The reduction of atmospheric nitrogen (N2) to ammonia (N2 fixation) by bacteria living symbiotically with legume hosts contributes approximately 50% of the biosphere’s available nitrogen. This association is established when soil bacteria (rhizobia) infect legume root nodules, differentiating into their N2-fixing bacteroid form and expressing the nitrogenase enzyme complex. In return for a supply of reduced N2, the plant provides the bacteroid carbon in the form of the C4 dicarboxylates malate, fumarate and succinate. Currently, it is unclear how bacteroids metabolise these organic acids to yield sufficient ATP and reductant to power nitrogenase. Although, evidence from some symbiotic interactions, such as those between Sinorhizobium meliloti and Medicago sativa (alfalfa), are consistent with the TCA cycle being the principle pathway to fuel N2 fixation, Bradyrhizobium japonicum is capable of fixing N2 in the absence of a fully functional TCA cycle on its host Glycine max (soybean). This suggests that in some symbioses, the TCA cycle may not be essential for N2 fixation.
While the role of the TCA cycle in N2 fixation has been well studied in a range of symbioses, its role in the association between Phaseolus vulgarius (common bean) and Rhizobium spp. has received relatively little attention. My research aims to create separate mutations in two essential TCA cycle genes in two different rhizobia strains, Rhizobium leguminosarum (8002) and Rhizobium tropicii (CIAT899), in an attempt to further characterise and understand the role of the TCA cycle in N2 fixation. A site directed approach will be applied to create mutations in both 8002 and 899, to create mutations in isocitrate dehydrogenase and 2-oxogluturate dehydrogenase, while a random mutagenesis approach will be applied to 899, allowing for characterisation of the role of other metabolic genes, including those targeted in the site-directed approach. Transconjugants will be screened for a metabolic phenotype as well as a symbiotic phenotype on their host plant, Phaseolus vulgarius to assess their impact on the N2 fixation process.