Dataset Information

Enrichment of anaerobic nitrate-dependent methanotrophic 'Candidatus Methanoperedens nitroreducens' archaea from an Italian paddy field soil.

ABSTRACT: Paddy fields are a significant source of methane and contribute up to 20% of total methane emissions from wetland ecosystems. These inundated, anoxic soils featuring abundant nitrogen compounds and methane are an ideal niche for nitrate-dependent anaerobic methanotrophs. After 2 years of enrichment with a continuous supply of methane and nitrate as the sole electron donor and acceptor, a stable enrichment dominated by 'Candidatus Methanoperedens nitroreducens' archaea and 'Candidatus Methylomirabilis oxyfera' NC10 phylum bacteria was achieved. In this community, the methanotrophic archaea supplied the NC10 phylum bacteria with the necessary nitrite through nitrate reduction coupled to methane oxidation. The results of qPCR quantification of 16S ribosomal RNA (rRNA) gene copies, analysis of metagenomic 16S rRNA reads, and fluorescence in situ hybridization (FISH) correlated well and showed that after 2 years, 'Candidatus Methanoperedens nitroreducens' had the highest abundance of (2.2 ± 0.4 × 108) 16S rRNA copies per milliliter and constituted approximately 22% of the total microbial community. Phylogenetic analysis showed that the 16S rRNA genes of the dominant microorganisms clustered with previously described 'Candidatus Methanoperedens nitroreducens ANME2D' (96% identity) and 'Candidatus Methylomirabilis oxyfera' (99% identity) strains. The pooled metagenomic sequences resulted in a high-quality draft genome assembly of 'Candidatus Methanoperedens nitroreducens Vercelli' that contained all key functional genes for the reverse methanogenesis pathway and nitrate reduction. The diagnostic mcrA gene was 96% similar to 'Candidatus Methanoperedens nitroreducens ANME2D' (WP_048089615.1) at the protein level. The 'Candidatus Methylomirabilis oxyfera' draft genome contained the marker genes pmoCAB, mdh, and nirS and putative NO dismutase genes. Whole-reactor anaerobic activity measurements with methane and nitrate revealed an average methane oxidation rate of 0.012 mmol/h/L, with cell-specific methane oxidation rates up to 0.57 fmol/cell/day for 'Candidatus Methanoperedens nitroreducens'. In summary, this study describes the first enrichment and draft genome of methanotrophic archaea from paddy field soil, where these organisms can contribute significantly to the mitigation of methane emissions.

SUBMITTER: Vaksmaa A

PROVIDER: S-EPMC5569662 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Enrichment of anaerobic nitrate-dependent methanotrophic 'Candidatus Methanoperedens nitroreducens' archaea from an Italian paddy field soil.

Vaksmaa Annika A Vaksmaa Annika A Guerrero-Cruz Simon S van Alen Theo A TA Cremers Geert G Ettwig Katharina F KF Lüke Claudia C Jetten Mike S M MSM

Applied microbiology and biotechnology 20170804 18

Paddy fields are a significant source of methane and contribute up to 20% of total methane emissions from wetland ecosystems. These inundated, anoxic soils featuring abundant nitrogen compounds and methane are an ideal niche for nitrate-dependent anaerobic methanotrophs. After 2 years of enrichment with a continuous supply of methane and nitrate as the sole electron donor and acceptor, a stable enrichment dominated by 'Candidatus Methanoperedens nitroreducens' archaea and 'Candidatus Methylomira ...[more]

PMID: 28779290

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Project description:"Candidatus Methanoperedens nitroreducens" is an archaeon that couples the anaerobic oxidation of methane to nitrate reduction. In natural and man-made ecosystems, this archaeon is often found at oxic-anoxic interfaces where nitrate, the product of aerobic nitrification, cooccurs with methane produced by methanogens. As such, populations of "Ca Methanoperedens nitroreducens" could be prone to regular oxygen exposure. Here, we investigated the effect of 5% (vol/vol) oxygen exposure in batch activity assays on a "Ca Methanoperedens nitroreducens" culture, enriched from an Italian paddy field. Metagenome sequencing of the DNA extracted from the enrichment culture revealed that 83% of 16S rRNA gene reads were assigned to a novel strain, "Candidatus Methanoperedens nitroreducens Verserenetto." RNA was extracted, and metatranscriptome sequencing upon oxygen exposure revealed that the active community changed, most notably in the appearance of aerobic methanotrophs. The gene expression of "Ca Methanoperedens nitroreducens" revealed that the key genes encoding enzymes of the methane oxidation and nitrate reduction pathways were downregulated. In contrast to this, we identified upregulation of glutaredoxin, thioredoxin family/like proteins, rubrerythrins, peroxiredoxins, peroxidase, alkyl hydroperoxidase, type A flavoproteins, FeS cluster assembly protein, and cysteine desulfurases, indicating the genomic potential of "Ca Methanoperedens nitroreducens Verserenetto" to counteract the oxidative damage and adapt in environments where they might be exposed to regular oxygen intrusion.IMPORTANCE "Candidatus Methanoperedens nitroreducens" is an anaerobic archaeon which couples the reduction of nitrate to the oxidation of methane. This microorganism is present in a wide range of aquatic environments and man-made ecosystems, such as paddy fields and wastewater treatment systems. In such environments, these archaea may experience regular oxygen exposure. However, "Ca Methanoperedens nitroreducens" is able to thrive under such conditions and could be applied for the simultaneous removal of dissolved methane and nitrogenous pollutants in oxygen-limited systems. To understand what machinery "Ca Methanoperedens nitroreducens" possesses to counteract the oxidative stress and survive, we characterized the response to oxygen exposure using a multi-omics approach.

Project description:The nitrogen and methane cycles are important biogeochemical processes. Recently, 'Candidatus Methanoperedens nitroreducens,' archaea that catalyze nitrate-dependent anaerobic oxidation of methane (AOM), were enriched, and their genomes were analyzed. Diagnostic molecular tools for the sensitive detection of 'Candidatus M. nitroreducens' are not yet available. Here, we report the design of two novel mcrA primer combinations that specifically target the alpha sub-unit of the methyl-coenzyme M reductase (mcrA) gene of 'Candidatus M. nitroreducens'. The first primer pair produces a fragment of 186-bp that can be used to quantify 'Candidatus M. nitroreducens' cells, whereas the second primer pair yields an 1191-bp amplicon that is with sufficient length and well suited for more detailed phylogenetic analyses. Six different environmental samples were evaluated with the new qPCR primer pair, and the abundances were compared with those determined using primers for the 16S rRNA gene. The qPCR results indicated that the number of copies of the 'Candidatus M. nitroreducens' mcrA gene was highest in rice field soil, with 5.6 ± 0.8 × 106 copies g-1 wet weight, whereas Indonesian river sediment had only 4.6 ± 2.7 × 102 copies g-1 wet weight. In addition to freshwater environments, sequences were also detected in marine sediment of the North Sea, which contained approximately 2.5 ± 0.7 × 104 copies g-1 wet weight. Phylogenetic analysis revealed that the amplified 1191-bp mcrA gene sequences from the different environments all clustered together with available genome sequences of mcrA from known 'Candidatus M. nitroreducens' archaea. Taken together, these results demonstrate the validity and utility of the new primers for the quantitative and sensitive detection of the mcrA gene sequences of these important nitrate-dependent AOM archaea. Furthermore, the newly obtained mcrA sequences will contribute to greater phylogenetic resolution of 'Candidatus M. nitroreducens' sequences, which have been only poorly captured by general methanogenic mcrA primers.

Project description: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.

Dataset Information

Enrichment of anaerobic nitrate-dependent methanotrophic 'Candidatus Methanoperedens nitroreducens' archaea from an Italian paddy field soil.

Publications

Enrichment of anaerobic nitrate-dependent methanotrophic 'Candidatus Methanoperedens nitroreducens' archaea from an Italian paddy field soil.

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