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Supporting information
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Nitrospina–like bacteria are the main drivers of nitrite oxidation in the seasonal upwelling
area of the Eastern South Pacific (central Chile ~36° S)
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Héctor A. Levipan, Verónica Molina and Camila Fernandez
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Experimental procedures
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Environmental setting and standard measurements
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Sampling was carried out in winter (August, non-upwelling period) and summer 2011
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(December, upwelling-favorable period) at the COPAS time series coastal Station 18 (36° 30’S,
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73° 07’W) located in the upwelling area off central Chile. Temperature, salinity and oxygen were
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measured with a CTD-O probe (model SBE-19; Sea-Bird Electronics, Bellevue, WA, USA)
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equipped with a PAR sensor. Discrete water samples were collected by using Niskin bottles
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according to the following resolution: 0, 5, 10, 20, 30, 50, and 80 m depth. Chlorophyll-a and
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nutrients (NO2−, NO3−) were determined in discrete seawater samples according to the method
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described by Holm-Hansen et al. (1965) and Strickland and Parsons (1972), respectively.
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Ammonium (NH4+) concentrations were determined by using a fluorometric method (Holmes et
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al., 1999). Prokaryote cell abundance was measured by flow cytometry (Marie et al., 2000) by
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using a FACSCaliburTM device (Becton Dickinson Biosciences, CA, USA).
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DNA extraction procedures
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Seawater (3 L) for DNA extraction was filtered through cellulose ester filters (pore size 0.22 μm;
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GSWP04700; Millipore) by using a peristaltic pump and it was stored frozen (-80 °C) until
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laboratory analyses. DNA was extracted by using the PowerSoil™ DNA Isolation Kit (MoBio
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Laboratories, Solana Beach, CA, USA) in accordance to the manufacturer’s specifications.
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Minor modifications were done as follows: (1) filters were thawed, cut into six or seven strips by
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using a sterile scalpel and tweezers, and then they were placed inside a bead-beating tube, (2)
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a step of alternative lyses consisting of a 10-min incubation at 70 °C (after adding solution C1)
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was included. (3) Samples were then homogenized twice at ~3000 rpm for 30 s by using a Mini-
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Beadbeater-8™ (Biospec Products, Bartlesville, OK) in order to ensure complete cellular
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disruption. The concentration and quality (A260/A280 ratio) of the eluted DNA was determined by
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absorption spectroscopy by using a Synergy Mx Microplate Reader (BioTek Instruments,
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Winooski, Vermont, USA).
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RNA extraction
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Seawater (50 mL) for RNA extraction was filtered by using a sterilized syringe (60 mL) and 25
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mm swinnex through Hydrophilic PVDF filters (pore size 0.22 μm, GVWP02500, Millipore) and
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stored frozen (-20 °C) with RNAlater® solution (Ambion, Austin, Texas) until laboratory analysis.
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RNA was extracted by using the Ambion® RNA extraction kit (AM1560, Ambion, Austin, TX,
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USA) in accordance to the manufacturer’s specifications including a mechanical disruption step
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by using 200-µm-diameter zirconium beads (Low Binding Zirconium Beads, OPS Diagnostics,
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Lebanon, NJ) and the homogenization procedure previously described. The concentration and
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quality (A260/A280 ratio) of RNA extracts was determined as with DNA extracts. Total RNA was
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treated with the TURBO DNA-free™ kit (Applied Biosystems, Austin, TX, USA) in order to
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remove traces of DNA that may have spoiled the subsequent retro-transcription and quantitative
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PCR experiments.
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454 high-throughput pyrosequencing of bacterial 16S rRNA genes and clone library construction
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and sequence analysis pipeline
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The in situ bacterial community composition, specially focused on the NOB guild, was studied by
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way of the high-throughput pyrosequencing method. Preparations of complementary DNA
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(cDNA) were generated by using random primers provided by the ImProm-II™ Reverse
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Transcription System (Promega Corp, Madison, WI, USA). Bacterial 16S rRNA gene
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pyrolibraries (V1-V3 region, ~500 bp) were generated from these cDNA preparations using the
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primers 28F (5’-GAGTTTGATCNTGGCTCAG) and 519R (5’-GTNTTACNGCGGCKGCTG) at the
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Research and Testing Laboratory (RTL, Texas, USA). RTL provides quality-checked, denoised
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and also preliminary classified by BLAST (with GeneBank) sequences (see details
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http://www.researchandtesting.com/docs/Data_Analysis_Methodology.pdf). We re-classify the
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16S rRNA sequences by using the RDP pyrosequencing pipeline and the RDP naïve Bayesian
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classifier (Wang et al., 2007) in order to select specific NOB groups.
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16S rRNA sequence reads associated to NOB were aligned and assigned to OTUs based on
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similarity thresholds (3% cut-off) by using Bosque program (v.1.8.1 http://bosque.udec.cl;
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Ramírez-Flandez and Ulloa, 2008). This procedure was also run with all the sequences used for
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comparison purposes in the different analyses are explained below. 16S rRNA gene-targeted
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Nitrospina-specific primers were compared with this alignment and modified in order to retrieve
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most of these sequences (See Table S1 and Figure S4). PCR products of selected DNA
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samples were cloned by using the pGEM®-T Easy Vector System (Promega, Madison, CA,
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USA) in accordance with the manufacturer’s instructions, and the sequencing was carried out by
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Macrogen Inc. (Seoul, Korea). A total of 11 Nitrospina-like clones from a sample collected at 50m
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depth during Aug 2011 were obtained with this approach. One of these clones (KF452035) was
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randomly chosen as a qPCR standard and was included in phylogenetic analysis.
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The richness of OTUs related to NOB was analyzed by using rarefaction curves and, richness
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and diversity indices (Colwell et al., 2004) by using the EstimateS software v.8.0
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(http://purl.oclc.org/estimates). These analyses included 16S rRNA gene sequences related to
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NOB that were retrieved in this study and other initiatives in the studied area such as the
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MI_LOCO (Microbial Initiative in Low Oxygen areas off Concepción and Oregon) metagenomic
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database (available from http://camera.calit2.net/index.shtm), and 16S rRNA gene sequences
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from the North Pacific (Mincer et al., 2007). The phylogenetic analyses were based on maximum
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likelihood using the FastTree 2.0 method (Price et al., 2010) with a bootstrap set of 1000. A
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consensus tree was generated by using branches bootstrap values higher than 50%. The clone
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sequences generated in this study were stored in the GenBank public database with the access
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numbers KF452035 to KF452045. The pyrolibraries were deposited in the European Nucleotide
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Archive (ENA) under study number ERP003739 with the following run access numbers:
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ERR324326, ERR324314, ERR324315 and ERR324327.
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Quantitative PCR (qPCR)
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Quantitative measurements of Nitrospina-like bacteria (presumably NOB) and ammonia-
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oxidizing bacteria and archaea were determined by using cDNA templates. The cDNA was
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synthesized with ImProm-II™ Reverse Transcription System using 20 ng of total RNA and the
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following specific reverse primers (Table S1): NitSSU_286R (Nitrospina-like bacteria; modified
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from NitSSU_282R reported by Mincer et al., 2007), amoA-2R (AOB; Rotthauwe et al., 1997)
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and ARCH-amoAR (AOA; Francis et al., 2005). Quantitative PCR reactions were carried out on
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a volume of 20 μL containing 1 μL of cDNA, 2X Fast SYBR® Green Master Mix (1X; Applied
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Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA), and forward and
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reverse primers (0.4 μM, final concentration; Table S1). In the case of Nitrospina-like bacteria,
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genomic DNA extracts were also used as a template (20 ng) for determining the copies number
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of 16S rRNA gene. Overall, the qPCR program consisted of an initial denaturation for 20 s at 95
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°C followed by 40 amplification cycles consisting of 95 °C for 3 s, 20 s annealing at a
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temperature chosen according to the group-specific primers used (see details in Table S1), and
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an extension of 20 s at 72 °C. We used the Nitrospina-like clone ST180811-50m11 (KF452035)
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to prepare 6-point standard curves in triplicate by using 10-fold dilutions series from 1 x 107
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copies. The efficiencies (E) and correlation coefficients (r2) for standard curves of Nitrospina-like
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bacteria ranging from 85.2 to 90.01% and 0.962 to 0.998, respectively. Archaeal and bacterial
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amoA gene copy numbers were determined in accordance to Molina et al. (2010) procedure, E
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and r2 values were as follows: general archaeal amoA (E = 90.1%, r2 = 0.997) and bacterial
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amoA (E = 80.3%, r2 = 0.998). All qPCR were performed by using a StepOne™ Real-Time PCR
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System (Applied Biosystems, Lincoln Centre Drive, Foster City, CA, USA) and the data were
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analyzed by using the StepOneTM software package (v.2.2.2; Foster city, CA, USA).
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Statistical analyses
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Spearman correlation coefficients were used to explore the relationship between environmental
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variables and molecular data. Differences between summer and wintertime were evaluated with
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Wilcoxon match pairs test. The analyses were performed with Statistica software (version 6.0).
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