Supplementary information
Material and Methods
Sampling. Water samples were obtained from the central Baltic Sea during a research
cruise onboard the RV Maria S. Merian in February 2006 (Baltic Sea monitoring station 271,
57°19.2’N; 20°03’E, Gotland Deep [Fig. 1]). Water samples from different depths around the
chemocline, which we define as the uppermost presence of sulfide, were collected in freeflow bottles attached to a conductivity, temperature, and depth (CTD)-rosette.
Physico-chemical structure of the water column. Chemical profiles of oxygen, hydrogen
sulfide, as well as ammonia, phosphate, nitrite, and nitrate were determined as described
elsewhere (Grasshoff et al. 1983).
Bacterioplankton sampling. For catalyzed reporter deposition fluorescence in situ
hybridization (CARD-FISH), 100 mL of water samples were directly transferred to glass
bottles and fixed with particle-free formaldehyde (2% final concentration) at 4°C for 2–5 h.
Portions of 40–45 mL were filtered onto white polycarbonate membrane filters (Millipore
GTTP, 0.2 µm pore size, 47 mm diameter). The filters were then rinsed with sterile seawater,
air dried, and stored at -80°C until further processing. For nucleic acid extraction, 1–2 L of
water samples were filtered onto white Durapore filters (Millipore GVWP, 0.22 µm pore size,
47 mm diameter) and stored frozen at -80°C.
Nucleic acid extraction and complementary DNA (cDNA) synthesis. RNA and DNA
was extracted from filters using an acidic phenol extraction protocol (Weinbauer et al. 2002).
Prior to RT-PCR, RNA extracts were purified from DNA by incubation with DNAse I (DNAfreeTM-Kit, Ambion, Austin, TX or amplification grade, Invitrogen) at 37°C for 30 min and
their concentrations were determined using a NanoDrop ND-1000 spectrophotometer
(NanoDrop Technologies). Different procedures were used to obtain cDNA. To retrieve 16S
rcDNA, 20 ng of template RNA was reverse transcribed using the iScriptTM cDNA Synthesis-
Kit (Bio-Rad) at 42°C. In addition to hexamers provided by the kit, the reverse primer 1492R
was also supplied (Tab. 1). To assess the diversity of amoA transcripts by denaturing gradient
gel electrophoresis (DGGE) or clone libraries, the Access One-Step-RT-PCR kit (Promega)
was used with Arch-amoAR, AOA-amoA-r, amoA-2R, or amoB-4R as reverse primers (Tab.
1). For quantitative PCR (qPCR) of amoA transcripts, 272 to 545 ng of total nucleic acids was
reverse transcribed into cDNA with random primers using Superscript III First-Strand
Synthesis supermix (Invitrogen). All reverse transcriptase steps followed the protocols of the
respective manufacturer. In each reverse transcription reaction, some RNA samples without
reverse transcriptase were used as controls in the PCR to check for DNA contamination. In
the case of a One-Step kit, the reverse transcriptase was heat-denaturized before use.
16S rRNA/rRNA gene based diversity analyses. (A) Single strand conformation
polymorphism (SSCP)-fingerprinting. Archaeal w036/039-primers (Tab. 1) were used for
16S rcDNA and 16S rRNA gene amplification from eight different depths throughout the
redoxcline (Fig. 2). PCR mixtures contained 1x PCR buffer, 2.5 mmol L-1 of MgCl2, 200
µmol L-1 of each dNTP, 400 nM of the forward and reverse primers and 0.025 U µL-1 of Taq
DNA polymerase (Eppendorf). Thermocycling started with an initial denaturation at 94°C for
5 min. A total of 25 (cDNA) or 35 (DNA) cycles, at 94°C for 30 s, 57°C for 60 s, 72°C for 2
min, were followed by a final elongation step at 72°C for 5 min. Generation and purification
of ssDNA and SSCP analysis were done according to Schwieger and Tebbe (Schwieger &
Tebbe 1998). Re-amplification of individual bands excised from the SSCP gels was
performed as described by Pöhler et al. (Pöhler et al. 2002). PCR products were purified using
the MiniElute Kit (Qiagen) according to the manufacturer’ protocol and sequenced by
SEQLAB (Göttingen, Germany).
(B) 16S rRNA/rRNA gene clone library construction and RFLP. Archaeal 16S rRNA
clone libraries were prepared from genomic DNA or RNA extracts originating from the
suboxic zone, suboxic-sulfidic interface and the sulfidic zone (Fig. 2). To amplify nearly-full-
length 16S rRNA from the total community RNA, the primer system Arch21F and 1492R
(Tab.1) was used for PCR. In order to complement 16S rRNA sequences generated by the
above described SSCP analyses, primer combinations w036/1492R and Arch21F/w039 were
also used. PCR mixtures contained 1x PCR buffer, primers (0.4 µmol L-1 each), 200 µmol L-1
of deoxynucleoside triphosphate, 500 µmol L-1 MgCl2, and 0.025 U µL-1 Taq DNA
polymerase (Fermentas). The reactions were incubated under the following conditions: initial
denaturation at 94˚C for 5 min, 25 (cDNA) or 35 (DNA) cycles of 94˚C for 30 s, 57˚C for 60
s, and 72°C for 50 s, followed by one cycle of 72˚C for 5 min. The PCR products were
purified as described above and cloned following the manufacturer’s instructions using the
pGEM-T-Easy Vector system (Promega) together with competent E. coli JM109 cells. For
restriction fragment length polymorphism (RFLP) and sequencing, the inserted fragment was
PCR-amplified with the vector-specific primers T7 and SP6 (Tab. 1). The unpurified PCR
products were digested with restriction enzymes Hin6I and MspI, the restricted fragments
separated on a 3% agarose gel and documented as described previously (Bond et al. 2000).
Identical RFLP patterns were grouped and representative cloned fragments sequenced by
SEQLAB (Göttingen, Germany) using the primers Arch21F, w036, w069, or 1492R.
(C) Phylogenetic analysis. 16S rRNA/rRNA gene sequences were examined for accuracy
using the software SeqMan (DNAstar) and checked for chimeras using the Bellerophon
program (Huber 2004). Phylogenetic affiliations of the partial sequences were initially
estimated with the program BLAST (Altschul 1990). Sequences were aligned using the ARB
software package (Ludwig et al. 2004) and those with a similarity greater than 99% grouped.
Basic trees were reconstructed based on related public sequences and generated clones of
approximately 1300 nucleotides. These were reduced to unambiguously alignable positions
using group specific filters. An evolutionary-distance dendrogram was constructed using
neighbor-joining (NJ), maximum likelihood (ML), and maximum parsimony (MP).
Eventually, partial SSCP sequences were individually imported using ARB Parsimony and
group specific filters.
Archaeal amoA mRNA/mRNA gene based diversity analyses. (A) DGGE
fingerprinting. Ten ng of DNA and 20 ng of RNA were used as template for the PCR and
One-Step-RT-PCR (see above), respectively, originating from eight different depths
throughout the redoxcline (Fig. 2). PCR mixtures contained 1 x PCR buffer, archaeal amoA
primers AOA-amoA-f and AOA-amoA-r (0.4 µmol L-1 each) (Tab. 1), 200 µmol L-1
deoxynucleoside triphosphate, 2 mmol L-1 MgCl2, and 0.025 U µL-1 Taq DNA polymerase
(Eppendorf). Thermocycling started with an initial denaturation step of 94°C for 5 min,
followed by 25 (cDNA) or 35 (DNA) cycles of denaturation at 94°C for 1 min, annealing at
58.5°C for 50 s, and elongation at 72°C for 90 s. The PCR ended with a denaturation step
followed by annealing at 60°C for 90 s and final elongation at 72°C for 10 min. DGGE
electrophoresis was performed according to (Coolen et al. 2004) using 9 % polyacrylamide
gels (ratio of acrylamide to bisacrylamide, 37.5:1) submerged in 0.5 x TAE buffer (20 mmol
L-1 Tris, 20 mmol L-1 acetic acid, 0.5 M EDTA; pH 7.4) at 60°C. Individual bands excised
from the DGGE gels were re-amplified using the same PCR protocol, purified using the
MiniElute Kit (Qiagen) as described by the manufacturer, and sequenced by SEQLAB
(Göttingen, Germany).
(B) amoA clone libraries. Archaeal and bacterial amoA clone libraries were prepared from 10
ng of genomic DNA or 20 ng of RNA (One-Step-RT-PCR, see above) extracts originating
from the suboxic zone, suboxic-sulfidic interface and the sulfidic zone (Fig. 2). For PCR, the
primer systems Arch-amoAF and Arch-amoAR, AmoA-1F and AmoA-2R, and AmoA-3F and
AmoB-4R were used (Table 1). PCR mixtures contained 1 x PCR buffer, the according primer
systems (0.4 µmol L-1 each), 200 µmol L-1 deoxynucleoside triphosphate, 2 mmol L-1 MgCl2,
and 0.025 U µL-1 Taq DNA polymerase (Fermentas). The archaeal PCR started with an initial
denaturation step of 94°C for 5 min, followed by 25 (cDNA) or 30 (DNA) cycles or of
denaturation at 94°C for 45 s, annealing at 53°C for 60 s, and elongation at 72°C for 60 s.
Thermocycling ended with a final elongation at 72°C for 15 min. The bacterial PCR started
with an initial denaturation step of 94°C for 5 min, followed by 25 (cDNA) or 30 cycles
(DNA) of denaturation at 94°C for 15 s, annealing at 55°C (betaproteobacteria) or 48°C
(gammaproteobacteria) for 20 s, and elongation at 72°C for 40 s. The PCR ended with a final
elongation at 72°C for 60 s. Cloning and sequencing steps were done as described for the 16S
rRNA procedure.
(C) Phylogenetic analyses. Phylogenetic analysis followed those described for the 16S
rRNA. Phylogenetic trees were reconstructed based on sequence information from DGGE
bands and clones, and related to public sequences. These were reduced to unambiguously
alignable positions using group specific filters, resulting in 166 valid characters. An
evolutionary-distance dendrogram was constructed using neighbor joining, maximum
likelihood, and maximum parsimony.
Gene probe design. Based on the above described 16S rRNA and rRNA gene clone
libraries, a specific probe against the crenarchaeal cells of the suboxic zone was designed with
the PROBE_FUNCTION tool of the ARB package. Probe specificity was validated by
employing the PROBE_MATCH tool of the ARB package, the SILVA database (Pruesse et al.
2007) and BLAST. Horseradish peroxidase (HRP)-labeled probes were synthesized by
Biomers (Ulm, Germany). The specificity of the newly designed probe S-*-Cren-0679-a-A-18
(Tab. 2), which was named according to the Oligonucleotide Probe Database nomenclature
(Alm et al. 1996), was experimentally tested by whole-cell hybridization with different
archaeal strains. With only one mismatch at the fourth base position of the gene probe, C.
“Nitrosopumilus maritimus” SCM1 served as positive control. Halorubrum lacusprofundi
(DSM5036T) and Pyrococcus furiosus (DSM3638T) served as negative controls (Tab. 2). All
strains were cultivated following the suggestions of the Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH.
Total cell numbers and quantification with CARD-FISH. Flow cytometric analyses
were done as described previously (Labrenz et al. 2007). All HRP-labeled probes, appropriate
sequences, target sites, and hybridization conditions used in this study are described in Table
3. Non-specific binding was determined using the NonEUB probe. By varying the formamide
concentration, the hybridization conditions for the newly designed probe Cren679 were
optimized (formamide concentrations tested: 0, 15, 20, 25, 30, 35, 50, 60%). A concentration
of 35% formamide in the hybridization buffer was found to be optimal for probe specificity.
The euryarchaeal CARD-FISH enumeration was carried out according to the proteinase K
protocol of Teira et al. (Teira et al. 2004), but this method was not successful for the Cren537
and Cren679 gene probes. Therefore, cell wall permeabilization treatments were performed as
described by Sekar et al. (Sekar et al. 2003). According to the procedure of Sekar et al.(Sekar
et al. 2003), filter sections were washed in pre-warmed washing buffer and the tyramide
signal was amplified with 5-(and 6-)carboxyfluorescein-labeled tyramides. The preparations
were counterstained with a previously described mixture of 4’,6’-diamidino-2-phenylindol
(DAPI), Citifluor, and VectaShield (Pernthaler et al. 2002). Stained filter sections were
examined with an epifluorescence microscope (Axioskop 2 mot plus, Zeiss) as described
previously (Grote et al. 2007). At least 1000 DAPI-stained cells in randomly distributed
microscopic fields were counted for each filter section.
Quantification of amoA gene transcripts by real-time PCR. Quantitative PCR standards
were prepared as described by Agogue et al. (Agogué et al. 2008). All qPCR analyses were
conducted on an iCycler iQ5 thermocycler (Bio-Rad) equipped with iCycler iQ software.
Archaeal or betaproteobacterial amoA transcripts were determined in triplicate on non-diluted,
1:5 and 1:25 diluted cDNA and RNA samples. The reaction mix contained 1 U of Pico Maxx
high fidelity DNA polymerase (Stratagene), 2µL of 10x Pico Maxx PCR buffer, 0.25 mmol L1
of dNTPs, 8µg of BSA, 0.2µmol L-1 of each primers, 1:50,000 diluted SYBRGreen
(Invitrogen), 10nM of fluorescein, 3mmol L-1 of MgCl2 and ultrapure sterile water (Sigma) to
a final volume of 19µL (Coolen et al. 2006, Agogué et al. 2008). One µL of diluted or nondiluted cDNA and RNA was added to the 19 µL of reaction mix. Archaeal amoA gene
transcripts were detected using the primers Arch-amoA-for and Arch-amoA-rev (Tab. 1) with
an annealing temperature of 58.5ºC. Betaproteobacterial amoA gene transcripts were detected
with the primers amoA-1F and amoAr NEW (Tab. 1) and an annealing temperature of 61.5ºC.
Thermocycling was performed with an initial denaturation at 95ºC for 4 min followed by 41
cycles at 95ºC for 30 s, primer annealing temperature for 40 s, extension at 72ºC for 30 s, and
80ºC for 25 s, with a plate read between each cycle. A melting curve was conducted between
60°C and 94ºC with a read every 0.5ºC held for 1 s between each read. The copy numbers of
the samples were calculated by quantifying simultaneously the standard with ten-fold serial
dilutions ranging from 107 to 101 copies. No inhibition of the PCR was determined on the
dilutions. Thus, the value obtained with the undiluted nucleic acids was used for calculation
of copy numbers of the respective genes. DNA contamination in the RNA samples was
always lower than 1% of the cDNA as determined by qPCR. Nevertheless, the value of
archaeal amoA copy numbers determined in the RNA sample was subtracted from the copy
numbers obtained in the cDNA.
Supplementary information TABLE 1. Horseradish-peroxidase-labeled oligonucleotide
probes and hybridization conditions used in this study.
Primers
Sequence (5’-3’)
Positiona
Specificity
% FA
EUB338
GCTGCCTCCCGTAGGAGT
338 - 355
Most Bacteria
55
EUB338II
GCAGCCACCCGTAGGTGT 338 - 355
Planctomycetales
55
EUB338III
GCTGCCACCCGTAGGTGT
338 - 355
Verrucomicrobiales
55
NONEUB
ACTCCTACGGGAGGCAGC
-
Negative control
55
Eury806
CACAGCGTTTACACCTAG
806 - 823
Euryarchaeota
20
Cren537
TGACCACTTGAGGTGCTG
537 - 554
Crenarchaeota
20
-“-
Cren679
TTTTACCCCTTCCTTCCG
679 - 696
35
This study
a
Escherichia coli 16S rRNA position
“C. N. maritimus”/
parts of MGI cluster
Reference
Amann et
al., 1990)
Daims et
al., 1999
-“Wallner et
al., 1993
Teira et
al., 2004
100
100
A
110
Chemocline
Chemocline
120
Depth [m]
Depth [m]
120
B
110
130
130
140
140
150
150
SSCP band 9
SSCP band 4
0
10
20
30
40
0
50
10
20
Crenarchaeotal distribution
C
Chemocline
140
16S rDNA
16S rRNA
D
100
Depth [m]
Depth [m]
50
Euryarchaeotal distribution
16S rDNA
16S rRNA
120
40
Relative concentration [%]
Relative abundance [%]
100
30
Chemocline
120
140
160
160
0
10
20
30
40
50
Relative abundance [%]
60
70
80
10
20
30
40
50
60
70
80
Relative abundance [%]
Supplementary information FIGURE 1. Molecular abundance of prominent archaeal
clusters throughout the redoxcline based on analyses of 16S rRNA gene (rDNA) and 16S
rcDNA SSCP fingerprints. Only bands with proportions of >2% were investigated.
Distribution and relative band abundances are shown for: A, crenarchaeotal SSCP band 4; B,
euryarchaeotal SSCP band 9; C, Crenarchaeota; D, Euryarchaeota.
SSCP gels were scanned into GelScan Software (BioSciTec) and documented as a tif-file. In
order to determine relative 16S rRNA gene and 16S rcDNA band abundances, the tif-files
were analyzed using the program Gelcompare II version 3.0 (Applied Maths, Belgium). Band
search parameters were: Minimum profiling 5.00, ”Gray zone” 0, Minimum area 2.00 and
Shoulder sensitivity 1.
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