The energy-converting hydrogenase Ech2 is important for the growth of the thermophilic acetogen Thermoanaerobacter kivui on ferredoxin-dependent substrates

Thermoanaerobacter kivui is the thermophilic acetogenic bacterium with the highest temperature optimum (66 degrees C) and with high growth rates on hydrogen (H-2) plus carbon dioxide (CO2). The bioenergetic model suggests that its redox and energy metabolism depends on energy-converting hydrogenases (Ech). Its genome encodes two Echs, Ech1 and Ech2, as sole coupling sites for energy conservation during growth on H-2 + CO2. During growth on other substrates, its redox activity, the (proton-gradient-coupled) oxidation of H-2 may be essential to provide reduced ferredoxin (Fd) to the cell. While Ech activity has been demonstrated biochemically, the physiological function of both Ech's is unclear. Toward that, we deleted the complete gene cluster encoding Ech2. Surprisingly, the ech2 mutant grew as fast as the wild type on sugar substrates and H-2 + CO2. Hence, Ech1 may be the essential enzyme for energy conservation, and either Ech1 or another enzyme may substitute for H-2-dependent Fd reduction during growth on sugar substrates, putatively the H-2-dependent CO2 reductase (HDCR). Growth on pyruvate and CO, substrates that are oxidized by Fd-dependent enzymes, was significantly impaired, but to a different extent. While triangle ech2 grew well on pyruvate after four transfers, triangle ech2 did not adapt to CO. Cell suspensions of triangle ech2 converted pyruvate to acetate, but no acetate was produced from CO. We analyzed the genome of five T. kivui strains adapted to CO. Strikingly, all strains carried mutations in the hycB3 subunit of HDCR. These mutations are obviously essential for the growth on CO but may inhibit its ability to utilize Fd as substrate. IMPORTANCE Acetogens thrive by converting H-2+CO2 to acetate. Under environmental conditions, this allows for only very little energy to be conserved (triangle G '<-20 kJ mol(-1)). CO2 serves as a terminal electron acceptor in the ancient Wood-Ljungdahl pathway (WLP). Since the WLP is ATP neutral, energy conservation during growth on H-2 + CO2 is dependent on the redox metabolism. Two types of acetogens can be distinguished, Rnf- and Ech-type. The function of both membrane-bound enzyme complexes is twofold-energy conversion and redox balancing. Ech couples the Fd-dependent reduction of protons to H-2 to the formation of a proton gradient in the thermophilic bacterium Thermoanaerobacter kivui. This bacterium may be utilized in gas fermentation at high temperatures, due to very high conversion rates and the availability of genetic tools. The physiological function of an Ech hydrogenase in T. kivui was studied to contribute an understanding of its energy and redox metabolism, a prerequisite for future industrial applications.