Microbial co-occurrence patterns in deep Precambrian bedrock fracture fluids

The bacterial and archaeal community composition and the possible carbon assimilation processes and energy sources of microbial communities in oligotrophic, deep, crystalline bedrock fractures is yet to be resolved. In this study, intrinsic microbial communities from groundwater of six fracture zones from 180 to 2300aEuro-m depths in Outokumpu bedrock were characterized using high-throughput amplicon sequencing and metagenomic prediction. Comamonadaceae-, Anaerobrancaceae- and Pseudomonadaceae-related operational taxonomic units (OTUs) form the core community in deep crystalline bedrock fractures in Outokumpu. Archaeal communities were mainly composed of Methanobacteriaceae-affiliating OTUs. The predicted bacterial metagenomes showed that pathways involved in fatty acid and amino sugar metabolism were common. In addition, relative abundance of genes coding the enzymes of autotrophic carbon fixation pathways in predicted metagenomes was low. This indicates that heterotrophic carbon assimilation is more important for microbial communities of the fracture zones. Network analysis based on co-occurrence of OTUs revealed possible "keystone" genera of the microbial communities belonging to Burkholderiales and Clostridiales. Bacterial communities in fractures resemble those found in oligotrophic, hydrogen-enriched environments. Serpentinization reactions of ophiolitic rocks in Outokumpu assemblage may provide a source of energy and organic carbon compounds for the microbial communities in the fractures. Sulfate reducers and methanogens form a minority of the total microbial communities, but OTUs forming these minor groups are similar to those found in other deep Precambrian terrestrial bedrock environments.Peer reviewe

Mitochondrial Genome of Phlebia radiata Is the Second Largest (156 kbp) among Fungi and Features Signs of Genome Flexibility and Recent Recombination Events

Creative Commons Attribution License (CC BY 4.0) Volume: 9Mitochondria are eukaryotic organelles supporting individual life-style via generation of proton motive force and cellular energy, and indispensable metabolic pathways. As part of genome sequencing of the white rot Basidiomycota species Phlebia radiata, we first assembled its mitochondrial genome (mtDNA). So far, the 156 348 bp mtDNA is the second largest described for fungi, and of considerable size among eukaryotes. The P. radiata mtDNA assembled as single circular dsDNA molecule containing genes for the large and small ribosomal RNAs, 28 transfer RNAs, and over 100 open reading frames encoding the 14 fungal conserved protein subunits of the mitochondrial complexes I, III, IV, and V. Two genes (atp6 and tRNA-IleGAU) were duplicated within 6.1 kbp inverted region, which is a unique feature of the genome. The large mtDNA size, however, is explained by the dominance of intronic and intergenic regions (sum 80% of mtDNA sequence). The intergenic DNA stretches harness short (≤200 nt) repetitive, dispersed and overlapping sequence elements in abundance. Long self-splicing introns of types I and II interrupt eleven of the conserved genes (cox1,2,3; cob; nad1,2,4,4L,5; rnl; rns). The introns embrace a total of 57 homing endonucleases with LAGLIDADGD and GYI-YIG core motifs, which makes P. radiata mtDNA to one of the largest known reservoirs of intron-homing endonucleases. The inverted duplication, intergenic stretches, and intronic features are indications of dynamics and genetic flexibility of the mtDNA, not fully recognized to this extent in fungal mitochondrial genomes previously, thus giving new insights for the evolution of organelle genomes in eukaryotes.Peer reviewe

Revealing the unexplored fungal communities in deep groundwater of crystalline bedrock fracture zones in Olkiluoto, Finland

The diversity and functional role of fungi, one of the ecologically most important groups of eukaryotic microorganisms, remains largely unknown in deep biosphere environments. In this study we investigated fungal communities in packer-isolated bedrock fractures in Olkiluoto, Finland at depths ranging from 296 m to 798 m below surface level. DNA- and cDNA-based high-throughput amplicon sequencing analysis of the fungal internal transcribed spacer (ITS) gene markers was used to examine the total fungal diversity and to identify the active members in deep fracture zones at different depths. Results showed that fungi were present in fracture zones at all depths and fungal diversity was higher than expected. Most of the observed fungal sequences belonged to the phylum Ascomycota. Phyla Basidiomycota and Chytridiomycota were only represented as a minor part of the fungal community. Dominating fungal classes in the deep bedrock aquifers were Sordariomycetes, Eurotiomycetes and Dothideomycetes from the Ascomycota phylum and classes Microbotryomycetes and Tremellomycetes from the Basidiomycota phylum, which are the most frequently detected fungal taxa reported also from deep sea environments. In addition some fungal sequences represented potentially novel fungal species. Active fungi were detected in most of the fracture zones, which proves that fungi are able to maintain cellular activity in these oligotrophic conditions. Possible roles of fungi and their origin in deep bedrock groundwater can only be speculated in the light of current knowledge but some species may be specifically adapted to deep subsurface environment and may play important roles in the utilization and recycling of nutrients and thus sustaining the deep subsurface microbial community

Transfer RNA genes in <i>P. radiata</i> mtDNA.

[1]<p>tRNAscan predicts the tRNA type from the anticodon.</p>[2]<p>This tRNA was determined to be Ile through comparative means (see below).</p>[3]<p>The bit score of tRNA-Cys was below 20, which is a typical cut-off value for a pseudogene. The gene was predicted with exceptionally low score from all Basidiomycota mtDNAs.</p

Schematic view of the C-termini regions of <i>P. radiata</i> mtDNA <i>atp6</i> and <i>cox2</i> genes.

<p>Green lines denote <i>atp6</i> coding sequence region and the last exon of <i>cox2</i>. Spheres/ovals represent LAGLIDADG 1 (green) and GIY-YIG (grey) homing endonuclease domains. <b>A)</b> Region of 201 bp (orange) with high 1 vs. 1 sequence similarity, corresponding to the 3′-end of the <i>atp6</i> gene. Codon Adaptation Index (CAI) values are shown for the <i>atp6</i> N-terminal region (blue) and for the regions of high sequence similarity. Reference codon usage is from: 1) <i>atp6</i> N-terminus, 2) <i>atp6</i> N-terminus and <i>atp8</i>-<i>9</i>, 3) <i>atp6</i> N-terminus, <i>atp8-9</i>, <i>cox1-3</i>, <i>nad1-5</i> and <i>nad4L</i>. <b>B)</b> Separated by three LAGLIDADG 1 domains and a GIY-YIG domain, two regions of high 1 vs. 1 sequence similarity (orange) exist for the last 66 bp of the <i>cox2</i> gene. Image was generated with ExPASy PROSITE MyDomains Image Creator (<a href="http://prosite.expasy.org/mydomains/" target="_blank">http://prosite.expasy.org/mydomains/</a>) and edited in Inkscape version 0.48.2 (<a href="http://inkscape.org/" target="_blank">http://inkscape.org/</a>).</p

Contribution of the various features of <i>P. radiata</i> mtDNA to genome size.

<p>Conserved coding sequence refers to the conserved fungal mitochondrial proteome ORFs, rRNAs and tRNAs. Significant ORFs refer to additional identified and hypothetical protein coding sequences with E-value<0.001 obtained by BLASTp queries. Freestanding refers to intronless. Intronic significant ORFs include homing endonucleases, also exon-to-exon-fused ORFs, excluding intergenic ORF HEGs (see text).</p

Gene map of <i>Phlebia radiata</i> mtDNA.

<p>Colour of the scale (kb) bar indicates orientation of transcription: clockwise (CW, white), counter-clockwise (CCW, grey). Bars mark protein-coding (blue) and RNA (red) genes, and alternative C-termini in <i>atp6</i> and <i>cox2</i> are depicted (orange). Intron type is indicated in colour: group I (light grey), group II (yellow), and uncertain (white). Within introns, the hypothetical and identified ORFs are indicated: over >200 amino-acid long ORFs (turquoise), and homing endonuclease domains GIY-YIG (black) and LAGLIDADG (dark grey). The transparent ribbon illustrates location of the 6,076 bp inversion-duplication. Asterisk indicates putative tRNA-Ile^CAU. The inner circle (scale at 12 o’clock in linear units) plots nucleotide bias (G/C skew) up to hexamers along each mtDNA position; G/C (red), A/T (blue), total strand bias (black), placing <i>oriC</i> around 11∶30 o’clock as the largest bias of G over C.</p

Phylogeny of fungal mitochondrial proteomes.

<p>The statistically most likely tree was derived by Bayesian inference from a multi-gene superalignment of mtDNA-encoded proteins (2 019 aa positions, 44 taxa). Posterior consensus support values are depicted for branching, and nodes receiving ≤0.8 support were collapsed into polytomies. The tree was rooted from mid-point. Colours refer to phyla or sub-phyla: turquoise, Chytridiomycota; yellow, Monoblepharidomycota; orange brown, Glomeromycota; dark blue, Ascomycota; pink, Ustilaginomycotina (Basidiomycota); red, Pucciniomycotina (Basidiomycota); bright green, Agaricomycotina (Basidiomycota); Blastocladiomycota node as outgroup. Scale bar indicates amino-acid substitutions per site. For species and mtDNA accession, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097141#pone-0097141-t005" target="_blank">Table 5</a>.</p