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Potentially active iron, sulfur and sulfate reducing bacteria in Skagerrak and Bothnian Bay
Carolina Reyesa#*, Dominik Schneiderb, Andrea Thürmerb, Ajinkya Kulkarnia, Marko Lipkac,
Saar Y. Sztejrenszusa‡, Michael E. Böttcherc, Rolf Danielb, Michael W. Friedricha
University of Bremen, Microbial Ecophysiology, Bremen, Germany a; University of Göttingen,
Department of Genomic and Applied Microbiology b; Göttingen, Germany
Leibniz-Institute for Baltic Sea Research, Geochemistry and Isotope Biogeochemistry Group,
Running Head: Active Microorganisms Baltic Sea-North Sea Sediments
#Address correspondence to Carolina Reyes, [email protected]
*Present Address: University of Vienna, Department of Environmental Geosciences, Vienna,
‡Present Address: University of Bremen, MARUM-Center of Marine Environmental Sciences,
Hydrothermal Geomicrobiology group, Bremen, Germany.
Table S1. 16S rRNA amplicon primers used to sequence the V3-V5 region.
Table S2. Primers used for quantifying dsrA gene abundances and preparing dsrAB standard
Table S3. Sequencing information for samples sequenced with V3-V5 primers.
Table S4. List of bacterial families and genera discussed in this study with members shown to
reduce Fe in pure culture.
Figure S1. Heatmap of bacterial families (f) and genera (g) detected by sequencing the V3-V5
region of the 16S rRNA gene. Samples that were sequenced were Bothnian Bay 3-4 cm (BB34),
Bothnian Bay 6-7 cm (BB67), Skagerrak 6-8 cm (SK68).
Figure S2. dsrA and 16S rRNA bacterial gene copy numbers detected in (A) BB and (B) SK
samples. Samples that were analyzed included Bothnian Bay 2-3 cm (BB23), 3-4 cm (BB34), 6-
7 cm (BB67), Skagerrak 8-10 cm (SK810) and 16-23 cm (SK1623).
16S rRNA cDNA Pyrosequencing
16S rRNA’s were PCR amplified using primers targeting the V3-V5 region as described in Table
S1. Samples that were sequenced included: Bothnian Bay 3-4 cm, 6-7 cm depths (BB34 and
BB67 respectively) and Skagerrak 6-8 cm depths (SK68). PCR was carried out using the Q5
High-Fidelity DNA Polymerase Kit (New England BioLabs, Frankfurt Am Main, Germany)
using a GeneAmp 9700 PCR system (Applied Biosystems, Darmstadt, Germany). Multiple PCR
reactions were performed for each sample. For bacterial 16S rRNA amplification, the following
program was used: 98 °C for 30 sec, 30 cycles [98 °C 10 s, 66 °C 30 s, 72 °C 30 s] 72 °C 2 min.
One or two µl of cDNA (undiluted or diluted 1:10 in 1x Tris-EDTA buffer) was amplified by
PCR as described above. Replicates of each sample were pooled together following the PCR step
in equal concentrations and purified. Amplicon products of the correct size (~650 bp) were
purified by gel excision using a 1 % low melting agarose gel. The PCR products were recovered
from the gels using the peqGOLD Gel Extraction Kit (PeqLab, VWR International GmbH,
Erlangen, Germany) following the manufacturer’s instructions and eluting with 50 µl elution
buffer. Purified amplicon concentrations were determined with NanoDrop Spectrophotometer
ND-1000 (PeqLab, VWR International GmbH, Erlangen, Germany) and Quant-iT Picogreen
dsDNA reagent (Invitrogen-Thermo Fisher Scientific, Steinheim, Germany) following the
manufacturer’s instructions. Fluorescence measurements were made using a Fluorimeter
(Fluoroskan Ascent FC, Thermo Labsystems, Milford, USA). Samples were prepared and
sequenced as described in Schneider et al. (2013).
Quantification of dsrA gene abundances
Abundances of sulfate reducing bacteria (SRB) were estimated by quantifying gene abundances
of dsrA, a gene encoding the alpha subunit of the key enzyme dissimilatory sulphite reductase,
from nucleic acid extracts obtained from various depths of the Bothnian Bay and Skagerrak
sediments. SRB abundances were estimated from depths where Fe-reductions rates were
observed to be high. Samples that were chosen were: BB23, BB34, BB67, SK810 and SK1623.
The qPCR preparation was done in a similar manner as mentioned in the methods section under
“Quantitative PCR” with a few changes. The dsrAB gene of Desulfovibrio burkinensis DSM
6830 was amplified using the primer pairs DSR1Fmix (a-h) and DSR4Rmix (a-g) (Table S2) in
order to be used as a standard template. The 50 µl reaction mixture consisted of 5 µl 10X PCR
buffer, 5 µl of 2 mM dNTP mix, 6 µl of 25 mM MgCl2, 0.5 µl of 20 mg/ml Bovine Serum
Albumin (Roche, Mannheim, Germany), 1 µl of each primer at a final concentration of 500 pM,
0.25 µl of 5 U/µl AmpliTaq polymerase (ThermoFisher, Steinheim, Germany), 27.25 µl nuclease
free water and 4 µl DNA template. PCR program was followed as per described in Pester et al.,
2010. The amplified product was run on a 1 % agarose gel and the PCR product (~1920 bp) was
excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).
Standard and samples were quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen-
ThermoFisher, Steinheim, Germany). qPCR was performed using the dsrA gene specific primers
DSR1-F+ and DSR-R (Table S2) at a final concentration of 400 pM and quantification of
samples was done using three biological replicates. The following qPCR program was used: 95°
C for 10 min, 40 cycles [95° C 15 s, 60° C 30 s, 72° C 40 s]. The amplification efficiency of the
qPCR was 89.9 % and the R2 was 0.997. For calculation of dsrA gene copy numbers the mass of
one gene copy of the standard used was 1184711 Da.
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