supporting material potentially active iron, sulfur and ...€¦ · supporting material potentially...
TRANSCRIPT
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.
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)
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