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The role of malonate in symbiotic nitrogen metabolism has long been controversial, although it is known to occur in legume roots, especially in the nodules. Here we report that malonate metabolism plays a key role in the differentiation of bacteroids Rhizobium legumi-nosarum bv. trifolii in clover nodules. An operon, mat, that consists of three consecutive genes (matABC) has been discovered. Mat encodes enzymes that catalyze the uptake and conversion of malonate to acetyl-CoA through malonyl-CoA. A mutant bacteria, which re-placed matB that encodes malonyl-CoA synthetase with a kanamycin resistant gene, was generated and infected with white clover. Clover growth was consid-erably reduced, even though nodules were formed. However, the nodules were filled with vacuoles, but not with bacteroids. This indicates that malonate me-tabolism is an important requirement for the forma-tion of mature nodules that are filled with bacteroids.