Catabolism of alpha -Ketoglutarate by a sucA Mutant of Bradyrhizobium japonicum: Evidence for an Alternative Tricarboxylic Acid Cycle

doi:10.1128/JB.182.10.2838-2844.2000

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Journal of Bacteriology, May 2000, p. 2838-2844, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Catabolism of $alpha$ -Ketoglutarate by a sucA Mutant of Bradyrhizobium japonicum: Evidence for an Alternative Tricarboxylic Acid Cycle

Laura S. Green,¹^,²^,^* Youzhong Li,² David W. Emerich,¹ Fraser J. Bergersen,² and David A. Day²^,

Biochemistry Department, University of Missouri, Columbia, Missouri,¹ and Division of Biochemistry and Molecular Biology, Australian National University, Canberra, Australian Capital Territory, Australia²

Received 3 January 2000/Accepted 3 March 2000

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acidsubstrates malate, succinate, and fumarate. However, a Bradyrhizobiumjaponicum sucA mutant that is missing $alpha$ -ketoglutarate dehydrogenaseis able to grow on malate as its sole source of carbon. This mutantalso fixes nitrogen in symbiosis with soybean, where dicarboxylicacids are its principal carbon substrate. Using a flow chambersystem to make direct measurements of oxygen consumption and ammoniumexcretion, we confirmed that bacteroids formed by the sucA mutantdisplayed wild-type rates of respiration and nitrogen fixation.Despite the absence of $alpha$ -ketoglutarate dehydrogenase activity,whole cells of the mutant were able to decarboxylate $alpha$ -[U-¹⁴C]ketoglutarate and [U-¹⁴C]glutamate at rates similar to those of wild-type B. japonicum,indicating that there was an alternative route for $alpha$ -ketoglutaratecatabolism. Because cell extracts from B. japonicum decarboxylated[U-¹⁴C]glutamate very slowly, the $gamma$ -aminobutyrate shunt is unlikelyto be the pathway responsible for $alpha$ -ketoglutarate catabolism inthe mutant. In contrast, cell extracts from both the wild typeand mutant showed a coenzyme A (CoA)-independent $alpha$ -ketoglutaratedecarboxylation activity. This activity was independent of pyridinenucleotides and was stimulated by thiamine PP_i. Thin-layer chromatographyshowed that the product of $alpha$ -ketoglutarate decarboxylation wassuccinic semialdehyde. The CoA-independent $alpha$ -ketoglutarate decarboxylase,along with succinate semialdehyde dehydrogenase, may form an alternativepathway for $alpha$ -ketoglutarate catabolism, and this pathway may enhanceTCA cycle function during symbiotic nitrogenfixation.

^* Corresponding author. Mailing address: Biochemistry Department, 117 Schweitzer Hall, University of Missouri, Columbia, MO65211. Phone: (573) 771-9076. Fax: (573) 882-5635. E-mail: greenl{at}missouri.edu.

Present address: Department of Biochemistry, University of Western Australia, Nedlands, Western Australia,Australia.

Journal of Bacteriology, May 2000, p. 2838-2844, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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Catabolism of -Ketoglutarate by a sucA Mutant of Bradyrhizobium japonicum: Evidence for an Alternative Tricarboxylic Acid Cycle

Catabolism of $alpha$ -Ketoglutarate by a sucA Mutant of Bradyrhizobium japonicum: Evidence for an Alternative Tricarboxylic Acid Cycle