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.

Archaea catalyze iron-dependent anaerobic oxidation of methane

“…All genes of the reverse methanogenesis pathway (28,29) were present in the enriched strain of AAA, in complete agreement with its closest relative, M. nitroreducens (5,24). Nitrate reduction could be accomplished by two candidate enzyme complexes, one encoded by the narGH genes (MPEBLZ_02035 and 2036), closely related to the ones of M. nitroreducens (81 and 83% identity, respectively), and the other by an nxrAB-related operon (MPEBLZ_01127 and 1129) absent from M. nitroreducens and similar to that in nitrite-oxidizing bacteria (68/78% similarity to Nitrolancea hollandica and 68/69% to Nitrobacter winogradskyi).…”

“…Nitrate reduction could be accomplished by two candidate enzyme complexes, one encoded by the narGH genes (MPEBLZ_02035 and 2036), closely related to the ones of M. nitroreducens (81 and 83% identity, respectively), and the other by an nxrAB-related operon (MPEBLZ_01127 and 1129) absent from M. nitroreducens and similar to that in nitrite-oxidizing bacteria (68/78% similarity to Nitrolancea hollandica and 68/69% to Nitrobacter winogradskyi). The further reduction of nitrite to N 2 gas could be accomplished by the M. oxyfera-like microorganism as previously demonstrated (6) or, alternatively, ammonium could be formed by a NrfAH-like protein complex (MPEBLZ_01114 and 1115) transcribed by the AAA (24). As the likely genetic basis for Fe 3+ reduction, we identified a wealth of genes encoding heme c-containing proteins (c-type cytochromes), which are generally recognized to function as electron shuttles from the cell to soluble or solid electron acceptors such as iron minerals (27,30).…”

“…Nitrate reduction in the enrichment culture was largely (>95%) dependent on methane. All genes necessary for nitrate reduction to nitrite and nitrite reduction to ammonium are present in the genome of AAA (5,24). Together, these results suggest that AAA most likely couples methane oxidation to dissimilatory nitrate reduction to ammonium via nitrite, a trait rarely observed in archaea (25).…”

“…The enrichment of the biomass used here was described in brief previously (24). Biomass from the settler of an M. oxyfera-dominated reactor (26) containing about 10% (of the total microbial community) AAA was used to inoculate a continuous culture reactor (2-L working volume) to further enrich AAA.…”

“…Rather, the analysis of the partially reconstructed AAA genome (24) supported that the AAA alone were able to couple methane oxidation to the reduction of different electron acceptors. All genes of the reverse methanogenesis pathway (28,29) were present in the enriched strain of AAA, in complete agreement with its closest relative, M. nitroreducens (5,24).…”