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The Hydrogenase Chip: A Tiling Oligonucleotide DNA Microarray Technique
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for Characterizing Hydrogen Producing and Consuming Microbes in Microbial
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Communities
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Supplemental Methods Section
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Hydrogenase Chip Design Variations for Each Version
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For Hydrogenase Chip version one, 199 genes involved in formate metabolism were
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retrieved from IMG/M by searching for annotations of genes encoding pyruvate
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formate lyase, formate hydrogen lyase, and formate dehydrogenase. These genes
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were added to the design after all retrieved hydrogenase genes were included and
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space for more probes remained.
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For Hydrogenase Chip version two, 57 reductive dehalogenase (rdhA) genes were
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retrieved from the NCBI non-redundant nucleotide database to fill space after all
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hydrogenase genes were included.
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For Hydrogenase Chip version 3, additional gene sequences were obtained from
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three sources: by retrieving all annotated hydrogenase and reductive dehalogenase
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gene sequences from all “Dehalococcoides” genomes in IMG version 2.9 (these were
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then not subjected to CD-HIT clustering), by retrieving hydrogenase sequences from
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published clone libraries (Boyd et al. 2009., Xin et al. 2008), and from the uptake
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[NiFe]-hydrogenase sequence from the genome of Microcoleus chthonoplastes PCC
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7420. The reductive dehalogenase gene sequences were derived from all
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“Dehalococcoides” genomes in IMG version 2.9.
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Operation of Reductive Dechlorinating Chemostat
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The Point Mugu dehalogenating enriched microbial culture was maintained in a
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chemostat reactor following ten years of batch cultivation as described in Yu, Dolan,
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and Semprini (Yu et al., 2005). This batch culture was used to inoculate the first in a
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series of three identically-operated chemostats, the third of which (“PM-5L”)
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provided culture for this experiment after nearly 250 days of operation.
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The PM-5L chemostat consists of a 5L (nominal) GL-45 Kimax reactor fitted with a
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three-hole Teflon cap (Kontes Glass Co., Vineland, NJ) that is compatible with PEEK
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tubing and fittings. It is an all-liquid reactor stirred using a 2” Teflon-coated
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magnetic stir bar. The influent feed solution was introduced to the chemostat via
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PEEK tubing, which was connected to a Hamilton 100-ml gas tight syringe driven by
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an Orion M361 syringe pump (Thermo Electron Corp., Beverly, MA). A dilution rate
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of 0.0186/day was maintained. The influent feed was a base of sterile basal
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anaerobic medium described by Yang and McCarty (1998), adjusted to double the
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buffering capacity (1 g/L K2HPO3 and 3 g/L Na2CO3) and increase the reductant, H2S,
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to 20 mg/L. The medium was amended with PCE (saturated, 1.15 mM), and sodium
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lactate (4.3 mM) as electron donor. Sulfate was not added to the influent feed. The
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chemostat was equipped to anaerobically transfer culture to batch reactors through
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PEEK tubing by pressurizing the chemostat with furnace-treated, anaerobic N2 gas.
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At the time of the batch experiment, a pseudo-steady state had been reached in the
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chemostat. Electrons from lactate were distributed as follows: 67% to acetate, 8%
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biomass, 2% VC, 16% ethene, and 7% unaccounted for, with a residual liquid H2
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concentration of 3.25 nM.
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Chemical Analytical Methods
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Reactor headspace samples were used to monitor chlorinated aliphatic
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hydrocarbons (CAHs), ethene, and H2. CAHs and ethene were measured with an HP-
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6890 gas chromatograph (GC) equipped with a photoionization flame ionization
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detector and a 30m-0.53mmGS-Q column (J&W Scientific, Folsom, CA), with helium
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as the carrier gas (15 mL/min). The headspace samples were injected to the GC
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using a 100μL gastight syringe (Hamilton, Leno, NV). The GC oven was initially set
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at 150°C for 2 min, heated at 45°C/min to 220°C, and held at 220°C for 1.44 min.
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Hydrogen concentrations in headspace gas samples (100 μL) were determined
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using an HP-5890 GC series II with a thermal conductivity detector (TCD), operated
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isothermally at 220°C. Gas samples were chromatographically separated with a
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Carboxen 1000 column (15 ft-1/8 in, Supelco, Bellefonte, PA) using argon as the
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carrier gas at 15 mL/min. Liquid samples (0.25 mL) were taken and diluted ten
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times with deionized water to measure sulfate and acetate concentrations. Sulfate in
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the aqueous phase was monitored with a Dionex DX-500 ion chromatograph
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(Sunnyvale, CA) equipped with an electrical conductivity detector and a Dionex
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AS14 column. Acetate was measured with a Dionex-500 HPLC chromatograph
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equipped with UV/VIS detector and an Alltech Prevail Organic acid column. Sulfide
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measurements were obtained with 0.3mL samples using the methylene blue
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colometric HACH method 8131.
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Batch Cultures
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Batch experiments were carried out in 125mL Borosilicate glass bottles fitted with
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phenolic screw-on caps with gray chlorobutyl rubber septa (Wheaton Industries,
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Millville, NJ). 50mL of culture was anaerobically transferred from the PM-5L
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chemostat into six reactors via PEEK tubing. Each bottle was purged with a furnace
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treated 75:25 Ar/CO2 gas mixture (Airco, Inc. (Albany, OR)) for 15 minutes to
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remove any residual chloroethenes, ethene, or H2 following the transfer.
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To enrich different populations in the culture (dehalogenating, sulfate-reducing, and
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combination of sulfate-reducing and dehalogenating microbes) three sets of
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duplicates were prepared: “P” bottles were amended with 15 μmol neat PCE
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(99.9%, spectrophotometric grade from Acros Organics (Pittsburgh, PA)), “S” bottles
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with 16 μmol sulfate (from a 228 mM Na2SO4 solution in media), and dual- electron
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acceptor “SP” bottles with 16 and 15 μmol sulfate and PCE, respectively. Sulfate and
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PCE were added to achieve equivalent electron acceptor level assuming sulfate was
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reduced to sulfide and PCE was reduced to ethene. Hydrogen (82 μmols, 99%, Airco,
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Inc. (Albany, OR)) was injected to the headspace, creating an initial H2 liquid
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concentration around 15,000 nM.
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Reactors were incubated at 20°C with continuous shaking at 200 rpm. PCE and
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transformation products, H2, sulfate, and acetate concentrations were monitored
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over time using gas chromatography (GC) with a flame ionization detector, GC with
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a thermo conductivity detector, ion chromatography, and high-performance liquid
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chromatography, respectively. Hydrogen was added whenever aqueous
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concentrations approached 1000nM, and bottles were amended to their initial PCE
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and sulfate concentration after PCE and sulfate had been completely reduced to VC,
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ethene, and sulfide, respectively. After 44 days (and eight to ten amendments), all
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bottles were amended with electron acceptors to the same electron accepting
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capacity as in the SP bottles, i.e. 15μmol of both PCE and sulfate were added to all
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bottles. Bottles were sacrificed for molecular analysis 1.2 days into this dual
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substrate experiment. Microcosm cultures were transferred to 50 mL polypropylene
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centrifuge tubes, centrifuged for 30 minutes at 9000 rpm and 4°C. Supernatant was
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decanted, and the pellets were stored at -80°C until analysis. An initial sample for
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RNA an DNA extraction (denoted “C”) was also collected from the chemostat in this
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same manner at the time of harvesting culture for the batch experiments.
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Reductive Dechlorinating Liquid Culture DNA/RNA Co-Isolation
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Liquid batch and chemostat cultures were transferred to 50mL Falcon tubes, then
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centrifuged for 30 minutes at 9000 rpm and 4°C. Supernatant was decanted, and the
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pellets were stored at -80°C until analysis. Frozen cell pellets were resuspended in
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1mL of the lysis solution described below for microbial mat nucleic acid extraction,
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and RNA/DNA extraction also carried out according to the same protocol.
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Primer Design and PCR
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All primers were designed using Geneious (Biomatters, Auckland, New Zealand)
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based on manual identification of conserved regions of the sequence alignment, and
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design of primers producing the desired sequence length and annealing
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temperature.
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A 400bp fragment of dsrA was PCR-amplified using forward primer Dsr-1F-GC (5’-
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ACSCACTGGAAGCACG-3’) and reverse primer Dsr-DGGE-Rev (5’-
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CGGTGMAGYTCRTCCTG-3’) as described by Leloup et al. (2009). PCR was
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performed in 50µL reactions containing 25µL 2X DreamTaq Green PCR Master Mix
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(Fermentas), 200nM of each primer, and 1µL of DNA in solution extracted from
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sample S.
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Primers DMR-15600_F-717 (5’- MAARAACCCSCAYMCCCAG-3’) and DMR-15600_R-
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1720 (5’- GACRTGYACRSMRCAG-3’) targeting the hynA-1 gene in Desulfovibrio sp.
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were designed based on hynA-1 sequences from Desulfovibrio sp. related to
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Desulfovibrio magneticus (IMG identifiers 637123154, 637783027, 639819476,
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643139751, 643538766, 643581839, 644801811, 644840066, and 645564504).
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These primers were used at a concentration of 500nM each to PCR-amplify hynA-1
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from DNA sample S in 50µL reactions with 25µL 2X DreamTaq Green Master Mix,
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cycled with an initial 95ºC denaturation step for 3min, followed by 45 cycles with
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30s at 95ºC, 30s at 48ºC, 30s at 72ºC, then a final 72ºC extension step of 10min. Gel
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extraction of the fragment with the expected amplicon size of approximately
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1000bp was performed using the Wizard SV Gel and PCR Clean-up System according
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to manufacturers instructions (Promega).
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Primers HupL_F (5’- ATGCAGAAGATAGTAATTGAYC-3’) and HupL_R (5’-
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GCCAATCTTRAGTTCCATMR-3’) for amplification of a 1099bp fragment of the hupL
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gene from Dehalococcoides sp. used in the plasmid standard and primers
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HupL_Fq_56 (5’- AAGCCACCGTAGACGGCG-3’) and HupL_Rq_190 (5’-
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AGTGCCGTGRGAGGTGGG-3’) for a 134bp fragment for qPCR were designed based
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on sequence alignments of hupL from Dehalococcoides sp. genomes of strains 195,
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BAV-1, CBDB1, and VS (IMG gene object identifiers 637119679, 646445988,
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637702682, and 640529159). HupL_F and HupL_R were used at a concentration of
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500nM each to PCR-amplify hupL from DNA sample S in 50µL reactions with 25µL
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2X DreamTaq Green Master Mix, cycled with an initial 95ºC denaturation step for
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3min, followed by 45 cycles with 30s at 95ºC, 30s at 55ºC, 30s at 72ºC, then a final
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72ºC extension step of 10min.
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Cloning
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The hynA-1 and dsrA PCR products were cloned using the TOPO TA cloning kit
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(Invitrogen), then Sanger-sequenced using the M13F primer (Elim Biopharm,
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Hayward, CA, USA). Using Geneious software, vector and primer sequence was
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trimmed from the sequences. Similar existing sequences were retrieved using the
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functional gene pipeline version 6.1 (http://fungene.cme.msu.edu/) and NCBI
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BLAST (Altschul et al., 1990), then a muscle alignment (Edgar et al., 2004) and
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PHYML tree bootstrapped 100X (Guindon et al., 2003) were generated with the
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sequences and their closest relatives (Biomatters, Auckland, New Zealand). Of ten
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clones generated for dsrA, three representative dsrA sequences were submitted to
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GenBank (accession numbers HQ399561- HQ399563). For hynA-1, two clones were
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sequenced and submitted to GenBank with accession numbers HQ399559 and
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HQ399560.
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Reverse Transcription - Quantitative PCR of Dehalococcoides sp. hupL
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A 1/10 dilution series was used for quantification, with eight dilutions from a 1/10
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dilution of the purified plasmid containing Dehalococcoides sp. hupL to a 1/108
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dilution.
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cDNA was synthesized using the Superscript III First-Strand Reverse Transcription
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Kit (Invitrogen) with random hexamers according to manufacturer’s instructions,
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albeit with a 3-hour 50ºC incubation. For each sample, in order to confirm that
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DNase treatment of the RNA was complete, a negative control cDNA synthesis
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reaction with no reverse transcriptase was performed.
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Triplicate reactions were performed for each sample in 25µL reactions containing
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12.5µL iQ SYBR Green Supermix (Biorad), 500nM of each primer, and 5µL of a 1/10
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dilution of cDNA or standard plasmid dilution. Thermal cycling and fluorometry was
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performed using an iCycler iQ Real-Time PCR Detection System (Biorad), with a 3-
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minute intial denaturation step at 95ºC, followed by 40 cycles of 10 seconds of 95ºC
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denaturation and 45 seconds of annealing/extension at 61.5ºC.
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Microbial Mat DNA/RNA Co-Isolation
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The top 2mm layer of the mat cores collected during that study were placed in a
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1mL a lysis solution consisting of 10mM EDTA, 50mM Tris-HCl, 4M guanidine
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thiocyanate, 2% sodium dodecyl sulfate, and 130mM -mercaptoethanol in a 2mL
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tube containing 0.1g 150-212µm acid-washed glass beads (Sigma-Aldrich, St. Louis,
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MO, USA). Tubes were vortexed at 4ºC at maximum speed for five minutes, then
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1mL of pH 4.5 acid-phenol:chloroform:isoamyl alcohol in the ratio 125:24:1
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(Ambion, Austin, TX, USA) was added. The solution was briefly vortexed, incubated
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at room temperature for five minutes, then centrifuged at 16,000 g (Eppendorf,
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Hamburg, Germany) for five minutes. The aqueous phase was removed and mixed
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with 40µL of RNase-free 3M sodium acetate (Ambion) and 1.7mL of -20ºC 100%
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ethanol. The mixture was incubated at -20ºC for one hour, centrifuged at 16,000g,
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4ºC for 30 minutes. The liquid phase was decanted leaving a nucleic acid pellet that
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was air-dried for ten minutes then resuspended in 200µL of nuclease-free water.
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100µL of this solution was stored at -20ºC for DNA analysis. 10µL TURBO DNase
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buffer and 2µL TURBO DNase (Ambion) were added to the remaining 100µL, then
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incubated at 37ºC for 30 minutes. An additional 2µL of DNase was added then
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incubated for a further 30 minutes. 120µL of the acid-phenol:chloroform:IAA
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solution were added, then the mixture was vortexed briefly, incubated at room
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temperature for one minutes, then centrifuged at 16,000g for two minutes. The
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aqueous phase was removed and mixed with 10µL 3M sodium acetate solution and
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300µL ethanol, incubated at -20ºC for 30 minutes, then centrifuged at 4ºC, 16,000g
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for 30 minutes. The liquid was decanted, RNA solution resuspended in 50µL
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nuclease-free water. RNA and DNA were quantified in the solution using the Qubit
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fluorometer and broad-range double-stranded DNA and broad-range RNA Quant-it
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quantification kits (Invitrogen).
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DNA Labeling and Hybridization
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DNA was mixed with random hexamers (Invitrogen) at a concentration of 250ng/µL
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in 39µL of water, then incubated at 95ºC for 10 minutes. The mixture was placed on
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ice for 30 seconds, then 5µL of NEBuffer 2 (New England Biolabs (NEB), Ipswich,
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MA, USA), 2µL of a dNTP labeling mix (5mM dATP, cCTP, dGTP, dTTP (Invitrogen),
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1.67mM amino-allyl labeled dUTP (Fermentas, Vilnius, Lithuania), and 4µL of
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Klenow Fragment (3´→5´ exo–) at 5000 units/mL (NEB). This Klenow reaction
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mixture was incubated for 16 hours at 37ºC, then stopped by the addition of 5µL
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0.5M EDTA. The amino-allyl labeled DNA (aa-DNA) was purified using the QIAquick
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PCR Purification Kit (Qiagen) with a custom-made phosphate wash buffer in place of
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the Qiagen-supplied buffer PE (50mM KPO4, 80% ethanol, pH 8.5) then dried down
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at 45ºC in a SpeedVac (Thermo Scientific, Waltham, MA, USA). A Cy3 mono-reactive
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dye pack (GE Healthcare Biosciences, Piscataway, NJ, USA) was resuspended in 13µL
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Dimethyl-sulfoxide (DMSO). aa-DNA was resuspended in 10µL nuclease-free water,
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0.5µL 1M sodium bicarbonate solution and 3µL of dissolved Cy3 dye, then incubated
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in the dark at room temperature for 60 minutes. Cy3-labeled DNA was again
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purified using the QIAquick PCR Purification Kit. Labeled DNA was quantified and
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Cy3 incorporation determined using a Nanodrop (Thermo Scientific). Labeled DNA
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was then hybridized to the test design DNA microarray at 65ºC for 17 hours
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according to the manufacturer’s protocol for one-color gene expression analysis
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(Agilent Technologies, Santa Clara, CA, USA). The hybridized microarray was
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scanned using a Genepix 4000B Microarray Scanner (Molecular Devices, Sunnyvale,
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CA, USA) and median probe intensity value used for further analysis.
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RNA Labeling and Hybridization
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The RNA amplification and labeling protocol was based on the Whole-Community
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RNA Amplification protocol (Gao et al., 2007). cDNA synthesis was performed with
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the Superscript III First-Strand synthesis kit (Invitrogen) following the
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manufacturer’s instructions, with the following modifications: 7µL of dissolved RNA
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(containing at least 500ng RNA) was used as starting material, and the primer
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added was 2µL 0.5µg/µL T7N6S primer (5’-
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AATTGTAATACGACTCACTATAGGGNNNNNN-3’), and the reverse transcription was
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performed overnight (18 hours) at 50ºC. The second strand cDNA was synthesized
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by adding 1µL of Klenow Fragment (New England Biolabs), 1µL 50ng/µL random
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hexamer solution (Invitrogen) to the cDNA first strand synthesis solution then
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incubated for two hours at 37ºC. The resulting cDNA was purified using the
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QIAquick PCR Purification Kit, quantified using the Qubit fluorometer and Quant-it
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broad-range double-stranded DNA kit, then dried down in a SpeedVac. This cDNA
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was used as a template for amino-allyl labeled RNA using the MEGAscript T7 Kit
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(Ambion) according to the manufacturer’s instructions, with the following
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modification: the 2µL UTP solution was replaced with 3µL of a 50mM 3:1 amino-
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allyl UTP : UTP solution mixture (Ambion). The resulting amino-allyl labeled cRNA
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was purified using the RNeasy Mini Kit (Qiagen) and eluted in 30µL nuclease-free
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water. A Cy3 mono-reactive dye pack (GE Healthcare) was resuspended in 13µL
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DMSO. 1.5µL 1M sodium bicarbonate and 3µL reactive Cy3 in DMSO were mixed
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with the amino-allyl-labeled cRNA then incubated in the dark at room temperature
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for 60 minutes. The resulting Cy3-labeled cRNA was purified using the RNeasy Mini
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Kit. Cy3 dye incorporation and cRNA quantity were determined using a Nanodrop.
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Labeled cRNA fragmentation, hybridization at 65ºC for 17 hours, and washing were
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performed according to the DNA microarray manufacturer’s instructions (Agilent
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Technologies, Santa Clara, CA). The hybridized microarray was scanned using a
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Genepix 4000B Microarray Scanner (Molecular Devices, Sunnyvale, CA, USA).
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DNA Microarray Data Analysis Continued
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For the reductive dechlorinating soil columns, both lactate and propionate amended
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time points were normalized to the formate sample in order to facilitate a
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meaningful three-way comparison. During analysis of the microbial mat samples,
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both the DNA and RNA probe intensities from the 20:00 time point were normalized
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to their respective 12:00 probe intensities. All reductively dechlorinating batch
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cultures were normalized to the chemostat sample.
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Quantitative shifts in gene or transcript abundance between samples were
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determined by calculating ln(A/B) for each probe targeting a given gene, where A is
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the probe intensity from one sample and B from another, then finding the median of
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these numbers. This median value is referred to as the log intensity ratio. A log
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intensity ratio > 0 signifies a greater abundance in sample A, and a result below 0
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signifies greater abundance in sample B. Since the set of ln(A/B) for a given gene
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generally fails tests of normality, statistics to determine significant changes in gene
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or transcript abundance based on an assumption that a distribution is normal could
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not be applied. Instead, the median absolute deviation was used as an estimate of
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variability in the measurement, and the binomial test (R function binom.test()) was
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applied to test the null hypothesis that the genes are in equal abundance (50% of
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probes with ln(A/B) > 0). Genes were considered of significantly different
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abundances in the two samples if the binomial test resulted in a null hypothesis
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probability of less than 0.01.
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