Insights into the Reverse Oxidative Tricarboxylic Acid Cycle Enabling Autotrophic CO2 Fixation in the Thermophilic Bacterium Deferribacter autotrophicus

Deferribacter autotrophicus SL50T is a thermophilic anaerobic bacterium capable of autotrophic growth, but its carbon fixation mechanism has remained unclear. Here, radioisotopic assays and comparative proteomics were used to identify the pathway supporting CO2 assimilation. During autotrophic growth on hydrogen and ferrihydrite, the rate of 14C incorporation was sixfold higher than under heterotrophic conditions, coinciding with the logarithmic growth phase. Proteomic analysis detected over 1400 proteins in each growth mode and showed high abundance of all major tricarboxylic acid (TCA) cycle enzymes, with citrate synthase and malate dehydrogenase among the most strongly expressed. Genomic and proteomic data indicate that D. autotrophicus SL50T lacks phosphoenolpyruvate carboxylase and instead converts pyruvate directly to oxaloacetate via pyruvate:ferredoxin oxidoreductase. Unexpectedly, acetate kinase and phosphate acetyltransferase were expressed threefold higher during autotrophic growth than during growth on acetate. These results suggest a previously unrecognized feature of the reverse oxidative TCA (roTCA) cycle, in which excess acetyl-CoA is recycled through acetyl-CoA:succinate CoA-transferase and subsequently reintegrated into the cycle. Together, these findings provide strong evidence that D. autotrophicus SL50T fixes CO2 via the roTCA cycle and broadens the understanding of the metabolic diversity of anaerobic microorganisms.