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Investigation of carbon metabolism in "Dehalococcoides ethenogenes" 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 (>95% Re-type stereospecific), i.e., a novel Re-citrate synthase that is apparently different from the one recently reported in Clostridium kluyveri.

SUBMITTER: Tang YJ

PROVIDER: S-EPMC2725585 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Investigation of carbon metabolism in "Dehalococcoides ethenogenes" strain 195 by use of isotopomer and transcriptomic analyses.

Tang Yinjie J YJ Yi Shan S Zhuang Wei-Qin WQ Zinder Stephen H SH Keasling Jay D JD Alvarez-Cohen Lisa L

Journal of bacteriology 20090612 16

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 pathw ...[more]

PMID: 19525347

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Project description:Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing D. ethenogenes strain 195 to fixed nitrogen limitation (FNL). As expected, the nitrogen-fixing (nif) genes were differentially up-regulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly up-regulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally down regulated in response to FNL and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition while others were associated with general stress responses. A number of genes related to the biological functions of nucleotide synthesis, replication, transcription, translation, and post-translational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate cellular nitrogen status in the environment. Dehalococcoides ethenogenes strain 195 was grown with or without ammonium in batch cultures with trichloroethene as the electron acceptor, acetate as the carbon source, and hydrogen as the electron donor. Microarrays were used to compare the transcriptome during fixed nitrogen limitation and triplicate biological samples were prepared for each condition. Differentially expressed genes between the two growth conditions were identified using a two-tailed Student's t-test, a false discovery rate of less than 1%, an absolute hybridization signal intensity of 300 or greater in one of the two conditions, and an expression ratio of two-fold or greater.

Dataset Information

Investigation of carbon metabolism in "Dehalococcoides ethenogenes" strain 195 by use of isotopomer and transcriptomic analyses.

Publications

Investigation of carbon metabolism in "Dehalococcoides ethenogenes" strain 195 by use of isotopomer and transcriptomic analyses.

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