Denitrification is the complete reduction of nitrate or nitrite to N2, via the intermediates nitric oxide (NO) and nitrous oxide (N2O), and is coupled to energy conservation and growth under O2-limiting conditions. In Bradyrhizobium japonicum, this process occurs through the action of the napEDABC, nirK, norCBQD and nosRZDFYLX gene products. DNA sequences showing homology with nap, nirK, nor and nos genes have been found in the genome of the symbiotic plasmid pSymA of Sinorhizobium meliloti strain 1021. Whole-genome transcriptomic analyses have demonstrated that S. meliloti denitrification genes are induced under micro-oxic conditions. Furthermore, S. meliloti has also been shown to possess denitrifying activities in both free-living and symbiotic forms. Despite possessing and expressing the complete set of denitrification genes, S. meliloti is considered a partial denitrifier since it does not grow under anaerobic conditions with nitrate or nitrite as terminal electron acceptors. In the present paper, we show that, under micro-oxic conditions, S. meliloti is able to grow by using nitrate or nitrite as respiratory substrates, which indicates that, in contrast with anaerobic denitrifiers, O2 is necessary for denitrification by S. meliloti. Current knowledge of the regulation of S. meliloti denitrification genes is also included.

BackgroundDenitrification is defined as the dissimilatory reduction of nitrate or nitrite to nitric oxide (NO), nitrous oxide (N2O), or dinitrogen gas (N2). N2O is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Legume crops might contribute to N2O production by providing nitrogen-rich residues for decomposition or by associating with rhizobia that are able to denitrify under free-living and symbiotic conditions. However, there are limited direct empirical data concerning N2O production by endosymbiotic bacteria associated with legume crops. Analysis of the Ensifer meliloti 1021 genome sequence revealed the presence of the napEFDABC, nirK, norECBQD and nosRZDFYLX denitrification genes. It was recently reported that this bacterium is able to grow using nitrate respiration when cells are incubated with an initial O2 concentration of 2%; however, these cells were unable to use nitrate respiration when initially incubated anoxically. The involvement of the nap, nirK, nor and nos genes in E. meliloti denitrification has not been reported.ResultsE. meliloti nap, nirK and norC mutant strains exhibited defects in their ability to grow using nitrate as a respiratory substrate. However, E meliloti nosZ was not essential for growth under these conditions. The E. meliloti napA, nirK, norC and nosZ genes encode corresponding nitrate, nitrite, nitric oxide and nitrous oxide reductases, respectively. The NorC component of the E. meliloti nitric oxide reductase has been identified as a c-type cytochrome that is 16 kDa in size. Herein, we also show that maximal expression of the E. meliloti napA, nirK, norC and nosZ genes occurred when cells were initially incubated anoxically with nitrate.ConclusionThe E. meliloti napA, nirK, norC and nosZ genes are involved in nitrate respiration and in the expression of denitrification enzymes in this bacterium. Our findings expand the short list of rhizobia for which denitrification gene function has been demonstrated. The inability of E. meliloti to grow when cells are initially subjected to anoxic conditions is not attributable to defects in the expression of the napA, nirK, norC and nosZ denitrification genes.

Background and aims Plant and bacteria are able to synthesise proline, which acts as a compound to counteract the negative effects of osmotic stresses. Most methodologies rely on the extraction of compounds using destructive methods. This work describes a new proline biosensor that allows the monitoring of proline levels in a non-invasive manner in root exudates and nodules of legume plants. Methods The proline biosensor was constructed by cloning the promoter region of pRL120553, a gene with high levels of induction in the presence of proline, in front of the lux cassette in Rhizobium leguminosarum bv. viciae. Results Free-living assays show that the proline biosensor is sensitive and specific for proline. Proline was detected in both root exudates and nodules of pea plants. The luminescence detected in bacteroids did not show variations during osmotic stress treatments, but significantly increased during recovery. Conclusions This biosensor is a useful tool for the in vivo monitoring of proline levels in root exudates and bacteroids of symbiotic root nodules, and it contributes to our understanding of the metabolic exchange occurring in nodules under abiotic stress conditions.

The current world population together with the predictions of further growth suggest that it is necessary to increase crop yields worldwide. Legumes are the second most important food crop after cereals, and thanks to their ability to establish a symbiotic relationship with soil bacteria, the impact of the use of nitrogen fertilizers on the environment is reduced. This symbiosis gives rise to the process known as biological nitrogen fixation (BNF), which consists in the reduction of molecular nitrogen to ammonium, from which plants synthesize organic nitrogenous products essential for their nutrition. Unfortunately, BNF is a very sensitive process to biotic and abiotic stresses such as salinity, drought, or nutrient limitation, among others. The general aim of this work was to gain further insights in the regulation of BNF and the physiological and biochemical mechanisms that plant activate in response to abiotic stresses. In order to counteract the negative effects of osmotic stresses, plant and bacteria are able to synthesise osmoprotectant compounds to maintain cell viability, e.g. the amino acid proline. A real-time monitoring of proline utilisation in both plant and bacterial systems is a first key step towards understanding the multiple roles of this molecule under osmotic stress situations. Our results in chapter one showed that, in bacteroids, proline accumulation does not occur during the stress phase, but during recovery, once optimal plant growth conditions are re-established. In chapter two, a proteomic and metabolic study was performed to gain further insights about amino acid metabolism in pea nodules. In the classical model of nutrient exchange between symbionts, plant supplies energy in the form of dicarboxylates to the N2-fixing bacteroids in exchange for ammonium. However, this classic model was challenged upon the observation that mutations in the general ABC amino-acid transporters AapJQMP and BraDEFGC in Rhizobium leguminosarum resulted in N starvation symptoms in both pea and bean plants. The uptake of branched-chain amino acids (BCAAs) from the plant by the bacteroid was found to be essential for an effective BNF at least in R. leguminosarum species. Another experimental approach to further understand the role of amino acid metabolism in nodules is the application of compounds that inhibit the biosynthesis of BCAAs in plant cells such as group B herbicides. These approaches allowed us to verify how the blockage of BCAA transport between symbionts had a greater effect on nodule metabolism than the inhibition of BCAA biosynthesis. In fact, BCAA biosynthesis was also inhibited due to the aap/bra double mutation. In chapter two, we also evaluate the effect of water deficit on nodule proteome, since among the strategies that plants use in response to abiotic stresses there are several related to amino acid metabolism. This study highlights the relevance of low abundant amino acids, such as methionine, aromatic amino acids or γ-aminobutyric acid, in the response to water deficit. Finally, until now no attempt has been made to carry out an integral approach in which possible changes caused by drought in carbon (C) allocation, and in addition, the effect on the consumption or accumulation of metabolites in all plant organs be analysed. For this purpose, in chapter three, the effect of drought on both the [U-13C]-sucrose distribution and ureides, organic acids and carbohydrates content were analysed. We found that drought decreased 13C transport to sink tissues and changed the priority of C allocation between sink organs.