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Supplemental Material and Methods
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Chemicals
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MCPA (chemical purity ≥ 97 %) and [U-14C]-MCPA (chemical purity 99.9%, specific
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activity 64 mCi mmol-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and GE
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Healthcare UK limited (Buckinghamshire, UK), respectively. 3 mM MCPA and 8 µM [U-14C]-
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MCPA (0.5 mCi l-1) stock solutions were prepared by dissolving MCPA in double deionized
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water. MCPA solutions were sterilized by filtration (0.22 m pore diameter, Minisart® NML,
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Sartorius, Aubagne, France).
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Microcosm experiments with [U-14C]-MCPA
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Oxic microcosms with [U-14C]-MCPA were set up with bulk soil or drilosphere material
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from soil pre-exposed to MCPA and earthworms in soil columns to determine the fate of
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MCPA. 0.5-1 g fresh weight of bulk soil (0-2 cm and 8-10 cm depth) and drilosphere material
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(gut content, burrow wall and cast) were placed into 15 ml glass tubes sealed with rubber
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stoppers and plastic caps, and supplemented with both non-labeled MCPA and [U-14C]-
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MCPA to a final concentration of 20 µg MCPA gdw-1 and a specific activity of approximately
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0.02 µCi gdw -1. Moisture contents of samples in microcosms were 25-28%. Incubation was
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essentially at room temperature in the dark. Microcosms were flushed with > 5 x the volume
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of the gas phase with air after 28 days of incubation and CO2 was trapped in 1 ml of 0.5 M
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NaOH. Microcosms were destructively sampled after 6 months of incubation and extracted
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with 10 ml of 0.5 M NaOH. Non-extractable organic residues were determined by drying
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material at 60 °C for 48 h, combusting at 600 °C and trapping
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(Wiesenberg et al., 2010).
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(Wiesenberg et al., 2010).
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CO2 in 10 ml 1 M NaOH
Radioactivity was determined by scintillation counting
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Bioconcentration of MCPA in earthworms
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Soil columns containing MCPA were setup with and without earthworms in triplicates
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as described in the main text with the following modifications: The height of soil was 3 cm
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(approximating 0.7 kg non-dried soil) per column. Four or 5 earthworms with a total biomass
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of 5.3 to 5.8 g were added per column that received earthworms.
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concentration approximated 15 µg (g dry weight soil)-1.
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sampled after 40 days of incubation (i.e., when no MCPA was detected).
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concentrations of mixed soil samples (including burrow walls) as well as earthworm gut
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contents and tissues were analyzed.
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earthworms were washed, dissected, the gut was separated, and the tissue was washed with
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sterile water and cut into small pieces (2-5 mm). Gut and earthworm tissue were then used
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for MCPA extraction as described in the main text.
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The initial MCPA
Soil columns were destructively
MCPA
For MCPA extractions from earthworm tissue,
The proportion of the total amount of initial soil MCPA present in worms was:
CGut  mGut   CTissue  mTissue 
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BC 
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concentrations of MCPA in earthworm gut content, earthworm tissue, and in soil prior to
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incubation, respectively (all in µmol per g fresh weight); mGut, mTissue, and mSoil were the total
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amount of earthworm gut content, earthworm tissue, and soil, respectively, in g fresh weight.
C
Initial
Soil
 mSoil
Initial
, where BC is bioconcentration; CGut, CTissue, and CSoil
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Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis of tfdA-like genes
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tfdA-like genes were amplified and fluorescently labeled with primer set 3 targeting
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group 1-5 tfdA-like genes (Zaprasis et al. 2010). Purified PCR products were first digested
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with Mung Bean nuclease (New England Biolabs, Frankfurt am Main, Germany) to remove
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single-stranded extensions at terminal ends and minimize the influence of pseudo-terminal
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restriction fragments (Egert and Friedrich, 2003; Palmer et al., 2010). Alignments of tfdA-like
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genes from Scheyern soil were screened for restriction sites that differed group 1, 2 and 3
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tfdA-like genes.
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Restriction digestion was performed with (A) BsaHI, (B) MefI, PmlI and FspI (triple digest),
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(C) NsiI, or (D) StuI and XcmI (double digest) at 37 °C for over 4 hours according to the
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manufacturer’s protocol (Fermentas, St. Leon-Rot, Germany), and the reaction was stopped
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by incubation at 80 °C for 20 min.
Restriction enzymes were chosen accordingly to resolve such genes.
Digestion products were quantified with PicoGreen
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(Invitrogen, Karlsruhe, Germany), diluted, denatured, size separated on denaturing
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polyacrylamide gels with a NEN model 4300 DNA analyzer (Licor, Lincoln, NE), and
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analyzed as described (Hamberger et al., 2008; Palmer et al., 2010). In silico T-RFs were
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assigned to tfdA-like gene fragments obtained previously from the same soil with the ARB
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software package as described (Hamberger et al., 2008; Palmer et al., 2010)(Zaprasis et al.
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2010).
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Supplemental Results
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MCPA degradation capacity of field fresh soil and associated structural genes
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mRNAs of cadA and tfdA-like genes targeted by primer set 5 and 1 (Table 1),
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respectively, were not detected. tfdA-like genes determined by primer set 1 per 16S rRNA
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increased 2.6 fold from 1.3 x 10-3 to 3.2 x 10-3, indicating an enrichment of
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alphaproteobacterial Bradyrhizobium-related MCPA degraders during MCPA degradation.
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tfdA-like gene transcripts were detected with primer set 2 (Table 1, Fig. 1). Thus, primer set
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2 was utilized for all further tfdA-like gene analyses.
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Up to 8.6 x 102, 0.3 x 102, and 3.5 x 105 copies per ng DNA were detected for tfdA-
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like, cadA and 16S rRNA genes, respectively. tfdA-like gene copy numbers were 30 to 60 x
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those of cadA, indicating that tfdA-like gene outnumbered cadA hosting microorganisms.
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16S rRNA gene copy numbers per ng DNA were essentially the same throughout the
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incubation (data not shown), indicating that growth of total bacteria did not occur. However,
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copy numbers of tfdA-like and cadA genes per per ng DNA increased approximately 3 to 6
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fold concomitant to MCPA degradation (data not shown), indicating an enrichment of tfdA-
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like and cadA gene hosting MCPA degraders over time. The microcosm data indicated that
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Scheyern soil sustains MCPA degradation that is quantitatively linked to both tfdA-like and
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cadA gene hosting aerobes of the Alphaproteobacteria and potentially novel non-group 1
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tfdA-like gene hosting degraders.
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Bioconcentration of MCPA in earthworms
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Bioconcentration of MCPA in earthworms was 6.8 ± 1.2 % of initial MCPA in soil. 0.7
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± 0.4 % and 6.0 ± 2.2 % of initial MCPA in soil were recovered from gut and tissue,
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respectively. Such findings indicated that earthworms incorporate MCPA from soil primarily
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into their tissue. The differences in the amount of MCPA in soil with or without earthworms at
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all time points were compared to that present in earthworms (Fig. S1), assuming that
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bioconcentraion was constant during the whole incubation period.
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exceeded 6.8 % of initial MCPA from day 20 to 40, suggesting that earthworms stimulated
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MCPA degradation rather than merely accumulating MCPA.
Such differences far
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Diversity of tfdA-like genes in soil columns
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T-RFLP analysis of group 1-5 tfdA-like genes with BsaHI yielded 5 T-RFs indicative of
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alphaproteobacterial group 2 tfdA-like genes irrespective of worms, indicating that Scheyern
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soil and the drilosphere harbor a diverse MCPA degrader community (Fig. S5). T-RFLP
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analysis with BsaHI indicated group 2 and 4 tfdA-like genes and eventually group 1 class 1
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genes (Fig. S5 A). T-RFLP analysis with MefI, PmlI and FspI (triple digest) indicated group 2
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tfdA-like genes in soil and drilosphere (Fig. S5 B). T-RFLP analysis with NsiI yielded a single
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T-RF at 96 bp indicative of group 3 tfdA-like genes (Fig. S5 C). T-RFLP analysis with StuI
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and XcmI (double digest) targets class I and II/ III tfdA-like genes, which theoretically yield T-
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RFs of 239 and 187 bp, respectively. However, the 187 bp T-RF and a T-RF of 57 bp were
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detected in soil and drilosphere at low relative abundances, indicating class II/ III organisms
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of group 1 and group 2 organisms, respectively (Fig. S5 D). Although only few tfdA-group 2
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genes from Scheyern soil yielded a 187 bp T-RF in an in silico T-RFLP analysis, a
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contribution of such sequences to the 187 bp T-RF indicative of group 1 might not be
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excluded. Group 1 class 1 tfdA-like genes (T-RF of 239) were not detected, suggesting that
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the 32 bp T-RF in Fig. S5A was exclusively due to group 2 sequences. The collective T-
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RFLP data suggest that (i) alphaproteobacterial group 2 tfdA-like gene hosting organisms are
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diverse in Scheyern soil and drilosphere, and (ii) group 2-4 rather than group 1 tfdA-like gene
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hosting organisms might have contributed to MCPA degradation.
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Supplemental References
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Egert M, Friedrich MW (2003). Formation of pseudo-terminal restriction fragments, a PCR-
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related bias affecting terminal restriction fragment length polymorphism analysis of microbial
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community structure. Appl Environ Microbiol 69: 2555-2562.
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Hamberger A, Horn MA, Dumont MG, Murrell JC, Drake HL (2008). Anaerobic consumers of
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monosaccharides in a moderately acidic fen. Appl Environ Microbiol 74: 3112-3120.
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Palmer K, Drake HL, Horn MA (2010). Association of novel and highly diverse acid-tolerant
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denitrifiers with N2O fluxes of an acidic fen. Appl Environ Microbiol 76: 1125-1134.
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Wiesenberg GLB, Gocke M, Kuzyakov Y (2010). Fast incorporation of root-derived lipids and
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fatty acids into soil - Evidence from a short term multiple
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Organic Geochemistry In Press.
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CO2 pulse labelling experiment.