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Journal of Bacteriology, August 2009, p. 5224-5231, Vol. 191, No. 16
0021-9193/09/$08.00+0     doi:10.1128/JB.00085-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Investigation of Carbon Metabolism in "Dehalococcoides ethenogenes" Strain 195 by Use of Isotopomer and Transcriptomic Analyses ^,

Yinjie J. Tang,^1, Shan Yi,^2, Wei-Qin Zhuang,^2, Stephen H. Zinder,⁴ Jay D. Keasling,⁵ and Lisa Alvarez-Cohen^2,3*

Energy, Environmental and Chemical Engineering, Washington University, One Brookings Drive, Box 1180, St. Louis, Missouri 63130,¹ Department of Civil and Environmental Engineering, University of California, Berkeley, California 94720,² Earth Sciences Division, Lawrence Berkeley National Laboratory, Cyclotron Rd., Berkeley, California 94720,³ Section of Microbiology, Cornell University, Ithaca, New York 14853,⁴ Joint Bio-Energy Institute, Lawrence Berkeley National Laboratory, Emeryville, California 94608⁵

Received 23 January 2009/ Accepted 2 June 2009

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 ¹³C-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 ¹³C-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, CO₂ 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 ¹³C-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.

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* Corresponding author. Mailing address: 760 Davis Hall, Department of Civil and Environmental Engineering, University of California, Berkeley, CA 94720-1710. Phone: (510) 643-5969. Fax: (510) 642-7483. E-mail: alvarez{at}ce.berkeley.edu

Published ahead of print on 12 June 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

Y.J.T., S.Y., and W.-Q.Z. contributed equally to this study.

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Journal of Bacteriology, August 2009, p. 5224-5231, Vol. 191, No. 16
0021-9193/09/$08.00+0     doi:10.1128/JB.00085-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.