RESUMEN
Toxic selenite, commonly found in soil and water, can be transformed by microorganisms into selenium nanoparticles (SeNPs) as part of a detoxification process. In this study, a comprehensive investigation was conducted on the resistance and biotransformation of selenite in Sinorhizobium meliloti 1021 and the synergistic impact of SeNPs and the strain on alfalfa growth promotion was explored. Strain 1021 reduced 46% of 5 mM selenite into SeNPs within 72 h. The SeNPs, composed of proteins, lipids and polysaccharides, were primarily located outside rhizobial cells and had a tendency to aggregate. Under selenite stress, many genes participated in multidrug efflux, sulfur metabolism and redox processes were significantly upregulated. Of them, four genes, namely gmc, yedE, dsh3 and mfs, were firstly identified in strain 1021 that played crucial roles in selenite biotransformation and resistance. Biotoxic evaluations showed that selenite had toxic effects on roots and seedlings of alfalfa, while SeNPs exhibited antioxidant properties, promoted growth, and enhanced plant's tolerance to salt stress. Overall, our research provides novel insights into selenite biotransformation and resistance mechanisms in rhizobium and highlights the potential of SeNPs-rhizobium complex as biofertilizer to promote legume growth and salt tolerance.
Asunto(s)
Nanopartículas , Selenio , Sinorhizobium meliloti , Ácido Selenioso/metabolismo , Medicago sativa , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , BiotransformaciónRESUMEN
Polyamines (PAs) are ubiquitous polycations derived from basic l-amino acids whose physiological roles are still being defined. Their biosynthesis and functions in nitrogen-fixing rhizobia such as Sinorhizobium meliloti have not been extensively investigated. Thin layer chromatographic and mass spectrometric analyses showed that S. meliloti Rm8530 produces the PAs, putrescine (Put), spermidine (Spd) and homospermidine (HSpd), in their free forms and norspermidine (NSpd) in a form bound to macromolecules. The S. meliloti genome encodes two putative ornithine decarboxylases (ODC) for Put synthesis. Activity assays with the purified enzymes showed that ODC2 (SMc02983) decarboxylates both ornithine and lysine. ODC1 (SMa0680) decarboxylates only ornithine. An odc1 mutant was similar to the wild-type in ODC activity, PA production and growth. In comparison to the wild-type, an odc2 mutant had 45â% as much ODC activity and its growth rates were reduced by 42, 14 and 44â% under non-stress, salt stress or acid stress conditions, respectively. The odc2 mutant produced only trace levels of Put, Spd and HSpd. Wild-type phenotypes were restored when the mutant was grown in cultures supplemented with 1 mM Put or Spd or when the odc2 gene was introduced in trans. odc2 gene expression was increased under acid stress and reduced under salt stress and with exogenous Put or Spd. An odc1 odc2 double mutant had phenotypes similar to the odc2 mutant. These results indicate that ODC2 is the major enzyme for Put synthesis in S. meliloti and that PAs are required for normal growth in vitro.
Asunto(s)
Ornitina Descarboxilasa/metabolismo , Poliaminas/metabolismo , Sinorhizobium meliloti/crecimiento & desarrollo , Sinorhizobium meliloti/metabolismo , Aminoácidos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Medios de Cultivo , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Mutación , Ornitina Descarboxilasa/genética , Poliaminas/análisis , Putrescina/metabolismo , Sinorhizobium meliloti/enzimología , Espermidina/análogos & derivados , Espermidina/metabolismo , Transcripción GenéticaRESUMEN
The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 µM, V = 9.4 ± 0.5 µM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.
Asunto(s)
Membrana Celular/metabolismo , Nitrato Reductasas/metabolismo , Nitratos/metabolismo , Sinorhizobium meliloti/metabolismo , Azidas/farmacología , Cloratos/farmacología , Cianuros/farmacología , Cinética , Molibdeno/metabolismo , Nitrato Reductasas/antagonistas & inhibidores , Oxidación-Reducción , Sinorhizobium meliloti/enzimología , Relación Estructura-ActividadRESUMEN
To understand the mechanisms of high-pH-induced protection in Sinorhizobium meliloti, a cDNA-amplified fragment length polymorphism analysis of S. meliloti cells grown in minimal medium under alkali stress was undertaken. This revealed that the first four genes of a seven-gene cluster encode the characteristic components of a putative sugar ATP-binding cassette (ABC) transporter. A functional study suggested that this putative sugar ABC transporter might play a role in potassium transport regulation, which we therefore designated supABCD. The transcription of three potassium uptake genes, trkH, kdpA and kup1, in S. meliloti is significantly attenuated in the supA mutant in the presence of potassium. The supA mutant was unable to grow at elevated levels of potassium. The expression of supA, as determined by beta-galactosidase activity, was shown to be induced by potassium but not by sodium.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico , Regulación Bacteriana de la Expresión Génica , Respuesta al Choque Térmico , Potasio/metabolismo , Sinorhizobium meliloti/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Medios de Cultivo , ADN Complementario/genética , Concentración de Iones de Hidrógeno , Familia de Multigenes , Mutación , Polimorfismo de Longitud del Fragmento de Restricción , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/crecimiento & desarrollo , Sinorhizobium meliloti/metabolismoRESUMEN
The microsymbiont of alfalfa, Sinorhizobium meliloti, possesses phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, and phosphatidylcholine as major membrane phospholipids, when grown in the presence of sufficient accessible phosphorus sources. Under phosphate-limiting conditions of growth, S. meliloti replaces its phospholipids by membrane lipids that do not contain any phosphorus in their molecular structure and, in S. meliloti, these phosphorus-free membrane lipids are sulphoquinovosyl diacylglycerols (SL), ornithine-containing lipids (OL), and diacylglyceryl-N,N,N-trimethylhomoserines (DGTS). In earlier work, we demonstrated that neither SL nor OL are required for establishing a nitrogen-fixing root nodule symbiosis with alfalfa. We now report the identification of the two structural genes btaA and btaB from S. meliloti required for DGTS biosynthesis. When the sinorhizobial btaA and btaB genes are expressed in Escherichia coli, they cause the formation of DGTS in this latter organism. A btaA-deficient mutant of S. meliloti is unable to form DGTS but can form nitrogen-fixing root nodules on alfalfa, demonstrating that sinorhizobial DGTS is not required for establishing a successful symbiosis with the host plant. Even a triple mutant of S. meliloti, unable to form any of the phosphorus-free membrane lipids SL, OL, or DGTS is equally competitive for nodule occupancy as the wild type. Only under growth-limiting concentrations of phosphate in culture media did mutants that could form neither OL nor DGTS grow to lesser cell densities.
Asunto(s)
Medicago sativa/microbiología , Lípidos de la Membrana/metabolismo , Fósforo/metabolismo , Sinorhizobium meliloti/crecimiento & desarrollo , Sinorhizobium meliloti/metabolismo , Secuencia de Bases , Medios de Cultivo , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genes Bacterianos , Medicago sativa/metabolismo , Lípidos de la Membrana/química , Fijación del Nitrógeno , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Sinorhizobium meliloti/genética , Simbiosis/fisiologíaRESUMEN
The global response to phosphate starvation was analysed at the transcriptional level in two closely related strains of Sinorhizobium meliloti, Rm1021 and Rm2011. The Pho regulon is known to be induced by PhoB under conditions of phosphate limitation. Ninety-eight genes were found to be significantly induced (more than three-fold) in a phoB -dependent manner in phosphate-stressed cells, and phoB -independent repression of 86 genes was observed. Possible roles of these genes in the phosphate stress response are discussed. Twenty new putative PHO box sequences were identified in regions upstream of 17 of the transcriptional units that showed phoB -dependent, or partially phoB -dependent, regulation, indicating direct regulation of these genes by PhoB. Despite the overall similarity between the phosphate stress responses in Rm1021 and Rm2011, lower induction rates were found for a set of phoB -dependent genes in Rm1021. Moreover, Rm1021 exhibited moderate constitutive activation of 12 phosphate starvation-inducible, phoB -dependent genes when cells were grown in a complex medium. A 1-bp deletion was observed in the pstC ORF in Rm1021, which results in truncation of the protein product. This mutation is probably responsible for the expression of phosphate starvation-inducible genes in Rm1021 in the absence of phosphate stress.
Asunto(s)
Expresión Génica , Fosfatos/metabolismo , Sinorhizobium meliloti/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Anhidrasas Carbónicas/metabolismo , Quimiotaxis/fisiología , Galactanos/biosíntesis , Perfilación de la Expresión Génica , Glucanos/biosíntesis , Hierro/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/fisiología , Fósforo/metabolismo , Polisacáridos Bacterianos/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinorhizobium meliloti/metabolismoRESUMEN
A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica/métodos , Sinorhizobium meliloti/genética , Simbiosis/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Extensiones de la Superficie Celular/genética , Electroforesis en Gel Bidimensional , Endopeptidasas/genética , Endopeptidasas/metabolismo , Punto Isoeléctrico , Datos de Secuencia Molecular , Nitrogenasa/genética , Nitrogenasa/metabolismo , Fósforo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Homología de Secuencia de Aminoácido , Sinorhizobium meliloti/metabolismoRESUMEN
Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sit B and sit D genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.
Asunto(s)
Manganeso/farmacología , Sinorhizobium meliloti/crecimiento & desarrollo , Sinorhizobium meliloti/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Elementos Transponibles de ADN , Etilenodiaminas/farmacología , Genes Bacterianos/genética , Hierro/metabolismo , Quelantes del Hierro/farmacología , Medicago sativa/crecimiento & desarrollo , Medicago sativa/microbiología , Mutación , Análisis de Secuencia de ADN , Sideróforos/genética , Sideróforos/metabolismo , Sinorhizobium meliloti/genética , SimbiosisRESUMEN
Twenty one cysteine and 13 methionine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5. The cysteine auxotrophs were sulfite reductase mutants and each of these auxotrophs had a mutation in cysI/cysJ gene. The methionine auxotrophs were metA/metZ, metE and metF mutants. One hundred per cent co-transfer of Tn5-induced kanamycin resistance and auxotrophy from each Tn5-induced auxotrophic mutant indicated that each mutant cell most likely had a single Tn5 insertion. However, the presence of more than one Tn5 insertions in the auxotrophs used in our study cannot be ruled out. All cysteine and methionine auxotrophs induced nodules on alfalfa plants. The nodules induced by cysteine auxotrophs were fully effective like those of the parental strain-induced nodules, whereas the nodules induced by methionine auxotrophs were completely ineffective. The supplementation of methionine to the plant nutrient medium completely restored symbiotic effectiveness to the methionine auxotrophs. These results indicated that the alfalfa host provides cysteine but not methionine to rhizobia during symbiosis. Histological studies showed that the defective symbiosis of methionine auxotrophs with alfalfa plants was due to reduced number of infected nodule cells and incomplete transformation of bacteroids.
Asunto(s)
Cisteína/metabolismo , Metionina/metabolismo , Sinorhizobium meliloti/fisiología , Simbiosis , Elementos Transponibles de ADN , Medicago sativa/microbiología , Mutagénesis , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismoRESUMEN
Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific. All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B(12)-dependent homocysteine methyltransferase for methionine biosynthesis.
Asunto(s)
Biotina/metabolismo , Cobalto/metabolismo , Metionina/metabolismo , Sinorhizobium meliloti/crecimiento & desarrollo , Medios de Cultivo , Sinorhizobium meliloti/metabolismoRESUMEN
Identifying the types and distributions of organic substrates that support microbial activities around plant roots is essential for a full understanding of plant-microbe interactions and rhizosphere ecology. We have constructed a strain of the soil bacterium Sinorhizobium meliloti containing a gfp gene fused to the melA promoter which is induced on exposure to galactose and galactosides. We used the fusion strain as a biosensor to determine that galactosides are released from the seeds of several different legume species during germination and are also released from roots of alfalfa seedlings growing on artificial medium. Galactoside presence in seed wash and sterile root washes was confirmed by HPLC. Experiments examining microbial growth on alpha-galactosides in seed wash suggested that alpha-galactoside utilization could play an important role in supporting growth of S. meliloti near germinating seeds of alfalfa. When inoculated into microcosms containing legumes or grasses, the biosensor allowed us to visualize the localized presence of galactosides on and around roots in unsterilized soil, as well as the grazing of fluorescent bacteria by protozoa. Galactosides were present in patches around zones of lateral root initiation and around roots hairs, but not around root tips. Such biosensors can reveal intriguing aspects of the environment and the physiology of the free-living soil S. meliloti before and during the establishment of nodulation, and they provide a nondestructive, spatially explicit method for examining rhizosphere soil chemical composition.
Asunto(s)
Técnicas Biosensibles , Galactósidos/metabolismo , Sinorhizobium meliloti/metabolismo , Secuencia de Bases , Cartilla de ADN , Fabaceae/microbiología , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Plantas Medicinales , SimbiosisRESUMEN
Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.
Asunto(s)
Fabaceae/genética , Plantas Medicinales , Proteoma , Sinorhizobium meliloti/genética , Simbiosis , Electroforesis en Gel Bidimensional , Fabaceae/metabolismo , Fabaceae/microbiología , Nitrógeno/metabolismo , Sinorhizobium meliloti/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Rhizobium meliloti Orange 1 was isolated from aerobic sediments of a drainage ditch receiving oil refinery leakage. This bacterium has been shown to be capable of growing on dibenzothiophene as the sole carbon and energy source. This strain can also efficaciously nodulate alfalfa plants. In cultures with dibenzothiophene, Orange 1 produces six degradation intermediates. By means of analyses with UV-visible and GC-MS spectrometry, as well as nuclear magnetic resonance spectroscopy, three of these products were identified as 3-hydroxy-2-formyl-benzothiophene (product A), benzothienopyran-2-one (product B'), and dibenzothiophene-5-oxide (product D). This suggests that R. meliloti Orange 1 metabolizes dibenzothiophene via oxidative cleavage of the aromatic ring with a mechanism analogous to that described for naphthalene degradation.
Asunto(s)
Sinorhizobium meliloti/aislamiento & purificación , Sinorhizobium meliloti/metabolismo , Tiofenos/metabolismo , Biodegradación Ambiental , Cromatografía de Gases y Espectrometría de Masas , Residuos Industriales , Imagen por Resonancia Magnética , Petróleo/metabolismo , Sinorhizobium meliloti/crecimiento & desarrolloAsunto(s)
Fabaceae/microbiología , Plantas Medicinales , Polisacáridos/fisiología , Sinorhizobium meliloti/metabolismo , Simbiosis , Conformación de Carbohidratos , Secuencia de Carbohidratos , Membrana Celular/metabolismo , Datos de Secuencia Molecular , Polisacáridos/química , Polisacáridos/genética , Sinorhizobium meliloti/genéticaRESUMEN
Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a -24/-12 type promoter (hupSp). A regulatory region located in the -173 to -88 region was essential for promoter activity in R. leguminosarum. Activation of hupSp was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential -173 to -88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifA-dependent hupSp activity in heterologous hosts was also absolutely dependent on the RpoN sigma-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupSp is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H2 production and H2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H2-rich environment for rhizobia, the legume nodule.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidrogenasas/biosíntesis , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Rhizobium leguminosarum/enzimología , Rhizobium leguminosarum/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Fabaceae/microbiología , Regulación Enzimológica de la Expresión Génica , Hidrogenasas/genética , Klebsiella pneumoniae/metabolismo , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , Pisum sativum/microbiología , Plantas Medicinales , Proteínas Recombinantes de Fusión/biosíntesis , Eliminación de Secuencia , Sinorhizobium meliloti/metabolismo , Transcripción GenéticaRESUMEN
Calystegines, polyhydroxy nortropane alkaloids, are a recently discovered group of plant secondary metabolites believed to influence rhizosphere ecology as nutritional sources for soil microorganisms and as glycosidase inhibitors. Evidence is presented that calystegines mediate nutritional relationships under natural conditions and that their biological activities are closely correlated with their chemical structures and stereochemistry. Assays using synthetic (+)- and (-)-enantiomers of calystegine B2 established that catabolism by Rhizobium meliloti, glycosidase inhibition, and allelopathic activities were uniquely associated with the natural, (+)-enantiomer. Furthermore, the N-methyl derivative of calystegine B2 was not catabolized by R. meliloti, and it inhibited alpha-galactosidase, but not beta-glucosidase, whereas the parent alkaloid inhibits both enzymes. This N-methyl analog therefore could serve to construct a cellular or animal model for Fabry's disease, which is caused by a lack of alpha-galactosidase activity.
Asunto(s)
Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Nortropanos/aislamiento & purificación , Nortropanos/farmacología , Plantas Medicinales/química , alfa-Galactosidasa/antagonistas & inhibidores , Bacterias/efectos de los fármacos , Bacterias/metabolismo , ADN Bacteriano/biosíntesis , Glicósido Hidrolasas/antagonistas & inhibidores , Raíces de Plantas/química , Pseudomonas/efectos de los fármacos , Pseudomonas/crecimiento & desarrollo , Pseudomonas/metabolismo , Semillas/química , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/crecimiento & desarrollo , Sinorhizobium meliloti/metabolismo , Alcaloides Solanáceos , Relación Estructura-ActividadRESUMEN
The mutant Rhizobium meliloti M5N1CS (NCIMB 40472) produced a partially acetylated (1-->4)-beta-D-glucuronan during fermentation. The polysaccharide (EPS) extracted from broth during fermentation by microfiltration and ultrafiltration (using a 100 kDa cut-off membrane) was characterized by size exclusion chromatography. The weight-average molecular weight (Mw) of the EPS decreased from 7.5 x 10(5) to 5 x 10(5) between 27 and 75 h of fermentation. When MgSO4.7H2O (0.8 gl-1 per day) was present in the medium during the same period, the Mw of the EPS decreased to 1.5 x 10(5). Since EPS degradation was detected after microfiltration of the fermentation medium supplemented with magnesium, the Mw decrease of the EPS extracted during fermentation may be explained by enzymic degradation of the polymer activated by Mg2+ ions.