Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation.

Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen-fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH-N cycle as a novel metabolic pathway that co-catabolizes plant-provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH-N cycle including transaminases that interlink the co-catabolism of arginine and succinate. The CATCH-N cycle uses N2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism-two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH-N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCH-N cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant-associated organisms with new properties to improve nitrogen fixation.

Quantitative Selection Analysis of Bacteriophage φCbK Susceptibility in Caulobacter crescentus.

Classical molecular genetics uses stringent selective conditions to identify mutants with distinct phenotypic responses. Mutations giving rise to less pronounced phenotypes are often missed. However, to gain systems-level insights into complex genetic interaction networks requires genome-wide assignment of quantitative phenotypic traits. In this paper, we present a quantitative selection approach coupled with transposon sequencing (QS-TnSeq) to globally identify the cellular components that orchestrate susceptibility of the cell cycle model bacterium Caulobacter crescentus toward bacteriophage φCbK infection. We found that 135 genes representing 3.30% of the Caulobacter genome exhibit significant accumulation of transposon insertions upon φCbK selection. More than 85% thereof consist of new factors not previously associated with phage φCbK susceptibility. Using hierarchical clustering of dose-dependent TnSeq datasets, we grouped these genes into functional modules that correlate with different stages of the φCbK infection process. We assign φCbK susceptibility to eight new genes that represent novel components of the pilus secretion machinery. Further, we demonstrate that, from 86 motility genes, only seven genes encoding structural and regulatory components of the flagellar hook increase phage resistance when disrupted by transposons, suggesting a link between flagellar hook assembly and pili biogenesis. In addition, we observe high recovery of Tn5 insertions within regulatory sequences of the genes encoding the essential NADH:ubiquinone oxidoreductase complex indicating that intact proton motive force is crucial for effective phage propagation. In sum, QS-TnSeq is broadly applicable to perform quantitative and genome-wide systems-genetics analysis of complex phenotypic traits.