A Metagenomics-Based Metabolic Model of Nitrate-Dependent Anaerobic Oxidation of Methane by Methanoperedens-Like Archaea

Abstract

Methane oxidation is an important process to mitigate the emission of the greenhouse gas methane and further exacerbating of climate forcing. Both aerobic and anaerobic microorganisms have been reported to catalyze methane oxidation with only a few possible electron acceptors. Recently, new microorganisms were identified that could couple the oxidation of methane to nitrate or nitrite reduction. Here we investigated such an enrichment culture at the (meta) genomic level to establish a metabolic model of nitrate-driven anaerobic oxidation of methane (nitrate-AOM). Nitrate-AOM is catalyzed by an archaeon closely related to (reverse) methanogens that belongs to the ANME-2d clade, tentatively named Methanoperedens nitroreducens. Methane may be activated by methyl-CoM reductase and subsequently undergo full oxidation to carbon dioxide via reverse methanogenesis. All enzymes of this pathway were present and expressed in the investigated culture. The genome of the archaeal enrichment culture encoded a variety of enzymes involved in an electron transport chain similar to those found in Methanosarcina species with additional features not previously found in methane-converting archaea. Nitrate reduction to nitrite seems to be located in the pseudoperiplasm and may be catalyzed by an unusual Nar-like protein complex. A small part of the resulting nitrite is reduced to ammonium which may be catalyzed by a Nrf-type nitrite reductase. One of the key questions is how electrons from cytoplasmically located reverse methanogenesis reach the nitrate reductase in the pseudoperiplasm. Electron transport in M. nitroreducens probably involves cofactor F420 in the cytoplasm, quinones in the cytoplasmic membrane and cytochrome c in the pseudoperiplasm. The membrane-bound electron transport chain includes F420H2 dehydrogenase and an unusual Rieske/cytochrome b complex. Based on genome and transcriptome studies a tentative model of how central energy metabolism of nitrate-AOM could work is presented and discussed.

Keywords: ANME; anaerobic respiration; archaea; cytochrome c; electron transport; heterodisulfide reductase; methanogenesis; methanotrophy.

Figure 1

F₄₂₀ fluorescence of aggregated biomass in the nitrate-AOM enrichment culture. (A) phase contrast micrograph, (B) fluorescence micrograph with an excitation wavelength of 390 nm and an emission wavelength of 420 nm, (C) overlay of the phase contrast and the fluorescence micrograph showing that not all cells in the aggregates exhibit F₄₂₀ fluorescence.

Figure 2

Tentative metabolic pathway model of membrane-bound electron transport in Methanoperedens. Reverse methanogenesis produces F₄₂₀H₂ and the thiol cofactors CoM-SH and CoB-SH as well as reduced ferredoxin (Fd_red). F₄₂₀H₂ may be oxidized by the F₄₂₀H₂ dehydrogenase (Fqo) and electrons transferred to menaquinone (MQ, menaquinone; MQH₂, menaquinol). The heterodisulfide reductase (Hdr) reaction is reversed resulting in quinone reduction upon CoM-S-S-CoB (heterodisulfide) production. Menaquinol can be oxidized by a Rieske-cytochrome b complex comprising two additional cytochrome c subunits. Electrons are transferred to an unusual nitrate reductase (Nar) complex, presumably via soluble cytochrome c (cytc_ox/_red, oxidized/reduced cytochrome c), to reduce nitrate to nitrite. A small part of the nitrite can further be reduced to ammonium by nitrite reductase (Nrf) with menaquinol as electron donor. The fate of reduced ferredoxin is unclear. It could either be oxidized by Ech hydrogenase, by FrhB or FqoF (homologous to each other) alone or by the hypothesized confurcating HdrABC-FrhB enzyme complex. For more details, see text. Methyltransferase (Mtr) and A₁A_O ATP synthase make use of the proton motif force built up by the respiratory chain. This metabolic construction is solely based on genome analysis. HCO II, heme copper oxidase subunit II like proteins; cytb, cytochrome b; cytc, cytochrome c; FeS, iron-sulfur cluster; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; MPT, molybdopterin; NiFe, nickel-iron center.

Figure 3

Analysis of the lipid-soluble electron carriers of the nitrate-AOM enrichment culture. (A) HPLC elution profile as visualized by the absorption at 248 nm. Numbers indicate the peaks that were further characterized by UV/Vis spectroscopy. (B) UV-Vis spectra of standard compounds (left, methanophenazine; middle, menaquinone-4; right, ubiquinone-4) for comparison with spectra obtained from fractions separated by HPLC. Based on the comparison of the experimental spectra to the spectra obtained from the HPLC fractions, the different peaks were assigned to contain a representative from the classes of ubiquinones, menaquinones, or methanophenazines. None of the spectra resembled the standard spectrum for methanophenazine (left), seven spectra (obtained from peaks 1, 3, 4, 5, 6, 7,) resembled the standard spectrum of menaquinone-4 (middle) and one spectrum (obtained from peak 2) resembled the standard spectrum of ubiquinone-4. Different retention times within one molecule class indicate a difference in the prenoid chain length. Experimental spectra are displayed in Supplementary Figure S2.

Figure 4

Hypothesis of a novel cytoplasmic electron confurcating heterodisulfide reductase complex for ferredoxin and CoM-S-S-CoB recycling. The exergonic reduction of F₄₂₀ (E${}^{0_{{}^{\prime }}}$ = −360 mV) with reduced ferredoxin (E${}^{0_{{}^{\prime }}}$ ≈−500 mV) may be coupled to the endergonic electron transfer from the thiol cofactors CoM-SH and CoB-SH (E${}^{0_{{}^{\prime }}}$ = −143 mV) to F₄₂₀. This hypothesis is based on the metabolic reconstruction using genome and transcriptome sequencing and is not yet supported by biochemical experiments. FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; FeS, iron-sulfur cluster; FrhB, F₄₂₀-reducing hydrogenase subunit B; HdrABC, heterodisulfide reductase subunits A, B, C.