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Supplemental material to:
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Expression patterns of mRNAs for methanotrophy and thiotrophy in symbionts of the
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hydrothermal vent mussel Bathymodiolus puteoserpentis
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A. Wendeberg, F. U. Zielinski, C. Borowski, and N. Dubilier
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PCR amplification, cloning, and sequencing. Fragments of pmoA (500 bases, subunit A of
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pMMO) and aprA (390 bases, subunit A of APS-reductase) genes were PCR amplified from
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DNA isolated from B. puteoserpentis gill tissues. The PCR conditions for the pmoA
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amplification are the same as described previously (Costello and Lidstrom 1999). For aprA,
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PCR amplification with the MasterTaq kit (Eppendorf) and two degenerate primers was used:
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AprA-1-FW 5’ tggcagatcatgatymaygg 3’ and AprA-5-RV 5’ gcgccaacyggrccrta 3’ (Meyer
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and Kuever 2007). MgCl2 was added to a final concentration of 1.5 mM. The annealing
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temperature was 58ºC and 36 cycles were performed. Amplicons were cloned (TOPO TA,
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Invitrogen, Karlsruhe, Germany) and sequenced (Abiprism 3100 Genetic Analyzer, Perkin
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Elmer, Boston, MA, U.S.A.). The pmoA and aprA gene sequences were deposited at the
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EMBL database (Kulikova et al 2004) under accession numbers FR865039 and FR865040,
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respectively.
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Probe synthesis. Templates for probe synthesis were PCR amplified using primers with a T7
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polymerase promoter on either the forward primer for the control probe or the reverse primer
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for the mRNA targeted anti-sense probe (for detailed description see (Pernthaler and Amann
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2004)). PCR conditions were the same as above. Amplicons were precipitated and the correct
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length checked on a 1.5% [w/v] agarose gel. Concentrations of T7 templates were determined
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photometrically (NanoDrop, NanoDrop Technologies, Rockland, DE, USA). Anti-sense
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probes (also referred to as “probe”, reverse complement to mRNA) and control probes (same
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sequence as mRNA) for pmoA and aprA of B. puteoserpentis symbionts were synthesized
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using the following conditions: 20 µl of the transcription reaction mix was mixed in the
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following order: 2 µl RNase free water, 2 µl of 10 x nucleotide mix [10 mM ATP, 10 mM
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CTP, 10 mM GTP, 8 mM UTP, 2mM fluorescein-UTP], 2 µl dithiothreitol [100 mM], 2 µl 10
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x transcription buffer [400 mM TrisHCl, pH 8.0, 60 mM MgCl2, 20 mM spermidine], 10 µl
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T7 template DNA (100 ng µl-1) and 2 µl T7 polymerase [50 U µl-1, Epicentre, Madison, WI,
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USA]. This mix was incubated at 37°C for 2 h. To remove the template DNA, 1 µl of RNase
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free DNase [1 U µl-1, Epicentre] was added, the mix was incubated at 37°C for another 15
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min, and subsequently precipitated. Length and labeling of the transcripts were checked on a
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1.5% [w/v] agarose gel, and probe concentrations determined photometrically. The probe
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stock solutions were prepared by mixing hybridization buffer for mRNA FISH (described
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below) and probe to obtain a concentration of 50 ng µl-1. The probe stock was denatured at
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80°C for 5 min and stored at -20°C.
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Controls for mRNA FISH. PmoA and aprA PCR products were cloned and expressed in E.
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coli cells according to the manufacturer’s instructions (pBAD vector, Invitrogen, Karlsruhe,
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Germany). Overnight cultures of Top10 E. coli were diluted 1:100 and grown for 2 h at 37°C.
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Cells were induced with 0.2% L-arabinose for 3 h and fixed and stored as described
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previously (Pernthaler and Amann 2004). FISH with the respective anti-sense probes showed
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intense signals in the induced E. coli cells. Two negative controls for mRNA FISH were
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performed: (1) E. coli cells were left uninduced and hybridized with the anti-sense probe, and
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(2) the B. puteoserpentis sections were hybridized with the control probes. In both cases,
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FISH signal intensities were below background.
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Supplemental Figure Legends:
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Figure S1. Comparison of symbiont distribution (rRNA-FISH) in cross sections through
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whole juvenile mussels. The cross sections are through the posterior – anterior axis of the
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mussel (posterior end where the exhalent and inhalent siphons lie are on the top part of each
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image, the anterior end on the bottom part). The figure shows that both symbionts are
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distributed evenly throughout the gills based on dual-hybridization using probes specific to
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the thiotrophic (red) and methanotrophic (green) symbionts on cross-sections through juvenile
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2 (upper panels) and juvenile 1 (lower panels). Due to red-green overlay, gill filaments appear
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yellow when both symbionts are densely packed together. The white solid outline indicates
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the outer dimension of the mussels (position of the mantle). The white dotted outline indicates
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the outer dimensions of the gill demibranches. Asterisks show small parts of the sections that
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were ruptured during the staining or sectioning procedure.
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Figure S2. Comparison of symbiont distribution (rRNA-FISH) in cross sections of single
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gill filaments. Thiotrophic symbionts shown in red and methanotrophic symbionts in green.
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The figure shows that there are no obvious variations in the relative and absolute abundances
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of the two symbionts between the three individuals. In each image, two gill filaments with the
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cilia at their frontal edges are outlined (white dotted line).
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Figure S3. Comparison of symbiont abundance (rRNA-FISH) and expression patterns
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(mRNA-FISH) in B. puteoserpentis gill sections. The figure shows hybridizations of mRNA
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and rRNA of methanotrophic symbionts (upper panel) and thiotrophic symbionts (lower
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panel) of juvenile 6. There are clear differences between the two signals, particularly in the
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regions furthest from the ciliated gill epithelia where mRNA signals are weaker than rRNA
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signals. In each image, two gill filaments and the cilia at their frontal edge where seawater
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enters the gills are outlined (white dotted line).
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Supplementary References:
Costello AM, Lidstrom ME (1999). Molecular characterization of functional and phylogenetic
genes from natural populations of methanotrophs in lake sediments. Applied & Environmental
Microbiology 65: 5066-5074.
Kulikova T, Aldebert P, Althorpe N, Baker W, Bates K, Browne P et al (2004). The EMBL
Nucleotide Sequence Database. Nucleic Acids Research 32: D27-D30.
Meyer B, Kuever J (2007). Molecular analysis of the diversity of sulfate-reducing and sulfuroxidizing prokaryotes in the environment, using aprA as a functional marker gene. Applied &
Environmental Microbiology 73: 7664-7679.
Pernthaler A, Amann R (2004). Simultaneous fluorescence in situ hybridization of mRNA
and rRNA in environmental bacteria. Applied & Environmental Microbiology 70: 5426-5433.
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