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Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens.

ABSTRACT: 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.

SUBMITTER: Akiyama H

PROVIDER: S-EPMC5025649 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens.

Akiyama Hiroko H Hoshino Yuko Takada YT Itakura Manabu M Shimomura Yumi Y Wang Yong Y Yamamoto Akinori A Tago Kanako K Nakajima Yasuhiro Y Minamisawa Kiwamu K Hayatsu Masahito M

Scientific reports 20160916

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 N2 ...[more]

PMID: 27633524

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Project description:Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4 Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2 IMPORTANCE: In this work, we investigated the regulation of polyhydroxybutyrate synthesis in the soybean-nodulating bacterium Bradyrhizobium diazoefficiens and its influence in bacterial free-living and symbiotic lifestyles. We uncovered a new interplay between the synthesis of this carbon reserve polymer and the network responsible for microoxic metabolism through the interaction between the gene regulators phaR and fixK2 These results contribute to the understanding of the physiological conditions required for polyhydroxybutyrate biosynthesis. The interaction between these two main metabolic pathways is also reflected in the symbiotic phenotypes of soybeans inoculated with phaR mutants, which were more competitive for nodulation and enhanced dry matter production by the plants. Therefore, this knowledge may be applied to the development of superior strains to be used as improved inoculants for soybean crops.

Project description:Bradyrhizobium diazoefficiens, a soybean N2-fixing symbiont, possesses a dual flagellar system comprising a constitutive subpolar flagellum and inducible lateral flagella. Here, we analyzed the genomic organization and biosynthetic regulation of the lateral-flagellar genes. We found that these genes are located in a single genomic cluster, organized in two monocistronic transcriptional units and three operons, one possibly containing an internal transcription start site. Among the monocistronic units is blr6846, homologous to the class IB master regulators of flagellum synthesis in Brucella melitensis and Ensifer meliloti and required for the expression of all the lateral-flagellar genes except lafA2, whose locus encodes a single lateral flagellin. We therefore named blr6846 lafR (lateral-flagellar regulator). Despite its similarity to two-component response regulators and its possession of a phosphorylatable Asp residue, lafR behaved as an orphan response regulator by not requiring phosphorylation at this site. Among the genes induced by lafR is flbTL , a class III regulator. We observed different requirements for FlbTL in the synthesis of each flagellin subunit. Although the accumulation of lafA1, but not lafA2, transcripts required FlbTL, the production of both flagellin polypeptides required FlbTL Moreover, the regulation cascade of this lateral-flagellar regulon appeared to be not as strictly ordered as those found in other bacterial species.IMPORTANCE Bacterial motility seems essential for the free-living style in the environment, and therefore these microorganisms allocate a great deal of their energetic resources to the biosynthesis and functioning of flagella. Despite energetic costs, some bacterial species possess dual flagellar systems, one of which is a primary system normally polar or subpolar, and the other is a secondary, lateral system that is produced only under special circumstances. Bradyrhizobium diazoefficiens, an N2-fixing symbiont of soybean plants, possesses dual flagellar systems, including the lateral system that contributes to swimming in wet soil and competition for nodulation and is expressed under high energy availability, as well as under requirement for high torque by the flagella. The structural organization and transcriptional regulation of the 41 genes that comprise this secondary flagellar system seem adapted to adjust bacterial energy expenditures for motility to the soil's environmental dynamics.

Project description:UnlabelledAnalysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure. Various genetic tools and well-established protocols are available for this type of mutagenesis in model bacteria; however, these methods are not always efficiently applicable in less established systems. Here we describe the construction and application of versatile plasmid vectors pREDSIX and pTETSIX for marker replacement and markerless mutagenesis, respectively. Apart from an array of restriction sites optimized for cloning of GC-rich DNA fragments, the vector backbone contains a constitutively expressed gene for mCherry, enabling the rapid identification of clones originating from single or double HR events by fluorescence-assisted cell sorting (FACS). In parallel, we constructed a series of plasmids from which gene cassettes providing resistance against gentamicin, kanamycin, hygromycin B, streptomycin and spectinomycin, or tetracycline were excised for use with pREDSIX-based marker replacement mutagenesis. In proof-of-concept mutagenesis experiments, we demonstrated the potential for the use of the developed tools for gene deletion mutagenesis in the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens(formerly Bradyrhizobium japonicum) and three additional members of the alphaproteobacteria.ImportanceMutation and phenotypic analysis are essential to the study of gene function. Efficient mutagenesis protocols and tools are available for many bacterial species, including various model organisms; however, genetic analysis of less-well-characterized organisms is often impaired by the lack of efficient methods. Here we describe a set of novel genetic tools for facilitated mutagenesis of the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens and related alphaproteobacteria. We demonstrated their usefulness by generating several mutant strains lacking defined genes. Isolation of both antibiotic resistance gene-containing and markerless deletion mutants is greatly facilitated because undesired clones which contain the entire mutagenic plasmid integrated in the genome can be identified on the basis of their fluorescent phenotype derived from them Cherrygene carried by the vector backbone. The possibility to generate markerless mutants assists with the isolation of strains carrying multiple deletions, which can be crucial while studying functionally redundant genes.

Project description:Sulfur (S)-containing molecules play an important role in symbiotic nitrogen fixation and are critical components of nitrogenase and other iron-S proteins. S deficiency inhibits symbiotic nitrogen fixation by rhizobia. However, despite its importance, little is known about the sources of S that rhizobia utilize during symbiosis. We previously showed that Bradyrhizobium diazoefficiens USDA110T can assimilate both inorganic and organic S and that genes involved in organic S utilization are expressed during symbiosis. Here, we show that a B. diazoefficiens USDA110T mutant with a sulfonate monooxygenase (ssuD) insertion is defective in nitrogen fixation. Microscopy analyses revealed that the ΔssuD mutant was defective in root hair infection and that ΔssuD mutant bacteroids showed degradation compared to the wild-type strain. Moreover, the ΔssuD mutant was significantly more sensitive to hydrogen peroxide-mediated oxidative stress than the wild-type strain. Taken together, these results show that the ability of rhizobia to utilize organic S plays an important role in symbiotic nitrogen fixation. Since nodules have been reported to be an important source of reduced S used during symbiosis and nitrogen fixation, further research will be needed to determine the mechanisms involved in the regulation of S assimilation by rhizobia.IMPORTANCE Rhizobia form symbiotic associations with legumes that lead to the formation of nitrogen-fixing nodules. Sulfur-containing molecules play a crucial role in nitrogen fixation; thus, the rhizobia inside nodules require large amounts of sulfur. Rhizobia can assimilate both inorganic (sulfate) and organic (sulfonates) sources of sulfur. However, very little is known about rhizobial sulfur metabolism during symbiosis. In this report, we show that sulfonate utilization by Bradyrhizobium diazoefficiens is important for symbiotic nitrogen fixation in both soybean and cowpea. The symbiotic defect is probably due to increased sensitivity to oxidative stress from sulfur deficiency in the mutant strain defective for sulfonate utilization. The results of this study can be extended to other rhizobium-legume symbioses, as sulfonate utilization genes are widespread in these bacteria.

Dataset Information

Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens.

Publications

Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens.

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