Telmatocola sphagniphila gen. nov., sp. nov., a Novel Dendriform Planctomycete from Northern Wetlands

Members of the phylum Planctomycetes are common inhabitants of northern wetlands. We used barcoded pyrosequencing to survey bacterial diversity in an acidic (pH 4.0) Sphagnum peat sampled from the peat bog Obukhovskoye, European North Russia. A total of 21189 bacterial 16S rRNA gene sequences were obtained, of which 1081 reads (5.1%) belonged to the Planctomycetes. Two-thirds of these sequences affiliated with planctomycete groups for which characterized representatives have not yet been available. Here, we describe two organisms from one of these previously uncultivated planctomycete groups. One isolate, strain OB3, was obtained from the peat sample used in our molecular study, while another strain, SP2T (=DSM 23888T = VKM B-2710T), was isolated from the peat bog Staroselsky moss. Both isolates are represented by aerobic, budding, pink-pigmented, non-motile, spherical cells that are arranged in unusual, dendriform-like structures during growth on solid media. These bacteria are moderately acidophilic and mesophilic, capable of growth at pH 4.0–7.0 (optimum pH 5.0–5.5) and at 6–30°C (optimum 20–26°C). The preferred growth substrates are various heteropolysaccharides and sugars, the latter being utilized only if provided in low concentrations (≤0.025%). In contrast to other described planctomycetes, strains SP2T and OB3 possess weak cellulolytic potential. The major fatty acids are C16:1ω5c, C18:1ω5c, C16:0, and C18:0. Characteristic lipids are the n-C31 polyunsaturated alkene (9–10 double bonds) and C30:1/C32:1 (ω-1) hydroxy fatty acids. The G + C content of the DNA is 58.5–59.0 mol%. Strains SP2T and OB3 share identical 16S rRNA gene sequences, which exhibit only 86 and 87% similarity to those of Gemmata obscuriglobus and Zavarzinella formosa. Based on the characteristics reported here, we propose to classify these novel planctomycetes as representatives of a novel genus and species, Telmatocola sphagniphila gen. nov., sp. nov

Pyrosequencing-Based Assessment of the <i>Bacteria</i> Diversity in Surface and Subsurface Peat Layers of a Northern Wetland, with Focus on Poorly Studied Phyla and Candidate Divisions

<div><p>Northern peatlands play a key role in the global carbon and water budget, but the bacterial diversity in these ecosystems remains poorly described. Here, we compared the bacterial community composition in the surface (0–5 cm depth) and subsurface (45–50 cm) peat layers of an acidic (pH 4.0) <i>Sphagnum</i>-dominated wetland, using pyrosequencing of 16<b> </b>S rRNA genes. The denoised sequences (37,229 reads, average length ∼430 bp) were affiliated with 27 bacterial phyla and corresponded to 1,269 operational taxonomic units (OTUs) determined at 97% sequence identity. Abundant OTUs were affiliated with the <i>Acidobacteria</i> (35.5±2.4% and 39.2±1.2% of all classified sequences in surface and subsurface peat, respectively), <i>Alphaproteobacteria</i> (15.9±1.7% and 25.8±1.4%), <i>Actinobacteria</i> (9.5±2.0% and 10.7±0.5%), <i>Verrucomicrobia</i> (8.5±1.4% and 0.6±0.2%), <i>Planctomycetes</i> (5.8±0.4% and 9.7±0.6%), <i>Deltaproteobacteria</i> (7.1±0.4% and 4.4%±0.3%), and <i>Gammaproteobacteria</i> (6.6±0.4% and 2.1±0.1%). The taxonomic patterns of the abundant OTUs were uniform across all the subsamples taken from each peat layer. In contrast, the taxonomic patterns of rare OTUs were different from those of the abundant OTUs and varied greatly among subsamples, in both surface and subsurface peat. In addition to the bacterial taxa listed above, rare OTUs represented the following groups: <i>Armatimonadetes</i>, <i>Bacteroidetes</i>, <i>Chlamydia</i>, <i>Chloroflexi</i>, <i>Cyanobacteria</i>, <i>Elusimicrobia</i>, <i>Fibrobacteres</i>, <i>Firmicutes</i>, <i>Gemmatimonadetes</i>, <i>Spirochaetes</i>, AD3, WS1, WS4, WS5, WYO, OD1, OP3, BRC1, TM6, TM7, WPS-2, and FCPU426. OTU richness was notably higher in the surface layer (882 OTUs) than in the anoxic subsurface peat (483 OTUs), with only 96 OTUs common to both data sets. Most members of poorly studied phyla, such as the <i>Acidobacteria</i>, <i>Verrucomicrobia</i>, <i>Planctomycetes</i> and the candidate division TM6, showed a clear preference for growth in either oxic or anoxic conditions. Apparently, the bacterial communities in surface and subsurface layers of northern peatlands are highly diverse and taxonomically distinct, reflecting the different abiotic conditions in microhabitats within the peat profile.</p></div

Phylogenetic tree showing the positions of peat bog 16 S rRNA gene sequences within the phylum <b><i>Acidobacteria</i></b><b>.</b>

<p>Peat-derived reads are displayed in relation to 16<b> </b>S rRNA gene sequences from cultured representatives of subdivisions 1 and 3 of the <i>Acidobacteria</i> and from other environmental samples. Only OTUs represented by at least 100 reads in one of the two sequence data sets are shown (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063994#pone-0063994-g005" target="_blank"><u>Figure 5</u></a>). 16<b> </b>S rRNA gene sequences from <i>Geothrix fermentans</i> H-5^T (U41563) and <i>Holophaga foetida</i> TMBS4^T (X77215), both being members of the acidobacterial subdivision 8, were used as an outgroup (not shown). Green and brown bars indicate the number of 454 reads retrieved for that branch from surface and subsurface peat, respectively. The distance bar indicates 0.1 substitutions per nucleotide position.</p

Bacterial taxon richness in acidic peat as assessed by barcoded pyrosequencing of 16 S rRNA genes.

<p>The rarefaction curves show the relation between the increase in bacterial OTUs and the number of randomly sampled sequences, separately for each subsample: I, orange; II, red; III, purple; and IV, green [experimentally observed richness (A) versus that predicted by Chao1 (B)]. The curves for the subsamples from surface and subsurface peat are shown by solid and dashed lines, respectively.</p

Phylogenetic tree showing the positions of 16 S rRNA gene sequences within the phylum <b><i>Planctomycetes</i></b><b>.</b>

<p>Peat-derived reads are displayed in relation to 16<b> </b>S rRNA gene sequences from cultivated representatives of the <i>Planctomycetes</i> and from other environmental samples. Only OTUs represented by more than 10 reads in at least one of the two sequence data sets are shown. 16<b> </b>S rRNA gene sequences from <i>Geothrix fermentans</i> H-5^T (U41563) and <i>Holophaga foetida</i> TMBS4^T (X77215) were used as an outgroup (not shown). Green and brown bars indicate the number of 454 reads retrieved for the respective branch from surface and subsurface peat, respectively. The distance bar indicates 0.1 substitutions per nucleotide position.</p

Phylogenetic tree showing the positions of peat bog 16 S rRNA gene sequences within the phylum <b><i>Verrucomicrobia</i></b><b>.</b>

<p>Peat-derived reads are displayed in relation to 16<b> </b>S rRNA gene sequences from cultivated representatives of subdivisions 1–7 of the <i>Verrucomicrobia</i> and from other environmental samples. Only OTUs represented by more than 10 reads in at least one of the two sequence data sets are shown. 16<b> </b>S rRNA gene sequences from <i>Gemmata obscuriglobus</i> and five <i>Gemmata</i>-like planctomycetes (ABGO01000192, X81957, AF239694, AF239696, AF239697, AF239698) were used as an outgroup (not shown). Green and brown bars indicate the number of 454 reads retrieved for that branch from surface and subsurface peat, respectively. The distance bar indicates 0.1 substitutions per nucleotide position.</p

OTU distribution and relative sequence abundance of bacterial phyla and candidate divisions among the subsamples from subsurface peat.

<p>(A) Venn diagram showing the number of OTUs unique to a single subsample or shared by two, three, or all four subsamples. (B) Relative sequence abundance of bacterial phyla and candidate divisions based on OTUs that were detected either in all four subsamples (core OTUs) or only in a single subsample (unique OTUs). Phyla that were detected only in subsurface peat, but not in the surface layer, are marked by asterisks.</p

Taxonomic assignment of 16<b> </b>S rRNA gene sequences affiliated with the <i>Acidobacteria</i>, which were retrieved from surface and subsurface peat layers (the analysis was made in MOTHUR by using SILVA reference database, at a confidence threshold of 80%).

<p>Taxonomic assignment of 16<b> </b>S rRNA gene sequences affiliated with the <i>Acidobacteria</i>, which were retrieved from surface and subsurface peat layers (the analysis was made in MOTHUR by using SILVA reference database, at a confidence threshold of 80%).</p

Heat map showing the most abundant OTUs and their distribution in surface and subsurface peat.

<p>OTUs are defined at a 97% sequence identity threshold. The digits indicate the total number of 454 reads recovered for the particular OTU from either surface (oxic) or subsurface (anoxic) peat. Low and high sequence abundances are highlighted in blue and red, respectively.</p

Bacterial community composition in surface (green bars) and subsurface (brown bars) peat.

<p>(A) Depth profile of <i>Sphagnum</i>-dominated peat bog. (B) Major taxa detected with a relative sequence abundance ≥0.5% are displayed. Column “other bacteria” indicates the combined relative sequence abundance of all the rare phyla and candidate divisions [each <0.5%] and of the taxonomically unclassified sequences. The error bars indicate the standard deviation of relative sequence abundances between the four subsamples. The number of 454 reads assigned to a particular taxon was significantly different between surface and subsurface peat based on two-tailed t-test (<i>P</i><0.05), except for those four taxa indicated by asterisk.</p