Metabolic flux analysis of the nitrogen-fixing bacterium Azorhizobium caulinodans

Symbiotic nitrogen fixation in rhizobial-legume symbioses is important for agriculture and the establishment of a successful symbiosis depends on the metabolic integration of the rhizobia inside the host nodules. This study aims to understand the metabolic adaptations in a rhizobium during nitrogen fixation. The study uses 13C-metabolic flux analysis to derive carbon fluxes across the metabolic network of the model diazotrophic rhizobium, Azorhizobium caulinodans ORS571. The flux maps obtained for aerobic, micro-aerobic and nitrogen-fixing free-living cultures identified these metabolic adjustments as an increase in polyhydroxybutyrate synthesis and tricarboxylic acid cycle fluxes required to sustain growth and metabolism during micro-aerobic nitrogen fixation. Flux maps for bacteroids, isolated from the root and stem nodules of S. rostrata, highlighted reduced tricarboxylic acid cycle fluxes and increased flux through the methyl malonate semialdehyde pathway relative to the free-living N2-fixing cultures. Polyhydroxybutyrate was indispensable for nitrogen fixation and diazotrophic growth in free-living cultures, and for establishment of effective symbiosis with the host, but it was less important for the growth of non-nitrogen fixing free-living cultures under aerobic and micro-aerobic conditions. Deletion of polyhydroxybutyrate under aerobic conditions resulted in slow growth and caused changes in biomass synthesis, mainly affecting lipids and exo-polysaccharide. Under micro-aerobic conditions, deletion of polyhydroxybutyrate also resulted in slow growth and increased fluxes through the tricarboxylic acid cycle, gluconeogenesis, and the pentose phosphate pathway. There was a significant rise in lipid production that potentially substituted for polyhydroxybutyrate and maintained redox balance during micro-aerobic growth. The combined findings from the flux maps highlight the adjustments in central carbon metabolism associated with free-living and symbiotic nitrogen fixation. This knowledge is important for research aimed at improving the existing symbiosis and developing synthetic symbioses between rhizobia and non-legumes, which will reduce dependence on chemical nitrogen fertilizers.