain specificity. S. fredii HH103 mutants unable to produce KPS or exopolysaccharide (EPS) also showed reduced symbiotic capacity with Glycyrrhiza uralensis, an indeterminate nodule-forming legume. A HH103 exoA-rkpH double mutant unable to produce KPS and EPS was still able to form some nitrogen-fixing nodules on G. uralensis. Thus, here we describe for the first time a Sinorhizobium mutant strain, which produces neither KPS nor EPS is able to induce the formation of functional nodules in an indeterminate nodule-forming legume.

In this work, the role of the rkpU and rkpJ genes in the production of the K-antigen polysaccharides (KPS) and in the symbiotic capacity of Sinorhizobium fredii HH103, a broad host-range rhizobial strain able to nodulate soybean and many other legumes, was studied. The rkpJ- and rkpU-encoded products are orthologous to Escherichia coli proteins involved in capsule export. S. fredii HH103 mutant derivatives were contructed in both genes. To our knowledge, this is the first time that the role of rkpU in KPS production has been studied in rhizobia. Both rkpJ and rkpU mutants were unable to produce KPS. The rkpU derivative also showed alterations in its lipopolysaccharide (LPS). Neither KPS production nor rkpJ and rkpU expression was affected by the presence of the flavonoid genistein. Soybean (Glycine max) plants inoculated with the S. fredii HH103 rkpU and rkpJ mutants showed reduced nodulation and clear symptoms of nitrogen starvation. However, neither the rkpJ nor the rkpU mutants were significantly impaired in their symbiotic interaction with cowpea (Vigna unguiculata). Thus, we demonstrate for the first time to our knowledge the involvement of the rkpU gene in rhizobial KPS production and also show that the symbiotic relevance of the S. fredii HH103 KPS depends on the specific bacterium–legume interaction.

Sinorhizobium fredii HH103 produces cyclic β glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgs∷lacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs∷Ω) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.

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.