Supporting Material Potentially active iron, sulfur and sulfate reducing bacteria in Skagerrak and Bothnian Bay Sediments Carolina Reyes a# *, Dominik Schneider b , Andrea Thürmer b , Ajinkya Kulkarni a , Marko Lipka c , Saar Y. Sztejrenszus a‡ , Michael E. Böttcher c , Rolf Daniel b , Michael W. Friedrich a 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, Germany c 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.
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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
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.
Röling, W.F.M (2015) The family Geobacteraceae. In The Prokaryotes: Deltaproteobacteria and
Epsilonproteobacteria. Rosenberg, E., DeLong, E.F., Lory, S., Stackebrandt, E., Thompson,
F. (eds). Springer-Verlag, Berlin Heidelberg, pp.157-172.
Scala, D.J., Hacherl, E.L., Cowan, R., Young, L.Y. and Kosson, D.S. (2006) Characterization of
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)
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 ●
*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