RESUMEN

Cyclic-ß-glucans (CßG) consist of cyclic homo-polymers of glucose that are present in the periplasmic space of many Gram-negative bacteria. A number of studies have demonstrated their importance for bacterial infection of plant and animal cells. In this study, a mutant of Rhizobium (Sinorhizobium) sp. strain NGR234 (NGR234) was generated in the cyclic glucan synthase (ndvB)-encoding gene. The great majority of CßG produced by wild-type NGR234 are negatively charged and substituted. The ndvB mutation abolished CßG biosynthesis. We found that, in NGR234, a functional ndvB gene is essential for hypo-osmotic adaptation and swimming, attachment to the roots, and efficient infection of Vigna unguiculata and Leucaena leucocephala.

Asunto(s)

Adaptación Fisiológica , Nódulos de las Raíces de las Plantas/microbiología , Sinorhizobium/fisiología , Simbiosis , beta-Glucanos/química , Adhesión Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clonación Molecular , Medios de Cultivo/química , Escherichia coli/química , Escherichia coli/genética , Fabaceae/microbiología , Flagelos/química , Flagelos/fisiología , Genes Bacterianos , Proteínas Fluorescentes Verdes/química , Locomoción , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mutación , Ósmosis , Fenotipo , Nodulación de la Raíz de la Planta , Regiones Promotoras Genéticas , Sinorhizobium/química , Sinorhizobium/genética , Transcripción Genética , beta-Glucanos/aislamiento & purificación

RESUMEN

Sinorhizobium sp. BL3 is a salt-tolerant strain that can fix atmospheric nitrogen in symbiosis with leguminous host plants under salt-stress conditions. Since cell membranes are the first barrier to environmental change, it is interesting to explore the membrane proteins within this protective barrier under salt stress. The protein contents of membrane-enriched fractions obtained from BL3 were analyzed by nanoflow liquid chromatography interfaced with electrospray ionization tandem mass spectrometry. A total of 105 membrane proteins were identified. These proteins could be classified into 17 functional categories, the two biggest of which were energy production and conversion, and proteins not in clusters of orthologous groups (COGs). In addition, a comparative analysis of membrane proteins between salt-stressed and non-stressed BL3 cells was conducted using a membrane enrichment method and off-line SCX fractionation coupled to nanoLC-MS/MS. These techniques would be useful for further comparative analysis of membrane proteins that function in the response to environmental stress.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteómica , Sinorhizobium/genética , Cloruro de Sodio/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Sinorhizobium/química , Sinorhizobium/metabolismo

RESUMEN

Succinoglycan, a sinorhizobial exopolysaccharide produced by Sinorhizobium meliloti, is composed of an octasaccharide subunit. S. meliloti produces both high-molecular-weight and low-molecular-weight (M(r)<10,000) succinoglycans which consist of monomers, dimers, or trimers. Succinoglycan monomers were isolated and further purified in the monomer series (M1, M2, and M3) by the degree of succinylation. We used sinorhizobial octasaccharides (M1, M2, and M3) as chiral additives in capillary electrophoresis (CE) for chiral separation of catechin and also as chiral shift reagents with (13)C NMR spectroscopy for chiral discrimination of catechin. Chiral separation of catechin took place when sinorhizobial octasaccharides (M2 and M3) were added to the background electrolyte (BGE) in CE. NMR signal splittings were also observed in the interactions of sinorhizobial octasaccharides with the enantiomers of catechin. Both chiral separation and discrimination of catechin depend on the presence of succinate substituents of the linear monomeric octasaccharide in CE and NMR spectroscopy, suggesting that succinylation of sinorhizobial octasaccharide is decisive for the effective chiral separation and discrimination of catechin.

Asunto(s)

Catequina/química , Catequina/aislamiento & purificación , Oligosacáridos/química , Sinorhizobium/química , Electroforesis Capilar , Indicadores y Reactivos/química , Espectroscopía de Resonancia Magnética , Estereoisomerismo

RESUMEN

Succinoglycan monomers (M1, M2, and M3) are octasaccharides with acetyl, pyruvyl, and/or succinyl groups as substituents derived from Sinorhizobium meliloti 1021. The dissociation patterns of the octasaccharides caused by low-energy collision-activated dissociation (CAD) were investigated using triple quadrupole tandem mass spectrometry (MS) equipped with an electrospray ionization (ESI) source with increasing collision energy (CE) in negative ion mode. None of the succinoglycan monomers were fragmented at a CE of -25eV. When the CE was applied to -50 or -70eV, the loss of the terminal Gal residue and/or the succinyl group of the monomers was observed in the product ion scan mode. Interestingly, the acetyl and the pyruvyl groups in the succinoglycan monomers were not lost even when a CE of -70eV was applied, indicating that the substituents are more stable than the succinyl group in the octasaccharides.

Asunto(s)

Polisacáridos Bacterianos/análisis , Polisacáridos Bacterianos/química , Sinorhizobium/química , Polisacáridos Bacterianos/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem

RESUMEN

Comparative metabolic responses of Sinorhizobium sp. C4 were investigated. Comprehensive metabolites profiles, including polar metabolites, fatty acids, and polyhydroxyalkanoates were evaluated through untargeted metabolome analyses. Intracellular metabolomes during the degradation of phenanthrene were compared with those from natural carbon sources. Principal component analysis showed a clear separation of metabolomes of phenanthrene degradation from other carbon sources. Shift to more hydrophobic fatty acid was observed from the analysis of fatty acid methyl ester. Polyhydroxyalkanoate from strain C4 was composed mainly with 3-hydroxybutyric acid and small amount of 3-hydroxypentanoic acid, while the monomeric composition was independent on carbon sources. However, the amount of polyhydroxyalkanoates during degradation of phenanthrene was 50-210% less than those from other carbon sources. Among 207 gas chromatography-mass spectrometry peaks from the polar metabolite fraction, 60% of the peaks were identified and compared. Several intermediates in tricarboxylic acid cycles and glycolysis were increased during phenanthrene degradation. Accumulation of trehalose was also evident in the phenanthrene-treated bacterium. Some amino acid, including branched amino acids, glycine, homoserine, and valine, were also increased, while more than 70% of identified metabolites were decreased during the phenanthrene metabolism. Accumulation of sulfur amino acids and nicotinic acid suggested the possible oxidative stress conditions during phenanthrene metabolism.

Asunto(s)

Fenantrenos/metabolismo , Sinorhizobium/metabolismo , Aminoácidos/metabolismo , Biodegradación Ambiental , Ácidos Grasos/metabolismo , Fenantrenos/química , Polihidroxialcanoatos/metabolismo , Sinorhizobium/química , Sinorhizobium/crecimiento & desarrollo

RESUMEN

A biofilm reactor was used to investigate kinetic and thermodynamic aspects of the sorption of polycyclic aromatic hydrocarbons (PAH) as model compounds for hydrophobic organic contaminants (HOC) to intact microbial biofilms. Effective diffusion coefficients are in the range of 10(-10) cm2 x s(-1) resulting in equilibration times of more than 3 days for a biofilm of 100 microm thickness. Diffusion in the biofilm was strongly temperature-dependent and increased by a factor of 3 (phenanthrene) to 6 (fluoranthene, pyrene) between 5 and 35 degrees C. Drying and rewetting of the biofilm as well as the inclusion of Ca2+ ions and of humic acids all strengthened the biofilm rigidity and slowed down the diffusion of PAH. The later two factors also influenced the thermodynamics of the process as they supported the partitioning of PAH into the biofilm. Humic acid inclusion from solution into the biofilm illustrates that a microbial biofilm can act as a primer allowing for the buildup of a particulate organic phase from dissolved organic matter. PAH metabolites (3-hydroxy-phenanthrene and 1-hydroxy-2-naphthoic acid) showed lower partition coefficients as compared to their parent compounds and 3-hydroxy-phenanthrene also showed a higher diffusion constant, indicating that these transformation products would be easily released into the water phase upon formation during PAH biodegradation in a biofilm. These results allow the quantification of the influence of environmental conditions on a biofilm's function as a sink or as a diffusion barrier for PAH from aqueous solution, and they indicate the importance of kinetic aspects of this partitioning process.

Asunto(s)

Biopelículas , Contaminantes Ambientales/química , Hidrocarburos Policíclicos Aromáticos/química , Sinorhizobium/química , Adsorción , Calcio/química , Desecación , Difusión , Sustancias Húmicas , Interacciones Hidrofóbicas e Hidrofílicas , Temperatura

RESUMEN

Recent investigations indicate the relevance of extracellular polymeric substances (EPS) in terms of fouling of low-pressure membranes in advanced wastewater treatment. In this study, the high impact of the macromolecular fraction of effluent organic matter on fouling was confirmed in cross-flow ultrafiltration experiments using secondary effluent with and without autochthonous biopolymers. A method for the extraction of a natural mixture of EPS derived from the bacterium Sinorhizobium sp. is presented. Ultrafiltration of solutions of this bacterial EPS extract revealed a correlation between the concentration of EPS and the loss of permeate flux. However, in ultrafiltration tests using extracted bacterial EPS in a model solution as well as in secondary effluent without autochthonous biopolymers, the extent of membrane fouling was not identical with the fouling provoked by secondary effluent organic matter, although the biopolymer concentrations were comparable. The differences in the fouling behaviour of the extracted bacterial EPS and effluent organic matter are considered to be due to different compositions of the biopolymer fraction in terms of proteins, polysaccharides, and other organic colloids, indicating a particular impact of proteins on ultrafiltration membrane fouling.

Asunto(s)

Membranas Artificiales , Compuestos Orgánicos , Ultrafiltración/instrumentación , Eliminación de Residuos Líquidos/instrumentación , Eliminación de Residuos Líquidos/métodos , Contaminantes Químicos del Agua/química , Reactores Biológicos , Sinorhizobium/química , Sinorhizobium/metabolismo , Ultrafiltración/métodos , Purificación del Agua

RESUMEN

High performance anion exchange chromatography (HPAEC)-pulsed amperometric detection (PAD) is a performing technique for carbohydrate analysis, due to the selectivity and sensitivity of the detection. The identification occurs through retention times. In absence of standards, structural characterization of complex polysaccharides requests the coupling of HPAEC-PAD with electrospray ionization (ESI)-MS. This is a technological challenge, due to the non-volatility and high conductance of the eluents. Therefore, a desalting device has been installed on-line between the PAD and the MS. On-line HPAEC-MS has only been rarely described. We report here successful analysis of biological acidic oligosaccharides, allowing for the first time to demonstrate that membrane anchored 3-deoxy-D-manno-2 octulosonic acid (Kdo) homopolymers are consensus sinorhizobial capsular polysaccharide (KPS).

Asunto(s)

Cápsulas Bacterianas/análisis , Cromatografía por Intercambio Iónico/métodos , Sinorhizobium/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Cápsulas Bacterianas/aislamiento & purificación , Sensibilidad y Especificidad , Sinorhizobium fredii/química , Sinorhizobium meliloti/química

RESUMEN

The Sesbania has been widely used as green manure to improve the productivity of several crops. Sinorhizobium saheli strain (SB2) was isolated from the root nodule of Sesbania aculeata. The Tn5 mutants (300) of SB2 were generated and studied for their nodulation efficiencies in its specific and cross-infective host plants. The mutant, SB2M3, was found to have two- and four fold higher nodulation efficiency than wild type in parent host and nonspecific host plant, respectively. SB2M3 differed from SB2 in exopolysaccharide and lipopolysaccharide content. SB2M3 was halotolerant and could grow in alkaline pH at comparatively high temperatures. Hence, it may find an application in agritechnology.

Asunto(s)

Sesbania/microbiología , Sinorhizobium/aislamiento & purificación , Elementos Transponibles de ADN , Concentración de Iones de Hidrógeno , Lipopolisacáridos/análisis , Mutagénesis Insercional , Raíces de Plantas/microbiología , Polisacáridos Bacterianos/análisis , Sinorhizobium/química , Sinorhizobium/genética , Sinorhizobium/fisiología , Cloruro de Sodio/metabolismo

RESUMEN

Sesbania-rhizobia associations have immense significance in soil amelioration programs for diverse habitats. Diversity in symbiotic properties, LPS profiles, Sym plasmid and rhizobiophage sensitivity of 28 root- and stem-nodulating bacterial isolates of three Sesbania species (S. sesban, S. aegyptica and S. rostrata) inhabiting six ecologically distinct sites of semi-arid Delhi region was analyzed. The isolates were highly promiscuous among the symbiotic partners (Sesbania spp.). The root nodules formed by all the isolates were morphologically similar but they differed in their symbiotic efficiency and effectiveness. 16S rDNA sequence analyses revealed that root nodule isolates of sesbanias belong to diverse rhizobial taxa (Sinorhizobium saheli, S. meliloti, Rhizobium huautlense) whereas stem-nodule isolates were strictly Azorhizobium caulinodans. Sinorhizobium spp. seem to dominate as microsymbiont partner of Sesbania in the Delhi region. The genetic diversity revealed by cluster analyses based on NPC-PCR reflects sorting of isolates across the ecological gradient. Parallel diversity was also observed in the grouping based on LPS profiles and sym plasmid (NPC-PCR). Segregation of different rhizobial taxa into distinct types/clusters based on LPS and NPC-PCR analyses suggest its significance in the circumscription of the taxa. However, subtypes and subclusters showed their sorting across the ecological gradients. Sesbania rhizobia showed extremely high specificity to rhizobiophages. Enormous diversity in LPS profiles and high specificity of rhizobiophages might be the result of environmental selection pressures operating in ecologically distinct habitats. The ability of sesbanias to enter into effective symbioses with different rhizobial taxa and colonize diverse habitats with various biotic and abiotic stresses appears to contribute to its wide ecological amplitude.

Asunto(s)

Alphaproteobacteria/clasificación , Alphaproteobacteria/aislamiento & purificación , Fabaceae/microbiología , Alphaproteobacteria/química , Alphaproteobacteria/genética , Azorhizobium caulinodans/química , Azorhizobium caulinodans/clasificación , Azorhizobium caulinodans/genética , Azorhizobium caulinodans/aislamiento & purificación , Bacteriófagos/crecimiento & desarrollo , Biodiversidad , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Ribosómico/química , ADN Ribosómico/aislamiento & purificación , Ecosistema , Genes de ARNr , India , Lipopolisacáridos/análisis , Lipopolisacáridos/aislamiento & purificación , Datos de Secuencia Molecular , Filogenia , Raíces de Plantas/microbiología , Tallos de la Planta/microbiología , Plásmidos , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Rhizobium/química , Rhizobium/clasificación , Rhizobium/genética , Rhizobium/aislamiento & purificación , Análisis de Secuencia de ADN , Sinorhizobium/química , Sinorhizobium/clasificación , Sinorhizobium/genética , Sinorhizobium/aislamiento & purificación , Simbiosis

RESUMEN

The lipopolysaccharides (LPS) are major components of the outer membrane of Gram negative bacteria and, because of their location, are important mediators in the interaction between these bacteria and their environment and other organisms. The alpha-Proteobacterial family Rhizobiaceae includes the rhizobia and agrobacteria, microorganisms which establish symbiotic or parasitic relationships with plants. Mutants deficient in LPS biosynthesis show anomalous interactions with their hosts. The agronomical relevance of the relationship between rhizobia and agrobacteria with plants has promoted a large number of studies on the LPS from these bacteria. The complete structures of one or several domains of LPS from Rhizobiaceae have been determined in the last years. Additionally, several metabolic steps in the biosynthesis of these molecules have been elucidated. This review aims at the description of the more recent findings on the structure and biosynthesis of LPS in Rhizobium, Sinorhizobium and Agrobacterium.

Asunto(s)

Lipopolisacáridos/química , Rhizobiaceae/química , Secuencia de Carbohidratos , Lípido A/biosíntesis , Lípido A/química , Lipopolisacáridos/biosíntesis , Datos de Secuencia Molecular , Estructura Molecular , Antígenos O/química , Rhizobiaceae/genética , Rhizobiaceae/metabolismo , Rhizobium/química , Rhizobium/genética , Rhizobium/metabolismo , Nódulos de las Raíces de las Plantas/microbiología , Sinorhizobium/química , Sinorhizobium/genética , Sinorhizobium/metabolismo , Especificidad de la Especie

RESUMEN

We report the minimum amounts of polysaccharides (6.5 ng) that can be studied using capillary electrophoresis-laser-induced fluorescence (CE-LIF) and describe the separation of the various monosaccharides susceptible to occur in Sinorhizobium cell wall. On-gel hydrolysis of the polysaccharides is described. It allowed the easy and rapid determination of the monosaccharide composition of the excised lipopolysaccharides. The composition obtained fits with gas chromatography-mass spectrometry (GC-MS) results. This study underlines the simplicity of using CE-LIF, which remains an entirely "water medium" method, unlike GC-MS which is, in addition, less sensitive (at least 100-fold) than CE-LIF.

Asunto(s)

Electroforesis Capilar/métodos , Polisacáridos Bacterianos/aislamiento & purificación , Sinorhizobium/química , Cromatografía Líquida de Alta Presión , Electroforesis Capilar/normas , Cromatografía de Gases y Espectrometría de Masas , Monosacáridos/análisis , Oligosacáridos/aislamiento & purificación , Polisacáridos Bacterianos/química

RESUMEN

A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3', and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated beta-(1' --> 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amide-linked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, beta-hydroxybutyrate, is esterified to (omega - 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.

Asunto(s)

Amidas/química , Ácidos Grasos/química , Lípido A/química , Lipopolisacáridos/química , Rhizobium/química , Sinorhizobium/química , Secuencia de Carbohidratos , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

When the rhizosphere is starved of nitrogen, the soil bacteria Rhizobium are able to infect legume roots and invade root nodules, where they can fix atmospheric nitrogen. Nod boxes, the nod gene promoters located on the rhizobial symbiotic plasmid, are activated by means of flavonoids present in the legume root exudates, leading to the synthesis of lipochitooligomers: the Nod factors. Several recent works pointed out the importance of rhizobial surface polysaccharides in establishing the highly specific symbiosis between rhizobia and legumes. Lipopolysaccharides (LPSs) exhibit specific active roles in the later stages of the nodulation processes, such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen-fixing phenotype. The study reported here concerns the structural modifications affecting surface (lipo)polysaccharides when Sinorhizobium sp. NGR234 strains are grown with nod gene induction under nitrogen starvation. In the absence of induction, NGR234 only produces fast-migrating LPSs. When cultured in the presence of flavonoids, the same strain produces large quantities of a high-molecular-weight rhamnose-rich lipopolysaccharide (RLPS). Because the synthesis of this compound seems to be coded by the symbiotic plasmid under direct or indirect gene induction by flavonoids, this RLPS is thought to be biologically relevant.

Asunto(s)

Fabaceae/microbiología , Nitrógeno/metabolismo , Polisacáridos/química , Sinorhizobium/metabolismo , Simbiosis , Proteínas Bacterianas/química , Biomarcadores , Ácidos Grasos/análisis , Regulación Bacteriana de la Expresión Génica , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/química , Proteínas de Microtúbulos/genética , Estructura Molecular , Polisacáridos/biosíntesis , Ramnosa/metabolismo , Sinorhizobium/química , Sinorhizobium/genética , Activación Transcripcional

RESUMEN

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-alpha-D-Glcp-(1-->4)-3-O-methyl-beta-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 HH303 have 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.

Asunto(s)

Disacáridos/química , Fabaceae/microbiología , Glycine max/microbiología , Plantas Medicinales , Polisacáridos Bacterianos/química , Sinorhizobium/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , China , Clima Desértico , Cromatografía de Gases y Espectrometría de Masas , Metilación , Fijación del Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Polisacáridos Bacterianos/aislamiento & purificación , Semillas/microbiología , Sinorhizobium/crecimiento & desarrollo

RESUMEN

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.

Asunto(s)

Monosacáridos/química , Polisacáridos Bacterianos/química , Sinorhizobium/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Simulación por Computador , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Soluciones , Termodinámica

RESUMEN

Nitrogen fixation in symbiotic rhizobia is subject to multiple levels of gene regulation. In Sinorhizobium meliloti, the alfalfa symbiont, the FixLJ two-component regulatory system plays a major role in inducing nitrogen fixation and respiration gene expression in response to the low ambient O(2) concentration of the nodule. Here we report on the mode of action of the FixT protein, a recently identified repressor of nitrogen fixation gene expression in S. meliloti. First, we provide evidence that FixT prevents transcription of the intermediate key regulatory genes nifA and fixK by counteracting the activity of the FixLJ two-component system under otherwise inducing microoxic conditions. Second, we demonstrate that FixT acts as an inhibitor of the sensor hemoprotein kinase FixL, preventing the production or the accumulation of its phosphorylated form. FixT is thus a new example of a regulatory protein that blocks signal transduction in two-component systems at the level of the sensor kinase.

Asunto(s)

Proteínas Bacterianas/antagonistas & inhibidores , Hemoproteínas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas , Proteínas Represoras/metabolismo , Sinorhizobium/química , Proteínas Bacterianas/genética , Mapeo Cromosómico , ADN Bacteriano/química , Genes Bacterianos , Histidina Quinasa , Fijación del Nitrógeno/genética , Fosforilación , Proteínas Represoras/genética , Sinorhizobium/genética , Factores de Transcripción/genética , Transcripción Genética

RESUMEN

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 (1)H-NMR and (13)C-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.

Asunto(s)

Polisacáridos Bacterianos/química , Ácidos Siálicos/química , Sinorhizobium/química , Conformación de Carbohidratos , Electroforesis en Gel de Poliacrilamida , Cromatografía de Gases y Espectrometría de Masas , Espectroscopía de Resonancia Magnética , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción