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N 2 O is an important greenhouse gas influencing global warming, and agricultural land is the predominant (anthropogenic) source of N 2 O emissions. Here, we report the high N 2 O-reducing activity of Bradyrhizobium ottawaense , suggesting the potential for efficiently mitigating N 2 O emission from agricultural lands. Among the 15 B. ottawaense isolates examined, the N 2 O-reducing activities of most (13) strains were approximately five-fold higher than that of Bradyrhizobium diazoefficiens USDA110 T under anaerobic conditions. This robust N 2 O-reducing activity of B. ottawaense was confirmed by N 2 O reductase (NosZ) protein levels and by mitigation of N 2 O 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 USDA110 T , suggesting the high N 2 O-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 N 2 O-respiring conditions, which may contribute to the high nosZ expression . Our study indicates the potential of B. ottawaense for effective N 2 O reduction and unique regulation of nos gene expression towards the high performance of N 2 O mitigation in the soil.

Soybeans fix atmospheric N 2 through symbiosis with rhizobia. The relationship between rhizobia and soybeans, particularly those with high nitrous oxide (N 2 O)-reducing (N 2 OR) activities, can be leveraged to reduce N 2 O emissions from agricultural soils. However, inoculating soybeans with these rhizobia under field conditions often fails because of the competition from indigenous rhizobia that possess low or no N 2 OR activity. In this work, we utilize natural incompatibility systems between soybean and rhizobia to address this challenge. Specifically, Rj2 and GmNNL1 inhibit certain rhizobial infections in response to NopP, an effector protein. By combining a soybean line with a hybrid accumulation of the Rj2 and GmNNL1 genes and bradyrhizobia lacking the nopP gene, we develop a soybean-bradyrhizobial symbiosis system in which strains with high N 2 OR activity predominantly infect. Our optimize symbiotic system substantially reduces N 2 O emissions in field and laboratory tests, presenting a promising approach for sustainable agricultural practices. During plant cultivation, denitrification process can release greenhouse gas nitrous oxide (N 2 O) to atmosphere. Here, the authors develop a soybean–bradyrhizobial symbiosis system with enhanced capacity to reduce N 2 O emissions using the incompatibility between two soybean R genes and their effector present in bradyrhizobia.

Soybean hosts the symbiotic nitrogen-fixing soil bacterium Bradyrhizobium japonicum , that can produce the greenhouse gas nitrous oxide. This study shows that nitrous oxide emissions from soybean ecosystems can be biologically mitigated at a field scale by inoculation with strains of B. japonicum that have increased nitrous oxide reductase activity. Nitrous oxide (N 2 O) is a greenhouse gas that is also capable of destroying the ozone layer 1 . Agricultural soil is the largest source of N 2 O (ref.  2 ). Soybean is a globally important leguminous crop, and hosts symbiotic nitrogen-fixing soil bacteria (rhizobia) that can also produce N 2 O (ref.  3 ). In agricultural soil, N 2 O is emitted from fertilizer and soil nitrogen. In soybean ecosystems, N 2 O is also emitted from the degradation of the root nodules 4 . Organic nitrogen inside the nodules is mineralized to NH 4 + , followed by nitrification and denitrification that produce N 2 O. N 2 O is then emitted into the atmosphere or is further reduced to N 2 by N 2 O reductase (N 2 OR), which is encoded by the nosZ gene. Pure culture and vermiculite pot experiments showed lower N 2 O emission by nosZ + strains 5 and nosZ ++ strains (mutants with increased N 2 OR activity) 6 of Bradyrhizobium japonicum than by nosZ − strains. A pot experiment using soil confirmed these results 7 . Although enhancing N 2 OR activity has been suggested as a N 2 O mitigation option 8 , 9 , this has never been tested in the field. Here, we show that post-harvest N 2 O emission from soybean ecosystems due to degradation of nodules can be mitigated by inoculation of nosZ + and non-genetically modified organism nosZ ++ strains of B. japonicum at a field scale.

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 N 2 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.

Agricultural soil is the largest source of nitrous oxide (N 2 O), a greenhouse gas. Soybean is an important leguminous crop worldwide. Soybean hosts symbiotic nitrogen-fixing soil bacteria (rhizobia) in root nodules. In soybean ecosystems, N 2 O emissions often increase during decomposition of the root nodules. Our previous study showed that N 2 O reductase can be used to mitigate N 2 O emission from soybean fields during nodule decomposition by inoculation with nosZ ++ strains [mutants with increased N 2 O reductase (N 2 OR) activity] of Bradyrhizobium diazoefficiens . Here, we show that N 2 O 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 N 2 O 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.

Soybeans fix atmospheric N through symbiosis with rhizobia. The relationship between rhizobia and soybeans, particularly those with high nitrous oxide (N O)-reducing (N OR) activities, can be leveraged to reduce N O emissions from agricultural soils. However, inoculating soybeans with these rhizobia under field conditions often fails because of the competition from indigenous rhizobia that possess low or no N OR activity. In this work, we utilize natural incompatibility systems between soybean and rhizobia to address this challenge. Specifically, Rj2 and GmNNL1 inhibit certain rhizobial infections in response to NopP, an effector protein. By combining a soybean line with a hybrid accumulation of the Rj2 and GmNNL1 genes and bradyrhizobia lacking the nopP gene, we develop a soybean-bradyrhizobial symbiosis system in which strains with high N OR activity predominantly infect. Our optimize symbiotic system substantially reduces N O emissions in field and laboratory tests, presenting a promising approach for sustainable agricultural practices.

Comparative genomic hybridization (CGH) was performed with nine strains of Bradyrhizobium japonicum (a symbiotic nitrogen-fixing bacterium associated with soybean) and eight other members of the Bradyrhizobiaceae by DNA macroarray of B. japonicum USDA110. CGH clearly discriminated genomic variations in B. japonicum strains, but similar CGH patterns were observed in other members of the Bradyrhizobiaceae. The most variable regions were 14 genomic islands (4–97 kb) and low G+C regions on the USDA110 genome, some of which were missing in several strains of B. japonicum and other members of the Bradyrhizobiaceae. The CGH profiles of B. japonicum were classified into three genome types: 110, 122 and 6. Analysis of DNA sequences around the boundary regions showed that at least seven genomic islands were missing in genome type 122 as compared with type 110. Phylogenetic analysis for internal transcribed sequences revealed that strains belonging to genome types 110 and 122 formed separate clades. Thus genomic islands were horizontally inserted into the ancestor genome of type 110 after divergence of the type 110 and 122 strains. To search for functional relationships of variable genomic islands, we conducted linear models of the correlation between the existence of genomic regions and the parameters associated with symbiotic nitrogen fixation in soybean. Variable genomic regions including genomic islands were associated with the enhancement of symbiotic nitrogen fixation in B. japonicum USDA110.

Agricultural soils are an important source of nitrous oxide (N2O), which has greenhouse and ozone-depleting effects. Bradyrhizobium ottawaense SG09 is a nitrogen-fixing rhizobium with high N2O-reducing activity. Rhizobia form symbiotic nodules in leguminous plants. The initial physical attachment of bacteria to plant roots is a critical step in the establishment of symbiotic interactions. In this study, we performed microscopic analysis using DsRed-expressing B. ottawaense SG09. We revealed that B. ottawaense SG09 attached to both the root surface and root hairs via single cellular poles. This polar attachment was observed not only to the symbiotic host soybean, but also to non-leguminous plants, such as Arabidopsis, rice, corn, and wheat. We identified and analyzed the unipolar polysaccharide (upp) gene cluster, which is proposed to be involved in polar attachment of rhizobia, in the genome of B. ottawaense SG09. We established an Arabidopsis-based interaction assay and demonstrated that uppC and uppE play a critical role in attachment to both the root surface and root hairs.

Clear cell sarcoma (CCS) is a rare soft tissue sarcoma caused by the EWS/ATF1 fusion gene. Here, we established induced pluripotent stem cells (iPSCs) from EWS/ATF1 -controllable murine CCS cells harboring sarcoma-associated genetic abnormalities. Sarcoma-iPSC mice develop secondary sarcomas immediately after EWS/ATF1 induction, but only in soft tissue. EWS/ATF1 expression induces oncogene-induced senescence in most cell types in sarcoma-iPSC mice but prevents it in sarcoma cells. We identify Tppp3 -expressing cells in peripheral nerves as a cell-of-origin for these sarcomas. We show cell type-specific recruitment of EWS/ATF1 to enhancer regions in CCS cells. Finally, epigenetic silencing at these enhancers induces senescence and inhibits CCS cell growth through altered EWS/ATF1 binding. Together, we propose that distinct responses to premature senescence are the basis for the cell type-specificity of cancer development. The EWS-ATF1 fusion gene causes clear cell sarcoma (CCS). Here, the authors show that the downstream effects of EWS-ATF1 expression are strictly context dependent, and reveal the cell of origin for CCS to be Tppp3-expressing cells in peripheral nerves.

The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections.Key points• The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs.• Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs.• Inoculation of mutated SVPs induces antibodies with low cross-reactivity.