Project description:Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR encodes a transcription factor annotated as PHA (polyhydroxyalkanoate) accumulation regulator. We found that PhaR not only controls the PHA cycle but also acts as a global regulator of excess carbon allocation by controlling the expression of fixK2 and nifA genes, both encoding key transcription factors for microoxic and symbiotic metabolism in B. diazoefficiens. The function of PhaR was expanded by a multi-pronged approach that includes analysis of the effects of phaR mutation at transcriptional and protein levels of putative PhaR targets and direct control mediated by PhaR determined by EMSA assays. We also were able to identify PhaR, phasins and other proteins associated with PHA granules which confirmed a global function of PhaR in microoxia.
Project description:Metabolomics and transcriptomics of Bradyrhizobium diazoefficiens-induced root nodules Bradyrhizobium diazoefficiens is a nitrogen-fixing endosymbiont, which can grow inside root-nodule cells of the agriculturally important soybean and other host plants. Our previous studies described B. diazoefficiens host-specific global expression changes occurring during legume infection at the transcript and protein level. In order to further characterize nodule metabolism, we here determine by flow injection -time of flight mass spectrometry analysis the metabolome of i) nodules and roots from four different B. diazoefficiens host plants, ii) soybean nodules harvested at different time points during nodule development, and iii) soybean nodules infected by two strains mutated in key genes for nitrogen fixation, respectively. Ribose (soybean), tartaric acid (mungbean), hydroxybutanoyloxybutanoate (siratro) and catechol (cowpea) were among the metabolites found to be specifically elevated in one of the respective host plants. While the level of C4-dicarboxylic acids decreased during soybean nodule development, we observed an accumulation of trehalose-phosphate at 21 days post infection (dpi). Moreover, nodules from non-nitrogen-fixing bacteroids (nifA and nifH mutants) showed specific metabolic alterations; these were also supported by transcriptomics data that was generated for the two mutant strains and were helpful to separate for some examples the respective bacterial and plant contributions to the metabolic profile. The alterations included signs of nitrogen limitation in both mutants, and an increased level of a phytoalexin in nodules induced by the nifA mutant, suggesting that the tissue of these nodules exhibits defense and stress reactions.
Project description:The form and physiology of Bradyrhizobium diazoefficiens after the decline of symbiotic nitrogen fixation has been characterized. Proteomic analyses showed that the post-symbiotic B. diazoefficiens undergo metabolic remodeling as well defined groups of proteins declined, increased or remained unchanged from 56 to 119 days after planting suggesting a transition to a hemibiotrophic-like lifestyle. Enzymatic analysis showed distinct patterns in both the cytoplasm and the periplasm. Similar to the bacteroid, the post-symbiotic bacteria rely on a non-citric acid cycle supply of succinate and although viable, they did not demonstrate the ability to grow within the senescent nodule.
