Supplemental Materials. DNA preparation. Dehalogenimonas lykanthroporepellens strain BL-DC-9 T (=ATCC

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1 Supplemental Materials DNA preparation. Dehalogenimonas lykanthroporepellens strain BL-DC-9 T (=ATCC BAA-1523 = JCM 15061) was grown in defined basal medium amended with 0.5 mm 1,1,2- trichloroethane (1,1,2-TCA) as described previously (S4). Cells were harvested by centrifugation at 10,000 g for 15 min at 4 o C. Genomic DNA was extracted from the resulting cell pellet using an UltraClean Microbial DNA Isolation kit (MoBio, Carlsbad, CA). Plasmids containing a partial 16S rrna gene fragment from strain BL-DC-9 T were constructed using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Inserts were produced by performing PCR using universal bacterial primers 27f and 1392r (S2) with strain BL-DC-9 T genomic DNA as template. After growth of recombinant Escherichia coli cells, plasmid DNA was extracted and purified using an UltraClean Mini Plasmid Prep kit (MoBio). Plasmids carrying an insert comprised of a partial 16S rrna gene amplified from Dehalococcoides were likewise prepared. The sequence of the cloned Dehaolococcoides plasmid insert, designated as DHC-4, was deposited in GenBank under accession no. GQ It shares % 16S rrna gene sequence identity with Dehalococcoides strains reported previously (9 nt different from Dehalococcoides ethenogenes strain 195 (AF004928) in the region of 1435 nucleotide positions compared). Design of 16S rrna gene primers targeting Dehalogenimonas sp. Oligonucleotide primers intended to target 16S rrna gene sequences unique to Dehalogenimonas were designed using variable and hypervariable regions in the 16S rrna gene sequences of D. lykanthroporepellens strains BL-DC-8 and BL-DC-9 T. Candidate primer sequences were selected manually, and the uniqueness of candidate sequences was examined by comparison to sequences in the GenBank database and using the Ribosomal Database Project II on-line Probe Match program ( S-1

2 Quantitative real-time PCR (qpcr). Concentrations of plasmid DNA were measured spectrophotometrically at 260 nm. A conversion factor of 50 μg/ml double strand DNA corresponding to an absorbance of 1.0 was applied in calculations. Serial ten-fold dilutions of DNA standards were prepared, and the threshold cycle values at each dilution level were determined in qpcr. Calibration curves, prepared using three independent serial dilutions of each DNA standard, were constructed by plotting log-transformed gene copy numbers against threshold cycle values. PCR amplification efficiency and 16S rrna gene copy number per reaction was calculated as described previously (S3). A plasmid size of 5308 bp [pcr 4-TOPO plasmid (3954 bp) (Invitrogen) plus insert (1354 bp)] and one 16S rrna gene copy per plasmid was assumed in calculations. A simple linear regression model was built using SAS 9.0 to calculate the slope, intercept, correlation coefficient (R 2 ), and standard deviation. DNA-free water or plasmids without target gene inserts were used as negative controls. Plasmid DNA carrying a partial 16S rrna gene insert from strain BL-DC-9 T was used to prepare calibration curves for total bacteria 16S rrna gene copy numbers using primers Bac1055YF/Bac1392R. Each sample was analyzed in triplicate. Following amplification, melting curve analysis was performed with 1.0 min denaturation at 95ºC, 1.0 min annealing at 55ºC, then 80 cycles of 0.5ºC increments (10 sec each) beginning at 55ºC. PCR amplification efficiency using primer set X with plasmid DNA containing a partial 16S rrna gene insert from Dehalogenimonas strain BL-DC-9 T ranged from %. PCR amplification efficiency using universal bacterial primers Bac1055YF/ Bac1392R ranged from % (corresponding to calibration curve slope of -3.37±0.10), which is consistent with the previously reported value (slope of -3.34±0.14) using the same primer set and plasmid DNA containing Dehalococcoides strain FL2 16S rrna gene insert as template (S3). For Dehalococcoides, PCR efficiency using primer set J ranged from 76-82% (corresponding to S-2

3 slope of -3.96±0.12), similar to that reported (slope of -3.80±0.22) for primer set Dhc1200F/ Dhc1271R in conjunction with plasmid DNA containing Dehalococcoides strain BAV1 16S rrna gene as template (S3). Linear ranges of calibration curves, correlation coefficients, and minimum detection quantities are summarized in Table S1. Primer set X (this study, Table 2) targeting Dehalogenimonas and primer set J (Table 1) targeting Dehalococcoides were used in qpcr to evaluate concentrations of putative dehalogenating bacteria in groundwater samples. To allow calculation of putative dehalogenating bacteria relative to the total bacterial population size, universal bacterial primers Bac1055YF/ Bac1392R (S3) were also employed to quantify total bacteria 16S rrna gene copies. qpcr was performed using an icycler iq PCR thermal cycler detection system (Bio-Rad) and a SYBR GreenER qpcr supermix kit (Invitrogen). Each 25 μl reaction contained: 12.5 μl 2 supermix, 0.5 μm each primer, 2 μl DNA extract diluted by a factor of 10, and 10 μl DNA-free water. The PCR thermal protocol was 50ºC for 2 min (UDG incubation) and 95ºC for 8.5 min (UDG inactivation and DNA polymerase activation), followed by 40 cycles of 95ºC for 15 sec and annealing and extension for 60 sec. Annealing/ extension temperatures were 68, 65, and 64ºC for reactions with primer sets X, J, and Bac1055YF/ Bac1392R, respectively. Preliminary experiments demonstrated that these annealing temperatures did not produce amplicons when Dehalococcoides primer set J was used in reactions with BL-DC-9 T DNA as template or when Dehalogenimonas primer set X was used in reactions with plasmid DNA from Dehalococcoides clone DHC-4 as template (data not shown). S-3

4 REFERENCES S1. Duhamel, M., K. Mo, and E. A. Edwards Characterization of a highly enriched Dehalococcoides-containing culture that grows on vinyl chloride and trichloroethene. Appl. Environ. Microbiol. 70: S2. Lane, D.J S/23S rrna sequencing, p In E. Stackebrandt, and M. Goodfellow, (ed.), Nucleic acid techniques in bacterial systematics. John Wiley, Chichester, UK: S3. Ritalahti, K. M., B. K. Amos, Y. Sung, Q. Z. Wu, S. S. Koenigsberg, and F. E. Löffler Quantitative PCR targeting 16S rrna and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains. Appl. Environ. Microbiol. 72: S4. Yan, J., B. A. Rash, F. A. Rainey, and W. M. Moe Isolation of novel bacteria within the Chloroflexi capable of reductive dechlorination of 1,2,3-trichloropropane. Environ. Microbiol. 11: S-4

5 TABLE S1. Amplification efficiencies and quantification linear ranges of 16S rrna gene calibration curves Target gene Primers Template All bacterial 16S rrna genes Dehalogenimonas 16S rrna genes Dehalococcoides 16S rrna genes a Ritalahti et al. (S3) b This study c Duhamel et al. (S1) Bac1055YF Bac1392R a BL-DC-631f BL-DC-796r b 582f 728r c BL-DC-9 T plasmid BL-DC-9 T plasmid DHC-4 plasmid Linear range Amplification efficiency d Slope b y- intercept e R 2 (copies/25 µl reaction) %-102% -3.37± ± %-102% -3.30± ± %-82% -3.96± ± Detection limit (copies/ 25 µl reaction) d Amplification efficiency was calculated according to equation: = 10 1 η slope 1 e Mean values and standard deviations were calculated using simple linear regression model by SAS. S-5

6 FIG. S1. Specificity test of Dehalococcoides 16S rrna gene primer sets. Part A: lanes 1 and 14, DNA marker III 0.12~21.2 kbp (Roche, Germany); lane 2-13, PCR products generated using Dehalococcoides primer sets A-L (Table 1) with Dehalogenimonas strain BL-DC-9 T genomic DNA as template with PCR annealing temperature 55 o C. Part B: lanes 1 and 11, DNA marker III; lanes 2-4, Dehalococcoides primer set A with PCR annealing temperature 62 o C; lanes 5-7, Dehalococcoides primer set D with PCR annealing temperature 68 o C; lanes 8-10, Dehalococcoides primer set E with PCR annealing temperature 66 o C. Lanes 2, 5, 8: BL-DC-9 T genomic DNA as template; Lanes 3, 6, 9: plasmid DNA containing BL-DC-9 T insert as template; Lanes 4, 7, 10: plasmid DNA from clone DHC-4 as template (positive control). S-6

bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 bp A bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 bp B FIG. S2.

sample collected from well ID W-0823 as template (B). Lane 1, DNA marker III 0.12-21.

7 bp bp A bp bp B FIG. S2. Dehalogenimonas-targeting primer sets with strain BL-DC-9 T genomic DNA as template (A) and DNA extract from groundwater sample collected from well ID W-0823 as template (B). Lane 1, DNA marker III kbp (Roche, Germany); lanes 2-14, Dehalogenimonas-targeting primer sets M to X (Table 2); lane 15, negative control; lane 16, DNA marker VIII kbp (Roche, Germany). S-7

sets X and J, respectively. Lanes 1 and 11, DNA ladder (100 bp DNA ladder, Biolabs Inc.

8 FIG. S3. Detection of Dehalogenimonas (A) and Dehalococcoides (B) with nested PCR using primer sets X and J, respectively. Lanes 1 and 11, DNA ladder (100 bp DNA ladder, Biolabs Inc.); lanes 2 to 8, PCR products from wells W , W , W , W , W , W , W ; lane 9, positive control; lane 10, negative control. S-8

Discovery of a trans-dichloroethene Respiring Dehalogenimonas in the 1,1,2,2-

Supplemental materials Discovery of a trans-dichloroethene Respiring Dehalogenimonas in the 1,1,2,2- Tetrachloroethane-Dechlorinating WBC-2 Consortium Authors Marie J. Manchester 1, Laura A. Hug 2, Matt