RESUMEN

Paenibacillus sonchi genomovar Riograndensis is a nitrogen-fixing bacteria isolated from wheat that displays diverse plant growth-promoting abilities. Beyond conventional Mo-nitrogenase, this organism also harbors an alternative Fe-nitrogenase, whose many aspects related to regulation, physiology, and evolution remain to be elucidated. In this work, the origins of this alternative system were investigated, exploring the distribution and diversification of nitrogenases in the Panibacillaceae family. Our analysis showed that diazotrophs represent 17% of Paenibacillaceae genomes, of these, only 14.4% (2.5% of all Paenibacillaceae genomes) also contained Fe or V- nitrogenases. Diverse nif-like sequences were also described, occurring mainly in genomes that also harbor the alternative systems. The analysis of genomes containing Fe-nitrogenase showed a conserved cluster of nifEN anfHDGK across three genera: Gorillibacterium, Fontibacillus, and Paenibacillus. A phylogeny of anfHDGK separated the Fe-nitrogenases into three main groups. Our analysis suggested that Fe-nitrogenase was acquired by the ancestral lineage of Fontibacillus, Gorillibacterium, and Paenibacillus genera via horizontal gene transfer (HGT), and further events of transfer and gene loss marked the evolution of this alternative nitrogenase in these groups. The species phylogeny of N-fixing Paenibacillaceae separated the diazotrophs into five clades, one of these containing all occurrences of strains harboring alternative nitrogenases in the Paenibacillus genus. The pangenome of this clade is open and composed of more than 96% of accessory genes. Diverse functional categories were enriched in the flexible genome, including functions related to replication and repair. The latter involved diverse genes related to HGT, suggesting that such events may have an important role in the evolution of diazotrophic Paenibacillus. This study provided an insight into the organization, distribution, and evolution of alternative nitrogenase genes in Paenibacillaceae, considering different genomic aspects.

Asunto(s)

Nitrogenasa , Paenibacillus , Fijación del Nitrógeno/genética , Nitrogenasa/genética , Nitrogenasa/metabolismo , Paenibacillus/genética , Paenibacillus/metabolismo , Filogenia

RESUMEN

The genome resequencing of spontaneous glyphosate-resistant mutants derived from the soybean inoculant E109 allowed identifying genes most likely associated with the uptake (gltL and cya) and metabolism (zigA and betA) of glyphosate, as well as with nitrogen fixation (nifH). Mutations in these genes reduce the lag phase and improve nodulation under glyphosate stress. In addition to providing glyphosate resistance, the amino acid exchange Ser90Ala in NifH increased the citrate synthase activity, growth rate and plant growth-promoting efficiency of E109 in the absence of glyphosate stress, suggesting roles for this site during both the free-living and symbiotic growth stages.

Asunto(s)

Bradyrhizobium , Rhizobium , Alanina/metabolismo , Bradyrhizobium/metabolismo , Glicina/análogos & derivados , Mutación , Fijación del Nitrógeno , Nitrogenasa/genética , Rhizobium/genética , Rhizobium/metabolismo , Serina/metabolismo , Simbiosis , Glifosato

RESUMEN

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme's sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle's genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an 'Fe-only' nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.

Asunto(s)

Chlamydomonas reinhardtii/crecimiento & desarrollo , Cloroplastos/metabolismo , Ingeniería Genética/métodos , Nitrogenasa/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Genoma del Cloroplasto , Fijación del Nitrógeno , Nitrogenasa/metabolismo , Fotosíntesis , Biología Sintética

RESUMEN

BACKGROUND: Azopirillum brasilense is a plant-growth promoting nitrogen-fixing bacteria that is used as bio-fertilizer in agriculture. Since nitrogen fixation has a high-energy demand, the reduction of N2 to NH4+ by nitrogenase occurs only under limiting conditions of NH4+ and O2. Moreover, the synthesis and activity of nitrogenase is highly regulated to prevent energy waste. In A. brasilense nitrogenase activity is regulated by the products of draG and draT. The product of the draB gene, located downstream in the draTGB operon, may be involved in the regulation of nitrogenase activity by an, as yet, unknown mechanism. RESULTS: A deep in silico analysis of the product of draB was undertaken aiming at suggesting its possible function and involvement with DraT and DraG in the regulation of nitrogenase activity in A. brasilense. In this work, we present a new artificial intelligence strategy for protein classification, named ProClaT. The features used by the pattern recognition model were derived from the primary structure of the DraB homologous proteins, calculated by a ProClaT internal algorithm. ProClaT was applied to this case study and the results revealed that the A. brasilense draB gene codes for a protein highly similar to the nitrogenase associated NifO protein of Azotobacter vinelandii. CONCLUSIONS: This tool allowed the reclassification of DraB/NifO homologous proteins, hypothetical, conserved hypothetical and those annotated as putative arsenate reductase, ArsC, as NifO-like. An analysis of co-occurrence of draB, draT, draG and of other nif genes was performed, suggesting the involvement of draB (nifO) in nitrogen fixation, however, without the definition of a specific function.

Asunto(s)

Azospirillum brasilense/química , Azospirillum brasilense/enzimología , Proteínas Bacterianas/química , Biología Computacional/métodos , Nitrogenasa/química , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biología Computacional/instrumentación , Genes Bacterianos , Fijación del Nitrógeno , Nitrogenasa/genética , Nitrogenasa/metabolismo , Operón

RESUMEN

The vast majority of Pseudomonas species are unable to fix atmospheric nitrogen. Although several studies have demonstrated that some strains belonging to the genus Pseudomonas sensu stricto do have the ability to fix nitrogen by the expression of horizontally acquired nitrogenase, little is known about the mechanisms of nitrogenase adaptation to the new bacterial host. Recently, we transferred the nitrogen fixation island from Pseudomonas stutzeri A1501 to the non-nitrogen-fixing bacterium Pseudomonas protegens Pf-5, and interestingly, the resulting recombinant strain Pf-5 X940 showed an uncommon phenotype of constitutive nitrogenase activity. Here, we integrated evolutionary and functional approaches to elucidate this unusual phenotype. Phylogenetic analysis showed that polyhydroxybutyrate (PHB) biosynthesis genes from natural nitrogen-fixing Pseudomonas strains have been acquired by horizontal transfer. Contrary to Pf-5 X940, its derived PHB-producing strain Pf-5 X940-PHB exhibited the inhibition of nitrogenase activity under nitrogen-excess conditions, and displayed the typical switch-on phenotype observed in natural nitrogen-fixing strains after nitrogen deficiency. This indicates a competition between PHB production and nitrogen fixation. Therefore, we propose that horizontal transfer of PHB biosynthesis genes could be an ancestral mechanism of regulation of horizontally acquired nitrogenases in the genus Pseudomonas.

Asunto(s)

Nitrogenasa/genética , Pseudomonas/enzimología , Evolución Molecular , Transferencia de Gen Horizontal , Genes Bacterianos , Fijación del Nitrógeno/genética , Nitrogenasa/biosíntesis , Filogenia , Pseudomonas/genética , Pseudomonas stutzeri/enzimología , Pseudomonas stutzeri/genética

RESUMEN

Background In this study, the detection of nifH and nifD by a polymerase chain reaction assay was used to screen the potential photosynthetic bacteria capable of producing hydrogen from five different environmental sources. Efficiency of photo-hydrogen production is highly dependent on the culture conditions. Initial pH, temperature and illumination intensity were optimized for maximal hydrogen production using response surface methodology with central composite design. Results Rhodobacter sp. KKU-PS1 (GenBank Accession No. KC478552) was isolated from the methane fermentation broth of an UASB reactor. Malic acid was the favored carbon source while Na-glutamate was the best nitrogen source. The optimum conditions for simultaneously maximizing the cumulative hydrogen production (Hmax) and hydrogen production rate (Rm) from malic acid were an initial of pH 7.0, a temperature of 25.6°C, and an illumination intensity of 2500 lx. Hmax and Rm levels of 1264 ml H2/l and 6.8 ml H2/L-h were obtained, respectively. The optimum initial pH and temperature were further used to optimize the illumination intensity for hydrogen production. An illumination intensity of 7500 lx gave the highest values of Hmax (1339 ml H2/l) and Rm (12.0 ml H2/L-h) with a hydrogen yield and substrate conversion efficiency of 3.88 mol H2/mol malate and 64.7%, respectively. Conclusions KKU-PS1 can produce hydrogen from at least 8 types of organic acids. By optimizing pH and temperature, a maximal hydrogen production by this strain was obtained. Additionally, by optimizing the light intensity, Rm was increased by approximately two fold and the lag phase of hydrogen production was shortened.

Asunto(s)

Oxidorreductasas/metabolismo , Rhodobacter/metabolismo , Nitrogenasa/metabolismo , Oxidorreductasas/genética , Temperatura , Reacción en Cadena de la Polimerasa , Rhodobacter/aislamiento & purificación , Reactores Biológicos , Fermentación , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Nitrogenasa/genética

RESUMEN

PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP.

Asunto(s)

Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mutación , Proteínas PII Reguladoras del Nitrógeno/genética , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Análisis Mutacional de ADN , Expresión Génica , Nitrogenasa/genética , Proteínas PII Reguladoras del Nitrógeno/química , Unión Proteica , Conformación Proteica

RESUMEN

Posttranslational modification of proteins plays a key role in the regulation of a plethora of metabolic functions. Protein modification by mono-ADP-ribosylation was first described as a mechanism of action of bacterial toxins. Since these pioneering studies, the number of pathways regulated by ADP-ribosylation in organisms from all domains of life expanded significantly. However, in only a few cases the full regulatory ADP-ribosylation circuit is known. Here, we review the system where mono-ADP-ribosylation regulates the activity of an enzyme: the regulation of nitrogenase in bacteria. When the nitrogenase product, ammonium, becomes available, the ADP-ribosyltransferase (DraT) covalently links an ADP-ribose moiety to a specific arginine residue on nitrogenase switching-off nitrogenase activity. After ammonium exhaustion, the ADP-ribosylhydrolase (DraG) removes the modifying group, restoring nitrogenase activity. DraT and DraG activities are reversibly regulated through interaction with PII signaling proteins . Bioinformatics analysis showed that DraT homologs are restricted to a few nitrogen-fixing bacteria while DraG homologs are widespread in Nature. Structural comparisons indicated that bacterial DraG is closely related to Archaea and mammalian ADP-ribosylhydrolases (ARH). In all available structures, the ARH active site consists of a hydrophilic cleft carrying a binuclear Mg(2+) or Mn(2+) cluster, which is critical for catalysis.

Asunto(s)

Adenosina Difosfato Ribosa/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Eucariontes/enzimología , Nitrogenasa/metabolismo , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , Animales , Bacterias/química , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Eucariontes/química , Eucariontes/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Nitrogenasa/química , Nitrogenasa/genética , Procesamiento Proteico-Postraduccional

RESUMEN

TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

Asunto(s)

Proteínas Bacterianas/genética , Gluconacetobacter/genética , Proteínas de la Membrana/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Sideróforos/genética , Transporte Biológico/genética , Medios de Cultivo/química , Prueba de Complementación Genética , Gluconacetobacter/enzimología , Gluconacetobacter/metabolismo , Hierro/metabolismo , Mutagénesis Insercional , Mutación , Nitrogenasa/genética , Fenotipo , Sideróforos/análisis , Sideróforos/metabolismo

RESUMEN

Biological nitrogen fixation is widespread among the Eubacteria and Archae domains but completely absent in eukaryotes. The lack of lateral transfer of nitrogen-fixation genes from prokaryotes to eukaryotes has been partially attributed to the physiological requirements necessary for the function of the nitrogenase complex. However, symbiotic bacterial nitrogenase activity is protected by the nodule, a plant structure whose organogenesis can be trigged in the absence of bacteria. To explore the intrinsic potentiality of this plant organ, we generated rhizobium-independent nodules in alfalfa by overexpressing the MsDMI3 kinase lacking the autoinhibitory domain. These transgenic nodules showed similar levels of leghemoglobin, free oxygen, ATP, and NADPH to those of efficient Sinorhizobium meliloti B399-infected nodules, suggesting that the rhizobium-independent nodules can provide an optimal microenvironment for nitrogenase activity. Finally, we discuss the intrinsic evolutionary constraints on transfer of nitrogen-fixation genes between bacteria and eukaryotes.

Asunto(s)

Bacterias/genética , Eucariontes/genética , Nitrogenasa/genética , Nitrogenasa/metabolismo , Bacterias/clasificación , Bacterias/metabolismo , Eucariontes/clasificación , Eucariontes/metabolismo , Regulación de la Expresión Génica de las Plantas , Medicago sativa/genética , Medicago sativa/metabolismo , Fijación del Nitrógeno/genética , Filogenia , Nodulación de la Raíz de la Planta/genética , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Simbiosis

RESUMEN

Nitrogen is the second most critical factor for crop production after water. In this study, the beneficial rhizobacterium Pseudomonas protegens Pf-5 was genetically modified to fix nitrogen using the genes encoding the nitrogenase of Pseudomonas stutzeri A1501 via the X940 cosmid. Pf-5 X940 was able to grow in L medium without nitrogen, displayed high nitrogenase activity and released significant quantities of ammonium to the medium. Pf-5 X940 also showed constitutive expression and enzymatic activity of nitrogenase in ammonium medium or in nitrogen-free medium, suggesting a constitutive nitrogen fixation. Similar to Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii and Pseudomonas taetrolens but not Pseudomonas balearica and Pseudomonas stutzeri transformed with cosmid X940 showed constitutive nitrogenase activity and high ammonium production, suggesting that this phenotype depends on the genome context and that this technology to obtain nitrogen-fixing bacteria is not restricted to Pf-5. Interestingly, inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-deficient conditions. In conclusion, these results open the way to the production of effective recombinant inoculants for nitrogen fixation on a wide range of crops.

Asunto(s)

Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Fijación del Nitrógeno/genética , Nitrogenasa/metabolismo , Pseudomonas/genética , Microbiología del Suelo , Arabidopsis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Vectores Genéticos , Medicago sativa/crecimiento & desarrollo , Nitrógeno/metabolismo , Nitrogenasa/genética , Pseudomonas/enzimología , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Pseudomonas stutzeri/enzimología , Pseudomonas stutzeri/genética , Transformación Bacteriana , Zea mays/crecimiento & desarrollo

RESUMEN

The bark beetles of the genus Dendroctonus feed on phloem that is a nitrogen-limited source. Nitrogen fixation and nitrogen recycling may compensate or alleviate such a limitation, and beetle-associated bacteria capable of such processes were identified. Raoultella terrigena, a diazotrophic bacteria present in the gut of Dendroctonus rhizophagus and D. valens, exhibited high acetylene reduction activity in vitro with different carbon sources, and its nifH and nifD genes were sequenced. Bacteria able to recycle uric acid were Pseudomonas fluorescens DVL3A that used it as carbon and nitrogen source, Serratia proteomaculans 2A CDF and Rahnella aquatilis 6-DR that used uric acid as sole nitrogen source. Also, this is the first report about the uric acid content in whole eggs, larvae, and adults (male and female) samples of the red turpentine beetle (Dendroctonus valens). Our results suggest that the gut bacteria of these bark beetles could contribute to insect N balance.

Asunto(s)

Bacterias/aislamiento & purificación , Bacterias/metabolismo , Escarabajos/microbiología , Fijación del Nitrógeno , Animales , Bacterias/clasificación , Bacterias/genética , Proteínas Bacterianas/genética , Escarabajos/clasificación , Escarabajos/crecimiento & desarrollo , Femenino , Tracto Gastrointestinal/microbiología , Larva/clasificación , Larva/crecimiento & desarrollo , Larva/microbiología , Masculino , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Nitrogenasa/genética , Filogenia

RESUMEN

The toxin producing nitrogen-fixing heterocystous freshwater cyanobacterium Cylindrospermopsis raciborskii recently radiated from its endemic tropical environment into sub-tropical and temperate regions, a radiation likely to be favored by its ability to fix dinitrogen (diazotrophy). Although most heterocystous cyanobacteria differentiate regularly spaced intercalary heterocysts along their trichomes when combined nitrogen sources are depleted, C. raciborskii differentiates only two terminal heterocysts (one at each trichome end) that can reach >100 vegetative cells each. Here we investigated whether these terminal heterocysts are the exclusive sites for dinitrogen fixation in C. raciborskii. The highest nitrogenase activity and NifH biosynthesis (western-blot) were restricted to the light phase of a 12/12 light/dark cycle. Separation of heterocysts and vegetative cells (sonication and two-phase aqueous polymer partitioning) demonstrated that the terminal heterocysts are the sole sites for nifH expression (RT-PCR) and NifH biosynthesis. The latter finding was verified by the exclusive localization of nitrogenase in the terminal heterocysts of intact trichomes (immunogold-transmission electron microscopy and in situ immunofluorescence-light microscopy). These results suggest that the terminal heterocysts provide the combined nitrogen required by the often long trichomes (>100 vegetative cells). Our data also suggests that the terminal-heterocyst phenotype in C. raciborskii may be explained by the lack of a patL ortholog. These data help identify mechanisms by which C. raciborskii and other terminal heterocyst-forming cyanobacteria successfully inhabit environments depleted in combined nitrogen.

Asunto(s)

Cianobacterias/genética , Cianobacterias/metabolismo , Cylindrospermopsis/genética , Cylindrospermopsis/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Luz , Nitrógeno/metabolismo , Fijación del Nitrógeno , Nitrogenasa/genética , Nitrogenasa/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo

RESUMEN

Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained an Azotobacter vinelandii mutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.

Asunto(s)

Azotobacter/crecimiento & desarrollo , Biocombustibles , Chlorella/crecimiento & desarrollo , Microalgas/crecimiento & desarrollo , Fijación del Nitrógeno , Compuestos de Amonio Cuaternario/metabolismo , Scenedesmus/crecimiento & desarrollo , Azotobacter/genética , Azotobacter/aislamiento & purificación , Azotobacter/metabolismo , Biomasa , Biotecnología/métodos , Chlorella/genética , Chlorella/aislamiento & purificación , Chlorella/metabolismo , Medios de Cultivo , Agua Dulce/microbiología , Eliminación de Gen , Microalgas/genética , Microalgas/aislamiento & purificación , Microalgas/metabolismo , Mutación , Nitrogenasa/genética , Scenedesmus/genética , Scenedesmus/aislamiento & purificación , Scenedesmus/metabolismo , Simbiosis

RESUMEN

The fixation of atmospheric nitrogen by the prokaryotic enzyme nitrogenase is an energy- expensive process and consequently it is tightly regulated at a variety of levels. In many diazotrophs this includes post-translational regulation of the enzyme's activity, which has been reported in both bacteria and archaea. The best understood response is the short-term inactivation of nitrogenase in response to a transient rise in ammonium levels in the environment. A number of proteobacteria species effect this regulation through reversible ADP-ribosylation of the enzyme, but other prokaryotes have evolved different mechanisms. Here we review current knowledge of post-translational control of nitrogenase and show that, for the response to ammonium, the P(II) signal transduction proteins act as key players.

Asunto(s)

Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Nitrogenasa/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Transducción de Señal , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Nitrogenasa/genética , Proteínas PII Reguladoras del Nitrógeno/genética , Procesamiento Proteico-Postraduccional

RESUMEN

Bacteria belonging to the genus Bradyrhizobium are capable of establishing symbiotic relationships with a broad range of plants belonging to the three subfamilies of the family Leguminosae (=Fabaceae), with the formation of specialized structures on the roots called nodules, where fixation of atmospheric nitrogen takes place. Symbiosis is under the control of finely tuned expression of common and host-specific nodulation genes and also of genes related to the assembly and activity of the nitrogenase, which, in Bradyrhizobium strains investigated so far, are clustered in a symbiotic island. Information about the diversity of these genes is essential to improve our current poor understanding of their origin, spread and maintenance and, in this study, we provide information on 40 Bradyrhizobium strains, mostly of tropical origin. For the nodulation trait, common (nodA), Bradyrhizobium-specific (nodY/K) and host-specific (nodZ) nodulation genes were studied, whereas for fixation ability, the diversity of nifH was investigated. In general, clustering of strains in all nod and nifH trees was similar and the Bradyrhizobium group could be clearly separated from other rhizobial genera. However, the congruence of nod and nif genes with ribosomal and housekeeping genes was low. nodA and nodY/K were not detected in three strains by amplification or hybridization with probes using Bradyrhizobium japonicum and Bradyrhizobium elkanii type strains, indicating the high diversity of these genes or that strains other than photosynthetic Bradyrhizobium must have alternative mechanisms to initiate the process of nodulation. For a large group of strains, the high diversity of nod genes (with an emphasis on nodZ), the low relationship between nod genes and the host legume, and some evidence of horizontal gene transfer might indicate strategies to increase host range. On the other hand, in a group of five symbionts of Acacia mearnsii, the high congruence between nod and ribosomal/housekeeping genes, in addition to shorter nodY/K sequences and the absence of nodZ, highlights a co-evolution process. Additionally, in a group of B. japonicum strains that were symbionts of soybean, vertical transfer seemed to represent the main genetic event. In conclusion, clustering of nodA and nifH gives additional support to the theory of monophyletic origin of the symbiotic genes in Bradyrhizobium and, in addition to the analysis of nodY/K and nodZ, indicates spread and maintenance of nod and nif genes through both vertical and horizontal transmission, apparently with the dominance of one or other of these events in some groups of strains.

Asunto(s)

Proteínas Bacterianas/genética , Evolución Biológica , Bradyrhizobium/clasificación , Bradyrhizobium/genética , Transferencia de Gen Horizontal , Nitrogenasa/genética , Filogenia , Proteínas Bacterianas/metabolismo , Bradyrhizobium/aislamiento & purificación , Bradyrhizobium/fisiología , Fabaceae/microbiología , Fabaceae/fisiología , Datos de Secuencia Molecular , Fijación del Nitrógeno , Nitrogenasa/metabolismo , Nodulación de la Raíz de la Planta , Nódulos de las Raíces de las Plantas/microbiología , Simbiosis

RESUMEN

BACKGROUND: G. diazotrophicus and A. vinelandii are aerobic nitrogen-fixing bacteria. Although oxygen is essential for the survival of these organisms, it irreversibly inhibits nitrogenase, the complex responsible for nitrogen fixation. Both microorganisms deal with this paradox through compensatory mechanisms. In A. vinelandii a conformational protection mechanism occurs through the interaction between the nitrogenase complex and the FeSII protein. Previous studies suggested the existence of a similar system in G. diazotrophicus, but the putative protein involved was not yet described. This study intends to identify the protein coding gene in the recently sequenced genome of G. diazotrophicus and also provide detailed structural information of nitrogenase conformational protection in both organisms. RESULTS: Genomic analysis of G. diazotrophicus sequences revealed a protein coding ORF (Gdia0615) enclosing a conserved "fer2" domain, typical of the ferredoxin family and found in A. vinelandii FeSII. Comparative models of both FeSII and Gdia0615 disclosed a conserved beta-grasp fold. Cysteine residues that coordinate the 2[Fe-S] cluster are in conserved positions towards the metallocluster. Analysis of solvent accessible residues and electrostatic surfaces unveiled an hydrophobic dimerization interface. Dimers assembled by molecular docking presented a stable behaviour and a proper accommodation of regions possibly involved in binding of FeSII to nitrogenase throughout molecular dynamics simulations in aqueous solution. Molecular modeling of the nitrogenase complex of G. diazotrophicus was performed and models were compared to the crystal structure of A. vinelandii nitrogenase. Docking experiments of FeSII and Gdia0615 with its corresponding nitrogenase complex pointed out in both systems a putative binding site presenting shape and charge complementarities at the Fe-protein/MoFe-protein complex interface. CONCLUSIONS: The identification of the putative FeSII coding gene in G. diazotrophicus genome represents a large step towards the understanding of the conformational protection mechanism of nitrogenase against oxygen. In addition, this is the first study regarding the structural complementarities of FeSII-nitrogenase interactions in diazotrophic bacteria. The combination of bioinformatic tools for genome analysis, comparative protein modeling, docking calculations and molecular dynamics provided a powerful strategy for the elucidation of molecular mechanisms and structural features of FeSII-nitrogenase interaction.

Asunto(s)

Azotobacter vinelandii/enzimología , Gluconacetobacter/enzimología , Modelos Moleculares , Nitrogenasa/metabolismo , Oxígeno/metabolismo , Conformación Proteica , Secuencia de Aminoácidos , Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biología Computacional , Genómica , Gluconacetobacter/genética , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fijación del Nitrógeno , Nitrogenasa/química , Nitrogenasa/genética , Unión Proteica , Electricidad Estática

RESUMEN

Ammonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the P(II) family. In this study we have characterized in vitro complex formation between the AmtB and P(II) proteins (GlnB and GlnZ) from the diazotrophic plant-associative bacterium Azospirillum brasilense. AmtB-P(II) complex formation only occurred in the presence of adenine nucleotides and was sensitive to 2-oxoglutarate when Mg(2+) and ATP were present, but not when ATP was substituted by ADP. We have also shown in vitro complex formation between GlnZ and the nitrogenase regulatory enzyme DraG, which was stimulated by ADP. The stoichiometry of this complex was 1:1 (DraG monomer : GlnZ trimer). We have previously reported that in vivo high levels of extracellular ammonium cause DraG to be sequestered to the cell membrane in an AmtB and GlnZ-dependent manner. We now report the reconstitution of a ternary complex involving AmtB, GlnZ and DraG in vitro. Sequestration of a regulatory protein by the membrane-bound AmtB-P(II) complex defines a new regulatory role for Amt proteins in Prokaryotes.

Asunto(s)

Azospirillum brasilense/metabolismo , Proteínas Bacterianas/metabolismo , Nitrógeno/metabolismo , Nitrogenasa/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Azospirillum brasilense/enzimología , Azospirillum brasilense/genética , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Regulación Bacteriana de la Expresión Génica , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Nitrogenasa/genética , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

In this work, we further analyzed an Azospirillum brasilense Sp7 mutant (Sp7::Tn5-33) showing a pleiotrophic phenotype due to a Tn5 insertion into an open reading frame of 840 bp (orf280). The deduced amino acid sequence of this region has high similarity to a family of universal stress proteins. Because the most interesting property exhibited by the Sp7::Tn5-33 mutant was an enhanced in vitro nitrogen fixation activity, we addressed the question of whether it could benefit the host plant. We found that the increased nitrogenase activity at the free-living state of the mutant bacterium was correlated with an increased production of the nitrogenase reductase protein (NifH), in amounts approximately 1.5 times higher than the wild type. The mutant strain exhibited the same level of auxin production and the same colonization pattern of wheat roots as the wild type. We also observed that Sp7::Tn5-33 increased the total plant dry weight, although the N content did not differ significantly between wheat plants inoculated with mutant or wild-type strains.

Asunto(s)

Azospirillum brasilense/genética , Nitrogenasa/metabolismo , Azospirillum brasilense/crecimiento & desarrollo , Azospirillum brasilense/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glucuronidasa/metabolismo , Ácidos Indolacéticos/metabolismo , Mutación/genética , Fijación del Nitrógeno/genética , Fijación del Nitrógeno/fisiología , Nitrogenasa/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Factores de Tiempo , Triticum/metabolismo , Triticum/microbiología

RESUMEN

Herbaspirillum seropedicae Z67 is a nitrogen-fixing bacterium able to colonize the rhizosphere and the interior of several plants. As iron is a key element for nitrogen fixation, we examined the response of this microorganism to iron deficiency under nitrogen fixing conditions. We identified a H. seropedicae exbD gene that was induced in response to iron limitation and is involved in iron homeostasis. We found that an exbD mutant grown in iron-chelated medium is unable to fix nitrogen. Moreover, we provide evidence that expression of the nifH and nifA genes is iron dependent in a H. seropedicae genetic background.

Asunto(s)

Proteínas Bacterianas/metabolismo , Herbaspirillum/metabolismo , Hierro/metabolismo , Nitrogenasa/metabolismo , Oxidorreductasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Herbaspirillum/enzimología , Herbaspirillum/genética , Mutación , Fijación del Nitrógeno , Nitrogenasa/genética , Oxidorreductasas/genética , Factores de Transcripción/genética