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Supporting Material

Potentially active iron, sulfur and sulfate reducing bacteria in Skagerrak and Bothnian Bay

Sediments

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,

Warnemünde, Germanyc

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,

Austria.

‡Present Address: University of Bremen, MARUM-Center of Marine Environmental Sciences,

Hydrothermal Geomicrobiology group, Bremen, Germany.

Supporting Table

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

template.

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.

Supporting Figure

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).

Supporting Methods

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.

References

Bhushan, B., Halasz, A., and Hawari, J. (2006) Effect of iron (III), humic acids and

anthraquinone-2, 6-disulfonate on biodegradation of cyclic nitramines by Clostridum sp.

EDB2. J Appl Microbiol 100: 555-563.

Boone, D.R., Liu, Y., Zhao, Z.J., Balkwill, D.L., Drake, G.R., Stevens, T.O. and Aldrich, H.C.

(1995) Bacillus infernus sp. nov., and Fe(III) and Mn(IV)-reducing anaerobe from the deep

terrestrial subsurface. IJSB 45: 441-448.

Dobbin, P.S., Carter, J.P., Juan, C.G.-S.S., Hobe, M. von, Powell, A.K., and Richardson, D.J.

(1999) Dissimilatory Fe(III) reduction by Clostridium beijerinckii isolated from freshwater

sediment using Fe(III) maltol enrichment. FEMS Microbiol Lett 176: 131–138.

Dobbin, P.S., Warren, L.H., Cook, N.J., McEwan, A.G., Powell, A.K., and Richardson, D.J.

(1996) Dissimilatory iron(III) reduction by Rhodobacter capsulatus. Microbiol 142: 765–

774.

Gao, W., and Francis, A.J. (2013) Fermentation and hydrogen metabolism affect uranium

reduction by Clostridia. ISRN Biotech doi:10.5402/2013/657160

Kanso, S., Greene, A.C. and Patel, B.K. (2002) Bacillus subterraneus sp. nov., an iron-and

manganese-reducing bacterium from a deep subsurface Australian thermal aquifer. IJSEM

52: 869-874.

Knoblauch, C., Sahm, K., Jørgensen, B.B. (1999) Psychrophilic sulfate-reducing bacteria

isolated from permanently cold Arctic marine sediments: description of Desulfofrigus

oceanense gen. nov., sp. nov., Desulfofrigus fragile sp. nov., Desulfofaba gelida gen. nov.,

sp. nov., Desulfotalea psychrophila gen. nov., sp. nov. and Desulfotalea arctica sp.nov.

IJSEM 49: 1631-1643.

Kondo R, Nedwell DB, Purdy KJ, Silva SQ. (2004). Detection and enumeration of sulphate-

reducing bacteria in estuarine sediments by competitive PCR. Geomicrobiol 21: 145–157.

Lovley DR. Dissimilatory Fe(III) and Mn(IV) reducing prokaryotes. In: Rosenberg E, DeLong

EF, Stackebrandt E et al. (eds). Prokaryotes: Prokaryotic Physiology and Biochemistry.

United Kingdom: Springer, 2013, 287–305.

Loy A, Küsel K, Lehner A, Drake HL, Wagner M. (2004). Microarray and functional gene

analyses of sulfate-reducing prokaryotes in low-sulfate, acidic fens reveal cooccurrence of

recognized genera and novel lineages. Appl Environ Microbiol 70: 6998–7009.

Muyzer G, De Waal EC, Uitterlinden AG. (1993). Profiling of complex microbial populations by

denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified

genes coding for 16S rRNA. Appl Environ Microbiol 59: 695-700.

Muyzer G, Teske A, Wirsen CO, Jannasch HW. (1995). Phylogenetic relationships of

Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by

denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol 164: 165-

172.

Pester M, Bittner N, Deevong P, Wagner M, Loy A. (2010). A ‘rare biosphere’ microorganism

contributes to sulfate reduction in a peatland. ISME J 4: 1591–1602.

Pollock, J., Weber, K.A., Lack, J., Achenbach, L.A., Mormille, M.R. and Coates, J.D. (2007)

Alkaline iron (III) reduction by a novel alkaliphilic, halotolerant, Bacillus sp. isolated from

salt flat sediments of Soap Lake. Appl Microbiol Biotech 77: 927-934.

reductases supports an early origin of sulfate respiration. J Bacteriol 180: 2975–2982.