ABSTRACTCarboxydothermusspecies are some of the most studied thermophilic carboxydotrophs. Their varied carboxydotrophic growth properties suggest distinct strategies for energy conservation via carbon monoxide (CO) metabolism. In this study, we used comparative genome analysis of the genusCarboxydothermusto show variations in the CO dehydrogenase-energy-converting hydrogenase gene cluster, which is responsible for CO metabolism with H2production (hydrogenogenic CO metabolism). Indeed, the ability or inability to produce H2with CO oxidation is explained by the presence or absence of this gene cluster inCarboxydothermus hydrogenoformans,Carboxydothermus islandicus, andCarboxydothermus ferrireducens. Interestingly, despite its hydrogenogenic CO metabolism,Carboxydothermus pertinaxlacks the Ni-CO dehydrogenase catalytic subunit (CooS-I) and its transcriptional regulator-encoding genes in this gene cluster, probably due to inversion. Transcriptional analysis inC. pertinaxshowed that the Ni-CO dehydrogenase gene (cooS-II) and distantly encoded energy-converting-hydrogenase-related genes were remarkably upregulated with 100% CO. In addition, when thiosulfate was available as a terminal electron acceptor in 100% CO, the maximum cell density and maximum specific growth rate ofC. pertinaxwere 3.1-fold and 1.5-fold higher, respectively, than when thiosulfate was absent. The amount of H2produced was only 62% of the amount of CO consumed, less than expected according to hydrogenogenic CO oxidation (CO + H2O → CO2+ H2). Accordingly,C. pertinaxwould couple CO oxidation by Ni-CO dehydrogenase II with simultaneous reduction of not only H2O but also thiosulfate when grown in 100% CO.IMPORTANCEAnaerobic hydrogenogenic carboxydotrophs are thought to fill a vital niche by scavenging potentially toxic CO and producing H2as an available energy source for thermophilic microbes. This hydrogenogenic carboxydotrophy relies on a Ni-CO dehydrogenase-energy-converting hydrogenase gene cluster. This feature is thought to be common to these organisms. However, the hydrogenogenic carboxydotrophCarboxydothermus pertinaxlacks the gene for the Ni-CO dehydrogenase catalytic subunit encoded in the gene cluster. Here, we performed a comparative genome analysis of the genusCarboxydothermus, a transcriptional analysis, and a cultivation study in 100% CO to prove the hydrogenogenic CO metabolism. Results revealed thatC. pertinaxcould couple Ni-CO dehydrogenase II alternatively to the distal energy-converting hydrogenase. Furthermore,C. pertinaxrepresents an example of the functioning of Ni-CO dehydrogenase that does not always correspond to its genomic context, owing to the versatility of CO metabolism and the low redox potential of CO.