Glutamate is usually synthesized from acetyl coenzyme A (acetyl-CoA) via citrate, isocitrate, and 2-oxoglutarate. Genome analysis revealed that in Syntrophus aciditrophicus, the gene for Si-citrate synthase is lacking. An alternative pathway starting from the catabolic intermediate glutaconyl-CoA via 2-hydroxyglutarate could be excluded by genomic analysis. On the other hand, a putative gene (SYN_02536; NCBI gene accession no. CP000252.1) annotated as coding for isopropylmalate/citramalate/homocitrate synthase has been shown to share 49% deduced amino acid sequence identity with the gene encoding Re-citrate synthase of Clostridium kluyveri. We cloned and overexpressed this gene in Escherichia coli together with the genes encoding the chaperone GroEL. The recombinant homotetrameric enzyme with a C-terminal Strep-tag (4 × 72,892 Da) was separated from GroEL on a Strep-Tactin column by incubation with ATP, K(+), and Mg(2+). The pure Re-citrate synthase used only acetyl-CoA and oxaloacetate as the substrates. As isolated, the enzyme contained stoichiometric amounts of Ca(2+) (0.9 Ca/73 kDa) but achieved higher specific activities in the presence of Mn(2+) (1.2 U/mg) or Co(2+) (2.0 U/mg). To determine the stereospecificity of the enzyme, [(14)C]citrate was enzymatically synthesized from oxaloacetate and [1-(14)C]acetyl-CoA; the subsequent cleavage by Si-citrate lyase yielded unlabeled acetate and labeled oxaloacetate, demonstrating that the enzyme is a Re-citrate synthase. The production of Re-citrate synthase by S. aciditrophicus grown axenically on crotonate was revealed by synthesis of [(14)C]citrate in a cell extract followed by stereochemical analysis. This result was supported by detection of transcripts of the Re-citrate synthase gene in axenic as well as in syntrophic cultures using quantitative reverse transcriptase PCR (qRT-PCR).