Oxidation of acetate through reactions of the citric acid cycle by Geobacter sulfurreducens in pure culture and in syntrophic coculture

Abstract

Geobacter sulfurreducens strain PCA oxidized acetate to CO 2 via citric acid cycle reactions during growth with acetate plus fumarate in pure culture, and with acetate plus nitrate in coculture with Wolinella succinogenes. Acetate was activated by succinyl-CoA:acetate CoA-transferase and also via acetate kinase plus phosphotransacetylase. Citrate was formed by citrate synthase. Soluble isocitrate and malate dehydrogenases reduced NADP + and NAD + , respectively. Oxidation of 2-oxoglutarate was measured as benzyl viologen reduction and was strictly CoA-dependent; a low activity was also observed with NADP + . Succinate dehydrogenase and fumarate reductase both were membrane-bound. Succinate oxidation was coupled to NADP + reduction whereas fumarate reduction was coupled to NADPH and NADH oxidation. Coupling of succinate oxidation to NADP + or cytochrome(s) reduction required an ATP-dependent reversed electron transport. Net ATP synthesis proceeded exclusively through electron transport phosphorylation. During fumarate reduction, both NADPH and NADH delivered reducing equivalents into the electron transport chain, which contained a menaquinone. Overall, acetate oxidation with fumarate proceeded through an open loop of citric acid cycle reactions, excluding succinate dehydrogenase, with fumarate reductase as the key reaction for electron delivery, whereas acetate oxidation in the syntrophic coculture required the complete citric acid cycle.

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

“…In conclusion, this study demonstrates the usefulness of 13 C metabolic flux analysis as a tool for verifying genome annotation, characterizing the physiological state of microorganisms, and mapping the central metabolism in anaerobic bacteria. The results from our technique provide valuable information complementary to genome-based modeling approaches, resulting in a comprehensive understanding of central carbon metabolism in microorganisms.…”

“…A futile pathway (pyruvate 3 oxaloacetate 3 PEP) is also evident by isotopomer data. Although the annotated genome indicates the absence of a few key enzymes in gluconeogenesis and some amino acid synthesis pathways, our 13 C tracer experiments demonstrate that those pathways are actually complete and G. metallireducens may contain some undocumented metabolic routes; e.g., an unusual isoleucine biosynthesis pathway possibly via citramalate as the intermediate is suggested by isotopic data. In combination with physiological data on the environmentally relevant microbe G. metallireducens, this study helps our understanding of carbon assimilation in the survival of such organisms in the environment.…”

“…The free energy of both steps indicates a positive driving force for converting oxoglutarate to succinate. However, the succinyl-CoA synthetase activity is absent in G. metallireducens, and acetyl-CoA transferase instead is used to complete the reaction: succinyl-CoA (ϩ acetate) 3 succinate (ϩ acetyl-CoA) (13). The reason for the very high reversibility between oxoglutarate and succinate is likely that the accumulation of acetyl-CoA forces the reaction in the reverse direction and thus inhibits the rate of carbon metabolism through TCA cycle.…”

“…Growth of G. metallireducens while oxidizing acetate requires incorporation of CO 2 into biomass (acetyl-CoA ϩ CO 2 3 pyruvate and pyruvate ϩ CO 2 3 oxaloacetate), and therefore our model evaluated the fate of the labeled 13 (Table 1). This is consistent with the fact that the labeled 13 CO 2 produced from acetate oxidization is negligible compared to the 12 CO 2 from the headspace gases (N 2 -CO 2 ).…”

Laboratory evolution of Geobacter sulfurreducens for enhanced growth on lactate via a single-base-pair substitution in a transcriptional regulator

“…One potential difference in growth on lactate versus growth on acetate (Figure 4a) is the necessity for succinyl-CoA synthase for growth on lactate (Figure 4b). G. sulfurreducens oxidizes acetate via the tricarboxylic acid cycle (Galushko and Schink, 2000;Mahadevan et al, 2006). Acetate is activated to acetyl-CoA for introduction into the tricarboxylic acid cycle via succinyl-CoA:acetate-CoA-transferase (Galushko and Schink, 2000;Segura et al, 2008).…”