| Popis: |
In this thesis the metabolic flow of carbon and electrons enabling N2 fixation in the agriculturally relevant model organisms Rhizobium leguminosarum and Azorhizobium caulinodans was investigated. Building upon functional genomic data-sets including transcriptomics, metabolomics and mutagenic screens, the core metabolic reactions needed for dicarboxylate ORS571 and RL3841. Isocitrate dehydrogenase was found to be essential for N2-fixation and operation of the TCA-cycle in RL3841, contradicting previously published INSeq data. This was due to TA insertions in gene termini misleading the Hidden Markov Model used to asses INSeq data. As a result of this, new bioinformatic pipelines have been developed to avoid such errors. The redox sink poly-3-hydroxy butyrate (PHB) was shown to be non-essential for N2 fixation in ORS571. Previously published results showing PHB synthesis to be essential are actually due to missregulation of phaR translation in phaC mutants used to block PHB synthesis, rather than the loss of PHB as a redox store. This bring ORS571 in line with other species of rhizobia, where PHB defective mutants retained their ability to fix N2. Two alternative metabolic pathways, the malonate shunt in ORS571 and the isocitrate shunt in RL3841 have both been investigated and shown to be inoperative at biologically relevant levels in their respective organisms. In ORS571, the entirety of the carbon flux between succinate and acetyl-CoA has been demonstrated to proceed via NAD+ dependent malic enzyme (DME) and phosphoenolpyruvate carboxykinase, allowing existing models of carbon flux to be revised to remove the erroneous malonate shunt. In addition, DME has been demonstrated to be essential for N2 fixation, but dispensable for growth on succinate, indicating a disconnect between the requirements of N2 fixation and dicarboxylate metabolism. |
