Open Access Research article

Site-Specific Mobilization of Vinyl Chloride Respiration Islands by a Mechanism Common in Dehalococcoides

Paul J McMurdie1, Laura A Hug2, Elizabeth A Edwards23, Susan Holmes4 and Alfred M Spormann15*

Author Affiliations

1 Department of Civil and Environmental Engineering, Stanford University, Stanford, California, USA

2 Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada

3 Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

4 Department of Statistics, Stanford University, Stanford, California, USA

5 Department of Chemical Engineering, Stanford University, Stanford, California, USA

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BMC Genomics 2011, 12:287  doi:10.1186/1471-2164-12-287

Published: 2 June 2011

Additional files

Additional file 1:

Figure S1: Alignment and Predicted Secondary Structure of Putative tRNA-gly. These tRNA-gly are strongly conserved in 16 Dehalococcoides ssrA-GI integration modules. Bases are shaded according to the Vienna RNA conservation coloring schema in both the alignment (A) and secondary structure cartoon indicating the majority consensus with degeneracy (B). Secondary structure prediction was unanimous from three independent secondary structure prediction servers [80-82]. Free energy of the thermodynamic ensemble is -54.26 kcal/mol [80]. Substructure labels correspond to classical tRNA, including the apparent anti-codon 'DCC'.

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Additional file 2:

Figure S2: Primers Mapped onto an Alignment of 16 ssrA Integration Modules. (A) Annotated alignment of the 16 integration modules discussed in this study. Individual sequences are shown as a thick black line, with gaps indicated by a thin horizontal line. Plot of average nucleotide identity (14 bp window) for all 16 sequences is shown along the top of the alignment. Three main target locations for primer design are indicated with downward-pointing black triangles, numbered beginning at ssrA (left). (B) Zoomed-in view of the alignment at the three target locations for primer binding. The 75% Consensus sequence is depressed slightly at the region targeted by primers, which are annotated along the top. Exact position of putative tRNA-gly is also shown.

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Additional file 3:

Figure S3: Phylogenetic Tree of ssrA Versus 16S rRNA gene. The most likely of 100 bootstrap Maximum Likelihood trees with bootstrap support shown at nodes. Support not shown at nodes with poor or ambiguous support. (A) Phylogenetic tree of ssrA, the ~350 bp gene encoding tmRNA. (B) Similarly calculated tree based on the 16S rRNA gene (~1500 bp), reflected relative to typical tree orientation to emphasize topological similarity with (A). Other Chlorofiexi are included, with Staphylococcus aureus as an outgroup. Full name and accession number correspond to the following abbreviations: Dehalococcoides - Dhc; CBDB1 - Dhc CBDB1 NC_007356; GT - Dhc GT NC_013890; BAV1 - Dhc BAV1 NC_009455; 195 - Dhc ethenogenes 195 NC_002936; VS - Dhc VS NC_013552; Deha lyk - Dehalogenimonas lykanthroporepellens BL-DC-9 NC_014314; Staph aur - Staphylococcus aureus NC_002952; Rose cast - Ro-seiflexus castenholzii DSM 13941 NC_009767; Rose RS-1 - Roseiflexus sp. RS-1 NC_009523; Chlo aur - Chloroflexus aurantiacus J-10-fl NC_010175; Chlo agg - Chloroflexus aggregans DSM 9485 NC_011831.

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Additional file 4:

Table S1: Parameters and example calculations utilized in divergence age estimates. (Top table) Summary of age estimates for Dehalococcoides-related genetic divergence utilizing four different models for rate of evolution: (1) estimated universal bacterial rate of evolution in nature [74], (2) in vitro E. coli empirically derived rate of evolution [75], (3) empirical Dehalococcoides rate based on observed mutations in the whole genomes of strain 195 and its resequenced variant in the DONNA2 sister culture (see Methods), and (4) the 16S rRNA gene clock model. For ages based on the first two rates of evolution, we further considered six different values for doubling time that span a range relevant to Dehalococcoides, including four published values for Dehalococcoides growth in laboratory culture [4,6,88,96], other anaerobic bacterial growth rates [47], and values derived from environmental anaerobic systems [48,49], as well as one arbitrarily large value (130 days) intended to represent general substrate-limited conditions. The left two columns indicate the divergence being considered and the tree calculation method, respectively. Ages are presented in units of 1 million years. (Middle Two Tables) Referenced summary of growth rates utilized for the age estimate calculations. (Bottom Table) Sample calculation for length of time to a single mutation, given rates of evolution taken from literature and the averaged Dehalococcoides growth rate.

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Additional file 5:

Figure S4: KB-1 variant at vcr-GI module transition. (A) Cartoon representation of the vcr-GI observed in all 8 versions, as shown in Figure 3. (B) Alignment of the region at the transition between integration and vcrABC cargo modules, including reads in the KB-1 metagenome dataset that disagree with the main consensus at this location. All 3 of these variant reads are perfectly identical to the VS, GT, WL, and WBC-2 vcr-GIs at this position.

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Additional file 6:

Figure S5: Genetic Map of Putative Fixed rdhA Region Downstream of Direct Repeats. (Top) Genetic map output from a Mauve alignment of the portion of High Plasticity Region 2 (HPR2) downstream of any ssrA direct repeats in the Dehalococcoides genomes. Each sequence was first aligned at tRNA-Ala-3 previously defining the boundary of HPR2 closest to the Ori [9], with local collinear blocks (LCBs) indicating large collinear homologous region that are free from rearrangements, but not necessarily indels. Large gaps were manually inserted such that vertical positions also containing the identity graph indicate aligned positions within the LCB. The darker grey LCB is the putative 'fixed' region of HPR2 downstream of any ssrA direct repeats. The lighter grey LCB is a portion of the Dehalococcoides core genome that surrounds the Ori. Annotated genes are shown beneath each LCB, with genes on the forward and reverse strands drawn as rectangles above or below the midline, respectively. rdhA are shaded red for emphasis. Scale bar shown in top left corner. Note that two different contigs from the ANAS genome are included. (Bottom) Phylogenetic trees of three semi-core (missing strain BAV1) rdhA that share a syntenic neighborhood within the putative fixed region. Each orthologous rdhA group recapitulates the topology and approximate genetic distances of the whole-genome tree (Figure 4). HPR2 was deleted in strain BAV1 [9], save for a ~600 bp rdhA fragment (DehaBAV1_1302) that is the basis for the tree on the right-hand side.

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Additional file 7:

Figure S6: Genetic Map of a dsiB-Containing Deep-Sea Environmental Fosmid. The fosmid, EU016565, contains the most similar non-Dehalococcoides integration module(s) detected in the public database. EU016565 is part of an environmental shotgun sequencing dataset of genomic DNA obtained from a 4000 m sub-seafloor sediment [87]. Two partial Dehalococcoides ssrA integration modules are detectable, one of which contains an ssrA direct repeat at the expected location within a dsiB homolog. It also contains 4 of the 6 protein encoding genes typically found in integration modules as well as the putative tRNA embedded within mom homolog. The reverse-complement of EU016565 is displayed for consistent orientation with other figures. Light grey, dark grey, and black indicate protein encoding genes for which the annotation is hypothetical, identifiable, or part of the integration module, respectively.

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Additional file 8:

Figure S7: Geographic locations of Dehalococcoides strains and cultures mentioned in this article. The underlying map was created using Google Earth. Labels have a dark red border if they are cultures/strains for which high throughput sequencing data is available and vinyl chloride respiration is reported. Blue borders indicate the vinyl chloride respiring cultures for which genomic island data was obtained during this study. White stars indicate cultures/strains for which no high throughput sequencing data was available at the time of this publication. The origin of the Dehalococcoides isolate FL2 [88] and the Dehalococcoides enrichment culture 'Pinellas' [89] are also shown. The following isolated bacterial strains were discussed in the manuscript: Dehalococcoides ethenogenes 195 - Ithaca Wastewater Treatment Plant, Ithaca, NY, USA [6,90]; CBDB1 - Saale River, Jena, Germany [91-93]; BAV1 - Bachman Road Site, Oscada, MI, USA [94]; VS - Contaminated Site, Victoria, Texas, USA [95]; GT - Hydrite Chemical Co., Cottage Grove, WI, USA [17]; Dehalogenimonas lykanthroporepellens BL-DC-9 [46]. The following Dehalococcoides enrichments were discussed. An asterisk indicates that no high-throughput sequence data is currently available: KB-1 - Southern Ontario, Canada [25]; ANAS - Alameda Naval Air Station, CA, USA [27] *PM - Point Mugu Naval Weapon Facility, CA, USA [28]; *EV - Evanite contaminated site, Corvallis, Oregon, USA [28]; *WBC-2 - West Branch Canal Creek, Aberdeen Proving Ground, MD [29] *WL - contaminated site, Western Louisiana, USA [30].

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