Microbial Community Composition and Methanotroph Diversity of a Subarctic Wetland in Russia.

This study assessed the microbial diversity, activity, and composition of methane-oxidizing communities of a subarctic wetland in Russia,with mosaic cover of Sphagnum mosses and lichens of the genera Cladonia and Cetraria. Potential methane-oxidizing activity of peat sampled from lichen-dominated wetland sites was higher than that in the sites dominated by Sphagnum mosses. In peat from lichendominated sites, major bacterial groups identified by high-throughput sequencing of the 16S rRNA genes were the Acidobacteria (35.4-41.2% of total 16S rRNA gene reads), Alphaproteobacteria (19.1-24.2%), Gammaproteobacteria (7.9-11.1%), Actinobacteria (5.5-13.2%), Planctomycetes (7.2-9.5%), and Verrucomicrobia (5.1-9.5%). The distinctive feature of this community was high proportion of Subdivision 2 Acidobacteria, which are not char- acteristic for boreal Sphagnum peat bogs. Methanotrophic community composition was determined by mo- lecular analysis of the pmoA gene encoding particulate methane monooxygenase. Most (-80%) of all pmoA gene fragments revealed in peat from lichen-dominated sites belonged to the phylogenetic lineage represented by a microaerobic spiral-shaped methanotroph, "Candidatus Methylospira mobilis." Members of the genus Methylocystis, which are typical inhabitants of boreal Sphagnum peat bogs, represented only a minor group of indigenous methanotrophs. The specific feature of a methanotrophic community in peat from lichen-dominated sites was the presence of uncultivated USCa (Upland Soil Cluster alpha) methanotrophs, which are typical for acidic upland soils showing atmospheric methane oxidation. The methanotrophic community composition in lichen-dominated sites of a tundra wetland, therefore, was markedly different from that in bo- real Sphagnum peat bogs.

[Decline of Activity and Shifts in the Methanotrophic Community Structure of an Ombrotrophic Peat Bog after Wildfire].

This study examined potential disturbances of methanotrophic communities playing a key role in reducing methane emissions from the peat bog Tasin Borskoye, Vladimir oblast, Russia as a result of the 2007 wildfire. The potential activity of the methane-oxidizing filter in the burned peatland site and the abundance of indigenous methanotrophic bacteria were significantly reduced in comparison to the undisturbed site. Molecular analysis of methanotrophic community structure by means of PCR amplification and cloning of the pmoAgene encoding particulate methane monooxygenase revealed the replacement of typical peat-inhabiting, acidophilic type II methanotrophic bacteria with type I methanotrophs, which are less active in acidic environments. In summary, both the structure and the activity of the methane-oxidizing filter in burned peatland sites underwent significant changes, which were clearly pronounced even after 7 years of the natural ecosystem recovery. These results point to the long-term character of the disturbances caused by wildfire in peatlands.

[Abundance and diversity of methanotrophic Gammaproteobacteria in northern wetlands].

Numeric abundance, identity and pH preferences of methanotrophic Gammaproteobacteria (type I methanotrophs) inhabiting the northern acidic wetlands were studied. The rates of methane oxidation by peat samples from six-wetlands of European Northern Russia (pH 3.9-4.7) varied from 0.04 to 0.60 µg CH4 g(-1) peat h(-1). The number of cells revealed by hybridization with fluorochrome-labeled probes M84 + M705 specific for type I methanotrophs was 0.05-2.16 x 10(5) cells g(-1) dry peat, i.e. 0.4-12.5% of the total number of methanotrophs and 0.004-0.39% of the total number of bacteria. Analysis of the fragments of the pmoA gene encoding particulate methane monooxygenase revealed predominance of the genus Methylocystis (92% of the clones) in the studied sample of acidic peat, while the proportion of the pmoA sequences of type I methanotrophs was insignificant (8%). PCR amplification of the 16S rRNA gene fragments of type I methanotrophs with TypeIF-Type IR primers had low specificity, since only three sequences out of 53 analyzed belonged to methanotrophs and exhibited 93-99% similarity to those of Methylovulum, Methylomonas, and Methylobacter species. Isolates of type I methanotrophs obtained from peat (strains SH10 and 83A5) were identified as members of the species Methylomonaspaludis and Methylovulum miyakonense, respectively. Only Methylomonaspaludum SH10 was capable of growth in acidic media (pH range for growth 3.8-7.2 with the optimum at pH 5.8-6.2), while Methylovulum miyakonense 83A5 exhibited the typical growth characteristics of neutrophilic methanotrophs (pH range for growth 5.5-8.0 with the optimum at pH 6.5-7.5).

[Phylogeny of beta-xylanases from Planctomycetes].

Here, we present the results of a computational analysis of a group of hypothetical GH10 endo-beta-xylanases from the Planctomycetes, a bacterial phylum with poorly characterized functional capabilities. These proteins are encoded in all analyzed genomes of heterotrophic Planctomycetes and form a phylogenetically distinct and tight cluster. In addition, we determined nucleotide sequences for endo-beta-xylanase genes from five strains of Isosphaera-Singulisphaera group of the Planctomycetes. The trees constructed for the 16S rRNA genes and the inferred amino acid sequences of endo-beta-xylanases were highly congruent, thus suggesting the vertical transfer of endo-beta-xylanase genes and their functional importance in Planctomycetes.

[Filterable microbial forms in the Rybinsk water reservoir].

Molecular identification of the filterable forms of microorganisms in the water of the Rybinsk reservoir, one of the largest open water bodies in European Russia, was carried out. The number of ultrasmall microbial cells passing through 0.22 µm filters was 10(4) cells/mL. These were represented by both bacteria and archaea. Most bacterial 16S rRNA gene sequences retrieved from filtered water belonged to the Betaproteobacteria and exhibited high similarity (99.0-99.5%) to thos of bacteria of the genus Polynucleobacter. The archaeal 16S rRNA gene clone library was composed of the sequences of members of the Euryarchaeota, including the orders Methanobacteriales and Methanomicrobiales, as well as of two archaeal groups (LDS and RC-V) with no characterized representatives. The species composition of filterable bacteria from reservoir water wast different from that revealed previously in bogs and small lakes at catchment areas; The pool of filterable archaea in the reservoir exhibited, however, significant similarity to that for boggy catchment areas and was characterized by perdominance of the clade LDS. Available data indicate that this archaeal group is typical of the northern freshwater ecosystems, and the organisms of this group are represented by ultrasmall cells.

[Methanotrophic bacteria in cold seeps of the floodplains of northern rivers].

Small mud volcanoes (cold seeps), which are common in the floodplains of northern rivers, are a potentially important, although poorly studied sources of atmospheric methane. Field research on the cold seeps of the Mukhrina River (Khanty-Mansiysk Autonomous okrug, Russia) revealed methane fluxes from these structures to be orders of magnitude higher than from equivalent areas of the mid-taiga bogs. Microbial communities developing around the seeps were formed under conditions of high methane concentrations, low temperatures (3-5 degrees C), and near-neutral pH. Molecular identification of methane-oxidizing bacteria from this community by analysis of the pmoA gene encoding particulate methane monooxygenase revealed both type I and type II methanotrophs (classes Gammaproteobacteria and Alphaproteobacteria, respectively), with predomination of type I methanotrophs. Among the latter, microorganisms related to Methylobacterpsychrophilus and Methylobacter tundripaludum, Crenothrix polyspora (a stagnant water dweller), and a number of methanotrophs belonging to unknown taxa were detected. Growth characteristics of two isolates were determined. Methylobactersp. CMS7 exhibited active growth at 4-10 degrees C, while Methylocystis sp. SB12 grew better at 20 degrees C. Experimental results confirmed the major role ofmethanotrophic gammaproteobacteria in controlling the methane emission from cold river seeps.

[Analysis of the bacterial community developing in the course of Sphagnum moss decomposition].

Slow degradation of organic matter in acidic Sphagnum peat bogs suggests a limited activity of organotrophic microorganisms. Monitoring of the Sphagnum debris decomposition in a laboratory simulation experiment showed that this process was accompanied by a shift in the water color to brownish due to accumulation of humic substances and by the development of a specific bacterial community with a density of 2.4 x 10(7) cells ml(-1). About half of these organisms are metabolically active and detectable with rRNA-specific oligonucleotide probes. Molecular identification of the components of this microbial community showed the numerical dominance of bacteria affiliated with the phyla Alphaproteobacteria, Actinobacteria, and Phanctomycetes. The population sizes of Firmicutes and Bacteroidetes, which are believed to be the main agents of bacterially-mediated decomposition in eutrophic wetlands, were low. The numbers of planctomycetes increased at the final stage of Sphagnum decomposition. The representative isolates of Alphaproteobacteria were able to utilize galacturonic acid, the only low-molecular-weight organic compound detected in the water samples; the representatives of Planctomycetes were able to decompose some heteropolysaccharides, which points to the possible functional role of these groups of microorganisms in the community under study. Thus, the composition of the bacterial community responsible for Sphagnum decomposition in acidic and low-mineral oligotrophic conditions seems to be fundamentally different from that of the bacterial community which decomposes plant debris in eutrophic ecosystems at neutral pH.

[Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation methods].

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4-2.0 x 10(7) cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.

[Bacteria of the genus Burkholderia as a typical component of the microbial community of sphagnum peat bogs].

Bacteria of the genus Burkholderia are a typical component of the microbial complex of sphagnum peat bogs and constitute a substantial portion of the aerobic chemoorganotrophic isolates which are routinely obtained from these environments on acidic nutrient media. The ecophysiological characteristics of the 27 strains of such organisms, which were isolated from the peat of acidic sphagnum bogs of the boreal and tundra zones of Russia, Canada, and Estonia, were investigated in the present paper. The overwhelming majority of the Burkholderia strains isolated from these bogs were phylogenetically close to the species B. glathei, B. phenazinium, B. fungorum, and B. caryophylli, the typical inhabitants of soil and plant rhizosphere. The bog isolates utilized a broad range of substrates as carbon and energy sources, including organic acids, sugars, polyalcohols, and certain aromatic compounds. All the strains studied were capable of growth on nitrogen-free media. They developed in the pH ranges of 3.5 to 7.4 and from 3 to 37 degrees C, with the optima at pH 5-7 and 11-23 degrees C, respectively. They were therefore moderately acidophilic, psychroactive, dinitrogen-fixing microorganisms well adapted to the conditions of acidic northern sphagnum bogs.

[High abundance of planctomycetes in anoxic layers of a Sphagnum peat bog].

The depth distribution of planctomycete abundance has been examined in six different sites of the Sphagnum peat bog in Bakchar, Tomsk oblast, Russia. In situ hybridization of peat with the fluorescently labeled oligonucleotide probes PLA46 and PLA886, reported to be group-specific for representatives of the phylum Planctomycetes, revealed two distinct population maxima of these bacteria in all of the profiles examined. The first population maximum was detected in the uppermost, oxic layer of the bog profile, while the second maximum was located at a depth of 30 cm below the water table level. The population sizes of planctomycetes in the uppermost layer and at a depth of 30 cm were of the same order of magnitude and comprised 0.5-1.5 x 10(7) and 0.4-0.7 x 10(7) cells per g of wet peat, respectively. Only 25-30% of the total number of planctomycete cells in the anoxic layer could be detected if the probe PLA886, whose target specificity is restricted to taxonomically characterized aerobic planctomycetes of the genera Gemmata, Planctomyces, Pirellula, and Isosphaera, was used alone. Other planctomycete cells in this layer were detected only with the probe PLA46, which possesses a much wider scope. This suggests the affiliation of these organisms with a yet undescribed phylogenetic subgroup within the Planctomycetes.

[Estimation of the phylogenetic diversity of prokaryotic microorganisms in Sphagnum bogs with the use of fluorescence in situ hybridization (FISH)].

The microbial population of Sphagnum bogs of northern Russia was analyzed with respect to the presence and cell numbers of representatives of particular phylogenetic groups of prokaryotes by means of in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes with broad detection spectra. The total number of cells that hybridized with universal Archaea- or Bacteria-specific probes varied, in peat samples of different bogs, from 45 to 83% of the number of cells revealed by DAPI staining. Down the bog profiles, the total number of prokaryotes and the fraction of archaea among them increased. Application of a set of oligonucleotide probes showed that the number of microorganisms belonging to such phylogenetic lineages of the domain Bacteria as the phyla Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Acidobacteria, and Planctomycetes constituted, in total, 14.0-26.5% of the number of eubacteria detected in the samples. Among the bacteria identified in the peat samples, the most abundant were representatives of the classes Alphaproteobacteria and Betaproteobacteria and the phyla Acidobacteria, Bacteroidetes, and Actinobacteria.