Methylotetracoccus oryzae Strain C50C1 Is a Novel Type Ib Gammaproteobacterial Methanotroph Adapted to Freshwater Environments

Methane-oxidizing microorganisms perform an important role in reducing emissions of the greenhouse gas methane to the atmosphere. To date, known bacterial methanotrophs belong to the Proteobacteria, Verrucomicrobia, and NC10 phyla. Within the Proteobacteria phylum, they can be divided into type Ia, type Ib, and type II methanotrophs. Type Ia and type II are well represented by isolates. Contrastingly, the vast majority of type Ib methanotrophs have not been able to be cultivated so far. Here, we compared the distributions of type Ib lineages in different environments. Whereas the cultivated type Ib methanotrophs (Methylococcus and Methylocaldum) are found in landfill and upland soils, lineages that are not represented by isolates are mostly dominant in freshwater environments, such as paddy fields and lake sediments. Thus, we observed a clear niche differentiation within type Ib methanotrophs. Our subsequent isolation attempts resulted in obtaining a pure culture of a novel type Ib methanotroph, tentatively named “Methylotetracoccus oryzae” C50C1. Strain C50C1 was further characterized to be an obligate methanotroph, containing C_(16:1)ω9c as the major membrane phospholipid fatty acid, which has not been found in other methanotrophs. Genome analysis of strain C50C1 showed the presence of two pmoCAB operon copies and XoxF5-type methanol dehydrogenase in addition to MxaFI. The genome also contained genes involved in nitrogen and sulfur cycling, but it remains to be demonstrated if and how these help this type Ib methanotroph to adapt to fluctuating environmental conditions in freshwater ecosystems

Methane-Oxidizing Communities in Lichen-Dominated Forested Tundra Are Composed Exclusively of High-Affinity USCα Methanotrophs

Upland soils of tundra function as a constant sink for atmospheric CH4 but the identity of methane oxidizers in these soils remains poorly understood. Methane uptake rates of −0.4 to −0.6 mg CH4-C m−2 day−1 were determined by the static chamber method in a mildly acidic upland soil of the lichen-dominated forested tundra, North Siberia, Russia. The maximal CH4 oxidation activity was localized in an organic surface soil layer underlying the lichen cover. Molecular identification of methanotrophic bacteria based on retrieval of the pmoA gene revealed Upland Soil Cluster Alpha (USCα) as the only detectable methanotroph group. Quantification of these pmoA gene fragments by means of specific qPCR assay detected ~107pmoA gene copies g−1 dry soil. The pmoA diversity was represented by seven closely related phylotypes; the most abundant phylotype displayed 97.5% identity to pmoA of Candidatus Methyloaffinis lahnbergensis. Further analysis of prokaryote diversity in this soil did not reveal 16S rRNA gene fragments from well-studied methanotrophs of the order Methylococcales and the family Methylocystaceae. The largest group of reads (~4% of all bacterial 16S rRNA gene fragments) that could potentially belong to methanotrophs was classified as uncultivated Beijerinckiaceae bacteria. These reads displayed 96–100 and 95–98% sequence similarity to 16S rRNA gene of Candidatus Methyloaffinis lahnbergensis and “Methylocapsa gorgona” MG08, respectively, and were represented by eight species-level operational taxonomic units (OTUs), two of which were highly abundant. These identification results characterize subarctic upland soils, which are exposed to atmospheric methane concentrations only, as a unique habitat colonized mostly by USCα methanotrophs

Phylogenetic Analysis and In Situ Identification of Bacteria Community Composition in an Acidic Sphagnum Peat Bog

The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (≥95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3 × 10(7) and 1.1 × 10(7) cells g(−1) peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity

One Step Closer to Enigmatic USCα Methanotrophs: Isolation of a <i>Methylocapsa</i>-like Bacterium from a Subarctic Soil

The scavenging of atmospheric trace gases has been recognized as one of the lifestyle-defining capabilities of microorganisms in terrestrial polar ecosystems. Several metagenome-assembled genomes of as-yet-uncultivated methanotrophic bacteria, which consume atmospheric CH4 in these ecosystems, have been retrieved in cultivation-independent studies. In this study, we isolated and characterized a representative of these methanotrophs, strain D3K7, from a subarctic soil of northern Russia. Strain D3K7 grows on methane and methanol in a wide range of temperatures, between 5 and 30 °C. Weak growth was also observed on acetate. The presence of acetate in the culture medium stimulated growth at low CH4 concentrations (~100 p.p.m.v.). The finished genome sequence of strain D3K7 is 4.15 Mb in size and contains about 3700 protein-encoding genes. According to the result of phylogenomic analysis, this bacterium forms a common clade with metagenome-assembled genomes obtained from the active layer of a permafrost thaw gradient in Stordalen Mire, Abisco, Sweden, and the mineral cryosol at Axel Heiberg Island in the Canadian High Arctic. This clade occupies a phylogenetic position in between characterized Methylocapsa methanotrophs and representatives of the as-yet-uncultivated upland soil cluster alpha (USCα). As shown by the global distribution analysis, D3K7-like methanotrophs are not restricted to polar habitats but inhabit peatlands and soils of various climatic zones

Building a Cell House from Cellulose: The Case of the Soil Acidobacterium <i>Acidisarcina polymorpha</i> SBC82^T

Acidisarcina polymorpha SBC82T is a recently described representative of the phylum Acidobacteriota from lichen-covered tundra soil. Cells of this bacterium occur within unusual saccular chambers, with the chamber envelope formed by tightly packed fibrils. These extracellular structures were most pronounced in old cultures of strain SBC82T and were organized in cluster-like aggregates. The latter were efficiently destroyed by incubating cell suspensions with cellulase, thus suggesting that they were composed of cellulose. The diffraction pattern obtained for 45-day-old cultures of strain SBC82T by using small angle X-ray scattering was similar to those reported earlier for mature wood samples. The genome analysis revealed the presence of a cellulose biosynthesis locus bcs. Cellulose synthase key subunits A and B were encoded by the bcsAB gene whose close homologs are found in genomes of many members of the order Acidobacteriales. More distant homologs of the acidobacterial bcsAB occurred in representatives of the Proteobacteria. A unique feature of bcs locus in strain SBC82T was the non-orthologous displacement of the bcsZ gene, which encodes the GH8 family glycosidase with a GH5 family gene. Presumably, these cellulose-made extracellular structures produced by A. polymorpha have a protective function and ensure the survival of this acidobacterium in habitats with harsh environmental conditions

Atmospheric Methane Consumption and Methanotroph Communities in West Siberian Boreal Upland Forest Ecosystems

Upland forest ecosystems are recognized as net sinks for atmospheric methane (CH4), one of the most impactful greenhouse gases. Biological methane uptake in these ecosystems occurs due to the activity of aerobic methanotrophic bacteria. Russia hosts one-fifth of the global forest area, with the most extensive forest landscapes located in West Siberia. Here, we report seasonal CH4 flux measurements conducted in 2018 in three types of stands in West Siberian middle taiga–Siberian pine, Aspen, and mixed forests. High rates of methane uptake of up to −0.184 mg CH4 m−2 h−1 were measured by a static chamber method, with an estimated total growing season consumption of 4.5 ± 0.5 kg CH4 ha−1. Forest type had little to no effect on methane fluxes within each season. Soil methane oxidation rate ranged from 0 to 8.1 ng CH4 gDW−1 h−1 and was negatively related to water-filled pore space. The microbial soil communities were dominated by the Alpha- and Gammaproteobacteria, Acidobacteriota and Actinobacteriota. The major group of 16S rRNA gene reads from methanotrophs belonged to uncultivated Beijerinckiaceae bacteria. Molecular identification of methanotrophs based on retrieval of the pmoA gene confirmed that Upland Soil Cluster Alpha was the major bacterial group responsible for CH4 oxidation