Carbon monoxide pathway enzyme activities in a thermophilic anaerobic bacterium grown acetogenically and in a syntrophic acetate-oxidizing coculture

We recently isolated an acetate-oxidizing rodshaped eubacterium (AOR) which was capable of oxidizing acetate to CO₂ when grown in coculture with the hydrogenotrophic methanogen Methanobacterium sp. strain THF. The AOR was also capable of growing axenically on H₂CO₂ which it converted to acetate. Previous results for the acetate oxidizing coculture showed isotopic exchange between acetate and CO₂, suggesting that the AOR was using a pathway for acetate oxidation resembling a reveral of the acetogenic (carbon monoxide) pathway. In this study, it was found that production of ¹⁴CO₂ from ¹⁴CH₃COO^- by the coculture was inhibited by 200 M cyanide, while methanogenesis from H₂–CO₂ was unaffected, implying the involvement of carbon monoxide dehydrogenase (CODH) in acetate oxidation. CODH was present at 0.055 mol methyl viologen reduced min^-1 mg^-1 protein in extracts of Methanobacterium sp. strain THF, but was present in higher levels in the acetate oxidizing coculture and in the AOR grown axenically and on H₂–CO₂ (2.0 and 6.4 mol min^-1 mg^-1 protein respectively). Anaerobic activity stains for CODH in native polyacrylamide gels from the AOR coculture showed components co-migrating with bands from both organisms, as well as an additional band in extracts of the coculture. Formate dehydrogenase (FDH) was present in both the AOR coculture and monoculture but not in extracts of H₂–CO₂ grown cells of Methanobacterium sp. strain THF. Formyltetrahydrofolate (FTHF) synthetase was not detectable in extracts of the AOR monoculture or coculture, although it was found in high amounts in extracts of H₂–CO₂ grown cells of the thermophilic acetogen Acetogenium kivui. Extracts of H₂–CO₂ grown cells of the AOR showed a fluorescence spectrum typical of pterin derivatives. Bioassay for folates showed levels to be at anabolic rather than catabolic levels. It is possible that the AOR uses pterins distinct from folate for catabolism. Isocitrate dehydrogenase, a citric acid cycle enzyme, was also present in the AOR, but at anabolic levels and -ketoglutarate dehydrogenase was not detectable.Abbreviations (AOR) acetate-oxidizing rod - (CODH) carbon monoxide dehydrogenase - (FDH) formate dehydrogenase - (FTHF) formyltetrahydrofolate