RESUMO
BACKGROUND: The microbial population of the intestinal tract and its relationship to specific diseases has been extensively studied during the past decade. However, reports characterizing the bile microbiota are rare. This study aims to investigate the microbiota composition in patients with pancreaticobiliary cancers and benign diseases by 16S rRNA gene amplicon sequencing and to evaluate its potential value as a biomarker for the cancer of the bile duct, pancreas, and gallbladder. RESULTS: We enrolled patients who were diagnosed with cancer, cystic lesions, and inflammation of the pancreaticobiliary tract. The study cohort comprised 244 patients. We extracted microbiome-derived DNA from the bile juice in surgically resected gallbladders. The microbiome composition was not significantly different according to lesion position and cancer type in terms of alpha and beta diversity. We found a significant difference in the relative abundance of Campylobacter, Citrobacter, Leptotrichia, Enterobacter, Hungatella, Mycolicibacterium, Phyllobacterium and Sphingomonas between patients without and with lymph node metastasis. CONCLUSIONS: There was a significant association between the relative abundance of certain microbes and overall survival prognosis. These microbes showed association with good prognosis in cholangiocarcinoma, but with poor prognosis in pancreatic adenocarcinoma, and vice versa. Our findings suggest that pancreaticobiliary tract cancer patients have an altered microbiome composition, which might be a biomarker for distinguishing malignancy.
Assuntos
Adenocarcinoma , Neoplasias da Vesícula Biliar , Microbiota , Neoplasias Pancreáticas , Humanos , Microbiota/genética , Prognóstico , RNA Ribossômico 16S/genéticaRESUMO
A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions.
Assuntos
Aldose-Cetose Isomerases , Proteínas de Bactérias , Expressão Gênica , Geobacillus stearothermophilus/genética , Aldose-Cetose Isomerases/biossíntese , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Estabilidade Enzimática , Geobacillus stearothermophilus/enzimologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The nodulation of Bradyrhizobium japonicum Is-34 is restricted by Rj4 genotype soybeans (Glycine max). To identify the genes responsible for this incompatibility, Tn5 mutants of B. japonicum Is-34 that were able to overcome this nodulation restriction were obtained. Analysis of the Tn5 mutants revealed that Tn5 was inserted into a region containing the MA20_12780 gene. In addition, direct disruption of this gene using marker exchange overcame the nodulation restriction by Rj4 genotype soybeans. The MA20_12780 gene has a tts box motif in its upstream region, indicating a possibility that this gene encodes a type III secretion system (T3SS) effector protein. Bioinformatic characterization revealed that the MA20_12780 protein contains the small ubiquitin-like modifier (SUMO) protease domain of the C48 peptidase (ubiquitin-like protease 1 [Ulp1]) family. The results of the present study indicate that a putative T3SS effector encoded by the MA20_12780 gene causes the incompatibility with Rj4 genotype soybeans, and they suggest the possibility that the nodulation restriction of B. japonicum Is-34 may be due to Rj4 genotype soybeans recognizing the putative T3SS effector (MA20_12780 protein) as a virulence factor.
Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/metabolismo , Glycine max/microbiologia , Sistemas de Secreção Tipo III/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bradyrhizobium/química , Bradyrhizobium/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutagênese Insercional , Nodulação , Raízes de Plantas/microbiologia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Glycine max/genética , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/genéticaRESUMO
Microbiologically influenced corrosion (MIC) of metallic materials imposes a heavy economic burden. The mechanism of MIC of metallic iron (Fe(0)) under anaerobic conditions is usually explained as the consumption of cathodic hydrogen by hydrogenotrophic microorganisms that accelerates anodic Fe(0) oxidation. In this study, we describe Fe(0) corrosion induced by a nonhydrogenotrophic nitrate-reducing bacterium called MIC1-1, which was isolated from a crude-oil sample collected at an oil well in Akita, Japan. This strain requires specific electron donor-acceptor combinations and an organic carbon source to grow. For example, the strain grew anaerobically on nitrate as a sole electron acceptor with pyruvate as a carbon source and Fe(0) as the sole electron donor. In addition, ferrous ion and l-cysteine served as electron donors, whereas molecular hydrogen did not. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MIC1-1 was a member of the genus Prolixibacter in the order Bacteroidales. Thus, Prolixibacter sp. strain MIC1-1 is the first Fe(0)-corroding representative belonging to the phylum Bacteroidetes. Under anaerobic conditions, Prolixibacter sp. MIC1-1 corroded Fe(0) concomitantly with nitrate reduction, and the amount of iron dissolved by the strain was six times higher than that in an aseptic control. Scanning electron microscopy analyses revealed that microscopic crystals of FePO4 developed on the surface of the Fe(0) foils, and a layer of FeCO3 covered the FePO4 crystals. We propose that cells of Prolixibacter sp. MIC1-1 accept electrons directly from Fe(0) to reduce nitrate.
Assuntos
Bacteroidetes/classificação , Bacteroidetes/metabolismo , Corrosão , Ferro/metabolismo , Nitratos/metabolismo , Anaerobiose , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Carbono/metabolismo , Análise por Conglomerados , Cisteína/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Microbiologia Ambiental , Japão , Dados de Sequência Molecular , Compostos Orgânicos/metabolismo , Oxirredução , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
In a previous study by our group, CH4 oxidation and N2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.
Assuntos
Bactérias/isolamento & purificação , Bactérias/metabolismo , Metano/metabolismo , Oryza/microbiologia , Proteômica , Processos Autotróficos , Bactérias/classificação , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Fixação de Nitrogênio , Nitrogenase/genética , Nitrogenase/metabolismo , Oryza/crescimento & desenvolvimento , Filogenia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Microbiologia do SoloRESUMO
To investigate functional plant growth-promoting rhizobacteria in sugar beet, seasonal shifts in bacterial community structures in the lateral roots of sugar beet were examined using amplicon sequencing ana-lyses of the 16S rRNA gene. Shannon and Simpson indexes significantly increased between June and July, but did not significantly differ between July and subsequent months (August and September). A weighted UniFrac principal coordinate ana-lysis grouped bacterial samples into four clusters along with PC1 (43.8%), corresponding to the four sampling months in the order of sampling dates. Taxonomic ana-lyses revealed that bacterial diversity in the lateral roots was exclusively dominated by three phyla (Actinobacteria, Bacteroidetes, and Proteobacteria) in all samples examined. At the lower taxonomic levels, the dominant taxa were roughly classified into three groups. Therefore, the relative abundances of seven dominant genera (Janthinobacterium, Kribbella, Pedobacter, Rhodanobacter, Sphingobium, Sphingopyxis, and Streptomyces) were the highest in June and gradually decreased as sugar beet grew. The relative abundances of eight taxa (Bradyrhizobiaceae, Caulobacteraceae, Chitinophagaceae, Novosphingobium, Phyllobacteriaceae, Pseudomonas, Rhizobiaceae, and Sphingomonas) were mainly high in July and/or August. The relative abundances of six taxa (unclassified Comamonadaceae, Cytophagaceae, unclassified Gammaproteobacteria, Haliangiaceae, unclassified Myxococcales, and Sinobacteraceae) were the highest in September. Among the dominant taxa, 12 genera (Amycolatopsis, Bradyrhizobium, Caulobacter, Devosia, Flavobacterium, Janthinobacterium, Kribbella, Kutzneria, Pedobacter, Rhizobium, Rhodanobacter, and Steroidobacter) were considered to be candidate groups of plant growth-promoting bacteria based on their previously reported beneficial traits as biopesticides and/or biofertilizers.
Assuntos
Beta vulgaris , Beta vulgaris/microbiologia , RNA Ribossômico 16S/genética , Japão , Estações do Ano , Bactérias/genética , AçúcaresRESUMO
The effects of different types of additional fertilizations (a compound fertilizer and Chiyoda-kasei) on the root-associated microbes of napa cabbage grown in an Andosol field were investigated by molecular community ana-lyses. Most of the closest known species of the bacterial sequences whose relative abundance significantly differed among fertilizers were sensitive to nitrogen fertilization and/or related to the geochemical cycles of nitrogen. The fungal community on the roots of napa cabbage was dominated by two genera, Bipolaris and Olpidium. The relative abundance of these two genera was affected by the types of fertilizers to some extent and showed a strong negative correlation.
Assuntos
Brassica , Fertilizantes , Fertilizantes/análise , Fertilizantes/microbiologia , Japão , Nitrogênio/análise , Solo/químicaRESUMO
Clone libraries of bacterial 16S rRNA genes (a total of 1,980 clones) were constructed from the leaf blades, petioles, taproots, and lateral roots of sugar beet (Beta vulgaris L.) grown under different fertilization conditions. A principal coordinate analysis revealed that the structures of bacterial communities in above- and underground tissues were largely separated by PC1 (44.5%). The bacterial communities of above-ground tissues (leaf blades and petioles) were more tightly clustered regardless of differences in the tissue types and fertilization conditions than those of below-ground tissues (taproots and lateral roots). The bacterial communities of below-ground tissues were largely separated by PC2 (26.0%). To survey plant growth-promoting bacteria (PGPBs), isolate collections (a total of 665 isolates) were constructed from the lateral roots. As candidate PGPBs, 44 isolates were selected via clustering analyses with the combined 16S rRNA gene sequence data of clone libraries and isolate collections. The results of inoculation tests using sugar beet seedlings showed that eight isolates exhibited growth-promoting effects on the seedlings. Among them, seven isolates belonging to seven genera (Asticcacaulis, Mesorhizobium, Nocardioides, Sphingobium, Sphingomonas, Sphingopyxis, and Polaromonas) were newly identified as PGPBs for sugar beet at the genus level, and two isolates belonging to two genera (Asticcacaulis and Polaromonas) were revealed to exert growth-promoting effects on the plant at the genus level for the first time. These results suggest that a community analysis-based selection strategy will facilitate the isolation of novel PGPBs and extend the potential for the development of novel biofertilizers.
Assuntos
Bactérias/isolamento & purificação , Beta vulgaris/crescimento & desenvolvimento , Microbiota , Bactérias/classificação , Bactérias/genética , Beta vulgaris/microbiologia , DNA Bacteriano/genética , Folhas de Planta/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Plântula/crescimento & desenvolvimento , Plântula/microbiologia , Microbiologia do SoloRESUMO
Microbiologically influenced corrosion of steel in anaerobic environments has been attributed to hydrogenotrophic microorganisms. A sludge sample collected from the bottom plate of a crude-oil storage tank was used to inoculate a medium containing iron (Fe(0)) granules, which was then incubated anaerobically at 37 degrees C under an N(2)-CO(2) atmosphere to enrich for microorganisms capable of using iron as the sole source of electrons. A methanogen, designated strain KA1, was isolated from the enrichment culture. An analysis of its 16S rRNA gene sequence revealed that strain KA1 is a Methanococcus maripaludis strain. Strain KA1 produced methane and oxidized iron much faster than did the type strain of M. maripaludis, strain JJ(T), which produced methane at a rate expected from the abiotic H(2) production rate from iron. Scanning electron micrographs of iron coupons that had been immersed in either a KA1 culture, a JJ(T) culture, or an aseptic medium showed that only coupons from the KA1 culture had corroded substantially, and these were covered with crystalline deposits that consisted mainly of FeCO(3).
Assuntos
Ferro/metabolismo , Metano/metabolismo , Mathanococcus/classificação , Mathanococcus/metabolismo , Petróleo/microbiologia , Análise por Conglomerados , Corrosão , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Mathanococcus/genética , Mathanococcus/isolamento & purificação , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Batch methane fermentation was conducted using seed sludge collected from six methane fermentation facilities. Swine feces were centrifuged and autoclaved, followed by its use as a substrate for methanogenesis. This "swine feces supernatant medium" facilitates the cultivation of the microbes of the seed sludge, sampling of the digested liquid using a syringe, and subculturing of the digested liquid in a subsequent medium using a syringe. Through 15 subcultures, digested liquids with high and low methane production potential were obtained, which were named "H-DS" and "L-DS," respectively. On the day 10 of cultivation, chemical oxygen demand (COD) of H-DS significantly decreased by 31% and that of L-DS did not differ significantly compared with that on the day 0 of cultivation. Acetic acid concentration of H-DS (1009 mg/L) was significantly lower than that of L-DS (2686 mg/L). These chemical characteristics indicate that organics decomposition in L-DS was not successful and suggest that H-DS has high relative abundance of bacteria decomposing organic matter and methanogen utilizing acetic acid compared with those in L-DS. Microbial community analysis revealed that Shannon index of H-DS was significantly higher than that of L-DS, and the relative abundance of acetogenic bacteria (e.g., Syntrophomonas) and acetic acid-utilizing methanogen (Methanosarcina) in H-DS was significantly higher than that in L-DS. Thus, the high methane production potential of H-DS might be attributable to the smooth flow from acetogenesis to methanogenesis step in the methane fermentation, compared with the case of L-DS.
Assuntos
Fermentação , Esterco , Metano/química , Microbiota , Esgotos , Ácido Acético/química , Amônia/química , Animais , Análise da Demanda Biológica de Oxigênio , Reatores Biológicos , Biotecnologia , Ácidos Graxos Voláteis/análise , Concentração de Íons de Hidrogênio , Methanosarcina/metabolismo , Nitrogênio/química , RNA Ribossômico 16S/genética , Suínos , Eliminação de Resíduos Líquidos/métodosRESUMO
Bradyrhizobium elkanii BLY3-8 does not form nodules on the roots of Rj3-genotype soybean (cultivar D-51). This is a cultivar-specific nodulation restriction. The genes A6X20_40975 and A6X20_41030 in strain BLY3-8 were predicted to encode the transcriptional activator and apparatus of the type III secretion system (T3SS) (the proteins TtsI and RhcJ), respectively. Mutants disrupted in these genes overcame the nodulation restriction. These results suggest that an effector injected via T3SS into Rj3-genotype soybean is involved in nodulation restriction by Rj3-genotype soybean.
Assuntos
Bradyrhizobium/genética , Glycine max/microbiologia , Nodulação/genética , Sistemas de Secreção Tipo III/genética , Regulação Bacteriana da Expressão Gênica , Genótipo , Mutação , Glycine max/genética , SimbioseRESUMO
Halophilic enzymes contain a large number of acidic amino acids and marginal large hydrophobic amino acids, which make them highly soluble even under strongly hydrophobic conditions. This characteristic of halophilic enzymes provides potential for their industrial application. However, halophilic enzymes easily degrade when used for industrial applications compared with enzymes from other extremophiles because of their instability in low-salt environments. We aimed to clarify the stabilization mechanism of halophilic enzymes. We previously attempted to express halophilic alkaline phosphatase from Halomonas (HaALP) in non-halophilic E. coli. However, the expressed HaALP showed little activity. Therefore, we overexpressed HaALP in Gram-positive non-halophilic Brevibacillus choshinensis in this study, which was successfully expressed and purified in its active form. HaALP was denatured in 6 M urea, refolded using various salts and the non-ionic osmolyte trimethylamine N-oxide (TMAO), and assessed by native polyacrylamide gel electrophoresis. HaALP refolded in 3 M NaCl or 3 M TMAO containing Na+ ions. Hydrophobic interactions due to a high salt concentration or TMAO enhanced the formation of the folding intermediate (the monomer precursor), and only Na+ ions activated the dimer form. This insight into the stabilization mechanism of HaALP may lead to the development of industrial applications of halophilic enzymes under low-salt conditions.
Assuntos
Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/química , Brevibacillus/genética , Halomonas/metabolismo , Clonagem Molecular , Metilaminas/química , Dobramento de Proteína , Cloreto de Sódio/químicaRESUMO
L-Arabinose isomerase isolated from Geobacillus stearothermophilus (GSAI) was modified to improve its substrate specificity for D-galactose for the production of D-tagatose, a potential reduced-energy sweetener. Among the selected residues, mutation at residue 18 produced a mutant strain, H18T, which exhibited increased activity for D-galactose compared with the wild-type (WT) enzyme. Analysis of the substrate specificity of H18T showed a 45.4% improvement for D-galactose. Replacing histidine with threonine at residue 18 resulted in approximately 2.7-fold and 1.8-fold higher substrate binding and catalytic efficiency, respectively, for D-galactose. Further enhancement of the specific activity and catalytic efficiency of H18T for D-galactose by up to 2.7-fold and 4.3-fold, respectively, was achieved by adding borate during L-arabinose isomerase catalysis. Moreover, H18T showed thermostability and no destabilization was detected, which is promising for the industrial production of D-tagatose.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Galactose/metabolismo , Geobacillus stearothermophilus/enzimologia , Catálise , Clonagem Molecular , Escherichia coli/genética , Hexoses/metabolismo , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Estrutura Molecular , Conformação Proteica , Especificidade por Substrato , Temperatura , Treonina/metabolismoRESUMO
Genotype-specific incompatibility in legume-rhizobium symbiosis has been suggested to be controlled by effector-triggered immunity underlying pathogenic host-bacteria interactions. However, the rhizobial determinant interacting with the host resistance protein (e.g., Rj2) and the molecular mechanism of symbiotic incompatibility remain unclear. Using natural mutants of Bradyrhizobium diazoefficiens USDA 122, we identified a type III-secretory protein NopP as the determinant of symbiotic incompatibility with Rj2-soybean. The analysis of nopP mutations and variants in a culture collection reveal that three amino acid residues (R60, R67, and H173) in NopP are required for Rj2-mediated incompatibility. Complementation of rj2-soybean by the Rj2 allele confers the incompatibility induced by USDA 122-type NopP. In response to incompatible strains, Rj2-soybean plants activate defense marker gene PR-2 and suppress infection thread number at 2 days after inoculation. These results suggest that Rj2-soybeans monitor the specific variants of NopP and reject bradyrhizobial infection via effector-triggered immunity mediated by Rj2 protein.
Assuntos
Bradyrhizobium/fisiologia , Glycine max/microbiologia , Imunidade Vegetal , Simbiose/genética , Sistemas de Secreção Tipo III/fisiologia , Alelos , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Bradyrhizobium/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Genótipo , Mutação , Fenótipo , Fosforilação , Filogenia , Proteínas de Plantas/genética , Nodulação , Raízes de Plantas/microbiologia , Sistemas de Secreção Tipo III/genéticaRESUMO
Certain methanogens deteriorate steel surfaces through a process called microbiologically influenced corrosion (MIC). However, the mechanisms of MIC, whereby methanogens oxidize zerovalent iron (Fe0), are largely unknown. In this study, Fe0-corroding Methanococcus maripaludis strain OS7 and its derivative (strain OS7mut1) defective in Fe0-corroding activity were isolated. Genomic analysis of these strains demonstrated that the strain OS7mut1 contained a 12-kb chromosomal deletion. The deleted region, termed "MIC island", encoded the genes for the large and small subunits of a [NiFe] hydrogenase, the TatA/TatC genes necessary for the secretion of the [NiFe] hydrogenase, and a gene for the hydrogenase maturation protease. Thus, the [NiFe] hydrogenase may be secreted outside the cytoplasmic membrane, where the [NiFe] hydrogenase can make direct contact with Fe0, and oxidize it, generating hydrogen gas: Fe0 + 2 H+ â Fe2+ + H2. Comparative analysis of extracellular and intracellular proteomes of strain OS7 supported this hypothesis. The identification of the MIC genes enables the development of molecular tools to monitor epidemiology, and to perform surveillance and risk assessment of MIC-inducing M. maripaludis.
Assuntos
Genoma Bacteriano , Ilhas Genômicas , Hidrogenase/genética , Hidrogenase/metabolismo , Ferro/metabolismo , Mathanococcus/genética , Mathanococcus/metabolismo , Antibacterianos/farmacologia , Sequência de Bases , Corrosão , Ordem dos Genes , Instabilidade Genômica , Mathanococcus/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Teóricos , OxirreduçãoRESUMO
We report here the draft genome sequences of Bradyrhizobium elkanii strains BLY3-8 and BLY6-1, which are incompatible with Rj3 genotype soybean cultivars. The genome sequences of these strains will be useful to identify a causal gene for this incompatibility.
RESUMO
Methylobacterium inhabits the phyllosphere of a large number of plants. We herein report the results of comparative metagenome analyses on methylobacterial communities of soybean plants grown in an experimental field in Tohoku University (Kashimadai, Miyagi, Japan). Methylobacterium was identified as the most dominant genus (33%) among bacteria inhabiting soybean stems. We classified plant-derived Methylobacterium species into Groups I, II, and III based on 16S rRNA gene sequences, and found that Group I members (phylogenetically close to M. extorquens) were dominant in soybean-associated Methylobacterium. By comparing 29 genomes, we found that all Group I members possessed a complete set of genes for the N-methylglutamate pathway for methylamine utilization, and genes for urea degradation (urea carboxylase, urea amidolyase, and conventional urease). Only Group I members and soybean methylobacterial isolates grew in a culture supplemented with methylamine as the sole carbon source. They utilized urea or allantoin (a urea-related compound in legumes) as the sole nitrogen source; however, group III also utilized these compounds. The utilization of allantoin may be crucial in soybean-bacterial interactions because allantoin is a transported form of fixed nitrogen in legume plants. Soybean-derived Group I strain AMS5 colonized the model legume Lotus japonicus well. A comparison among the 29 genomes of plant-derived and other strains suggested that several candidate genes are involved in plant colonization such as csgG (curli fimbriae). Genes for the N-methylglutamate pathway and curli fimbriae were more abundant in soybean microbiomes than in rice microbiomes in the field. Based on these results, we discuss the lifestyle of Methylobacterium in the legume phyllosphere.
Assuntos
Glycine max/microbiologia , Metagenoma , Metagenômica , Metilaminas/metabolismo , Methylobacterium/genética , Methylobacterium/metabolismo , Ureia/metabolismo , Alantoína/metabolismo , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genótipo , Japão , Methylobacterium/classificação , Filogenia , Caules de Planta/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
It has been hypothesized that nitrogen fixation occurs in the human gut. However, whether the gut microbiota truly has this potential remains unclear. We investigated the nitrogen-fixing activity and diversity of the nitrogenase reductase (NifH) genes in the faecal microbiota of humans, focusing on Papua New Guinean and Japanese individuals with low to high habitual nitrogen intake. A (15)N2 incorporation assay showed significant enrichment of (15)N in all faecal samples, irrespective of the host nitrogen intake, which was also supported by an acetylene reduction assay. The fixed nitrogen corresponded to 0.01% of the standard nitrogen requirement for humans, although our data implied that the contribution in the gut in vivo might be higher than this value. The nifH genes recovered in cloning and metagenomic analyses were classified in two clusters: one comprising sequences almost identical to Klebsiella sequences and the other related to sequences of Clostridiales members. These results are consistent with an analysis of databases of faecal metagenomes from other human populations. Collectively, the human gut microbiota has a potential for nitrogen fixation, which may be attributable to Klebsiella and Clostridiales strains, although no evidence was found that the nitrogen-fixing activity substantially contributes to the host nitrogen balance.
Assuntos
Proteínas de Bactérias/metabolismo , Microbioma Gastrointestinal , Nitrogênio/metabolismo , Oxirredutases/metabolismo , Acetileno/química , Acetileno/metabolismo , Adulto , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Clostridiales/enzimologia , Clostridiales/genética , Clostridiales/isolamento & purificação , Bases de Dados Factuais , Fezes/microbiologia , Feminino , Humanos , Klebsiella/enzimologia , Klebsiella/genética , Klebsiella/isolamento & purificação , Masculino , Metagenômica , Nitrogênio/química , Fixação de Nitrogênio , Isótopos de Nitrogênio/metabolismo , Oxirredutases/classificação , Oxirredutases/genética , Filogenia , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , Análise de Sequência de DNA , Adulto JovemRESUMO
We report the draft genome sequence of Bradyrhizobium japonicum Is-1, which is incompatible with Rj2 genotype soybeans. The estimated genome size of this strain is 8.9 Mb. Genome sequence information of this strain will help to identify a causal gene for this incompatibility.
RESUMO
We analyzed a metagenome of the bacterial community associated with the taproot of sugar beet (Beta vulgaris L.) in order to investigate the genes involved in plant growth-promoting traits (PGPTs), namely 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, indole acetic acid (IAA), N2 fixation, phosphate solubilization, pyrroloquinoline quinone, siderophores, and plant disease suppression as well as methanol, sucrose, and betaine utilization. The most frequently detected gene among the PGPT categories encoded ß-1,3-glucanase (18 per 10(5) reads), which plays a role in the suppression of plant diseases. Genes involved in phosphate solubilization (e.g., for quinoprotein glucose dehydrogenase), methanol utilization (e.g., for methanol dehydrogenase), siderophore production (e.g. isochorismate pyruvate lyase), and ACC deaminase were also abundant. These results suggested that such PGPTs are crucially involved in supporting the growth of sugar beet. In contrast, genes for IAA production (iaaM and ipdC) were less abundant (~1 per 10(5) reads). N2 fixation genes (nifHDK) were not detected; bacterial N2 -fixing activity was not observed in the (15)N2 -feeding experiment. An analysis of nitrogen metabolism suggested that the sugar beet microbiome mainly utilized ammonium and nitroalkane as nitrogen sources. Thus, N2 fixation and IAA production did not appear to contribute to sugar beet growth. Taxonomic assignment of this metagenome revealed the high abundance of Mesorhizobium, Bradyrhizobium, and Streptomyces, suggesting that these genera have ecologically important roles in the taproot of sugar beet. Bradyrhizobium-assigned reads in particular were found in almost all categories of dominant PGPTs with high abundance. The present study revealed the characteristic functional genes in the taproot-associated microbiome of sugar beet, and suggest the opportunity to select sugar beet growth-promoting bacteria.