Competition for electrons favours N2 O reduction in denitrifying Bradyrhizobium isolates.

Bradyrhizobia are common members of soil microbiomes and known as N2 -fixing symbionts of economically important legumes. Many are also denitrifiers, which can act as sinks or sources for N2 O. Inoculation with compatible rhizobia is often needed for optimal N2 -fixation, but the choice of inoculant may have consequences for N2 O emission. Here, we determined the phylogeny and denitrification capacity of Bradyrhizobium strains, most of them isolated from peanut-nodules. Analyses of genomes and denitrification end-points showed that all were denitrifiers, but only ~1/3 could reduce N2 O. The N2 O-reducing isolates had strong preference for N2 O- over NO3 - -reduction. Such preference was also observed in a study of other bradyrhizobia and tentatively ascribed to competition between the electron pathways to Nap (periplasmic NO3 - reductase) and Nos (N2 O reductase). Another possible explanation is lower abundance of Nap than Nos. Here, proteomics revealed that Nap was instead more abundant than Nos, supporting the hypothesis that the electron pathway to Nos outcompetes that to Nap. In contrast, Paracoccus denitrificans, which has membrane-bond NO3 - reductase (Nar), reduced N2 O and NO3 - simultaneously. We propose that the control at the metabolic level, favouring N2 O reduction over NO3 - reduction, applies also to other denitrifiers carrying Nos and Nap but lacking Nar.

A common mechanism for efficient N2 O reduction in diverse isolates of nodule-forming bradyrhizobia.

Bradyrhizobia are abundant soil bacteria, which can form nitrogen-fixing symbioses with leguminous plants, including important crops such as soybean, cowpea and peanut. Many bradyrhizobia can denitrify, but studies have hitherto focused on a few model organisms. We screened 39 diverse Bradyrhizobium strains, isolated from legume nodules. Half of them were unable to reduce N2 O, making them sources of this greenhouse gas. Most others could denitrify NO3 - to N2 . Time-resolved gas kinetics and transcription analyses during transition to anaerobic respiration revealed a common regulation of nirK, norCB and nosZ (encoding NO2 - , NO and N2 O reductases), and differing regulation of napAB (encoding periplasmic NO3 - reductase). A prominent feature in all N2 -producing strains was a virtually complete hampering of NO3 - reduction in the presence of N2 O. In-depth analyses suggest that this was due to a competition between electron transport pathways, strongly favouring N2 O over NO3 - reduction. In a natural context, bacteria with this feature would preferentially reduce available N2 O, produced by themselves or other soil bacteria, making them powerful sinks for this greenhouse gas. One way to augment such populations in agricultural soils is to develop inoculants for legume crops with dual capabilities of efficient N2 -fixation and efficient N2 O reduction.

Host Range and Symbiotic Effectiveness of N2O Reducing Bradyrhizobium Strains.

Emissions of the potent greenhouse gas N2O is one of the environmental problems associated with intensive use of synthetic N fertilizers, and novel N2O mitigation strategies are needed to minimize fertilizer applications and N2O release without affecting agricultural efficiencies. Increased incorporation of legume crops in agricultural practices offers a sustainable alternative. Legumes, in their symbiosis with nitrogen fixing bacteria, rhizobia, reduce the need for fertilizers and also respond to the need for increased production of plant-based proteins. Not all combinations of rhizobia and legumes result in efficient nitrogen fixation, and legume crops therefore often need to be inoculated with compatible rhizobial strains. Recent research has demonstrated that some rhizobia are also very efficient N2O reducers. Several nutritionally and economically important legumes form root nodules in symbiosis with bacteria belonging to Bradyrhizobium. Here, the host-ranges of fourteen N2O reducing Bradyrhizobium strains were tested on six legume hosts; cowpea, groundnut, mung bean, haricot bean, soybean, and alfalfa. The plants were grown for 35 days in pots in sterile sand supplemented with N-free nutrient solution. Cowpea was the most promiscuous host nodulated by all test strains, followed by groundnut (11 strains) and mungbean (4 strains). Three test strains were able to nodulate all these three legumes, while none nodulated the other three hosts. For cowpea, five strains increased the shoot dry weight and ten strains the shoot nitrogen content (pairwise comparison; p < 0.05). For groundnut the corresponding results were three and nine strains. The symbiotic effectiveness for the different strains ranged from 45 to 98% in cowpea and 34 to 95% in groundnut, relative to fertilized controls. The N2O reduction capacity of detached nodules from cowpea plants inoculated with one of these strains confirmed active N2O reduction inside the nodules. When released from senescent nodules such strains are expected to also act as sinks for N2O produced by denitrifying organisms in the soil microbial community. Our strategy to search among known N2O-reducing Bradyrhizobium strains for their N2-fixation effectiveness successfully identified several strains which can potentially be used for the production of legume inoculants with the dual capacities of efficacious N2-fixation and N2O reduction.

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of tree and annual legume species growing in Ethiopia.

Bacteria belonging to the genus Bradyrhizobium nodulate various leguminous woody plants and herbs, including economically important crops such as soybean, peanut and cowpea. Here we analysed 39 Bradyrhizobium strains originating from root nodules of the leguminous trees and crops Acacia saligna, Faidherbia albida, Erythrina brucei, Albizia gummifera, Millettia ferruginea, Cajanus cajan, Vigna unguiculata and Phaseolus vulgaris, growing in southern Ethiopia. Multilocus sequence analyses (MLSA) of the 16S rRNA, glnII, recA, gyrB and dnaK genes and the ITS region grouped the test strains into seven well-supported genospecies (I-VII), six of which occupied distinct positions excluding all hitherto defined Bradyrhizobium species. Analyses of the nodA, nodC and nifH genes suggested different evolutionary history of the chromosomal and symbiosis-related genes. Our study corroborates earlier findings that Ethiopia is a hotspot for rhizobial biodiversity, justifying further search for novel strains from this region and calling for intensified research on the ecology and biochemistry of these organisms.