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, creyes6@gmail.com

*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.

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g_Pelobacter

g_Rhodoferax

g_Geobacter

g_Desulfuromonas

f_Desulfuromonadaceae

g_Desulfovibrio

f_Desulfobulbaceae

f_Desulfobacteraceae

g_Rhodobacter

g_Clostridium

BB34 BB67 SK68

Fam

ilies

and

Gen

era

Samples

% R

elative Abun

dance

Figure S1. Heatmap of families (f) and genera (g) detected by sequencing the V3-V5 region of the 16S rRNA gene. Samples that were sequenced were Bothnian Bay (BB) samples 3-4 and 6-7 cm and Skagerrak (SK) 6-8 cm. See supplementary text for the methods used in the sequencing step.ND means taxa were not detected in the sample.

NDND

ND

ND

ND

ND

ND

ND

2-3 cm

3-4 cm

6-7 cm

Bothnian Bay

0 5 x 108 1 x 109 1.5 x 109 2 x 109

gene copies/g sediment

gene copies/g sediment

8-10 cm

16-23 cm

0 5 x 107 1 x 108 2 x 108

Skagerrak

Figure S2. dsr 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

dsrA(DNA)

16S rRNA

dsrA (DNA)

A

B

Table S1. 16S rRNA amplicon primers used to sequence the V3-V5 region.

Target

group

Sample Froward Primer Reverse Primer Variable Region References

Bacteria Bac341f Bac907r V3-V5 Muyzer et al.

1993; Muyzer et

al. 1995

BB34 ccatctcatccctgcgtgtctccgacTCAGACG

AGTGCGTTACGGRAGGCAGCA

G

cctatcccctgtgtgccttggcagtcTCAGC

CGTCAATTCMTTTGAGT

BB67 ccatctcatccctgcgtgtctccgacTCAGAGA

CGCACTCTACGGRAGGCAGCA

G

cctatcccctgtgtgccttggcagtcTCAGC

CGTCAATTCMTTTGAGT

SK68 ccatctcatccctgcgtgtctccgacTCAGATC

AGACACGTACGGRAGGCAGCA

G

cctatcccctgtgtgccttggcagtcTCAGC

CGTCAATTCMTTTGAGT

Lower case letters indicate the adapter sequences; underlined letters indicate MIDs, italicized letters indicate the barcode key; bold

letters indicate the 16S rRNA gene sequence.

Table S2. Primers used for quantifying dsrA gene abundances and preparing dsrAB standard

template.

Primer Sequences(5’-3’) Reference

For qPCR

DSR1-F+ ACSCACTGGAAGCACGGCGG Kondo et al., 2004

DSR-R GTGGMRCCGTGCAKRTTGG Kondo et al., 2004

DSR1Fmix (a-h) and DSR4Rmix (a-g)

DSR1F ACSCACTGGAAGCACG Wagner et al., 1998

DSR1´4R GTGTAGCAGTTACCGCA Wagner et al., 1998

DSR1Fa ACCCAYTGGAAACACG Loy et al., 2004

DSR4Ra GTGTAACAGTTTCCACA Loy et al., 2004

DSR1Fb GGCCACTGGAAGCACG Loy et al., 2004

DSR4Rb GTGTAACAGTTACCGCA Loy et al., 2004

DSR1Fc ACCCATTGGAAACATG Zverlov et al.,2005

DSR4Rc GTGTAGCAGTTKCCGCA Zverlov et al.,2005

DSR1Fd ACTCACTGGAAGCACG Zverlov et al., 2005

DSR4Rd GTGTAGCAGTTACCACA Zverlov et al., 2005

DSR1Fe GTTCACTGGAAACACG Pester et al., 2010

DSR4Re GTGTAACAGTTACCACA Zverlov et al., 2005

DSR1Ff AGCCACTGGAAACACG Pester et al., 2010

DSR4Rf GTATAGCARTTGCCGCA Pester et al., 2010

DSR1Fg GGCCACTGGAAACATG Pester et al., 2010

DSR4Rg GTGAAGCAGTTGCCGCA Pester et al., 2010

DSR1Fh GGCTATTGGAAGCACG Pester et al., 2010

TableS3.SequencinginformationforsamplessequencedwithV3‐V5primers.16S rRNA region Site Depth

(cm) Samples Number of Sequences Average Sequence Length (bp)

V3-V5 Bothnian Bay (At4) 3-4 BB34 21,468 535±18.6 6-7 BB67 10,283 540±14.8 Skagerrak (Geo 2a) 6-8 BB68 6,011 542±14.5

TableS4

Table S4. List of bacterial families and genera discussed in this study with members shown to reduce Fe in pure culture. Family Genus Species Reference DIR* IR** Geobacteraceae Geobacter "Geobacter akaganeitreducens" Lovley 2013 ●

"Geobacter arculus" Lovley 2013 ● "Geobacter chapellel" (strain 172) Lovley 2013 ● "Geobacter grbicium" (strain TACTP-2) Lovley 2013 ● "Geobacter humireducens" (strain JW3) Lovley 2013 ● "Geobacter hydrogenophilus" (strain

H2) Lovley 2013 ●

Geobacter metallireducens Lovley 2013 ● Geobacter sulfurreducens Lovley 2013 ● "Geopsychrobacter electrodiphilus" Weber 2006 ● Geothermobacter ehrilichii Weber 2006 ● Geobacter argillaceus Röling 2015 ● Geobacter bemidjiensis Röling 2015 ● Geobacter bremensis Röling 2015 ● Geobacter daltonii Röling 2015 ● Geobacter grbiciae Röling 2015 ● Geobacter pelophilus Röling 2015 ● Geobacter pickeringii Röling 2015 ● Geobacter psychrophilus Röling 2015 ● Geobacter toluenoxydans Röling 2015 ● Geobacter thiogenes Röling 2015 ● Geobacter uraniireducens Röling 2015 ● Pelobacter propionicus Lovley 2013 ●

Pelobacteraceae Pelobacter Pelobacter carbinolicus Lovley 2013 ● Pelobacter venetianus Lovley 2013 ●

TableS4

Family Genus Species Reference DIR* IR** Rhodobacteraceae Rhodobacter Rhodobacter capsulatus Dobbins et al., 1996 ●

Sinorhodobacter ferrireducens Yang et al., 2013 ● ●

Bacilliaceae Bacillus Bacillus cereus Lovley 2013 ● Bacillus circulans Lovley 2013 ● Bacillus mesentericus Lovley 2013 ● Bacillus polymyxa Lovley 2013 ● Bacillus pumilus Lovley 2013 ● Bacillus sp. Lovley 2013 ● Bacillus subtilis Lovley 2013 ● Bacillus megaterium isolate AC46b1 Scala et al., 2006 ● Bacillus sp. TUT1008 Scala et al., 2006 ● Bacillus sp. strain SFB Pollock et al., 2007 ● Bacillus subterraneus Kanso et al., 2002 ● Bacillus infernus Boone et al., 1995 ●

Clostridiaceae Clostridium Clostridium butyricum Lovley 2013 ● Clostridium polymyxa Lovley 2013 ● Clostridium saccarobutyricum Lovley 2013 ● Clostridium sporogenes Lovley 2013 ● Clostridium beijerinckii Dobbin et al., 1999 ● Clostridium sp. EDB2 Bhushan et al., 2005 ● Clostridium aff. Estertheticum Scala et al., 2006 ● Clostridium hydroxybenzoicum Scala et al., 2006 ● Clostridium sp. BC1 Gao and Francis 2012 ● Clostridium celerecrescens García-Balboa et al.,

2010 ●

Clostridium sp.strain FGH Shah et al., 2014 ●

TableS4

Family Genus Species Reference DIR* IR**

Desulfuromonadaceae Desulfuromonas Desulfuromonas acetexigens Lovley 2013 ● Desulfuromonas acetoxidans Lovley 2013 ● Desulfuromonas chloroethenica Lovley 2013 ● Desulfuromonas palmitatis Lovley 2013 ● Desulfuromusa Desulfuromusa bakii Lovley 2013 ● Desulfuromusa kysingii Lovley 2013 ● Desulfuromusa succinoxidans Lovley 2013 ●

Desulfobulbaceae Desulfobulbus Desulfobulbus propionicus Lovely 2013 ● Desulfotalea Desulfotalea psychrophila LSvS4 Knoblauch et al., 1999 ● Desulfotalea arctica LSv514 Knoblauch et al., 1999 ●

Desulfobacteraceae Desulfobacter Desulfobacter postgatei Lovley 2013 ● Desulfobacterium Desulfobacterium autotrophicum Lovley 2013 ●

*DIR refers to dissimilatory iron reduction **IR refers to iron reduction The symbol • is used to indicate if the microorganism was found to perform DIR or IR in pure culture experiments