Investigation of Carbon Metabolism in “ <i>Dehalococcoides ethenogenes</i> ” Strain 195 by Use of Isotopomer and Transcriptomic Analyses

ABSTRACT Members of the genus “ Dehalococcoides ” are the only known microorganisms that can completely dechlorinate tetrachloroethene and trichloroethene to the innocuous end product, ethene. This study examines the central metabolism in “ Dehalococcoides ethenogenes ” strain 195 via 13 C-labeled tracer experiments. Supported by the genome annotation and the transcript profile, isotopomer analysis of key metabolites clarifies ambiguities in the genome annotation and identifies an unusual biosynthetic pathway in strain 195. First, the 13 C-labeling studies revealed that strain 195 contains complete amino acid biosynthesis pathways, even though current genome annotation suggests that several of these pathways are incomplete. Second, the tricarboxylic acid cycle of strain 195 is confirmed to be branched, and the Wood-Ljungdahl carbon fixation pathway is shown to not be functionally active under our experimental conditions; rather, CO 2 is assimilated via two reactions, conversion of acetyl-coenzyme A (acetyl coenzyme A [acetyl-CoA]) to pyruvate catalyzed by pyruvate synthase (DET0724-0727) and pyruvate conversion to oxaloacetate via pyruvate carboxylase (DET0119-0120). Third, the 13 C-labeling studies also suggested that isoleucine is synthesized from acetyl-CoA and pyruvate via citramalate synthase (CimA, EC 2.3.1.182), rather than from the common pathway via threonine ammonia-lyase (EC 4.3.1.19). Finally, evidence is presented that strain 195 may contain an undocumented citrate synthase (&gt;95% Re -type stereospecific), i.e., a novel Re -citrate synthase that is apparently different from the one recently reported in Clostridium kluyveri .