RESUMO
A stimuli-responsible reversible structural transformation is of key importance in biological systems. We now report a unique water-mediated reversible transformation among three discrete double-stranded dinuclear titanium(IV) achiral meso- and chiral rac-helicates linked by a mono(µ-oxo) or a bis(µ-hydroxo) bridge between the titanium ions through hydration/dehydration or its combination with a water-mediated dynamic cleavage/re-formation of the titanium-phenoxide (Ti-OPh) bonds. The bis(µ-hydroxo) bridged titanium(IV) meso-helicate prepared from two tetraphenol strands with titanium(IV) oxide was readily dehydrated in CD3CN containing a small amount of water upon heating, accompanied by Ti-OPh bond cleavage/re-formation catalyzed by water, resulting in the formation of the mono(µ-oxo)-bridged rac-helicate, which reverted back to the original bis(µ-hydroxo)-bridged meso-helicate upon hydration in aqueous CD3CN. These reversible transformations between the meso- and rac-helicates were also promoted in the presence of a catalytic amount of an acid, which remarkably accelerated the reactions at lower temperature. Interestingly, in anhydrous CD3CN, the bis(µ-hydroxo)-bridged meso-helicate was further slowly converted to a different helicate, while its meso-helicate framework was maintained, namely the mono(µ-oxo)-bridged meso-helicate, through dehydration upon heating and its meso to meso transformation was significantly accelerated in the presence of cryptand[2.2.1], which contributes to removing Na+ ions coordinated to the helicate. Upon cooling, the backward meso to meso transformation took place via hydration. Hence, three different, discrete double-stranded chiral rac- and achiral meso-titanium(IV) helicates linked by a mono(µ-oxo) or a bis(µ-hydroxo) bridge were successfully generated in a controllable manner by a change in the water content of the reaction media.
RESUMO
A one-handed double-stranded spiroborate helicate exhibits a unique reversible extension-contraction motion coupled with a twisting motion in one direction triggered by binding and release of a Na+ ion while retaining its handedness. Here we report that an extended meso-helicate was also produced together with the racemo-helicate, and the meso-helicate was readily converted to the racemo-helicate assisted by a Na+ ion as a template in the presence of water. The thermodynamic analyses of the ion-triggered springlike motion of the racemo-helicate using a series of monovalent cations with different sizes revealed that the association constants of the extended racemo-helicate decreased in the following order: Li+ > Na+ > NH4+ > Ag+ ≥ K+ > Cs+ > Rb+, which roughly agrees with the reverse order of their ionic radii except for the NH4+ ion due to the more elongated contracted helicates when complexed with larger cations as supported by the crystal and DFT calculated structures. The one-handed contracted helicates showed characteristic CD spectra depending on the cation species due to the differences in their contracted helical structures, and its absolute handedness of the spiroborate helicate was determined by X-ray crystallography. The kinetic studies of the springlike motions of the racemo-helicate showed that the exchange rate between the extended and contracted helicates tend to increase in the following order: Li+ < Na+ < K+ ≤ NH4+ < Rb+ < Cs+ < Ag+ as anticipated from the association constants, being in good agreement with the order of the cation sizes except for Ag+.
RESUMO
Extra-slow-growing bradyrhizobia from root nodules of field-grown soybeans harbor abundant insertion sequences (ISs) and are termed highly reiterated sequence-possessing (HRS) strains. We analyzed the genome organization of HRS strains with the focus on IS distribution and symbiosis island structure. Using pulsed-field gel electrophoresis, we consistently detected several plasmids (0.07 to 0.4 Mb) in the HRS strains (NK5, NK6, USDA135, 2281, USDA123, and T2), whereas no plasmids were detected in the non-HRS strain USDA110. The chromosomes of the six HRS strains (9.7 to 10.7 Mb) were larger than that of USDA110 (9.1 Mb). Using MiSeq sequences of 6 HRS and 17 non-HRS strains mapped to the USDA110 genome, we found that the copy numbers of ISRj1, ISRj2, ISFK1, IS1632, ISB27, ISBj8, and IS1631 were markedly higher in HRS strains. Whole-genome sequencing showed that the HRS strain NK6 had four small plasmids (136 to 212 kb) and a large chromosome (9,780 kb). Strong colinearity was found between 7.4-Mb core regions of the NK6 and USDA110 chromosomes. USDA110 symbiosis islands corresponded mainly to five small regions (S1 to S5) within two variable regions, V1 (0.8 Mb) and V2 (1.6 Mb), of the NK6 chromosome. The USDA110 nif gene cluster (nifDKENXSBZHQW-fixBCX) was split into two regions, S2 and S3, where ISRj1-mediated rearrangement occurred between nifS and nifB. ISs were also scattered in NK6 core regions, and ISRj1 insertion often disrupted some genes important for survival and environmental responses. These results suggest that HRS strains of soybean bradyrhizobia were subjected to IS-mediated symbiosis island shuffling and core genome degradation.
Assuntos
Bradyrhizobium/crescimento & desenvolvimento , Bradyrhizobium/genética , Elementos de DNA Transponíveis , Genoma Bacteriano , Ilhas Genômicas , Glycine max/microbiologia , Nódulos Radiculares de Plantas/microbiologia , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Filogenia , Plasmídeos , Análise de Sequência de DNARESUMO
The soybean endosymbiont Bradyrhizobium japonicum is able to scavenge the greenhouse gas N2O through the N2O reductase (Nos). In previous research, N2O emission from soybean rhizosphere was mitigated by B. japonicumâ Nos(++) strains (mutants with increased Nos activity). Here, we report the mechanism underlying the Nos(++) phenotype. Comparative analysis of Nos(++) mutant genomes showed that mutation of bll4572 resulted in Nos(++) phenotype. bll4572 encodes NasS, the nitrate (NO3(-))-sensor of the two-component NasST regulatory system. Transcriptional analyses of nosZ (encoding Nos) and other genes from the denitrification process in nasS and nasST mutants showed that, in the absence of NO3(-) , nasS mutation induces nosZ and nap (periplasmic nitrate reductase) via nasT. NO3(-) addition dissociated the NasS-NasT complex in vitro, suggesting the release of the activator NasT. Disruption of nasT led to a marked decrease in nosZ and nap transcription in cells incubated in the presence of NO3(-). Thus, although NasST is known to regulate the NO3(-)-mediated response of NO3(-) assimilation genes in bacteria, our results show that NasST regulates the NO3(-) -mediated response of nosZ and napE genes, from the dissimilatory denitrification pathway, in B. japonicum.
Assuntos
Proteínas de Bactérias/genética , Bradyrhizobium/enzimologia , Bradyrhizobium/genética , Regulação Bacteriana da Expressão Gênica , Nitrato Redutase/genética , Nitratos/metabolismo , Oxirredutases/genética , Bradyrhizobium/metabolismo , Desnitrificação , Regulação Enzimológica da Expressão Gênica , Genoma Bacteriano , Proteínas de Membrana Transportadoras/genética , Mutação , Oxirredutases/metabolismo , Periplasma/enzimologia , Proteínas de Ligação a RNA/genética , Rizosfera , Glycine max/microbiologiaRESUMO
To understand the mechanisms underlying the increased N2O reductase activity in the Bradyrhizobium japonicum 5M09 mutant from enrichment culture under N2O respiration, we analyzed the expression of genes encoding denitrification reductases and regulators. Our results suggest a common regulation of nap (encoding periplasmic nitrate reductase) and nos (encoding N2O reductase).
Assuntos
Bradyrhizobium/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Redes Reguladoras de Genes/fisiologia , Nitrato Redutase/metabolismo , Oxirredutases/metabolismo , Bradyrhizobium/enzimologia , Bradyrhizobium/crescimento & desenvolvimento , Redes Reguladoras de Genes/genética , Reação em Cadeia da Polimerase em Tempo Real , beta-GalactosidaseRESUMO
Agromonas oligotrophica (Bradyrhizobium oligotrophicum) S58(T) is a nitrogen-fixing oligotrophic bacterium isolated from paddy field soil that is able to grow in extra-low-nutrient environments. Here, the complete genome sequence of S58 was determined. The S58 genome was found to comprise a circular chromosome of 8,264,165 bp with an average GC content of 65.1% lacking nodABC genes and the typical symbiosis island. The genome showed a high level of similarity to the genomes of Bradyrhizobium sp. ORS278 and Bradyrhizobium sp. BTAi1, including nitrogen fixation and photosynthesis gene clusters, which nodulate an aquatic legume plant, Aeschynomene indica, in a Nod factor-independent manner. Although nonsymbiotic (brady)rhizobia are significant components of rhizobial populations in soil, we found that most genes important for nodule development (ndv) and symbiotic nitrogen fixation (nif and fix) with A. indica were well conserved between the ORS278 and S58 genomes. Therefore, we performed inoculation experiments with five A. oligotrophica strains (S58, S42, S55, S72, and S80). Surprisingly, all five strains of A. oligotrophica formed effective nitrogen-fixing nodules on the roots and/or stems of A. indica, with differentiated bacteroids. Nonsymbiotic (brady)rhizobia are known to be significant components of rhizobial populations without a symbiosis island or symbiotic plasmids in soil, but the present results indicate that soil-dwelling A. oligotrophica generally possesses the ability to establish symbiosis with A. indica. Phylogenetic analyses suggest that Nod factor-independent symbiosis with A. indica is a common trait of nodABC- and symbiosis island-lacking strains within the members of the photosynthetic Bradyrhizobium clade, including A. oligotrophica.
Assuntos
Bradyrhizobium/genética , Bradyrhizobium/metabolismo , Fabaceae/microbiologia , Fixação de Nitrogênio/genética , Simbiose , Composição de Bases , Sequência de Bases , Bradyrhizobium/classificação , DNA Bacteriano/genética , Genoma Bacteriano/genética , Fotossíntese/genética , Filogenia , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Microbiologia do SoloRESUMO
The rhcJ and ttsI mutants of Bradyrhizobium japonicum USDA122 for the type III protein secretion system (T3SS) failed to secrete typical effector proteins and gained the ability to nodulate Rj2 soybean plants (Hardee), which are symbiotically incompatible with wild-type USDA122. This suggests that effectors secreted via the T3SS trigger incompatibility between these two partners.
Assuntos
Sistemas de Secreção Bacterianos/genética , Bradyrhizobium/fisiologia , Glycine max/microbiologia , Glycine max/fisiologia , Nodulação , Simbiose , Bradyrhizobium/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Técnicas de Inativação de Genes , Genes Bacterianos , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
N2O is an important greenhouse gas influencing global warming, and agricultural land is the predominant (anthropogenic) source of N2O emissions. Here, we report the high N2O-reducing activity of Bradyrhizobium ottawaense, suggesting the potential for efficiently mitigating N2O emission from agricultural lands. Among the 15 B. ottawaense isolates examined, the N2O-reducing activities of most (13) strains were approximately five-fold higher than that of Bradyrhizobium diazoefficiens USDA110T under anaerobic conditions. This robust N2O-reducing activity of B. ottawaense was confirmed by N2O reductase (NosZ) protein levels and by mitigation of N2O emitted by nodule decomposition in laboratory system. While the NosZ of B. ottawaense and B. diazoefficiens showed high homology, nosZ gene expression in B. ottawaense was over 150-fold higher than that in B. diazoefficiens USDA110T, suggesting the high N2O-reducing activity of B. ottawaense is achieved by high nos expression. Furthermore, we examined the nos operon transcription start sites and found that, unlike B. diazoefficiens, B. ottawaense has two transcription start sites under N2O-respiring conditions, which may contribute to the high nosZ expression. Our study indicates the potential of B. ottawaense for effective N2O reduction and unique regulation of nos gene expression towards the high performance of N2O mitigation in the soil.
Assuntos
Bradyrhizobium , Óxido Nitroso , Óxido Nitroso/análise , Oxirredutases/genética , Oxirredutases/metabolismo , Bradyrhizobium/genética , Bradyrhizobium/metabolismo , Solo , Expressão Gênica , Microbiologia do Solo , DesnitrificaçãoRESUMO
The genome-wide expression profiles of Bradyrhizobium japonicum in response to soybean (Glycine max (L.) Merr.) seed extract (SSE) and genistein were monitored with time at a low temperature (15 degrees C). A comparison with the expression profiles of the B. japonicum genome previously captured at the common growth temperature (30 degrees C) revealed that the expression of SSE preferentially induced genomic loci, including a large gene cluster encoding the type III secretion system (T3SS), were considerably delayed at 15 degrees C, whereas most nodulation (nod) gene loci, including nodD1 and nodW, were rapidly and strongly induced by both SSE and genistein. Induction of the T3SS genes was progressively activated upon the elevation of temperature to 30 degrees C and positively responded to culture population density. In addition, genes nolA and nodD2 were dramatically induced by SSE, concomitantly with the expression of T3SS genes. However, the deletion mutation of nodD2 but not nolA led to elimination of the T3SS genes expression. These results indicate that the expression of the T3SS gene cluster is tightly regulated with integration of environmental cues such as temperature and that NodD2 may be involved in its efficient induction in B. japonicum.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bradyrhizobium/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Temperatura , Bradyrhizobium/crescimento & desenvolvimento , Células Clonais , Perfilação da Expressão Gênica , Loci Gênicos/genética , Família Multigênica , Nodulação/genética , Deleção de Sequência , Simbiose/genéticaRESUMO
Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium for Glycine max, has complex respiratory electron transport chains. Bll4880 contained a copper-binding motif for metallochaperone, H(M)X(10)MX(21)HXM. A mutant strain, Bj4880, induced nodules with lower acetylene reduction activity. A double mutant, Bj4880-1131, which had inserted mutations both in blr1131, a gene of the Sco1-like protein, and in bll4880, induced nodules of significant Fix(-) phenotype and low cytochrome c oxidase (Cco) activity in the bacteroid. Our data suggest that bll4880 protein is involved in copper ion delivery to Cco through blr1131 protein, and the expression of both proteins was induced under microaerobic conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/genética , Cobre/metabolismo , Metalochaperonas/metabolismo , Fixação de Nitrogênio , Nódulos Radiculares de Plantas/microbiologia , Proteínas de Bactérias/genética , Bradyrhizobium/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metalochaperonas/genética , Mutagênese Insercional , Mutação , Glycine max/microbiologiaRESUMO
Bradyrhizobium japonicum, a symbiotic nitrogen-fixing soil bacterium, has multiple gene copies for aromatic degradation on the genome and is able to use low concentrations of vanillate, a methoxylated lignin monomer, as an energy source. A transcriptome analysis indicated that one set of vanA1B, pcaG1H1, and genes for C(1) compound catabolism was upregulated in B. japonicum USDA110 cells grown in vanillate (N. Ito, M. Itakura, S. Eda, K. Saeki, H. Oomori, T. Yokoyama, T. Kaneko, S. Tabata, T. Ohwada, S. Tajima, T. Uchiumi, E. Masai, M. Tsuda, H. Mitsui, and K. Minamisawa, Microbes Environ. 21:240-250, 2006). To examine the functions of these genes in vanillate degradation, we tested cell growth and substrate consumption in vanA1B, pcaG1H1, and mxaF mutants of USDA110. The vanA1B and pcaG1H1 mutants were unable to grow in minimal media containing 1 mM vanillate and protocatechuate, respectively, although wild-type USDA110 was able to grow in both media, indicating that the upregulated copies of vanA1B and pcaG1H1 are exclusively responsible for vanillate degradation. Mutating mxaF eliminated expression of gfa and flhA, which contribute to glutathione-dependent C(1) metabolism. The mxaF mutant had markedly lower cell growth in medium containing vanillate than the wild-type strain. In the presence of protocatechuate, there was no difference in cell growth between the mxaF mutant and the wild-type strain. These results suggest that the C(1) pathway genes are required for efficient vanillate catabolism. In addition, wild-type USDA110 oxidized methanol, whereas the mxaF mutant did not, suggesting that the metabolic capability of the C(1) pathway in B. japonicum extends to methanol oxidation. The mxaF mutant showed normal nodulation and N(2) fixation phenotypes with soybeans, which was not similar to symbiotic phenotypes of methylotrophic rhizobia.
Assuntos
Bradyrhizobium/metabolismo , Carbono/metabolismo , Ácido Vanílico/metabolismo , Proteínas de Bactérias/genética , Meios de Cultura/química , Deleção de Genes , Perfilação da Expressão Gênica , Hidroxibenzoatos/metabolismo , Redes e Vias Metabólicas/genética , Metanol/metabolismo , Modelos Biológicos , Fixação de Nitrogênio , Oxirredução , Nodulação , Glycine max/microbiologiaRESUMO
We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N(2)O) reductase (N(2)OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N(2)O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N(2)O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N(2)OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N(2)OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.
Assuntos
Proteínas de Bactérias/biossíntese , Bradyrhizobium/enzimologia , DNA Polimerase III/genética , Proteínas Mutantes/biossíntese , Oxirredutases/biossíntese , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Proteínas Mutantes/genética , Óxido Nitroso/metabolismo , Oxirredutases/genética , PlasmídeosRESUMO
We investigated the effects of the water status (flooded or non-flooded) and presence of the nosZ gene in bradyrhizobia on the bradyrhizobial community structure in a factorial experiment that examined three temperature levels (20°C, 25°C, and 30°C) and two soil types (andosol and gray lowland soil) using microcosm incubations. All microcosms were inoculated with Bradyrhizobium japonicum USDA6T, B. japonicum USDA123, and B. elkanii USDA76T, which do not possess the nosZ gene, and then half received B. diazoefficiens USDA110Twt (wt for the wild-type) and the other half received B. diazoefficiens USDA110ΔnosZ. USDA110Twt possesses the nosZ gene, which encodes N2O reductase; 110ΔnosZ, a mutant variant, does not. Changes in the community structure after 30- and 60-d incubations were investigated by denaturing-gradient gel electrophoresis and an image analysis. USDA6T and 76T strains slightly increased in non-flooded soil regardless of which USDA110T strain was present. In flooded microcosms with the USDA110Twt strain, USDA110Twt became dominant, whereas in microcosms with the USDA110ΔnosZ, a similar change in the community structure occurred to that in non-flooded microcosms. These results suggest that possession of the nosZ gene confers a competitive advantage to B. diazoefficiens USDA110T in flooded soil. We herein demonstrated that the dominance of B. diazoefficiens USDA110Twt within the soil bradyrhizobial population may be enhanced by periods of flooding or waterlogging systems such as paddy-soybean rotations because it appears to have the ability to thrive in moderately anaerobic soil.
Assuntos
Bradyrhizobium/genética , Inundações , Glycine max/microbiologia , Oxirredutases/genética , Microbiologia do Solo , Genes BacterianosRESUMO
The hydrogen uptake (Hup) system of Bradyrhizobium diazoefficiens recycles the H2 released by nitrogenase in soybean nodule symbiosis, and is responsible for H2-dependent chemolithoautotrophic growth. The strain USDA110 has two hup gene clusters located outside (locus I) and inside (locus II) a symbiosis island. Bacterial growth under H2-dependent chemolithoautotrophic conditions was markedly weaker and H2 production by soybean nodules was markedly stronger for the mutant of hup locus I (ΔhupS1L1) than for the mutant of hup locus II (ΔhupS2L2). These results indicate that locus I is primarily responsible for Hup activity.
Assuntos
Bradyrhizobium/genética , Bradyrhizobium/metabolismo , Crescimento Quimioautotrófico , Hidrogênio/metabolismo , Família Multigênica , Simbiose , Bradyrhizobium/crescimento & desenvolvimento , Bradyrhizobium/fisiologia , Técnicas de Inativação de Genes , Ilhas Genômicas , Nódulos Radiculares de Plantas/microbiologia , Glycine max/microbiologiaRESUMO
Agricultural soil is the largest source of nitrous oxide (N2O), a greenhouse gas. Soybean is an important leguminous crop worldwide. Soybean hosts symbiotic nitrogen-fixing soil bacteria (rhizobia) in root nodules. In soybean ecosystems, N2O emissions often increase during decomposition of the root nodules. Our previous study showed that N2O reductase can be used to mitigate N2O emission from soybean fields during nodule decomposition by inoculation with nosZ++ strains [mutants with increased N2O reductase (N2OR) activity] of Bradyrhizobium diazoefficiens. Here, we show that N2O emission can be reduced at the field scale by inoculation with a mixed culture of indigenous nosZ+ strains of B. diazoefficiens USDA110 group isolated from Japanese agricultural fields. Our results also suggested that nodule nitrogen is the main source of N2O production during nodule decomposition. Isolating nosZ+ strains from local soybean fields would be more applicable and feasible for many soybean-producing countries than generating mutants.
Assuntos
Biodegradação Ambiental , Bradyrhizobium/metabolismo , Óxido Nitroso/análise , Solo/química , Ecossistema , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mutação , Nitrogênio/análise , Fixação de Nitrogênio , Raízes de Plantas/microbiologia , Brotos de Planta/microbiologia , Microbiologia do Solo , Glycine max/microbiologiaRESUMO
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
Bradyrhizobium japonicum strains that have the nosZ gene, which encodes N2O reductase, are able to mitigate N2O emissions from soils (15). To examine the distribution of nosZ genotypes among Japanese indigenous soybean bradyrhizobia, we isolated bradyrhizobia from the root nodules of soybean plants inoculated with 32 different soils and analyzed their nosZ and nodC genotypes. The 1556 resultant isolates were classified into the nosZ+/nodC+ genotype (855 isolates) and nosZ-/nodC+ genotype (701 isolates). The 11 soil samples in which nosZ- isolates significantly dominated (P < 0.05; the χ(2) test) were all Andosols (a volcanic ash soil prevalent in agricultural fields in Japan), whereas the 17 soil samples in which nosZ+ isolates significantly dominated were mainly alluvial soils (non-volcanic ash soils). This result was supported by a principal component analysis of environmental factors: the dominance of the nosZ- genotype was positively correlated with total N, total C, and the phosphate absorption coefficient in the soils, which are soil properties typical of Andosols. Internal transcribed spacer sequencing of representative isolates showed that the nosZ+ and nosZ- isolates of B. japonicum fell mainly into the USDA110 (BJ1) and USDA6 (BJ2) groups, respectively. These results demonstrated that the group lacking nosZ was dominant in Andosols, which can be a target soil type for an N2O mitigation strategy in soybean fields. We herein discussed how the nosZ genotypes of soybean bradyrhizobia depended on soil types in terms of N2O respiration selection and genomic determinants for soil adaptation.
Assuntos
Variação Genética , Glycine max/microbiologia , Oxirredutases/genética , Nódulos Radiculares de Plantas/microbiologia , Microbiologia do Solo , Solo/química , Carbono/análise , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Genótipo , Japão , Dados de Sequência Molecular , Nitrogênio/análise , Fosfatos/análise , Filogenia , Análise de Sequência de DNARESUMO
The early molecular dialogue between soybean and the bacterium Bradyrhizobium japonicum is crucial for triggering their symbiotic interaction. Here we found a single large genomic locus that is widely separated from the symbiosis island and was conspicuously induced within minutes after the addition of genistein. This locus (named BjG30) contains genes for the multidrug efflux pump, TetR family transcriptional regulator, and polyhydroxybutyrate (PHB) metabolism. The induction of BjG30 by genistein was competitively inhibited by daidzein, although both genistein and daidzein are soybean-derived inducers of nodulation (nod) genes. Such a differential expression pattern is also observed in some legume-derived flavonoids, which structurally differ in the hydroxy/deoxy group at the 5-position. In addition, not only did the induction start far in advance of nodW and nodD1 after the addition of genistein, but the levels showed distinct concentration dependence, indicating that the induction pattern of BjG30 is completely different from that of nod genes. The deletion of genes encoding either the multidrug efflux pump or PHB metabolism, especially the former, resulted in defective nodulation performance and nitrogen-fixing capability. Taken together, these results indicate that BjG30, and especially its multidrug efflux pump, may play a key role in the early stage of symbiosis by balancing the dual functions of genistein as both a nod gene inducer and toxicant.
Assuntos
Proteínas de Bactérias/genética , Bradyrhizobium/fisiologia , Regulação Bacteriana da Expressão Gênica , Genisteína/metabolismo , Glycine max/metabolismo , Glycine max/microbiologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Bactérias/metabolismo , Bradyrhizobium/genética , Isoflavonas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , SimbioseRESUMO
A model system developed to produce N(2)O emissions from degrading soybean nodules in the laboratory was used to clarify the mechanism of N(2)O emission from soybean fields. Soybean plants inoculated with nosZ-defective strains of Bradyrhizobium japonicum USDA110 (ΔnosZ, lacking N(2)O reductase) were grown in aseptic jars. After 30 days, shoot decapitation (D, to promote nodule degradation), soil addition (S, to supply soil microbes), or both (DS) were applied. N(2)O was emitted only with DS treatment. Thus, both soil microbes and nodule degradation are required for the emission of N(2)O from the soybean rhizosphere. The N(2)O flux peaked 15 days after DS treatment. Nitrate addition markedly enhanced N(2)O emission. A (15)N tracer experiment indicated that N(2)O was derived from N fixed in the nodules. To evaluate the contribution of bradyrhizobia, N(2)O emission was compared between a nirK mutant (ΔnirKΔnosZ, lacking nitrite reductase) and ΔnosZ. The N(2)O flux from the ΔnirKΔnosZ rhizosphere was significantly lower than that from ΔnosZ, but was still 40% to 60% of that of ΔnosZ, suggesting that N(2)O emission is due to both B. japonicum and other soil microorganisms. Only nosZ-competent B. japonicum (nosZ+ strain) could take up N(2)O. Therefore, during nodule degradation, both B. japonicum and other soil microorganisms release N(2)O from nodule N via their denitrification processes (N(2)O source), whereas nosZ-competent B. japonicum exclusively takes up N(2)O (N(2)O sink). Net N(2)O flux from soybean rhizosphere is likely determined by the balance of N(2)O source and sink.
Assuntos
Bradyrhizobium/metabolismo , Glycine max/microbiologia , Óxido Nitroso/metabolismo , Rizosfera , Nódulos Radiculares de Plantas/microbiologia , Bradyrhizobium/enzimologia , Bradyrhizobium/genética , Desnitrificação , Fixação de Nitrogênio , Brotos de Planta/metabolismo , Microbiologia do SoloRESUMO
Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.