RESUMO
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.
Assuntos
Nitrogenase , Paenibacillus , Fixação de Nitrogênio/genética , Nitrogenase/genética , Nitrogenase/metabolismo , Paenibacillus/genética , Paenibacillus/metabolismo , FilogeniaRESUMO
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.
Assuntos
Bradyrhizobium , Rhizobium , Alanina/metabolismo , Bradyrhizobium/metabolismo , Glicina/análogos & derivados , Mutação , Fixação de Nitrogênio , Nitrogenase/genética , Rhizobium/genética , Rhizobium/metabolismo , Serina/metabolismo , Simbiose , GlifosatoRESUMO
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.
Assuntos
Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cloroplastos/metabolismo , Engenharia Genética/métodos , Nitrogenase/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Genoma de Cloroplastos , Fixação de Nitrogênio , Nitrogenase/metabolismo , Fotossíntese , Biologia SintéticaRESUMO
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.
Assuntos
Azospirillum brasilense/química , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/química , Biologia Computacional/métodos , Nitrogenase/química , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional/instrumentação , Genes Bacterianos , Fixação de Nitrogênio , Nitrogenase/genética , Nitrogenase/metabolismo , ÓperonRESUMO
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.
Assuntos
Nitrogenase/genética , Pseudomonas/enzimologia , Evolução Molecular , Transferência Genética Horizontal , Genes Bacterianos , Fixação de Nitrogênio/genética , Nitrogenase/biossíntese , Filogenia , Pseudomonas/genética , Pseudomonas stutzeri/enzimologia , Pseudomonas stutzeri/genéticaRESUMO
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.
Assuntos
Oxirredutases/metabolismo , Rhodobacter/metabolismo , Nitrogenase/metabolismo , Oxirredutases/genética , Temperatura , Reação em Cadeia da Polimerase , Rhodobacter/isolamento & purificação , Reatores Biológicos , Fermentação , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Nitrogenase/genéticaRESUMO
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.
Assuntos
Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mutação , Proteínas PII Reguladoras de Nitrogênio/genética , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Análise Mutacional de DNA , Expressão Gênica , Nitrogenase/genética , Proteínas PII Reguladoras de Nitrogênio/química , Ligação Proteica , Conformação ProteicaRESUMO
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.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Eucariotos/enzimologia , Nitrogenase/metabolismo , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Animais , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Eucariotos/química , Eucariotos/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Nitrogenase/química , Nitrogenase/genética , Processamento de Proteína Pós-TraducionalRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Gluconacetobacter/genética , Proteínas de Membrana/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Sideróforos/genética , Transporte Biológico/genética , Meios de Cultura/química , Teste de Complementação Genética , Gluconacetobacter/enzimologia , Gluconacetobacter/metabolismo , Ferro/metabolismo , Mutagênese Insercional , Mutação , Nitrogenase/genética , Fenótipo , Sideróforos/análise , Sideróforos/metabolismoRESUMO
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.