Catabolism of alpha-ketoglutarate by a sucA mutant of Bradyrhizobium japonicum: Evidence for an alternative tricarboxylic acid cycle

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succin ate, and fumarate. However, a Bradyrhizobium japonicum sucA mutant that is missing alpha-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with so ybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the sucA mutant displayed 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-C-14]ketoglutarate and [U-C-14]g lutamate at rates similar to those of wild-type B. japonicum, indicating th at there was an alternative route for alpha-ketoglutarate catabolism. Becau se cell extracts from B. japonicum decarboxylated [U-C-14]glutamate very sl owly, the gamma-aminobutyrate shunt is unlikely to be the pathway responsib le for alpha-ketoglutarate catabolism in the mutant. In contrast, cell extr acts from both the wild type and mutant showed a coenzyme A (CoA)-independe nt alpha-ketoglutarate decarboxylation activity. This activity was independ ent of pyridine nucleotides and was stimulated by thiamine PPi. Thin-layer chromatography showed that the product of alpha-ketoglutarate decarboxylati on was succinic semialdehyde. The CoA-independent alpha-ketoglutarate decar boxylase, along with succinate semialdehyde dehydrogenase, may form an alte rnative pathway for alpha-ketoglutarate catabolism, and this pathway may en hance TCA cycle function during symbiotic nitrogen fixation.