Distribution of dehalococcoidia in the anaerobic deep water of a remote meromictic crater lake and detection of dehalococcoidia-derived reductive dehalogenase homologous genes

Here we describe the natural occurrence of bacteria of the class Dehalococcoidia (DEH) and their diversity at different depths in anoxic waters of a remote meromictic lake (Lake Pavin) using 16S rRNA gene amplicon sequencing and quantitative PCR. Detected DEH are phylogenetically diverse and the majority of 16S rRNA sequences have less than 91% similarity to previously isolated DEH 16S rRNA sequences. To predict the metabolic potential of detected DEH subgroups and to assess if they encode genes to transform halogenated compounds, we enriched DEH-affiliated genomic DNA by using a specific-gene capture method and probes against DEH-derived 16S rRNA genes, reductive dehalogenase genes and known insertion sequences. Two reductive dehalogenase homologous sequences were identified from DEH-enriched genomic DNA, and marker genes in the direct vicinity confirm that gene fragments were derived from DEH. The low sequence similarity with known reductive dehalogenase genes suggests yet-unknown catabolic potential in the anoxic zone of Lake Pavin

Genomic fragments obtained from Lake Pavin using the specific-gene capture approach targeting insertion sequences.

<p>The genomic fragments were recovered at 68 m depth using the capture approach targeting IS911, an insertion sequence (IS) belonging to the IS3 family. In total, 18 genomic fragments with IS are grouped together into five distinct clusters.</p

Physical parameter measurement along Lake Pavin water column.

<p>Vertical profiles of dissolved oxygen (DO) and temperature measured in March 2012 using the submersible YSI GRANT 3800 multiparameter probe. Redox potential (Eh) measured in June 2013 in reference to a standard electrode and expressed in millivolts (mV) (Didier Jézéquel personal communication).</p

Quantification of ‘the total density of’ bacterial 16S rRNA genes and of two DEH-related phylotypes in Lake Pavin water column.

<p>Bacteria are represented by filled triangles. For DEH-related phylotypes: OTU1 is represented by filled diamonds and a dashed line and OTU2, by filled squares and a dotted line. Shown are mean values and standard deviations of triplicate PCR reactions.</p

Phylogenetic tree of <i>Dehalococcoidia</i> (DEH) 16S rRNA gene sequences highlighting the phylogenetic position of the 11 OTUs obtained from Lake Pavin water column.

<p>The phylogenetic analysis was performed using the neighbor-joining method; branches with bootstrap values (1000 replicates) of at least 50% are marked with a dot. The tree was constructed using MEGA version 6.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145558#pone.0145558.ref038" target="_blank">38</a>]. The branches were coloured using iTOL tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145558#pone.0145558.ref045" target="_blank">45</a>]. Sequences from this study fall into three classes defined by Wasmund <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145558#pone.0145558.ref015" target="_blank">15</a>]: GIF-9B in dark blue, MSBL5 in light blue and Ord-DEH in orange (<i>Dehalogenimonas</i>) and in yellow (<i>Dehalococcoides</i>). An additional class not previously described is presented in grey as an annotated new subgroup. Sequences of cultured DEH are highlighted in red. Sequences derived from single-cell genome DEH-J10 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145558#pone.0145558.ref016" target="_blank">16</a>] and an aquifer sediment metagenome-derived genome RBG-2 are highlighted in blue. The scale bar represents 1% sequence divergence.</p

Genomic fragments obtained using the specific-gene capture approach targeting an <i>rdhA</i> sequence identified in a Lake Pavin metagenome [39].

<p>The genomic fragments were recovered from the 68 m-depth sample by specific-gene capture approach using probes targeting a <i>rdhA</i> gene (first and second capture experiments) and a hypothetical protein (second capture experiment) identified in Lake Pavin water column. Capt_rdase, RdaseH8-1, RdaseH8-2, RdaseH8-3 and RdaseH8-4: capture probes.</p

Vertical distribution and relative abundance of the main bacterial phyla.

<p>Sample names starting with “Pav” indicate the samples were amplified from gDNA, names starting with “cDNA” indicate that the samples were processed by reverse transcription from mRNA. The number in the name indicates sample depth.</p