A novel biosensor to monitor proline in pea root exudates and nodules under osmotic stress and recovery.

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.

Genetic basis for denitrification in Ensifer meliloti.

BACKGROUND: Denitrification 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. RESULTS: E. 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. CONCLUSION: The 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.

Denitrification in Sinorhizobium meliloti.

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.