Supplementary Materials and methods
Sediment processing
Intact sediment cores were sliced, fixed and further processed immediately in the
field or upon arrival in the lab within a few hours as described previously (Lenk et al.
2011). Similarly, intact cores were taken for DNA extraction and selected sediment
horizons were frozen at – 20°C until further use.
CARD-FISH and FISH on sediments and enrichment cultures
We quantified RCB in sediments from other sites including Sylt, Baltic Sea,
arctic, Mediterranean and deep-sea sediments according to the protocol given by Ishii et
al. (2004). For details see Supplementary Table 1. For quantification of RCB in the
enrichment culture, 1 ml of medium was fixed with formaldehyde for 1 h at room
temperature at a final concentration of 3.7%. Cells were hybridized on slides as described
elsewhere (Llobet-Brossa et al. 1998) using the following probes ROS537, EPS549 (Lin
et al. 2006), GAM42a (Manz et al. 1992), ALF968 and NON338 (Wallner et al. 1993).
SoxB gene library from Janssand sediment
A soxB gene library was established using DNA of previously sampled surface
sediment (0–3 cm) from Janssand site (Lenk et al. 2011). Primers and PCR conditions for
amplification of an approximately 1000 bp fragment were chosen according to (Petri et
al. 2001) using 30 cycles. Generally, PCR reactions contained 50 pmol of each primer,
6.25 nmol of each dNTP, 1× Master Taq Buffer and 1 U of Taq DNA Polymerase
(Eppendorf, Hamburg, Germany) and were adjusted to a total volume of 25 μl with sterile
PCR water. Cloning and sequencing was performed as described previously (Lenk et al.
2011).
Phylogenetic analyses of 16S rRNA, SoxB and DsrAB
The 16S rRNA sequences were analyzed with the ARB software (Ludwig et al.
2004) using the SILVA 16S rRNA SSU Reference database release 102 (Pruesse et al.
2007). For selected clones almost full length sequences were generated (>1400 bp).
Sequences were analyzed for chimera formation using the Bellerophon software (Huber
et al. 2004). Tree reconstruction was based on 1075 sequences with >1200 nucleotides.
The phylogenetic tree was constructed using the maximum likelihood method RAxML
(Stamatakis et al. 2008), Neighbor joining and maximum Parsimony employing a 50%
conservation filter. Partial sequences were subsequently added to the tree without
allowing changes in the overall topology. Operational taxonomic units (OTUs) were
grouped based on 97% sequence identity using the ARB similarity matrix. Fosmid and
PCR derived sequences of the dsrAB and soxB genes were imported into ARB databases
containing a respective nucleotide/amino acid alignment. The dsrAB sequences were
manually aligned using a previously established dsrAB/DsrAB reference database (Lenk
et al. 2011). Deduced amino acid sequences of environmental soxB genes of this study
and publicly available sequences were aligned using the BioEdit program package prior
to import of the alignment in ARB. DsrAB and SoxB amino acid sequences were used to
reconstruct phylogenetic trees using ARB PhyML and RAxML (Stamatakis et al. 2008)
with insertion/deletion filters and the JTT amino acid substitution matrix. OTUs were
grouped according to branching patterns based and 90% amino acid sequence identity.
dsrA–targeted in situ hybridization (geneFISH) combined with 16S rRNA CARD-FISH
After synthesis the dsDNA probes were purified with the GeneClean Turbo kit
(Q-Biogene). Formaldehyde fixed cells of the enrichment culture were filtered on
palladium/gold coated polycarbonate filters. Filters were incubated in 0.01 M HCL for 10
min at room temperature to inactivate endogenous peroxidases, followed by incubation in
10 mg/ml lysozyme for 1 h at 37°C to permeabilize cell walls. CARD-FISH for 16S
rRNA with probes ROS537 and GAM42a was performed as described earlier (Ishii et al.
2004) using Alexa488-labeled tyramide for signal amplification. Following hybridization
of the 16S rRNA gene, inactivation of horse radish peroxidase enzymes (HRP)
introduced with the 16S rRNA targeting probe was achieved by incubations of the filters
in 3% H2O2 in PBS for 30 min followed by incubation in 0.1 M HCL for 10 min. Total
inactivation of the HRP was checked on control filters subjected to an additional
tyramide signal amplification step using Alexa 594 dye. No deposition of the dye
occurred as visible signals were absent during microscopic observation. To digest mRNA
the filters were incubated in RNase solution (0.5 U μl-1 RNase I, Ambion), 30 μg ml-1
RNase A (Sigma), 0.1 M Tris-HCl pH 8 for 4-5 h at 37 °C. Filter sections, with either
probe dsr285_RCB or probe NonPoly350 were incubated in hybridization buffer
containing 45% formamide. After initial denaturation at 85°C for 25 min hybridization
lasted for 18–22 h at 50°C followed by binding of the anti-Dig HRP-conjugated antibody
(Fab fragments) and signal amplification with a Alexa 594-labeled tyramide. Filter
sections were embedded in SlowFadeGold antifade reagent (Invitrogen), containing 1
μg/ml 4´,6-diamidino-2-phenylindole (DAPI). Microscopy was performed on an
epifluorescence microscope (Axioplan, Carl Zeiss), equipped with the following
fluorescence filters: DAPI (365/10 nm excitation, 420 LP emissions, FT 395 Beam
Splitter), Alexa488 (472/30 excitation, 520/35 emission, 495 Beam Splitter) and
Alexa594 (562/40 excitation, 624/40 emission, 593 Beam Splitter).
Amplification of 16S rRNA, dsrAB and soxCD genes from the enrichment culture
DGGE analysis was performed with primers GC-ROSEO536Rf and GRb735r
specific for Roseobacter-clade bacteria (Rink et al. 2007). For construction of the 16S
rRNA gene library a cell suspension was repeatedly frozen and thawn and served as DNA
template. PCR, cloning and sequencing for the bacterial 16S rRNA and dsrAB genes
were performed as described previously (Lenk et al. 2011).
SoxCD genes from the enrichment culture were amplified using novel primers
that were designed based on fosmid ws101A12 and published RCB soxCD sequences.
Forward primer soxC2f targets soxC (5’-TCGATCCCGATGGAAAAGG-3’), reverse
primer soxD2r targets soxD (5’-TGCCGAAGGGCATCGAGC-‘3). PCR was conducted
as follows: initial denaturation for 5 min at 95°C, followed by 30 cycles of 30 sec at
95°C, annealing for 30 sec at 51°C and elongation for 2 min at 72°C. PCR products were
sequenced directly without cloning with the Big Dye Terminator v3.1 Cycle Sequencing
Kit (Applied Biosystems) according to the manufacturers' instructions. The phylogenetic
analyses of 16S rRNA genes and DsrAB are detailed in Supplementary Methods.
Fosmid sequencing and annotation.
Short insert shotgun libraries were generated with 1.5 and 2.5 kb inserts. Endsequencing was performed on recombinant plasmids using BigDye 3.1 chemistry and
3730XL capillary sequencers (ABI, Darmstadt, Germany) up to a 10-fold sequencing
coverage at least. Reads were assembled using PhredPhrap (http://www.phrap.org) and
imported into Consed (Gordon et al. 1998), edited and verified. Finishing experiments
were performed by primer-walking on fosmid and bridging shotgun clones to improve
sequence quality and gap closure. ORF prediction was performed with Glimmer3
(Delcher et al. 1999) and Metagene (Noguchi et al. 2006). Metagenomic analysis of
predicted protein coding sequences was performed with JCoast (Richter et al. 2008)
using the software MicHanThi (Quast 2006) for automatic annotation. The annotation of
proteins highlighted within the scope of this study was manually refined.
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