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Legumes can contribute to emissions of the potent greenhouse gas nitrous oxide (NO) directly by some rhizobia species that are able to denitrify under free-living conditions and in symbiotic association with the plant. In this study, the capacity of Phaseolus vulgaris-Rhizobium etli symbiosis to emit NO in response to nitrate (NO) has been demonstrated for the first time. We found that bacteroidal assimilatory nitrate reductase (NarB) as well as nitrite reductase (NirK) and nitric oxide reductase (cNor) denitrifying enzymes contribute to nitric oxide (NO) and NO formation in nodules. We also show that R. etli NarK is involved in NO extrusion and links NO reduction by NarB in the cytoplasm with NirK and cNor denitrification activities in the periplasm. The knowledge generated in this work will be instrumental for exploring strategies and sustainable practices in agricultural soil management to increase legume crop yield and mitigate greenhouse gas emissions.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

En este trabajo se estudió el efecto del compost de alperujo sobre la nodulación en la simbiosis leguminosa-rizobio, concretamente entre plantas de soja (Glycine maxL.) y su simbionte natural (Bradyrhizobium diazoefficiens). Para eso, se añadieron varias dosis de compost de alperujo, el principal residuo orgánico de la industria extractiva del aceite de oliva, a plantas de soja cultivadas en condiciones controladas de invernadero durante 4 semanas. Al finalizar el experimento se determinó el peso seco de la biomasa vegetal (parte aérea y raíz), el número y peso fresco de los nódulos aparecidos, así como el contenido en leghemoglobina de los mismos. Los resultados obtenidos sugieren que el compost de alperujo mejora significativamente el crecimiento de las plantas al mejorar la eficiencia simbiótica de la asociación leguminosa-rizobio

In this work, we analyzed the symbiotic performance and diversity of rhizobial strains isolated from the endemic shrubby legume Chamaecytisus albidus grown in soils of three different agroforestry ecosystems representing arid and semi-arid forest areas in Morocco. The analysis of the rrs gene sequences from twenty-four representative strains selected after REP-PCR fingerprinting showed that all the strains belong to the genus Bradyrhizobium. Following multi-locus sequence analysis (MLSA) using the rrs, gyrB, recA, glnII, and rpoB housekeeping genes, five representative strains, CA20, CA61, CJ2, CB10, and CB61 were selected for further molecular studies. Phylogenetic analysis of the concatenated glnII, gyrB, recA, and rpoB genes showed that the strain CJ2 isolated from Sahel Doukkala soil is close to Bradyrhizobium canariense BTA-1 (96.95%); that strains CA20 and CA61 isolated from the Amhach site are more related to Bradyrhizobium valentinum LmjM3, with 96.40 and 94.57% similarity values; and that the strains CB10 and CB60 isolated from soil in the Bounaga site are more related to Bradyrhizobium murdochi CNPSo 4020 and Bradyrhizobium. retamae Ro19, with which they showed 95.45 and 97.34% similarity values, respectively. The phylogenetic analysis of the symbiotic genes showed that the strains belong to symbiovars lupini, genistearum, and retamae. All the five strains are able to nodulate Lupinus luteus, Retama monosperma, and Cytisus monspessilanus, but they do not nodulate Glycine max and Phaseolus vulgaris. The inoculation tests showed that the strains isolated from the 3 regions improve significantly the plant yield as compared to uninoculated plants. However, the strains of Bradyrhizobium sp. sv. retamae isolated from the site of Amhach were the most performing. The phenotypic analysis showed that the strains are able to use a wide range of carbohydrates and amino acids as sole carbon and nitrogen source. The strains isolated from the arid areas of Bounaga and Am

FixK2 is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK2 gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK2 activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK2, we replaced C183 for aspartic acid. Purified C183D FixK2 protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK2, expression of a fixNOQP’-‘lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK2 encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK2 gene expression levels and FixK2 protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK2 encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK2 in vivo.

Out of 70 bacterial strains isolated from root nodules of Lupinus albus and L. angustifolius grown in the soils from the Maamora forest in Morocco, 56 isolates possessed the nodC symbiotic gene, as determined by nodC-PCR, and they were able to renodulate their original hosts. The phenotypic analysis showed that many strains had great potential for using different carbon compounds and amino acids as sole carbon and nitrogen sources. The majority of strains grew in media with pH values between 6 and 8. Only one strain isolated from L. angustifolius was able to grow at low pH values, whereas fourteen strains nodulating L. albus grew at pH 5. No strain developed at 40 °C, and eighteen strains grew at NaCl concentrations as high as 855 mM. A total of 17 strains solubilized phosphates, whereas 20 produced siderophores and seven produced IAA. Only three strains, Lalb41, Lang10 and Lang16, possessed all three plant growth promoting activities. The strains were grouped into eight genetic groups by rep-PCR. Analysis of the 16S rRNA sequences of eight strains representing the different groups showed that they were members of the genus Bradyrhizobium. The sequencing of the five housekeeping genes atpD, glnII, dnaK, gyrB and recA, from the eight representative strains, and the phylogenetic analysis of their concatenated sequences, showed that both plants were nodulated by different Bradyrhizobium species. Accordingly, two strains, Lalb41 and Lalb5.2, belonged to B. lupini, whereas two strains, Lalb2 and Lang17.2, were affiliated to B. cytisi, and one strain, Lang2, was close to B. canariense. The fourth group of strains, Lalb25, Lang14.3 and Lang8.3, which had similarity values of less than 96% with their closest named species, B. cytisi, may belong to two new genospecies in the genus Bradyrhizobium. All the strains nodulated Lupinus cosentinii, L. luteus, Retama sphaerocarpa, R. monosperma, Chamaecytisus albus, but not Vachellia gummifera, Phaseolus vulgaris or Glycine max. The

Aims: The purpose of this work was to analyze the agronomic and environmental performance of soybean plants inoculated with the Bradyrhizobium strains widely used as soybean biofertilizers in South America and to determine if these strains possess any functional or taxonomic trait associated with the NO emission. Methods: Bradyrhizobium japonicum E109 and CPAC 15, B. diazoefficiens USDA 110 and CPAC 7, and B. elkanii SEMIA 5019 and SEMIA 587 were used to inoculate soybean seeds. The field experiment was carried out in a soil without history of soybean cultivation in the Argentinian Humid Pampa. The natural N abundance method was applied to estimate N-fixation, and NO production was evaluated using gas chromatography. Among other physiological parameters, shoot dry weight, shoot N content, and crop yield were estimated after harvest. Results: B. japonicum inoculation produced the greatest increases in soybean growth and crop yield but also led to higher NO emissions compared to all other inoculated treatments. Plants inoculated with B. diazoefficiens released the lowest amount of NO, and their growth and yield were the least affected. Inoculation with B. elkanii resulted in intermediate NO emission fluxes and crop yield compared with B. japonicum and B. diazoefficiens. Conclusions: We found that soybean inoculation with strains of B. japonicum and B. elkanii that lack the nosZ gene led to the highest NO emissions under field conditions, but also to the highest crop yield, while inoculation with strains that carry out complete denitrification, nosZ-containing B. diazoefficiens, showed lower NO emission and lower crop yield.

The greenhouse gas nitrous oxide (N2O) has strong potential to drive climate change. Soils are a major source of N2O, with microbial nitrification and denitrification being the primary processes involved in such emissions. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model microorganism to study denitrification, a process that depends on a set of reductases, encoded by the napEDABC, nirK, norCBQD, and nosRZDYFLX genes, which sequentially reduce nitrate (NO3) to nitrite (NO2), nitric oxide (NO), N2O, and dinitrogen (N2). In this bacterium, the regulatory network and environmental cues governing the expression of denitrification genes rely on the FixK2 and NnrR transcriptional regulators. To understand the role of FixK2 and NnrR proteins in N2O turnover, we monitored real-time kinetics of NO3, NO2, NO, N2O, N2, and oxygen (O2) in a fixK2 and nnrR mutant using a robotized incubation system. We confirmed that FixK2 and NnrR are regulatory determinants essential for NO3 respiration and N2O reduction. Furthermore, we demonstrated that N2O reduction by B. diazoefficiens is independent of canonical inducers of denitrification, such as the nitrogen oxide NO3, and it is negatively affected by acidic and alkaline conditions. These findings advance the understanding of how specific environmental conditions and two single regulators modulate N2O turnover in B. diazoefficiens.

Nitrous oxide (NO) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of NO in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing NO emissions from agricultural soils.