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Genetic Characterization of a Single Bifunctional Enzyme for Fumarate Reduction and Succinate Oxidation in Geobacter sulfurreducens and Engineering of Fumarate Reduction in Geobacter metallireducens

Jessica E. Butler,^* Richard H. Glaven, Abraham Esteve-Núñez,^¶ Cinthia Núñez, Evgenya S. Shelobolina, Daniel R. Bond, and Derek R. Lovley

Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003

Received 9 September 2005/ Accepted 18 October 2005

The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.

^¶ Present address: Centro de Astrobiología, Instituto Nacional de Técnica Aerospacial, Madrid, Spain.

Present address: Departamento de Microbiologia Molecular, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos, Mexico.

Present address: Department of Geology and Geophysics, University of Wisconsin, Madison, Wis.

Present address: Department of Microbiology, Biotechnology Institute, University of Minnesota, St. Paul, Minn.

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