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Applied and Environmental Microbiology, July 2005, p. 3858-3865, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3858-3865.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of Citrate Synthase from Geobacter sulfurreducens and Evidence for a Family of Citrate Synthases Similar to Those of Eukaryotes throughout the Geobacteraceae

Daniel R. Bond,^1*, Tünde Mester,^1, Camilla L. Nesbø,² Andrea V. Izquierdo-Lopez,¹ Frank L. Collart,³ and Derek R. Lovley¹

Department of Microbiology, University of Massachusetts—Amherst, Amherst, Massachusetts 01003,¹ Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada,² Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439³

Received 19 November 2004/ Accepted 27 January 2005

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (k_cat = 8.3 s^–1; K_m = 14.1 and 4.3 µM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (K_i = 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K⁺ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.

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* Corresponding author. Mailing address: BioTechnology Institute, University of Minnesota, 140 Gortner, 1479 Gortner Ave., St. Paul, MN 55108. Phone: (612) 624-8619. Fax: (612) 625-1700. E-mail: dbond{at}umn.edu.

These authors contributed equally to this work.

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Applied and Environmental Microbiology, July 2005, p. 3858-3865, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3858-3865.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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