Three separate pathways in <i>Rhizobium leguminosarum</i> maintain phosphatidylcholine biosynthesis, which is required for symbiotic nitrogen fixation with clover

Understanding the molecular mechanisms of symbiotic nitrogen fixation has important implications for sustainable agriculture. The presence of the phospholipid phosphatidylcholine (PC) in the membrane of rhizobia is critical for the establishment of productive nitrogen-fixing root nodules on legume plants. The reasons for the PC requirement are unknown. Here, we employed <i>Rhizobium leguminosarum</i> and clover as model system for a beneficial plant-microbe interaction. We found that <i>R. leguminosarum</i> produces PC by three distinct pathways. The relative contribution of these pathways to PC formation was determined in an array of single, double, and triple mutant strains. Several of the PC biosynthesis enzymes were purified and biochemically characterized. Most importantly, we demonstrated the essential role of PC formation by <i>R. leguminosarum</i> in nitrogen fixation and pinpointed a specific enzyme indispensable for plant-microbe interaction. Our study offers profound insights into bacterial PC biosynthesis and its pivotal role in biological nitrogen fixation.