Proteome profiling of Paenibacillus sonchi genomovar Riograndensis SBR5T under conventional and alternative nitrogen fixation.

Paenibacillus sonchi SBR5T is a Gram-positive, endospore-forming facultative aerobic diazotrophic bacterium that can fix nitrogen via an alternative Fe-only nitrogenase (AnfHDGK). In several bacteria, this alternative system is expressed under molybdenum (Mo)-limiting conditions when the conventional Mo-dependent nitrogenase (NifHDK) production is impaired. The regulatory mechanisms, metabolic processes, and cellular functions of N2 fixation by alternative and/or conventional systems are poorly understood in the Paenibacillus genus. We conducted a comparative proteomic profiling study of P. sonchi SBR5T grown under N2-fixing conditions with and without Mo supply through an LC-MS/MS and label-free quantification analysis to address this gap. Protein abundances revealed overrepresented processes related to anaerobiosis growth adaption, Fe-S cluster biosynthesis, ammonia assimilation, electron transfer, and sporulation under N2-fixing conditions compared to non-fixing control. Under Mo limitation, the Fe-only nitrogenase components were overrepresented together with the Mo-transporter system, while the dinitrogenase component (NifDK) of Mo­nitrogenase was underrepresented. The dinitrogenase reductase component (NifH) and accessory proteins encoded by the nif operon had no significant differential expression, suggesting post-transcriptional regulation of nif gene products in this strain. Overall, this was the first comprehensive proteomic analysis of a diazotrophic strain from the Paenibacillaceae family, and it provided insights related to alternative N2-fixation by Fe-only nitrogenase. SIGNIFICANCE: In this work, we try to understand how the alternative nitrogen fixation system, presented by some diazotrophic bacteria, works. For this, we used the SBR5 lineage of P. sonchi, which presents the alternative system in which the nitrogenase cofactor is composed only of iron. In addition, we tried to unravel the proteome of this strain in different situations of nitrogen fixation, since, for Gram-positive bacteria, these systems are little known. The results achieved, through LC-MS/MS and label-free quantitative analysis, showed an overrepresentation of proteins related to different processes involved with growth under stressful conditions in situations of nitrogen deficiency, in addition to suggesting that some encoded proteins by the nif operon may be regulated at post-transcriptional levels. Our findings represent important steps toward the elucidation of nitrogen fixation systems in Gram-positive diazotrophic bacteria.

Comparative Genomics Reveal the High Conservation and Scarce Distribution of Nitrogen Fixation nif Genes in the Plant-Associated Genus Herbaspirillum.

The genus Herbaspirillum gained the spotlight due to the several reports of diazotrophic strains and promising results in plant-growth field assays. However, as diversity exploration of Herbaspirillum species gained momentum, it became clearer that the plant beneficial lifestyle was not the only form of ecological interaction in this genus, due to reports of phytopathogenesis and nosocomial infections. Here we performed a deep search across all publicly available Herbaspirillum genomes. Using a robust core genome phylogeny, we have found that all described species are well delineated, being the only exception H. aquaticum and H. huttiense clade. We also uncovered that the nif genes are only highly prevalent in H. rubrisubalbicans; however, irrespective to the species, all nif genes share the same gene arrangement with high protein identity, and are present in only two main types, in inverted strands. By means of a NifHDKENB phylogenetic tree, we have further revealed that the Herbaspirillum nif sequences may have been acquired from the same last common ancestor belonging to the Nitrosomonadales order.

Comparative study of symbiotic activity of legumes when using Risotorphin and Epin-extra / Estudo comparativo da atividade simbiótica de leguminosas em uso de Risotorfina e Epin-extra

In a vegetation experiment with soybean plants of the Svapa and Mageva varieties and in a field experiment with bean plants of the Geliada and Shokoladnitsa varieties, we studied the effect of pre-sowing treatment of the seeds of these plants with Rizotorfin and Epin-extra on the nitrogenase activity of the nodules of these plants and their ultrastructure. Analysis of the ultrastructure of the nodule tissue of beans and soybeans was carried out in the flowering phase. It was found that the highest indices of the mass and number of nodules and the activity of nitrogenase in them were found in bean plants of the Heliada cultivar when the seeds were treated with Epin-extra against the background of inoculation with Rizotorfin, where the largest area of symbiosomes, volutin and their number was noted in the nodules. Beans of the Shokoladnitsa variety showed the protective effect of Rizotorfin. In the nodules of soybean plants of the Svapa variety, the seeds of which were treated with Epin-extra against the background of inoculation with Rizotorfin, the presence of a large number of symbiosomes, bacteroids, volutin inclusions with a larger area and a minimum number of inclusions of poly-ß-hydroxybutyric acid (PHB) was noted, and the highest indicators of symbiotic activity. Soybean plants of the Mageva variety showed the protective effect of Rizotorfin. The efficiency of the symbiotic system was determined by the number and weight of nodules and the activity of the nitrogenase enzyme.

Em um experimento de vegetação com plantas de soja das variedades Svapa e Mageva e em um experimento de campo com plantas de feijão das variedades Geliada e Shokoladnitsa, o efeito do tratamento pré-semeadura das sementes dessas plantas com Rizotorfin e Epin-extra sobre a atividade nitrogenase dos nódulos dessas plantas e sua ultraestrutura foi examinado. A análise da ultraestrutura do tecido do nódulo de feijão e soja foi realizada na fase de floração. Dessa forma, constatou-se que os maiores índices de massa, número de nódulos e a atividade da nitrogenase foram encontrados em plantas de feijão da cultivar Heliada quando as sementes foram tratadas com Epin-extra no contexto da inoculação com Rizotorfina, em que o maior área de simbiossomas, volutina e seu número foi anotado nos nódulos. O feijão da variedade Shokoladnitsa mostrou o efeito protetor do Rizotorfin. Nos nódulos de plantas de soja da variedade Svapa, cujas sementes foram tratadas com Epin-extra no contexto da inoculação com Rizotorfin, a presença de um grande número de simbiossomas, bacteróides, inclusões de volutina com uma área maior e um número mínimo de inclusões de ácido poli-ß-hidroxibutírico (PHB) foi notado, bem como os indicadores mais altos de atividade simbiótica. As plantas de soja da variedade Mageva apresentaram o efeito protetor do Rizotorfin. A eficiência do sistema simbiótico foi determinada pelo número e peso dos nódulos e pela atividade da enzima nitrogenase.

A complete biomimetic iron-sulfur cubane redox series.

Synthetic iron-sulfur cubanes are models for biological cofactors, which are essential to delineate oxidation states in the more complex enzymatic systems. However, a complete series of [Fe4S4]n complexes spanning all redox states accessible by 1-electron transformations of the individual iron atoms (n = 0-4+) has never been prepared, deterring the methodical comparison of structure and spectroscopic signature. Here, we demonstrate that the use of a bulky arylthiolate ligand promoting the encapsulation of alkali-metal cations in the vicinity of the cubane enables the synthesis of such a series. Characterization by EPR, 57Fe Mössbauer spectroscopy, UV-visible electronic absorption, variable-temperature X-ray diffraction analysis, and cyclic voltammetry reveals key trends for the geometry of the Fe4S4 core as well as for the Mössbauer isomer shift, which both correlate systematically with oxidation state. Furthermore, we confirm the S = 4 electronic ground state of the most reduced member of the series, [Fe4S4]0, and provide electrochemical evidence that it is accessible within 0.82 V from the [Fe4S4]2+ state, highlighting its relevance as a mimic of the nitrogenase iron protein cluster.

Genetic variation in symbiotic islands of natural variant strains of soybean Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens differing in competitiveness and in the efficiency of nitrogen fixation.

Soybean is the most important legume cropped worldwide and can highly benefit from the biological nitrogen fixation (BNF) process. Brazil is recognized for its leadership in the use of inoculants and two strains, Bradyrhizobium japonicum CPAC 15 (=SEMIA 5079) and Bradyrhizobium diazoefficiens CPAC 7 (=SEMIA 5080) compose the majority of the 70 million doses of soybean inoculants commercialized yearly in the country. We studied a collection of natural variants of these two strains, differing in properties of competitiveness and efficiency of BNF. We sequenced the genomes of the parental strain SEMIA 566 of B. japonicum, of three natural variants of this strain (S 204, S 340 and S 370), and compared with another variant of this group, strain CPAC 15. We also sequenced the genome of the parental strain SEMIA 586 of B. diazoefficiens, of three natural variants of this strain (CPAC 390, CPAC 392 and CPAC 394) and compared with the genome of another natural variant, strain CPAC 7. As the main genes responsible for nodulation (nod, noe, nol) and BNF (nif, fix) in soybean Bradyrhizobium are located in symbiotic islands, our objective was to identify genetic variations located in this region, including single nucleotide polymorphisms (SNPs) and insertions and deletions (indels), that could be potentially related to their different symbiotic phenotypes. We detected 44 genetic variations in the B. japonicum strains and three in B. diazoefficiens. As the B. japonicum strains have gone through a longer period of adaptation to the soil, the higher number of genetic variations could be explained by survival strategies under the harsh environmental conditions of the Brazilian Cerrado biome. Genetic variations were detected in genes enconding proteins such as a dephospho-CoA kinase, related to the CoA biosynthesis; a glucosamine-fructose-6-phosphate aminotransferase, key regulator of the hexosamine biosynthetic pathway; a LysR family transcriptional regulator related to nodulation genes; and NifE and NifS proteins, directly related to the BNF process. We suggest potential genetic variations related to differences in the symbiotic phenotypes.

Multiple ways to evade the bacteriostatic action of glyphosate in rhizobia include the mutation of the conserved serine 90 of the nitrogenase subunit NifH to alanine.

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.

Relieving your stress: PGPB associated with Andean xerophytic plants are most abundant and active on the most extreme slopes.

Introduction: Plants interact with plant growth-promoting bacteria (PGPB), especially under stress condition in natural and agricultural systems. Although a potentially beneficial microbiome has been found associated to plants from alpine systems, this plant- PGPB interaction has been scarcely studied. Nevados de Chillán Complex hold one of the southernmost xerophytic formations in Chile. Plant species living there have to cope with drought and extreme temperatures during the growing season period, microclimatic conditions that become harsher on equatorial than polar slopes, and where the interaction with PGPB could be key for plant survival. Our goal was to study the abundance and activity of different PGPB associated to two abundant plant species of Andean xerophytic formations on contrasting slopes. Methods: Twenty individuals of Berberis empetrifolia and Azorella prolifera shrubs were selected growing on a north and south slope nearby Las Fumarolas, at 2,050 m elevation. On each slope, microclimate based on temperature and moisture conditions were monitored throughout the growing period (oct. - apr.). Chemical properties of the soil under plant species canopies were also characterized. Bacterial abundance was measured as Log CFU g-1 from soil samples collected from each individual and slope. Then, the most abundant bacterial colonies were selected, and different hormonal (indoleacetic acid) and enzymatic (nitrogenase, phosphatase, ACC-deaminase) mechanisms that promote plant growth were assessed and measured. Results and Discussion: Extreme temperatures were observed in the north facing slope, recording the hottest days (41 vs. 36°C) and coldest nights (-9.9 vs. 6.6°C). Moreover, air and soil moisture were lower on north than on south slope, especially late in the growing season. We found that bacterial abundance was higher in soils on north than on south slope but only under B. empetrifolia canopy. Moreover, the activity of plant growth-promoting mechanisms varied between slopes, being on average higher on north than on south slope, but with plant species-dependent trends. Our work showed how the environmental heterogeneity at microscale in alpine systems (slope and plant species identity) underlies variations in the abundance and plant growth promoting activity of the microorganisms present under the plant canopy of the Andean xerophytic formations and highlight the importance of PGPB from harsh systems as biotechnological tools for restoration.

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants.

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.

Synthesis of a Nitrogenase PN -Cluster Model with [Fe8 S7 (µ-Sthiolate )2 ] Core from the All-Ferric [Fe4 S4 (Sthiolate )4 ] Cubane Synthon.

Constructing synthetic models of the nitrogenase PN -cluster has been a long-standing synthetic challenge. Here, we report an optimal nitrogenase PN -cluster model [{(TbtS)(OEt2 )Fe4 S3 }2 (µ-STbt)2 (µ6 -S)] (2) [Tbt=2,4,6-tris{bis(trimethylsilyl)methyl}phenyl] that is the closest synthetic mimic constructed to date. Of note is that two thiolate ligands and one hexacoordinated sulfide are connecting the two Fe4 S3 incomplete cubanes similar to the native PN -cluster, which has never been achieved. Cluster 2 has been characterized by X-ray crystallography and relevant physico-chemical methods. The variable temperature magnetic moments of 2 indicate a singlet ground state (S=0). The Mössbauer spectrum of 2 exhibits two doublets with an intensity ratio of 3:1, which suggests the presence of two types of iron sites. The synthetic pathway of the cluster 2 could indicate the native PN -cluster maturation process as it has been achieved from the Fe4 S4 cubane Fe4 S4 (STbt)4 (1).

Genetics and regulation of nitrogen fixation in Paenibacillus brasilensis PB24.

Biological nitrogen fixation (BNF), performed by diazotrophic prokaryotes, is responsible for reducing dinitrogen (N2) present in the biosphere into biologically available forms of nitrogen. Paenibacillus brasilensis PB24 is a diazotrophic Gram-positive bacterium and is considered ecologically and industrially important because it is able to produce antimicrobial substances and 2,3-butanediol. However, the genetics and regulation of its nitrogen fixing (nif) genes have never been assessed so far. Therefore, the present study aimed to (i) identify the structural and regulatory genes related to BNF in the PB24 genome, (ii) perform comparative genomics analysis of the nif operon among different Paenibacillus species and (iii) study the expression of these genes in the presence and absence of NH4. Strain PB24 showed a nif operon composed of nine genes (nifBHDKENXhesAV), with a conserved synteny (with small variations) among the Paenibacillus species evaluated. BNF regulatory genes, glnK and amtB (encoding GlnK signal transduction protein and AmtB transmembrane protein, respectively) and glnR and glnA genes (encoding the transcription factor GlnR and glutamine synthetase) were found in the PB24 genome. Primers were designed for qPCR amplification of the nitrogenase structural (nifH, nifD and nifK) and regulatory (glnA and amtB) BNF genes. The structural gene expression in PB24 was up- and downregulated in the absence and presence of NH4, respectively. The gene expression levels indicated a GlnR-mediated repression of genes associated with ammonium import (amtBglnK) and BNF (nif genes). Additionally, the regulatory mechanism of GlnR in P. brasilensis PB24 differed from the other Paenibacillus evaluated, considering the different distribution of binding sites recognized by GlnR.

Corn Responsiveness to Azospirillum: Accessing the Effect of Root Exudates on the Bacterial Growth and Its Ability to Fix Nitrogen.

Corn has shown different degrees of positive response to inoculation with the nitrogen- fixing bacteria of the genera Azospirillum. Part of it has been attributed to the plant genotypic variation, including the root exudates, that are used by the bacteria as energy source. In this study, we grew two corn hybrids that differ for their response to Azospirillum, to investigate the effect of different exudates profiles on the bacteria growth and nitrogenase activity. Employing high performance liquid chromatography, we identified nine amino acids (asparagine, aspartic acid, serine, glutamic acid, valine, phenylalanine, threonine, tryptophan and alanine), six sugars (glucose, sucrose, xylose, arabinose, fructose and galactose) and four organic acids (citrate, malate, succinate and fumarate). The less responsive corn genotype showed reduced plant growth (root volume, shoot dry mass and shoot N content), a lower concentration of Azospirillum cells within the root tissues, a higher content of asparagine and glucose and a reduced amount of metabolites that serve as bacterial energy source (all organic acids + five sugars, excluding glucose). The genotypes did not interfere in the ability of Azospirillum to colonize the substrate, but the metabolites released by the less responsive one reduced the nitrogenase activity.

Zinc toxicity in seedlings of three trees from the Fabaceae associated with arbuscular mycorrhizal fungi.

Due to diverse human activities zinc (Zn) may reach phytotoxic levels in the soil. Here, we evaluated the differential sensibility of three Brazilian tree species from the Fabaceae to increasing soil Zn concentrations and its physiological response to cope with excess Zn. A greenhouse experiment was conducted with the species: Mimosa caesalpiniaefolia, Erythrina speciosa and Schizolobium parahyba, and the addition of 0, 200, 400 and 600 mg Zn kg-1 to the soil. Plants were harvested after three months of cultivation, and growth, root symbiosis, biochemical markers and elemental composition were analyzed. Soil Zn addition reduced seedling growth, irrespective of the species, with a strong reduction in M. caesalpiniaefolia. Regarding root symbiosis, in N2-fixing species, nitrogenase activity was reduced by the highest Zn concentrations. Zn addition caused plants nutritional imbalances, mainly in roots. The content of photosynthetic pigments in leaves decreased up to 40%, suggesting that high Zn contents interfered with its biosynthesis, and altered the content of foliar polyamines and free amino acids, depending on the species and the soil Zn concentration. Zn toxicity in M. caesalpiniaefolia plants was observed at available soil Zn concentrations greater than 100 mg kg-1 (DTPA-extractable), being the most sensitive species and E. speciosa was moderately sensitive. S. parahyba was a moderately tolerant species, which seems to be related to polyamines accumulation and to mycorrhizal association. This last species has the potential for revegetation of areas with moderately high soil Zn concentration and for phytostabilization purposes. Future research evaluating the tolerance to multiple metal stress under field conditions should confirm S. parayba suitability in Zn contaminated areas of tropical regions.

Double-Cubane [8Fe9S] Clusters: A Novel Nitrogenase-Related Cofactor in Biology.

Three different types of electron-transferring metallo-ATPases are able to couple ATP hydrolysis to the reduction of low-potential metal sites, thereby energizing an electron. Besides the Fe-protein known from nitrogenase and homologous enzymes, two other kinds of ATPase with different scaffolds and cofactors are used to achieve a unidirectional, energetic, uphill electron transfer to either reduce inactive Co-corrinoid-containing proteins (RACE-type activators) or a second iron-sulfur cluster-containing enzyme of a unique radical enzymes family (archerases). We have found a new cofactor in the latter enzyme family, that is, a double-cubane cluster with two [4Fe4S] subclusters bridged by a sulfido ligand. An enzyme containing this cofactor catalyzes the ATP-dependent reduction of small molecules, including acetylene. Thus, enzymes containing the double-cubane cofactor are analogous in function and share some structural features with nitrogenases.

Hydrogen-uptake genes improve symbiotic efficiency in common beans (Phaseolus vulgaris L.).

Hydrogen-uptake (Hup) activity is implicated in the mitigation of energy losses associated with the biological nitrogen fixation process, and has been related to productivity increases in some legume hosts. However, in common bean (Phaseolus vulgaris L.) the expression of hydrogenase is rare. In this study an 18-kb hup gene cluster from Rhizobium leguminosarum bv. viciae encoding a NiFe hydrogenase was successfully transferred to three common bean rhizobial strains lacking hydrogenase activity (Hup-) but symbiotically very effective and used in commercial inoculants in Brazil: one strain originally from Colombia (Rhizobium tropici CIAT 899), and two strains from Brazil (R. tropici H 12 and Rhizobium freirei PRF 81). The inclusion of NiCl2 in the nutrient solution did not increase hydrogenase activity, indicating that common bean plants allow efficient nickel provision for hydrogenase synthesis in the bacteroids. The symbiotic performance-evaluated by nodulation, plant growth, N accumulation and seed production-of wild-type and Hup+ derivative strains was compared in experiments performed with cultivar Carioca under greenhouse conditions, in sterile substrate and in non-sterile soil. Statistically significant increases in one or more parameters were observed for all three Hup+ derivatives when compared to the respective wild-type strain. Differences were found mainly with the Brazilian strains, reaching impressive increases in nodule efficiency and seed total N content. The results highlight the potential of using Rhizobium Hup+ strains for the design of more energy-efficient inoculants for the common bean crop.

ATP-dependent substrate reduction at an [Fe8S9] double-cubane cluster.

Chemically demanding reductive conversions in biology, such as the reduction of dinitrogen to ammonia or the Birch-type reduction of aromatic compounds, depend on Fe/S-cluster-containing ATPases. These reductions are typically catalyzed by two-component systems, in which an Fe/S-cluster-containing ATPase energizes an electron to reduce a metal site on the acceptor protein that drives the reductive reaction. Here, we show a two-component system featuring a double-cubane [Fe8S9]-cluster [{Fe4S4(SCys)3}2(µ2-S)]. The double-cubane-cluster-containing enzyme is capable of reducing small molecules, such as acetylene (C2H2), azide (N3-), and hydrazine (N2H4). We thus present a class of metalloenzymes akin in fold, metal clusters, and reactivity to nitrogenases.

Bacillus spp: una alternativa para la promoción vegetal por dos caminos enzimáticos / Bacillus spp: An alternative for plant promotion by two enzymatic pathways

Resumen Objetivo. Se realizó una revisión sobre las características de las fitasas y nitrogenasas de Bacillus spp. y sus opciones de uso como alternativa biofertilizante. El género Bacillus es secretor de proteínas y metabolitos eficientes para el control de plagas y enfermedades, promueve el crecimiento vegetal a través de la solubilización de fósforo y la producción de reguladores de crecimiento como el ácido indol acético; así mismo participa en la fijación de nitrógeno cuando hace parte de consorcios microbianos. Como biofertilizante es una opción amigable para el suelo y el ambiente que da respuesta a la necesidad de implementar la agricultura sostenible.

Abstract Objective. We conducted a review of the characteristics of the phytase and nitrogenase of Bacillus sp. and their potential use as an alternative biological fertilizer. The genus Bacillus is an efficient secretor of proteins and metabolites; to control pests and diseases, promote plant growth through the solubilization of phosphorus and production of growth regulators as acetic indole. Likewise it is involved in nitrogen fixation when it is part of microbial consortia. As biological fertilizer is a friendly option for the soil and the environment that responds to the need to implement sustainable agriculture.

Flooding of the root system in soybean: biochemical and molecular aspects of N metabolism in the nodule during stress and recovery.

Nitrogen fixation of the nodule of soybean is highly sensitive to oxygen deficiency such as provoked by waterlogging of the root system. This study aimed to evaluate the effects of flooding on N metabolism in nodules of soybean. Flooding resulted in a marked decrease of asparagine (the most abundant amino acid) and a concomitant accumulation of γ-aminobutyric acid (GABA). Flooding also resulted in a strong reduction of the incorporation of (15)N2 in amino acids. Nodule amino acids labelled before flooding rapidly lost (15)N during flooding, except for GABA, which initially increased and declined slowly thereafter. Both nitrogenase activity and the expression of nifH and nifD genes were strongly decreased on flooding. Expression of the asparagine synthetase genes SAS1 and SAS2 was reduced, especially the former. Expression of genes encoding the enzyme glutamic acid decarboxylase (GAD1, GAD4, GAD5) was also strongly suppressed except for GAD2 which increased. Almost all changes observed during flooding were reversible after draining. Possible changes in asparagine and GABA metabolism that may explain the marked fluctuations of these amino acids during flooding are discussed. It is suggested that the accumulation of GABA has a storage role during flooding stress.

ProClaT, a new bioinformatics tool for in silico protein reclassification: case study of DraB, a protein coded from the draTGB operon in Azospirillum brasilense.

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.

Exploring the Ancestral Mechanisms of Regulation of Horizontally Acquired Nitrogenases.

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.

Long-term non-invasive and continuous measurements of legume nodule activity.

Symbiotic nitrogen fixation is a process of considerable economic, ecological and scientific interest. The central enzyme nitrogenase reduces H(+) alongside N2 , and the evolving H2 allows a continuous and non-invasive in vivo measurement of nitrogenase activity. The objective of this study was to show that an elaborated set-up providing such measurements for periods as long as several weeks will produce specific insight into the nodule activity's dependence on environmental conditions and genotype features. A system was developed that allows the air-proof separation of a root/nodule and a shoot compartment. H2 evolution in the root/nodule compartment can be monitored continuously. Nutrient solution composition, temperature, CO2 concentration and humidity around the shoots can concomitantly be maintained and manipulated. Medicago truncatula plants showed vigorous growth in the system when relying on nitrogen fixation. The set-up was able to provide specific insights into nitrogen fixation. For example, nodule activity depended on the temperature in their surroundings, but not on temperature or light around shoots. Increased temperature around the nodules was able to induce higher nodule activity in darkness versus light around shoots for a period of as long as 8 h. Conditions that affected the N demand of the shoots (ammonium application, Mg or P depletion, super numeric nodules) induced consistent and complex daily rhythms in nodule activity. It was shown that long-term continuous measurements of nodule activity could be useful for revealing special features in mutants and could be of importance when synchronizing nodule harvests for complex analysis of their metabolic status.