Supporting Information
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Abundance of anammox bacteria in different wetland soils
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Sylvia Humbert, Jakob Zopfi, and Sonia-Estelle Tarnawski
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Experimental procedures
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Library of environmental clones
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Environmental clones from a previous study (Humbert et al., 2011) served for primer design,
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method testing, and qPCR calibration curves (Table1). They contained partial 16S rRNA gene
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sequences obtained by PCR using primers Amx368f/Amx820r (Schmid et al., 2003; Schmid et al.,
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2005) and DNA from soil samples or soil anammox enrichment cultures. Extracted plasmid DNA
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was
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Inc.,Wilmington, USA) and stored at -20°C before use.
quantified
using
NanoDropTM
1000
Spectrophotometer
(Thermo
Fisher
Scientific
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Soil samples and DNA extraction
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Wetland soil samples for DNA extraction were collected in the Camargue (F, 43°29’37’’N,
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4°38’57’’E), the «Grande Cariçaie» (CH, 46°58’32’’N, 7°02’36’’E), the Piora Valley (CH,
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46°32’53’’N, 8°42’04’’E), the Rhone Valley (CH, 46°17’52’’N, 7°55’12’’E), in Bellefontaine (F,
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46°34’8.76''N, 6°04’53.28''E), and the shores of Lake Neuchâtel (CH, 46°55’60’’N, 6°50’21’’E) and
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eutrophic Lake Loclat (CH, 47°01’07’’N, 6°59’57’’E). The soil of this last site was identified as a
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REDUCTISOL TYPIQUE
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standard taxonomic soil classification system (WRB, 2006). Further details on the sampling sites
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can be found in Table 2 and elsewhere (Humbert et al., 2010). This set of soil samples covers the
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range of all currently known anammox genera (Table 2).
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Genomic DNA was extracted from about 0.7-1 g of fresh soil. A bead-beating apparatus (FP120
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FastPrepTM cell disruptor, Savant Instruments Inc., New York, USA) was used in combination with
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the FastDNA Spin Kit for Soil (Bio101, Qbiogene Inc., Carlsbad, USA) according to the
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manufacturer’s instructions. Amplifiability of the extracted DNA was tested by PCR using primers
(AFES, 2009), corresponding to a Gleysol according to the international
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338f and 518r targeting bacterial 16S rRNA genes (Ovreas et al., 1997). Soil DNA extracts were
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quantified using NanoDropTM 1000 (Thermo Fisher Scientific Inc.) and stored at -80°C until use.
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Design and evaluation of anammox-specific primers
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Anammox-specific primers were designed based on twelve 16S rRNA gene sequences of
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anammox bacteria retrieved from GenBank (Table 1). In the analysis, there were additionally
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included 19 different partial 16S rRNA gene sequences of anammox-related environmental clones
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(maximum similarity with corresponding candidate genus ≥ 94%) and 11 of non-anammox clones
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(similarity with anammox cluster ≤ 84%) from a previous study (Humbert et al., 2010, Table 1).
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Sequences were aligned using the ClustalW implementation of MEGA4 (Tamura et al., 2007). The
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most promising oligonucleotide sequences were selected for their degree of conservation and
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specificity for all anammox bacteria genera. Identified candidate sequences were 5’-
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GTCRGGAGTTADGAAATG-3’ for the degenerate forward primer A438f (E. coli position 438-455)
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and 5’-ACCAGAAGTTCCACTCTC-3’ for the reverse primer A684r (E. coli position 667-684) with
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compatible melting temperatures of 55.1°C - 59.3°C for A438r and 56,7°C for A684f (calculated
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with Oligolyzer 3.1 online application, Integrated DNA Technologies, Inc.).
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Primer specificity was assessed in silico with the ‘Probe Match’ tool of RDP-II (Cole et al., 2005)
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and verified experimentally by testing i) anammox and non-anammox 16S rRNA genes of
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environmental clones (Table 1), ii) soil DNA extracts from where the clones were obtained and, iii)
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anammox enrichment culture DNA.
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The optimized qPCR reaction mixture consisted of: 0.5X "SensiMixPlus SYBR®" qPCR master mix
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(Quantace Ltd, London, UK), 1200 nM of forward primer A438f, 300 nM of reverse primer A684r,
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and 10% v/v of template DNA. Amplification was done on a Rotor-gene™ 3000 (Corbett Research,
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Sydney, Australia) in 10 µl reaction volume. Template DNA was diluted in nuclease-free water
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(Qiagen, Hilden, Germany) in order to reach final concentrations of 0.1 to 1.5 ng/µl in the PCR
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mixture. All dilutions of DNA samples were run in triplicate. Amplification conditions were as
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follows: initial activation of the hot-start polymerase at 95°C for 15 min, followed by 40 cycles of
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denaturation at 95°C for 30 s, annealing at 55.5°C for 15 s and elongation at 72°C for 30 s. Control
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reactions without template were included in every qPCR run. A melting curve analysis was
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performed after the last amplification step of each run to detect primer dimers and unspecific
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amplifications.
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Calibration and concentration calculations
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Standard curves were established with 10-fold serial dilutions of plasmid DNA harbouring a 482 bp
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16S rRNA gene fragment. The number of gene copies in a known amount of plasmid DNA was
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determined according to Ritalahti et al. (2006). Standard curves in the range of 4.6 101 to 4.6 108
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copies/µl DNA were run in triplicate. Cycle threshold (CT) and calibration curve parameters
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(amplification efficiency, slope, and R2 correlation between CT and log numbers of gene molecules)
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were determined using Rotor-Gene software 6 (Corbett Research, Sydney, Australia). Differences
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in gene copy numbers were tested using Student’s t-test and considered significant for P ≤ 0.05.
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Cloning, sequencing, and phylogenetic analysis
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PCR products obtained for Bellefontaine (25 cm depth) and Lake Loclat shore (30-40 cm depth,
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October) soil samples were analyzed further using a cloning sequencing approach. The 248 bp
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qPCR amplicons were ligated into pCR®2.1-TOPO® vector and transformed into E. coli TOP10F’
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using TOPO TA cloning® kit following the manufacturer’s instructions (Invitrogen, Carlsbad, CA).
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After blue/white screening, 50 transformants per sample were randomly picked to constitute a
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clone library. Inserted DNA was amplified using primers T7 and Sp6. PCR products of 31 clones
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per sample were purified using MSB Spin PCRapace (Invitek, Berlin, Germany) and sequenced
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(Eurofins MWG Operon, Ebersberg, Germany). The most closely related sequences were
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identified using BLAST (Altschul et al., 1997). Phylogenetic analyses were done in MEGA4
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(Tamura et al., 2007) as described previously (Humbert et al., 2010). Nucleotide sequences were
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deposited in the EMBL sequence database under accession numbers FN908027 to FN908044.
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