Metabolic control of nitrogen fixation in rhizobium-legume symbioses

Abstract

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD+ ratio than sugars. Modeling and 13C metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing dicarboxylates as the energy and electron source donors for N2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses.

Genome-Scale Metabolic Modelling of Lifestyle Changes in Rhizobium leguminosarum

“…We next defined a biomass function based on evidence from the literature ( Table S1 ). Because our previous work showed the dependence of carbon polymer synthesis on environmental conditions ( 23 ), demand reactions for polymers such as glycogen, polyhydroxybutyrate (PHB), and exopolysaccharides were included in the model to allow for their flexible accumulation. The final model contained 1,224 genes, 1,257 reactions, and 984 metabolites ( Table 1 ), and was named iCS1224 according to standard naming conventions.…”

“…To extract a model specific for nitrogen-fixing bacteroids, we used dRNA-Seq data derived from the middle of nodules ( 67 ), which contains fully differentiated bacteroids performing nitrogen fixation ( 66 ). In addition, a list of 38 genes that were present in the model and encoded proteins significantly upregulated in bacteroids compared to free-living bacteria ( 23 ) and the dct genes ( 75 ) were specified to ensure inclusion of those genes in the bacteroid model. Nitrogenase activity was set as the objective function while low levels of protein and fatty acid production were enforced through demand reactions.…”

“…Gene essentiality data from the INSeq screen were not included for model contextualization due to the aforementioned difficulty of determining the developmental stage where a gene is essential inside the nodule environment. In contrast to our previously reconstructed model for bacteroid metabolism (iCS323) ( 23 ), the bacteroid model was thus obtained using a top-down approach to constrain iCS1224 rather than assembling individual pathways in a bottom-up manner, and uptake of a wider range of nutrients was enabled. The bacteroid model contained 307 reactions and 308 metabolites and achieved significant correlation with the transcriptome data (Spearman’s Rho = 0.348, P < 0.001) when nitrogenase activity was constrained to 65% of its maximum.…”

“…Due to the gene-protein-reaction associations contained in metabolic models, they also provide a convenient framework for contextualizing genome-scale data obtained by omics technologies, such as transcriptomics or proteomics ( 21 ). Most metabolic models of rhizobial species so far have focused on fully differentiated bacteroids ( 22 – 26 ). One in silico study of Sinorhizobium meliloti has addressed the differences in metabolism for free-living growth in the bulk soil, growth of the rhizosphere, and symbiotic nitrogen fixation during the bacteroid stage ( 9 ).…”

Maintaining osmotic balance in legume nodules

“…Plants control O 2 levels in nodules with an O 2 diffusion barrier and the synthesis of O 2 binding haem proteins, the leghaemoglobins ( Ott et al , 2005 ). Such a tight control of oxygen supply by legumes while providing dicarboxylates as the energy and electron source donors for N 2 fixation, with their high O 2 requirement for metabolism, promotes ammonia secretion rather than assimilation into the central amino acid glutamate by bacteroids ( Schulte et al , 2021 ). However, modelling, in agreement with experimental studies, also shows that as the O 2 supply becomes even more limited, alanine as well as ammonia will be secreted by bacteroids.…”