A gene cluster consisting of three consecutive genes, matABC, was isolated using a probe prepared from amino acid sequence information of Rhizobium trifolii malonyl-CoA synthetase, and was subsequently sequenced. The sequences of matA and matB were overlapped by four base pairs, whereas the intergenic region between matB and matC had 95 base pairs. The upstream region contained DNA sequences which are typical for an Escherichia coli sigma70 promoter, and no other open reading frame was found within 400 bp downstream of matC. The ribosome-binding sites were found 7 to 12 base pairs upstream of each gene. MatA gene encoded a polypeptide of 462 amino acid residues, with deduced molecular mass of 51414 Da. A glutathione-S-transferase-MatA fusion protein has been purified and MatA was shown to have an intrinsic malonyl-CoA decarboxylase activity (Km = 0.47 mM; Vmax = 52 micromol x min(-1) x mg(-1)). MatB encoded a polypeptide of 504 amino acid residues with deduced molecular mass of 54612 Da. MatB was also purified from E. coli transformant carrying the gene cluster. The enzyme was essentially indistinguishable from the wild-type malonyl-CoA synthetase of R. trifolii by the criteria of polyacrylamide gel electrophoresis and biochemical properties. MatC encoded a 46453-Da protein with a high content of hydrophobic residues. The deduced amino acid sequences of matC showed identity to some extent with anaerobic C4-dicarboxylate carrier proteins from E. coli (25%) and Haemophilus influenzae (17%). MatC protein appears to be an integral membrane protein that could function as a malonate carrier. The formation of acetyl-CoA and malonyl-CoA from malonate was confirmed by thin-layer chromatographic analysis. These results strongly suggest that the gene cluster encodes proteins involved in the malonate-metabolizing system, malonate-->malonyl-CoA-->acetyl-CoA, in R. trifolii and that the metabolic pathway in the malonate-rich clover nodule might play an important role in symbiosis.