In methanogenic Archaea, the final step of methanogenesis generates methane and a heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB). Reduction of this heterodisulfide by heterodisulfide reductase to regenerate HS-CoM and HS-CoB is an exergonic process. Thauer et al. [Thauer, et al. 2008 Nat Rev Microbiol 6:579-591] recently suggested that in hydrogenotrophic methanogens the energy of heterodisulfide reduction powers the most endergonic reaction in the pathway, catalyzed by the formylmethanofuran dehydrogenase, via flavin-based electron bifurcation. Here we present evidence that these two steps in methanogenesis are physically linked. We identify a protein complex from the hydrogenotrophic methanogen, Methanococcus maripaludis, that contains heterodisulfide reductase, formylmethanofuran dehydrogenase, F 420 -nonreducing hydrogenase, and formate dehydrogenase. In addition to establishing a physical basis for the electronbifurcation model of energy conservation, the composition of the complex also suggests that either H 2 or formate (two alternative electron donors for methanogenesis) can donate electrons to the heterodisulfide-H 2 via F 420 -nonreducing hydrogenase or formate via formate dehydrogenase. Electron flow from formate to the heterodisulfide rather than the use of H 2 as an intermediate represents a previously unknown path of electron flow in methanogenesis. We further tested whether this path occurs by constructing a mutant lacking F 420 -nonreducing hydrogenase. The mutant displayed growth equal to wild-type with formate but markedly slower growth with hydrogen. The results support the model of electron bifurcation and suggest that formate, like H 2 , is closely integrated into the methanogenic pathway.energy conservation | Archaea | formate dehydrogenase | formylmethanofuran dehydrogenase | F 420 -nonreducing hydrogenase T he biochemical steps in methanogenesis from CO 2 are well known, but the interactions that lead to net energy conservation are not well understood. The steps in the pathway are diagrammed in Fig. 1 (1). The first step involves the reduction of CO 2 and covalent attachment to a unique cofactor, methanofuran (MFR), via the action of formylmethanofuran dehydrogenase (Fwd) to generate formyl-MFR. This represents an energy-consuming step in the pathway and is dependent on reduced ferredoxin, thought to be produced at the expense of a chemiosmotic membrane potential via the energy-conserving hydrogenase, Eha. Next, the formyl group is transferred to another carrier, tetrahydromethanopterin (H 4 MPT), and is then reduced to generate methyl-H 4 MPT. The methyl group is then transferred to yet another carrier, coenzyme M (HS-CoM), by methyl-H 4 MPT-CoM methyltransferase (Mtr) to generate methyl-S-CoM. At this point, Na + ions are translocated across the cell membrane. The final step involves reduction of the methyl group to CH 4 and capture of HS-CoM by coenzyme B (HS-CoB) to form a CoM-S-S-CoB heterodisulfide. To regenerate HS-CoM and HS-CoB, another enzyme is used, heterodisulfid...

A coordinated study of water chemistry, sediment mineralogy, and sediment microbial community was conducted on four >73 degrees C springs in the northwestern Great Basin. Despite generally similar chemistry and mineralogy, springs with short residence time (approximately 5-20 min) were rich in reduced chemistry, whereas springs with long residence time (>1 day) accumulated oxygen and oxidized nitrogen species. The presence of oxygen suggested that aerobic metabolisms prevail in the water and surface sediment. However, Gibbs free energy calculations using empirical chemistry data suggested that several inorganic electron donors were similarly favorable. Analysis of 298 bacterial 16S rDNAs identified 36 species-level phylotypes, 14 of which failed to affiliate with cultivated phyla. Highly represented phylotypes included Thermus, Thermotoga, a member of candidate phylum OP1, and two deeply branching Chloroflexi. The 276 archaeal 16S rDNAs represented 28 phylotypes, most of which were Crenarchaeota unrelated to the Thermoprotei. The most abundant archaeal phylotype was closely related to "Candidatus Nitrosocaldus yellowstonii", suggesting a role for ammonia oxidation in primary production; however, few other phylotypes could be linked with energy calculations because phylotypes were either related to chemoorganotrophs or were unrelated to known organisms.

Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H 2 metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H 2 suggests that its role is anaplerotic. Indeed, H 2 via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for. M ethanogenesis is an anaerobic respiration carried out by a phylogenetically related group of Archaea within the phylum Euryarchaeota. Methanogens are divided into two metabolic types, those without and those with cytochromes (1). Methanogens without cytochromes use H 2 as an electron donor and are termed hydrogenotrophic. Some species can substitute H 2 with formate, and a few can use secondary alcohols. CO 2 is the electron acceptor and is reduced to methane. Methanogens with cytochromes reduce certain methyl compounds or the methyl carbon of acetate to methane and are called methylotrophic. Many can also use H 2 and CO 2 , as can hydrogenotrophic methanogens.Although the pathways of methanogenesis have long been known, an understanding of energy conservation has been slower to emerge. Methanogens with and without cytochromes both export Na + when a methyl group is transferred from the carrier tetrahydromethanopterin (H 4 MPT) to coenzyme M (CoM) (Fig. 1). The Na + gradient across the membrane is used directly for ATP synthesis or is converted by an antiporter to a proton gradient. However, for methanogenesis from CO 2 , the initial reduction of CO 2 to a formyl group attached to methanofuran (MFR) is endergonic. How energy is provided to drive this reaction is not well understood. Methanogens with and without cytochromes have membrane-associated energy-converting hydrogenases that couple the reduction of low-potential ferredoxins (Fd) to a chemiosmotic membrane gradient (2). If such a Fd donates electrons for CO 2 reduction, an energy-converting hydrogenase is the conduit of ener...

High representation by ammonia-oxidizing archaea (AOA) in marine systems is consistent with their high affinity for ammonia, efficient carbon fixation, and copper (Cu)-centric respiratory system. However, little is known about their response to nutrient stress. We therefore used global transcriptional and proteomic analyses to characterize the response of a model AOA, Nitrosopumilus maritimus SCM1, to ammonia starvation, Cu limitation and Cu excess. Most predicted protein-coding genes were transcribed in exponentially growing cells, and of ~74% detected in the proteome, ~6% were modified by N-terminal acetylation. The general response to ammonia starvation and Cu stress was downregulation of genes for energy generation and biosynthesis. Cells rapidly depleted transcripts for the A and B subunits of ammonia monooxygenase (AMO) in response to ammonia starvation, yet retained relatively high levels of transcripts for the C subunit. Thus, similar to ammonia-oxidizing bacteria, selective retention of amoC transcripts during starvation appears important for subsequent recovery, and also suggests that AMO subunit transcript ratios could be used to assess the physiological status of marine populations. Unexpectedly, cobalamin biosynthesis was upregulated in response to both ammonia starvation and Cu stress, indicating the importance of this cofactor in retaining functional integrity during times of stress.