Metagenomic Binning Recovers a Transcriptionally Active Gammaproteobacterium Linking Methanotrophy to Partial Denitrification in an Anoxic Oxygen Minimum Zone

Abstract

Diverse planktonic microorganisms play a crucial role in mediating methane flux from the ocean to the atmosphere. The distribution and composition of the marine methanotroph community is determined partly by oxygen availability. The low oxygen conditions of oxygen minimum zones (OMZs) may select for methanotrophs that oxidize methane using inorganic nitrogen compounds (e.g., nitrate, nitrite) in place of oxygen. However, environmental evidence for methane-nitrogen linkages in OMZs remains sparse, as does our knowledge of the genomic content and metabolic capacity of organisms catalyzing OMZ methane oxidation. Here, binning of metagenome sequences from a coastal anoxic OMZ recovered the first near complete (95%) draft genome representing the methanotroph clade OPU3. Phylogenetic reconstruction of concatenated single copy marker genes confirmed the OPU3-like bacterium as a divergent member of the type Ia methanotrophs, with an estimated genome size half that of other sequenced taxa in this group. The proportional abundance of this bacterium peaked at 4% of the total microbial community at the top of the anoxic zone in areas of nitrite and nitrate availability but low methane concentrations. Genes mediating dissimilatory nitrate and nitrite reduction were identified in the OPU3 genome, and transcribed in conjunction with key enzymes catalyzing methane oxidation to formaldehyde and the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation, suggesting partial denitrification linked to methane oxidation. Together, these data provide the first field-based evidence for methanotrophic partial denitrification by the OPU3 cluster under anoxic conditions, supporting a role for OMZs as key sites in pelagic methane turnover.

Anaerobic methane oxidation is an important sink for methane in the ocean's largest oxygen minimum zone

“…For the Methylococcales ASVs, the peaks in relative abundance near the oxic‐anoxic interface at ~ 100 m depth at Sta. 10 and, for ASV 1150 (OPU3), at 50 m at T‐North are consistent with a previous observation of peaks in abundance of the OPU1 and OPU3 clades at the upper and lower boundary of the low‐oxygen core in the southernmost part of the ETNP OMZ (Tavormina et al ), as well as with evidence that denitrifying Methylococcales in OMZs predominantly inhabit oxycline or upper OMZ depths where low oxygen may be available for methane oxidation (Padilla et al ). The highest relative abundance of ASV 1471 (OPU1) at both stations and of ASV 1150 (OPU3) at Sta.…”

“…In addition, as energy conservation is coupled to the reduction of both nitrate and nitrite during denitrification, the scavenging of nitric oxide would not necessarily inhibit methane oxidation. Indeed, nitric oxide appeared to be the end product of nitrate reduction in Methylococcales methanotrophs identified through metagenomics near the oxic‐anoxic interface of an OMZ‐like coastal basin (Padilla et al ). A metabolism of this type is therefore less likely to explain the observed effect of PTIO.…”

“…DNA and RNA were extracted from replicate (subsamples from the same Niskin bottle) Sterivex™ filters as described previously (Padilla et al , ). For DNA extractions, cells were lysed by adding lysozyme (2 mg in 40 μ L of lysis buffer per filter) directly to the Sterivex™ cartridge, sealing the cartridge, and incubating for 45 min at 37°C.…”

“…For the latter case, we focused on methane monooxygenase and methyl‐coenzyme‐M reductase‐encoding genes ( pmo/mmo and mcr ) as markers of methanotrophy, as well as known genes of dissimilatory nitrogen metabolisms (see below). Prior to this analysis, metatranscriptome sequences were trimmed and merged as previously described (Padilla et al ). Ribosomal RNA genes were identified and removed using riboPicker with default settings (Schmieder et al ).…”

Fixed-Nitrogen Loss Associated with Sinking Zooplankton Carcasses in a Coastal Oxygen Minimum Zone (Golfo Dulce, Costa Rica)

“…15 N-tracer was added as Na 15 NO 2 (98 atom% 15 N; SigmaAldrich) at a final concentration of ∼3 µmol L −1 , which allowed to trace both aerobic and anaerobic pathways of the microbial N-cycle. In situ NO − 2 concentrations in GD were up to 0.72 µmol L −1 at the time of the current study (Padilla et al, 2017). The pre-incubated carcasses were added using a plastic pipette.…”