SUPPLEMENTARY INFORMATION
Supplementary Methods
Chemical analyses
100 µL headspace samples of chloroethenes, ethene, methane, and hydrogen were measured by
gas chromatograph as described previously (Freeborn et al., 2005; Lee et al., 2006). Organic
acids, including lactate and acetate, were analyzed with a high-performance liquid
chromatograph as described previously (Freeborn et al., 2005).
QPCR Analysis
2 mL liquid samples were collected throughout the 14-day cycle for cell density measurements.
Cells were harvested by centrifugation (14,000 × g, 10 min). Qiagen DNeasy Blood and Tissue
Kit was used to extract DNA from the cell pellets according to the manufacturer’s instructions
for gram-positive bacteria. QPCR with universal Bacterial 16S rRNA gene primers and probe
(forward primer 5’-TCCTACGGGAGGCAGCAG-3’, probe 5’CGTATTACCGCGGCTGCTGG-3’, and reverse primer 5’-GTTTAVDGCRTRGACTACCA3’), with Dhc 16S rRNA gene primers and probe (forward primer 5’GGTAATACGTAGGAAGCAAGC-3’, probe 5’-ACATCCAACTTGAAAGACCAC-3’, and
reverse primer 5’-CCGGTTAAGCCGGGAAATT-3’), and with universal Archaeal 16S rRNA
gene primers and probe (forward primer 5’-ATTAGATACCCSBGTAGTCC-3’, probe 5’AGGAATTGGCGGGGGAGCAC-3’, and reverse primer 5’-GCCATGCACCWCCTCT-3’) was
carried out as previously described (Johnson et al., 2005a).
DNA and RNA extraction for microarray analysis
Cells were collected from 30 mL culture samples by vacuum filtration, stored at –80°C, and total
nucleic acids were extracted as previously described (West et al., 2008) with minor
modifications as follows. The ratio of phenol (pH 8.0)-chloroform-isoamylalcohol used for
extraction was adjusted to 25:24:1 (vol:vol), and the DNA/RNA pellet was resuspended in 50 µL
of nuclease-free water. DNA and RNA were separated and purified following manufacturer’s
instructions with the AllPrep DNA/RNA Mini Kit (Qiagen). RNA was treated with on-column
RNAse-free DNase (Qiagen), and additional DNA contamination was removed with the DNAfree kit (Ambion, Austin, TX) according to the manufacturers’ instructions. Purified DNA and
RNA were stored at –80°C prior to further use.
The masses of DNA and RNA per volume were quantified using a fluorometer (model TD-700,
Turner Designs, Sunnyvale, CA) and the Quant-iT PicoGreen dsDNA and Quant-iT RiboGreen
RNA reagents (Invitrogen Molecular Probes, Carlsbad, CA), respectively, according to the
manufacturer's instructions.
Microarray sample preparation and application
DNA and RNA were prepared for application to microarrays with minimal modifications to the
protocols outlined in section 3 of the Affymetrix GeneChip Expression Analysis Technical
Manual (Affymetrix, Santa Clara, CA). Briefly, 10 µg of community total RNA was used as
starting material for each RNA microarray analysis as recommended, and cDNA was
synthesized. Based on previous microarray experience with the ANAS culture (West et al.,
2008), 1 µg of community gDNA was applied to each DNA microarray. The cDNA and gDNA
was fragmented and labeled. Hybridized arrays were stained and washed according to standard
Affymetrix protocols and were scanned using an Affymetrix Scan3000 scanner.
Microarray data analysis
The hybridization signal intensities for each probe set were computed using Affymetrix
GeneChip software and the MAS5 algorithm (Affymetrix, 2001). The data set of each microarray
was normalized by scaling the signal intensity of every probe by setting the spiked-in positive
controls to a target signal intensity average of 2500, thus allowing for comparisons between
microarray chips. The GeneChip MAS5 software was used to implement the Wilcoxon signed
rank-based algorithm to evaluate the “presence” or “absence” of individual target sequences and
return a statistical P value (Affymetrix, 2001; Liu et al., 2002). DNA for any particular ORF was
considered “present” if each replicate probe set for that ORF had a signal intensity greater than
140 and a P value less than 0.05 in the DNA chips. The average coefficient of variation for a
probe set across DNA replicates was 12%. Of the 3186 probe sets that were not identified as
“present” by DNA analysis using these criteria, two gave high signal intensities from the RNA
analysis; the two anomalous probe sets target genes DehaBAV1_0548 and DhcVS_884 which
are both annotated only as hypothetical proteins. All probe sets that were considered “present”
by DNA microarray analysis were considered in the RNA analysis. Average signal intensity was
calculated for each RNA chip, and each chip was globally scaled to the average of the replicate
chips to compare intensities across time-points. RNA for any particular ORF was considered
“present” if the average signal intensity of the probe sets for that ORF across replicates was
greater than the highest signal intensity measured for the negative controls (signal intensity
>120) for at least one time-point in the experiment.
In this paper, the term “gene expression” is used to refer to the presence of RNA transcripts as
indicated by ORF signal intensity. “Up-regulation” and “down-regulation” respectively describe
increases and decreases in transcription as indicated by signal intensity changes over time.
All microarray data analyses were performed within the R statistical programming environment
(www.r-project.org) using packages available from Bioconductor, version 2.4.0 for Windows
(www.bioconductor.org). To test the hypothesis that a gene was differentially expressed during
the experiment, one-way analysis of variance (ANOVA) was applied using the “ftest” function
from the “multtest” package (Gentleman et al., 2004). The P values returned from the ANOVA
test were then adjusted to correct for multiple hypothesis testing by applying the Benjamini and
Hochberg (1995) procedure with the “rawp2adjp” function from the “multtest” package
(Gentleman et al., 2004). This procedure was used to control the false discovery rate below 5%.
Genes designated as differentially expressed were further restricted to those with changes in
expression between any two sampling time-points of greater than twofold. Differentially
expressed genes meeting these criteria were sorted by hierarchical clustering (Eisen et al., 1998)
using the “heatplot” function from the “made4” package (Culhane et al., 2005).
REFERENCES
Affymetrix, Inc., 2001. Statistical algorithms reference guide. Technical report. Affymetrix,
Santa Clara, CA.
Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: a practical and powerful
approach to multiple testing. J R Stat Soc Ser B 57:289–300.
Culhane A, Thioulouse CJ, Perrie`re G, Higgins DG. 2005. MADE4: an R package for
multivariate analysis of gene expression data. Bioinformatics 21:2789–2790.
Freeborn RA, West KA, Bhupathiraju VK, Chauhan S, Rahm BG, Richardson RE, AlvarezCohen L. 2005. Phylogenetic analysis of TCE-dechlorinating consortia enriched on a variety of
electron donors. Environ Sci Technol 39:8358-8368.
Eisen MB, Spellman PT, Brown PO, Botstein D. 1998. Cluster analysis and display of genomewide expression patterns. Proc Natl Acad Sci USA 95:14863–14868.
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y,
Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M,
Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JYH, Zhang J. 2004. Bioconductor:
open software development for computational biology and bioinformatics. Genome Biol 5:R80.
Lee PKH, Johnson DR, Holmes VF, He J, Alvarez-Cohen L. 2006. Reductive dehalogenase gene
expression as a biomarker for physiological activity of Dehalococcoides spp. Appl Environ
Microbiol 72:6161-6168.
Liu WM, Mei R, Di X, Ryder TB, Hubbell E, Dee S, Webster TA, Harrington CA, Ho MH, Baid
J, Smeekens SP. 2002. Analysis of high density expression microarrays with signed-rank call
algorithms. Bioinformatics 18:1593-1599.
Johnson DR, Lee PKH, Holmes VF, Alvarez-Cohen L. 2005. An internal reference technique for
accurately quantifying specific mRNAs by real-time PCR with application to the tceA reductive
dehalogenase gene. Appl Environ Microbiol 71:3866-3871.
West KA, Johnson DR, Hu P, DeSantis TZ, Brodie EL, Lee PKH, Feil H, Andersen GL, Zinder
SH, Alvarez-Cohen L. 2008. Comparative genomics of Dehalococcoides ethenogenes 195 and
an enrichment culture containing unsequenced Dehalococcoides strains. Appl Environ Microbiol
74:3533-3540.