Formate-Dependent H ₂ Production by the Mesophilic Methanogen Methanococcus maripaludis

Abstract

Fdh1 isozyme, indicating that it was the primary Fdh. In contrast, a mutant containing a deletion of the gene encoding the Fdh2 isozyme possessed near-wild-type activities, indicating that this isozyme did not play a major role. H 2 production by a mutant containing a deletion of the coenzyme F 420 -reducing hydrogenase Fru was also severely reduced, suggesting that the major pathway of H 2 production comprised Fdh1 and Fru. Because a ⌬fru-⌬frc mutant retained 10% of the wild-type activity, an additional pathway is present. Mutants possessing deletions of the gene encoding the F 420 -dependent methylene-H 4 MTP dehydrogenase (Mtd) or the H 2 -forming methylene-H 4 MTP dehydrogenase (Hmd) also possessed reduced activity, which suggested that this second pathway was comprised of Fdh1-Mtd-Hmd. In contrast to H 2 production, the cellular rates of methanogenesis were unaffected in these mutants, which suggested that the observed H 2 production was not a direct intermediate of methanogenesis. In conclusion, high rates of formatedependent H 2 production demonstrated the potential of M. maripaludis for the microbial production of H 2 from formate.Many hydrogenotrophic methanogens use H 2 or formate for the reduction of CO 2 to obtain energy for growth. Methanococcus maripaludis, the model microorganism in this study, is a hydrogenotrophic, formate-utilizing, mesophilic methanogen. It is common in salt marsh sediments, from which it was isolated (12). An extraordinarily active H 2 consumer, M. maripaludis is exceptionally well equipped with enzymes responsible for H 2 metabolism. M. maripaludis contains genes for seven different hydrogenases, whose expression depends upon the growth conditions (18). It possesses two membrane-bound, energy-converting [Ni-Fe] hydrogenases, designated Eha and Ehb, that are involved in the reduction of low-potential ferredoxins for anabolism (16,26). There are also four cytoplasmic [Ni-Fe] hydrogenases, including two coenzyme F 420 -reducing (Fru and Frc) and two coenzyme F 420 -nonreducing (Vhu and Vhc) hydrogenases. One hydrogenase of each type (Fru and Vhu) contains a selenocysteinyl residue. The other hydrogenases (Frc and Vhc) contain cysteinyl residues at homologous positions (18). The selenocysteine-containing isozymes are abundant during cultivation in medium containing selenium, and the cysteine-containing isozymes (Frc and Vhc) are produced only upon selenium limitation (27). Lastly, the cells contain a cytoplasmic [Fe-S] cluster-free hydrogenase, the H 2 -forming methylenetetrahydromethanopterin (methylene-H 4 MPT) dehydrogenase (Hmd), which in other species has been shown to play an important role at high levels of H 2 or under nickel limitation (1, 2, 29).When formate is the substrate, it is oxidized for the reduction of CO 2 to methane. The key enzyme for formate utilization is formate dehydrogenase, Fdh. The genome of M. maripaludis harbors two sets of genes encoding Fdh, fdhA1B1 and fdhA2B2 (33). Both Fdhs contain selenocysteinyl residues. While fdhA1B1 are found in an appa...

Essential anaplerotic role for the energy-converting hydrogenase Eha in hydrogenotrophic methanogenesis

“…Indeed, experiments with cell suspensions have already shown that rates of methanogenesis can substantially exceed rates of H 2 production from formate (5). As a further test, our approach here was to genetically remove formate-hydrogen lyase activity, so that H 2 would not be produced from formate.…”

“…In the conventional view, during growth on formate, H 2 generated from formate serves as the electron donor. Indeed, H 2 is generated from formate and recycled in a poorly understood manner (5,11). However, if there is no direct requirement for H 2 in methanogenesis (5, 7), most of the hydrogenases encoded in the genome ought to be dispensable during growth on formate.…”

“…To measure H 2 production by cell suspensions, cultures (5 mL) were grown on formate to OD 660 between 0.5 and 0.6, and cells were pelleted, washed, and resuspended anaerobically in the same volume of assay buffer (modified from ref. 5, 50 mM Mops pH 7.0, 400 mM NaCl, 20 mM KCl, 20 mM MgCl 2 , 1 mM CaCl 2 , 5 mM DTT, and 1 mM bromoethanesulfonate). Tubes were then flushed with N 2 for 10-30 min to remove residual H 2 .…”

Nickel–Iron–Selenium Hydrogenases – An Overview

“…The Frc enzyme shows similar activities of F420 reduction from H 2 or H 2 production from reduced F420 [36] . The latter activity is important, for example, during growth on formate [37] . [39] .…”

A systems level predictive model for global gene regulation of methanogenesis in a hydrogenotrophic methanogen

“…These microorganisms contain very high levels of different types of hydrogenases and consume H 2 at very high rates. Furthermore, under certain conditions the H 2 uptake system can be induced to produce H 2 at elevated proportions by using formate as an electron donor Lupa et al 2008;Costa et al 2013a). Certain species of methanogens can produce H 2 by fixing nitrogen, and therefore have the potential to produce H 2 using the nitrogenase system.…”