Distinct Physiological Roles of the Three [NiFe]-Hydrogenase Orthologs in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H(2)) and play a key role in the energy metabolism of microorganisms in anaerobic environments. The hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which assimilates organic carbon coupled with the reduction of elemental sulfur (S(0)) or H(2) generation, harbors three gene operons encoding [NiFe]-hydrogenase orthologs, namely, Hyh, Mbh, and Mbx. In order to elucidate their functions in vivo, a gene disruption mutant for each [NiFe]-hydrogenase ortholog was constructed. The Hyh-deficient mutant (PHY1) grew well under both H(2)S- and H(2)-evolving conditions. H(2)S generation in PHY1 was equivalent to that of the host strain, and H(2) generation was higher in PHY1, suggesting that Hyh functions in the direction of H(2) uptake in T. kodakarensis under these conditions. Analyses of culture metabolites suggested that significant amounts of NADPH produced by Hyh are used for alanine production through glutamate dehydrogenase and alanine aminotransferase. On the other hand, the Mbh-deficient mutant (MHD1) showed no growth under H(2)-evolving conditions. This fact, as well as the impaired H(2) generation activity in MHD1, indicated that Mbh is mainly responsible for H(2) evolution. The copresence of Hyh and Mbh raised the possibility of intraspecies H(2) transfer (i.e., H(2) evolved by Mbh is reoxidized by Hyh) in this archaeon. In contrast, the Mbx-deficient mutant (MXD1) showed a decreased growth rate only under H(2)S-evolving conditions and exhibited a lower H(2)S generation activity, indicating the involvement of Mbx in the S(0) reduction process. This study provides important genetic evidence for understanding the physiological roles of hydrogenase orthologs in the Thermococcales.