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Journal of Bacteriology, February 2007, p. 940-949, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.00948-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Pathway Confirmation and Flux Analysis of Central Metabolic Pathways in Desulfovibrio vulgaris Hildenborough using Gas Chromatography-Mass Spectrometry and Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry{triangledown} ,{dagger}

Yinjie Tang,1,2,{ddagger} Francesco Pingitore,1,2,{ddagger} Aindrila Mukhopadhyay,1,2,{ddagger} Richard Phan,1,3 Terry C. Hazen,1,3 and Jay D. Keasling1,2,4*

Virtual Institute of Microbial Stress and Survival,1 Physical Biosciences Division,2 Earth Sciences Division, Lawrence Berkeley National Laboratory,3 Departments of Chemical Engineering and Bioengineering, University of California, Berkeley, California4

Received 29 June 2006/ Accepted 5 November 2006

Flux distribution in central metabolic pathways of Desulfovibrio vulgaris Hildenborough was examined using 13C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both gas chromatography-mass spectrometry (GC-MS) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) indicate the lack of an oxidatively functional tricarboxylic acid (TCA) cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80% of the lactate was converted to acetate and that the reactions involved are the primary route of energy production [NAD(P)H and ATP production]. Independently of the TCA cycle, direct cleavage of acetyl coenzyme A to CO and 5,10-methyl tetrahydrofuran also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or the reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports [the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (S)-citrate synthase]. These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris and also demonstrate that FT-ICR MS is a powerful tool for isotopomer analysis, overcoming the problems with both GC-MS and nuclear magnetic resonance spectroscopy.

* Corresponding author. Mailing address: Berkeley Center for Synthetic Biology, 717 Potter St., Berkeley, CA 94720. Phone: (510) 495-2620. Fax: (510) 495-2630. E-mail: keasling{at}berkeley.edu.

{triangledown} Published ahead of print on 17 November 2006.

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

{ddagger} Y.T., F.P., and A.M. contributed equally to this study.

Journal of Bacteriology, February 2007, p. 940-949, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.00948-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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