Sulfur and methane oxidation by a single microorganism

Abstract

Natural and anthropogenic wetlands are major sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic bacteria at the oxic-anoxic interface, a zone of intense redox cycling of carbon, sulfur, and nitrogen compounds. Here, we report on the isolation of an aerobic methanotrophic bacterium, 'Methylovirgula thiovorans' strain HY1, which possesses metabolic capabilities never before found in any methanotroph. Most notably, strain HY1 is the first bacterium shown to aerobically oxidize both methane and reduced sulfur compounds for growth. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are responsible for methane and methanol oxidation, respectively. Various pathways for respiratory sulfur oxidation were present, including the Sox-rDsr pathway and the S₄I system. Strain HY1 employed the Calvin-Benson-Bassham cycle for CO₂ fixation during chemolithoautotrophic growth on reduced sulfur compounds. Proteomic and microrespirometry analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of the respective substrates. Methane and thiosulfate could therefore be independently or simultaneously oxidized. The discovery of this versatile bacterium demonstrates that methanotrophy and thiotrophy are compatible in a single microorganism and underpins the intimate interactions of methane and sulfur cycles in oxic-anoxic interface environments.

Keywords: facultative methanotrophy; mixotrophy; thiotrophy; wetland.

Fig. 1.

Phylogenomic tree and distribution of distinctive metabolic traits in methane- and sulfur-oxidizing bacteria in the classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Chlorobia. The tree includes 37 genomes and 2 metagenome-assembled genomes. Representative genomes of Sox-containing Alphaproteobacterial and Gammaproteobacterial methanotrophs were included. The tree was constructed based on 27 concatenated ribosomal proteins with FastTree implemented within Anvi’o phylogenomics workflow (details are in Materials and Methods). Black circles indicate 70% bootstrap support for nodes along the tree. A homology-based search for functional genes was performed by using BLAST (124), OrthoFinder (125), and manual examination (details are in Materials and Methods). Solid and open squares indicate the presence and absence of the genes, respectively.

Fig. 2.

Growth and time course of methane and thiosulfate oxidation by batch cultures of strain HY1. (A) Time courses of methane oxidation and the concomitant growth of strain HY1. Due to oxygen depletion, methane was not completely oxidized. (B) Time courses of thiosulfate oxidation, sulfate production, and the concomitant growth of strain HY1. The stoichiometry of thiosulfate consumption to sulfate production was nearly equal to the predicted 1:2. Specific analytical assays for methane, sulfate, and thiosulfate in batch cultures are described in SI Appendix, Analytical Methods. Error bars represent ±1 SD of three biological replicates. V/v, vol/vol.

Fig. 3.

Biomass production in strain HY1 grown on methane and thiosulfate. (A) The growth yield was calculated as milligrams of cellular protein produced per culture volume (mg⋅protein⋅L⁻¹) after the complete oxidation of the substrate(s). Strain HY1 was grown in 100 mL of LSM medium at pH 5.0 with methane (15%, vol/vol), thiosulfate (4 mM), and methane+thiosulfate (15%, vol/vol; 4 mM), respectively, in 160-mL serum vials. For the complete oxidation of substrates, 60% (vol/vol) oxygen was supplied. (B) The molar growth yield (Y_x/m) was calculated as gram of dry cell weight per mol of substrate consumed (g dry cell weight⋅mol⁻¹⋅substrate). Error bars represent ±1 SD of three biological replicates.

Fig. 4.

Proposed central carbon and energy metabolism in strain HY1 and differential protein abundance between methane-grown and thiosulfate-grown cells. The color scale indicates whether proteins have higher abundance in methane-grown (red) or thiosulfate-grown (blue) cells. The intensity of the color in each protein indicates the relative fold change difference (log₂FC). Methane oxidation: Methane is oxidized to methanol by the soluble methane monooxygenase, sMMO (MHY1_02902–2908). The produced methanol is oxidized to formaldehyde via the lanthanide-dependent MDH, XoxF (MHY1_02202 was the most abundant MDH). Formaldehyde oxidation to formate then proceeds via the tetrahydromethanopterin (H₄MPT) pathway, and C1 incorporation into the serine cycle is mediated by the tetrahydrofolate (H₄F) carbon-assimilation pathway. Sulfur oxidation: In the periplasm, two thiosulfate molecules are oxidized to tetrathionate by thiosulfate dehydrogenase, DoxDA (MHY1_01298), and then TetH (MHY1_02468) hydrolyzes tetrathionate to sulfate and disulfane monosulfonic acid, which most probably decomposes spontaneously to thiosulfate and sulfur. Sulfane sulfur derived from thiosulfate and sulfide via SoxYZAB (MHY1_00063–66) and Sqr (MHY1_02376), respectively, is transported into the cytoplasm via PmpAB (MHY1_00234–235 and MHY1_01361–1362; only MHY1_01361–1362 are indicated here), then transferred to the DsrEFH (MHY1_00081–83) and DsrC (MHY1_00084) via the sulfur-transporting complex [rhodanese (MHY1_01281)-TusA (MHY1_00072)-DsrE2A (MHY1_00073)]. The persulfurated DsrC is oxidized to DsrC and sulfite by DsrAB sulfite reductase (MHY1_00079–80), thereby releasing electrons to the iron–sulfur flavoprotein, DsrL (MHY1_00087). Sulfite is probably transported to the periplasm by a TauE-like exporter (MHY1_01299). The sulfite:cytochrome c oxidoreductase, SorAB (MHY1_p00095–0096), encoded in the megaplasmid, might be involved in sulfite oxidation. It is speculated that the TauD/DsrQ protein (MHY1_00078) catalyzes the release of sulfite during the breakdown of sulfonates.