Rhizobium leguminosarum strains unable to grow on rhamnose as a sole carbon source are less competitive for nodule occupancy. To determine if the ability to use rhamnose as a sole carbon source affects competition for nodule occupancy in Sinorhizobium meliloti, Tn5 mutants unable to use rhamnose as a sole carbon source were isolated. S. meliloti mutations affecting rhamnose utilization were found in two operons syntenous to those of R. leguminosarum. Although the S. meliloti Tn5 mutants were complemented using an R. leguminosarum cosmid that contains the entire wild-type rhamnose catabolic locus, complementation did not occur if the cosmids carried Tn5 insertions within the locus. Through a series of heterologous complementation experiments, enzyme assays, gene fusion, and transport experiments, we show that the S. meliloti regulator, RhaR, is dominant to its R. leguminosarum counterpart. In addition, the data support the hypothesis that the R. leguminosarum kinase is capable of directly phosphorylating rhamnose and rhamnulose, whereas the S. meliloti kinase does not possess rhamnose kinase activity. In nodule competition assays, S. meliloti mutants incapable of rhamnose transport were shown to be less competitive than the wild-type and had a decreased ability to bind plant roots in the presence of rhamnose. The data suggests that rhamnose catabolism is a general determinant in competition for nodule occupancy that spans across rhizobial species. Full article

Isolated from desert, the nitrogen-fixing bacterium Ensifer aridi LMR001 is capable of survival under particularly harsh environmental conditions. To obtain insights in molecular mechanisms involved in stress adaptation, a recent study using RNAseq revealed that the RpoE2-mediated general stress response was activated under mild saline stress but appeared non-essential for the bacterium to thrive under stress and develop the symbiosis. Functions associated with the stress response included the metabolisms of trehalose, methionine, and inositol. To explore the roles of these metabolisms in stress adaptation and symbiosis development, and the possible regulatory mechanisms involved, mutants were generated notably in regulators and their transcriptions were studied in various mutant backgrounds. We found that mutations in regulatory genes nesR and sahR of the methionine cycle generating S-adenosylmethionine negatively impacted symbiosis, tolerance to salt, and motility in the presence of NaCl. When both regulators were mutated, an increased tolerance to detergent, oxidative, and acid stresses was found, suggesting a modification of the cell wall components which may explain these phenotypes and support a major role of the fine-tuning methylation for symbiosis and stress adaptation of the bacterium. In contrast, we also found that mutations in the predicted trehalose transport and utilization regulator ThuR and the trehalose phosphate phosphatase OtsB-encoding genes improved symbiosis and growth in liquid medium containing 0.4 M of NaCl of LMR001ΔotsB, suggesting that trehalose metabolism control and possibly trehalose-6 phosphate cellular status may be biotechnologically engineered for improved symbiosis under stress. Finally, transcriptional fusions of gfp to promoters of selected genes and expression studies in the various mutant backgrounds suggest complex regulatory interplay between inositol, methionine, and trehalose metabolic pathways. Full article