Supporting information for manuscript:
Uncultured Desulfobacteraceae and Crenarchaeotal group C3 incorporate 13C-acetate in
coastal marine sediment
Hyunsoo Na, Mark Alexander Lever, Kasper Urup Kjeldsen, Frederik Schulz,
Bo Barker Jørgensen
Appendix S1. Supplementary methods
Sampling site
Sediment samples were collected from Aarhus Bay, station M5 (56°06´1977´´N
10°27´4822´´E), in May, 2010. The sulfate-rich zone at this site extends roughly to 60
centimeters below seafloor (cmbsf). Samples used for this study were from 10–20 cmbsf (1418 mM sulfate), a depth interval where sulfate reduction is the dominant terminal
remineralization process (Langerhuus et al., 2012). Sediments were kept in intact Rumohr Lot
(Meischner and Rumohr, 1974) cores at 4 °C until further processing. Immediately before
being used for the experiments, the sediment was thoroughly homogenized in a sealed airtight bag flushed with oxygen-free N2. After homogenization the porewater sulfate
concentration was 23 mM.
Incubation of sediments
Replicates of 1 cm3 of sediment were anaerobically incubated in Hungate tubes containing 9
mL of marine medium consisting of (per litre of distilled water): 25 g NaCl, 3 g MgCl2·6H2O,
0.5 g KCl, 0.15 g CaCl2·2H2O, 0.26 g NH4Cl, 0.03 g KH2PO4, 1.42 g Na2SO4, 1 mL trace
element solution (Widdel and Bak, 1992) and 0.001 g resazurin. The pH was adjusted to 7.5.
The medium was autoclaved and made anoxic by cooling under a sterile-filtered N2
atmosphere. Then the following solutions were added to the sterile medium from separate
sterile stocks: 30 mM final concentration NaHCO3, 0.12 g L-1 Na2S·9H2O, 0.1 g L-1 yeast
extract, and 1 mL L-1 vitamin mixture solution (Widdel and Bak 1992). As a carbon source,
either unlabeled (12C) or uniformly labeled 13C sodium acetate (Sigma-Aldrich) was added
after filtration through 0.22 µm pore size polyethersulfone membrane filters (Cronus biotech
Ltd.), to final concentrations of 10 mM, 1 mM or 0.1 mM. After adding the sediment
inoculum, headspaces were flushed with sterile-filtered N2/CO2 (9:1, vol/vol), and tubes were
sealed with butyl rubber stoppers. For each sulfate and acetate treatment, 3 biological
replicates were prepared resulting in a total of 18 tubes (Table S3). The sediments were
incubated statically for a period of 8.5 months at 15 °C in the dark.
Chemical analyses
Chemical parameters were monitored in sediment suspensions or headspace gas subsamples
aseptically withdrawn by N2-flushed syringes after 1, 5, 6 and 8.5 months of incubation
(Table S3). Sulfate, acetate and dissolved inorganic carbon (DIC) were analyzed in
supernatants of sediment suspensions upon centrifugation at 10,000 × g for 15 min. Sulfate
was measured by ion chromatography as described previously (Tarpgaard et al., 2011). Total
and 12C/13C-labeled acetate were measured using 2-dimensional ion chromatography-mass
1
spectrometry (IC-IC-MS; Dionex ICS-3000-MSQ, with AS 24 as the first column to separate
the volatile fatty acids from chloride, and AS 11 HC as the second column), as described by
(Glombitza et al., 2014). Subsamples for DIC were processed as described previously
(Tarpgaard et al., 2011). Total DIC was measured by gas chromatography (SRI 310C GC,
SRI Instruments Europe GmbH) using a thermal conductivity detector, and 13C-DIC was
measured using a Delta V plus Isotope Ratio Mass Spectrometer (Thermo Scientific).
Methane was measured by gas chromatography (SRI 310C GC, SRI Instruments Europe
GmbH) using a flame ionization detector.
DNA extraction and separation of 13C-DNA
Enrichments were sampled for DNA extraction after 1, 2, 3, 5, 6 and 8.5 months of incubation
(Table S3). DNA was extracted from the sediment pellets after centrifugation (as described
for chemical analyses), using the PowerLyzerTM UltraClean® Microbial DNA Isolation kit
(MoBio, Carlsbad, CA, US), following the manufacturer’s instructions. For SIP, DNA
extracts (10-65 ng DNA per sample) were mixed with up to 0.75 mL of gradient buffer and 5
mL of sterile CsCl stock solution (~1.83 g/mL, dissolved in gradient buffer) (Lueders 2009)
to obtain a final buoyant density (BD) of 1.673 g ml-1. The resultant mixtures were loaded
into 5.1-ml polyallomer tubes. Ultracentrifugation was done at 177,000 × g, in a Vti 65.2
vertical rotor (Beckman Coulter) at 20 °C for 38 hours. Next, 17-21 fractions were collected
from each gradient mixture using a syringe pump (Zevenaar, The Netherlands) as described
previously (Neufeld et al., 2007). BDs of each fraction were measured using a refractometer
(Refracto 30 PX, Mettler Toledo). DNA was recovered from the collected fractions (Neufeld
et al., 2007) and DNA concentrations were measured using a Nanodrop-3300
fluorospectrophotometer (Thermo Scientific, Wilmington, DE, US) with 1:20 diluted
Picogreen reagent (Invitrogen). Serial dilutions of the lambda DNA/HindIII ladder (New
England Biolabs) ranging in concentrations from 0.001 ng µL-1 to 5 ng µL-1 were used as
standards for the measurements.
Quantification of 16S rRNA genes by quantitative PCR (qPCR)
Total numbers of 16S rRNA gene copies were quantified by SyBrGreen-based qPCR assays,
on a LightCycler 480 instrument (Roche) using the primer pair Bac8Fmod (modified from
Loy et al., 2002)-Bac338Rabc (Daims et al., 1999) for Bacteria and Arch806F (Takai and
Horikoshi 2000)-Arch958R (DeLong, 1992) for Archaea (Table S4). A total reaction volume
of 20 µL contained 10 µL of LightCycler 480 SYBR Green I Master (Roche), 2 µL of bovine
serum albumin (10 µg µL-1), 1 µL of each primer (10 µM), and 1 µL of template DNA.
Thermal cycling was comprised of an initial activation step (95 °C for 5 min), 45 cycles of
amplification (95 °C for 30 sec, 55 °C for 30 sec, 72 °C for 15 sec) and fluorescence
measurement (80 °C for 5 sec). Each run was finalized by a melting curve analysis (55–95 °C,
1 min) to ensure specific amplification.
PCR amplification of 16S rRNA genes for Ion Torrent sequencing
2
For phylogenetic analyses, prokaryotic 16S rRNA genes from total DNA and from selected
heavy SIP fractions were PCR amplified using a Veriti Thermal Cycler (Applied Biosystems)
and a primer pair that targets both bacterial and archaeal 16S rRNA genes, Uni519F and
Uni802R (Table S4), modified from Øvreås et al. (1997) and Claesson et al. (2009),
respectively. The reaction mixture (25 µL) included 12.5 µL of 2× KAPA HiFi HotStart
Ready Mix (Kapabiosystems, Boston, MA, US) and 10 µM of each primer. Thermal cycling
consisted of 95 °C for 5 min, then 14 cycles of 98 °C for 20 sec, 49 °C for 15 sec, 72 °C for
15 sec, with final extension at 72 °C for 5 min. The PCR products were barcoded individually
in a subsequent PCR using 1 µL of initial PCR products (1:25 dilution) as templates. The
initial 5 thermal cycles for the barcoding PCR were the same as for the original template (see
above) and were followed by 12 cycles with annealing at 61 °C.
Ion Torrent sequencing and phylogenetic analysis of 16S rRNA gene sequences
The barcoded 16S rRNA gene PCR amplicons were purified using Agencourt AMPure XP kit
(Beckman Coulter GmbH, Krefeld, Germany) and quantified by Agilent 2100 Bioanalyzer
(Agilent Technologies, Germany GmbH). After quantification, the samples were pooled in an
equimolar mixture to a final concentration of 100 ng in a 79 µL volume. Adapter ligation was
carried out using Ion Plus Fragment Library Kit/Adapters (Life Technologies, USA).
Templates for sequencing were prepared according to the manufacturer’s instructions on the
Ion OneTouch System (Life Technologies, USA), using Ion OneTouchTM 200 Template Kit
v2 (Life Technologies, USA). Sequencing was performed on the Ion Torrent Personal
Genome Machine (PGM) System (Life Technologies, USA) following the manufacturer’s
protocol.
Sequence analyses were performed using the Mothur software package (v.1.28.0., Schloss et
al. 2009, 2011), referring to the Schloss standard operating procedure for processing 16S
rRNA gene amplicon sequences generated by 454 pyrosequencing (Schloss et al. 2011,
version September 2012), as follows. Sequencing reads passing the PGM system default
quality filtering were further quality filtered to remove barcodes, primers, sequences shorter
than 80 bp and sequences containing homopolymers longer than 8 bp. The sequences were
aligned according to the SILVA (Quast et al., 2012) reference alignment provided by the
Mothur project containing both bacterial and archaeal sequences. Sequences not aligning to
the expected 16S rRNA gene region were removed from further analyses. The sequences were
pre-clustered allowing 1 mismatch to remove further sequencing errors. Chimeric sequences
were removed using Uchime implemented in Mothur (Edgar et al., 2011). After processing
the sequences from a total of 38 samples, 1765 sequences were obtained per sample on
average (range: 1285 - 4677 sequences per sample) with an average length of 179 bp (range:
90 - 200 bp). After constructing a distance matrix, the sequences were clustered into
operational taxonomic units (OTUs) at 97 % similarity using the average neighbor clustering
method. Representative sequences of a total of 12,070 identified OTUs were selected and
taxonomically assigned using the RDP classifier function (Wang et al. 2007).
For phylogenetic analysis of deltaproteobacterial OTUs, representative sequences were
aligned using the SINA Web Aligner of the SILVA rRNA database project (Pruesse et al.
2012), and then imported into the Silva SSURef release 111 ARB database (Ludwig et al.
2004; Quast et al. 2012). Phylogenetically closely related sequences identified by BLAST
search of the NCBI nr database (http://www.ncbi.nlm.nih.gov) were aligned and imported as
well. A reference tree including near full-length 16S rRNA gene sequences that are closely
related to the Ion Torrent sequence reads was calculated by maximum likelihood analysis
3
using FastTree2 (Generalized Time-Reversible model, CAT approximation with 20 rate
categories) (Price et al., 2010). The resultant tree was re-imported into ARB and the short Ion
Torrent sequence reads were added by ‘ARB parsimony quick add marked’ option.
The Ion Torrent sequencing data have been deposited at NCBI Sequence Read Archive under
Accession SRP026290.
PCR amplification and cloning of dissimilatory (bi)sulfite reductase (dsrB) genes
DsrB fragments were PCR amplified using the SpeedSTARTM DNA Polymerase (Takara Bio
Inc.). Two primer pairs, each consisting of a mixture of forward and reverse primers targeting
the same position in the dsrB gene, were used: dsrB F1a-h and dsrB 4RSIa-f to target all dsrB
except those of xenologous Firmicutes (i.e. Firmicutes carrying a deltaproteobacterial-type
dsrB), and dsrB F2a-i and dsrB 4RSIb,e to target dsrB genes of xenologous Firmicutes (Lever
et al. 2013; Table S4). PCR mixtures were prepared according to the manufacturer’s
instructions. Thermal cycling consisted of 98 °C for 2 min, then 35 cycles of 98 °C for 10 sec,
56 °C for 30 sec, 72 °C for 1 min, with final extension at 72 °C for 10 min.
The PCR products were purified with the GenEluteTM PCR Clean-Up kit (Sigma) and cloned
using the TOPO TA Cloning® Kit (Invitrogen, Carlsbad, USA) according to the
manufacturer’s protocol.
DsrB sequencing and phylogeny
Cloned dsrB PCR amplicons (approximately 350 nt long) were sequenced via Sanger dideoxy
method (GATC Biotech, Germany). Sequences were analyzed in ARB using a dsrAB
sequence database containing >8000 aligned dsrAB sequences as reference. The top BLAST
hits of the cloned sequences were included in the analyses. The cloned dsrB sequences were
clustered into OTUs based on 90 % sequence similarity, as suggested previously (Kjeldsen et
al. 2007). Representative sequences of the OTUs were translated into amino acid sequences
and aligned in ARB. Closely related DsrAB sequences (full length) were exported to
FastTree2 for maximum likelihood tree calculation (WAG (Whelan and Goldman, 2001),
CAT approximation, Gamma20-based likelihood). The generated phylogenetic tree was
imported into ARB, and the representative OTU sequences were added into the tree by ‘ARB
parsimony quick add marked’ option. The dsrB sequences have been submitted to the
GenBank/EMBL/DDBJ database under the accession numbers AB857175 to AB857214.
4
Supplementary tables
Table S1. Sulfate, total- and 13C-acetate, 13C-dissolved inorganic carbon (DIC), total DIC and
total methane concentrations in sediments incubated for 8.5 months. The column on the far
left shows the treatment of sediments. The measured values from duplicate incubactions are
shown. Total DIC concentration in the native sediment was ~13.1 mM, and bicarbonate
concentration in the medium was ~33.3 mM. ND, no data available.
13
Treatment
Sulfate
(mM)
Total acetate
(mM)
Cacetate
(mM)
10 mM 12C-acetate
0.02
0.523
0
0-0.54
ND
0.51-2.30
10 mM 13C-acetate
0.01
0.109-0.101
0
10.43-11.38
35.7-40.4
2.31-2.95
1 mM 12C-acetate 4.36-4.37
0.019-0.041
0
0-0.46
42.0
0.03-0.04
1 mM 13C-acetate 4.41-4.53
0.012-0.015
0
1.49-1.84
31.0
0.02-0.05
0.1 mM 12C-acetate 5.20-5.21
0.012-0.017
0
0.35-0.44
32.0-40.0
0.04-0.05
0.1 mM 13C-acetate
0.008-0.014
0-0.003
0.51
31.3-32.7
0.02-0.02
5.36
13
C-DIC
Total DIC
accumulation (mM)
(mM)
Total methane
(mM)
Table S2. Phylogenetic affiliation, relative abundance of 16S rRNA gene sequences retrieved
from sediment before incubation (T0), after 3 months of incubation on 10 mM acetate (total
community, T3), and SIP fractions (12C-DNA, 13C-DNA). For each OTU, values from
biological replicates are shown in consecutive rows. For archaeal taxa, their relative
abundances were separately calculated after extracting the archaeal sequences from the
datasets before subsampling. Relative abundance change over time was calculated by
subtracting the average relative abundance at T3 (total community) from that at T0. Relative
abundance ratios were calculated with averaged values. NA, not available.
5
Change over
time
Relative abundance (%)
Phylogenetic affiliations
Total
community
12
T3-T0 (%)
C-DNA
(SIP)/12C-DNA
(SIP)
13
T0
10838
0.0
0.0
9.0
9.4
11.6
12.2
1.4
0.7
30.1
42.1
10.5
3.0
34.6
Desulfovibrionales
Desulfovibrionaceae
Desulfovibrio
10709
0.0
0.0
3.2
2.0
0.5
3.3
1.0
0.1
2.4
2.2
2.2
1.2
4.3
Desulfuromonadales
Desulfuromonadaceae
Desulfuromusa
8806
0.0
0.1
0.3
0.5
0.3
0.6
0.6
0.2
0.9
1.8
0.4
3.1
3.4
Gammaproteobacteria
Alteromonadales
Shwanellaceae
Shewanella
10854
0.0
0.0
0.1
0.0
0.8
0.1
0.1
0.0
0.4
0.0
0.2
0.5
3.9
6
C-DNA (SIP)
13
C-DNA
(SIP)/total
community
(13C1, 13C2)
OTU no.
Bacteria
Proteobacteria
Deltaproteobacteria
Desulfobacterales
Desulfobacteraceae
Unclassified
C-DNA (SIP)
13
Relative abundance ratios
Change over
time
Relative abundance (%)
Relative abundance ratios
13
Phylogenetic affiliations
Bacteroidetes
Flavobacteria
Flavobacteriales
Unclassified
Unclassified
Firmicutes
Clostridia
Clostridiales
Clostridiales Family XII.
Incertae Sedis
Fusibacter
Clostridiaceae
Uncassified
OTU no.
T0
11125
0.0
0.2
11142
0.0
0.0
11190
10464
Total
community
12
C-DNA (SIP)
13
C-DNA (SIP)
T3-T0 (%)
C-DNA
(SIP)/total
community
(13C1, 13C2)
C-DNA
(SIP)/12C-DNA
(SIP)
13
0.3
0.1
0.5
0.3
0.5
1.1
0.5
0.4
0.0
0.0
0.2
0.1
0.2
0.3
NA
0.1
0.1
0.5
0.0
0.6
0.6
3.0
0.0
0.0
2.5
2.8
1.2
2.7
0.4
0.2
1.2
0.1
2.3
0.3
2.5
0.0
0.0
0.7
0.4
1.2
1.1
0.0
0.0
0.6
0.0
0.8
0.3
NA
7
Change over
time
Relative abundance (%)
Relative abundance ratios
13
Phylogenetic affiliations
Fusobacteria
Fusobacteriia
Fusobacteriales
Fusobacteriaceae
Ilyobacter
Unclassified
Archaea
Crenarchaeota
Group C3
OTU no.
T0
8134
0.0
0.0
10661
0.0
0.0
675
0.2
0.0
1158
0.2
0.0
Total
community
12
C-DNA (SIP)
13
C-DNA (SIP)
T3-T0 (%)
C-DNA
(SIP)/total
community
(13C1, 13C2)
C-DNA
(SIP)/12C-DNA
(SIP)
13
0.1
0.3
0.2
0.1
1.4
1.8
7.3
2.3
0.0
0.0
0.1
0.0
0.2
0.4
NA
0.3
0.0
0.8
0.0
3.2
0.1
2.8
0.0
1.6
0.0
0.0
0.8
3.2
0.0
0.0
0.0
2.9
0.3
NA
NA
0.0
2.9
0.9
NA
NA
8
Table S3. Sediment incubations with different acetate treatments. For each treatment, 3
biological samples were prepared, either with 12C- or 13C-acetate. Subsamples were taken at
selected time points for chemical analysis (C) and gDNA extraction (D). Density-gradient
ultracentrifugation and density fractionation of gDNA extracts (U) were done for all the
samples at the end of incubation, and for a subset of samples at intermediate time points
(indicated as superscripts; U1-2 means ultracentrifugation was done for 2 of the replicates
while U means it was done for all replicates).
Treatment
Acetate
(mM)
Incubation time (month)
Isotope
&
Replicate
0
1
2
3
5
6
8.5
10
12
C1-3
C
C, D, U1-2
D, U1-2
D, U1-2
C, D, U1
C, D
C, D, U
10
13
C1-3
C
C, D, U1-2
D, U1-2
D, U1-2
C, D, U1
C, D
C, D, U
1
12
C1-3
C
C, D
D
D
C, D, U
C, D
C, D, U
1
13
C1-3
C
C, D
D
D
C, D, U
C, D
C, D, U
0,1
12
C1-3
C
C, D
D
D
C, D
C, D
C, D, U
0,1
13
C1-3
C
C, D
D
D
C, D
C, D
C, D, U
Unincubated Replicates
C, D, U
sediment
1-3
Table S4. PCR primers used in this study.
Primer Name
Sequence (5’ – 3’)
Reference
Bac8Fmod
AGAGTTTGATYMTGGCTCAG
Loy et al., 2002
Bac338Rabc
GCWGCCWCCCGTAGGWGT
Daims et al., 1999
Arch806F
ATTAGATACCCSBGTAGTCC
Takai and Horikoshi, 2000
Arch958R
YCCGGCGTTGAMTCCAATT
DeLong, 1992
Uni519F
CAGCMGCCGCGGTAA
Øvreås et al., 1997
Uni802R
TACNVGGGTATCTAATCC
Claesson et al., 2009
dsrB F1a-h/4RSI1a
Primer mixture
Lever et al., 2013
dsrB F2a-i/4RSII1b
Primer mixture
Lever et al., 2013
9
Supplementary figures
Fig. S1. Microbial activity in the sediment slurries. (A) Changes in sulfate (upper row) and
acetate (lower row) concentrations upon incubation of sediments on 10 mM (left column), 1
mM (middle column) and 0.1 mM acetate (right column). The data values from sediments
treated with 12C-acetate are shown in empty circles and the ones with 13C-acetate are shown in
filled triangles. All data points represent the average values from two biological replicates,
and error bars indicate the range of two measured values. Samples from 10 mM and 1 mM
12
C and 13C-labeled acetate treatments at 1-month time point were lost during measurements.
(B) Changes in the number of bacterial and archaeal 16S rRNA gene copies per gram of
sediment, as measured by qPCR. T0, unincubated sediment; T3, after 3 months of incubation.
All data points represent average values from two biological replicates, and error bars indicate
the range of two measured values.
10
Fig. S2. Non-metric multidimensional scaling (nMDS) plots showing the ordination patterns
of microbial communities. Distances between points reflect the difference based on the BrayCurtis dissimilarity. The dots of the same color-coding represent the total communities of
biological replicates with the same treatment (2 dots for T0, 4 dots for each 10 mM, 1 mM,
0.1 mM acetate treatment; two of which were amended with 12C-acetate (12C1, 12C2) and two
with 13C-acetate (13C1, 13C2)) and are connected to make clusters. Each grey cross
corresponds to an OTU based on 97 % sequence similarity. T0, unincubated sediment; T3,
after 3 months of incubation.
Fig. S3. Maximum-likelihood tree of DsrB amino acid sequences showing the relationship of
40 OTUs from enrichments on acetate (in boldface) and selected sequences of cultured and
uncultured sulfate-reducing prokaryotes. Sequences identities of <70 % were assigned to be
separate clusters (cluster A-Q). Percentage values next to the OTU number indicate the
relative abundance of the OTU in different samples (shown in different color codes).
Thermodesulfovibrio islandicus (AF334599) was used as an outgroup. The scale bar
represents 10 % estimated sequence divergence.
11
12
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