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Supplementary Information
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Table S1: Sampling details. Tube worm specimens were sampled during the two cruises I:
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AT15-28 (“Fix08 I”, December 2007/January 2008) and II: AT15-38 (“Fix08 II”, October
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2008) at different EPR vent sites. AD: Alvin Dive. The in situ chemistry measurements
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(“Chemistry”) were performed during the cruise AT15-28 at 9°50’N, Tica vent.
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Table S2: Single gene homogeneity. Selected genes with relevant metabolic functions and
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the ITS and 16S rRNA sequences were compared a) between all three symbiont metagenomes
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pyrosequenced in this study (R1: Riftia 1 symbiont, R2: Riftia 2 symbiont, T: Tevnia
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symbiont) and b) between R1, R2, T and the Sanger-sequenced metagenome of 1: Cand. E.
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persephone as published by Robidart et al. (2008), using the Geneious ProTM tool.
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Homogeneity is given as percentage of average pairwise identities, i. e. the proportion of
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homologous base pairs at the same column with regard to the total number of pairs in the
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alignment. The homogeneity of all key metabolic gene sequences (excluding 16S rRNA and
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ITS genes) from R1, R2 and T averages 99.9 %, resulting in an average heterogeneity of 0.1
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%. The key gene alignment of R1, R2, T and the previously published Cand. E. persephone
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metagenome resulted in an average of 99.6% homogeneity (0.4% heterogeneity). Five of the
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genes were not identified in the Cand. E. persephone metagenome. For accession numbers see
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Figures S2 and S3.
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Table S3: Genes of particular interest. Selected representative genes encoding relevant
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metabolic key enzymes, proteins involved in oxidative stress response and in cell surface-
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associated processes, and proteins which are putatively related to symbiont-host interactions
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are listed with their respective GenBank accession numbers for the metagenomes of the Riftia
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1 symbiont, the Riftia 2 symbiont and the Tevnia symbiont. Asterisk: Protein is also involved
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in organic carbon metabolism. 1) Two or more partial coding sequences for one gene; 2) two or
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more individual copies in separate locations of the metagenomes. n. a.: “not annotated”,
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nucleotide sequence was manually identified in the metagenome but was not detected during
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the initial automatic annotation. (Note: This list is not exhaustive but presents exemplary
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features of the three metagenomes.)
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Table S4: Comparison of intracellular soluble proteins. Protein names with corresponding
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function, Enzyme Commission (E.C.) number, isoelectric point (pI), molecular weight (MW)
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and GenBank accession number are listed for all identified proteins with at least 1.5-fold
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change of spot volume (ratio) when comparing the Riftia symbiont and the Tevnia symbiont
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protein gels (Figure 2). Spot volume values are expressed in percentages (%Vol) of the total
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proteome on the respective symbiont reference map (pI range 4 – 7). Only proteins exhibiting
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relative volumes (%Vol) of at least 0.1 were included. Negative ratio values (light gray cells)
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correspond to comparatively higher spot volumes on the Tevnia symbiont master gel, whereas
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positive ratios (dark gray cells) indicate larger spot volumes for the Riftia symbiont proteins.
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Proteins were considered as unambiguously identified, if their MS identification was based on
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at least two individual peptides, a minimum score of 75 and a sequence coverage of at least
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30%. Asterisk: Protein is also involved in organic carbon metabolism.
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Figure S1: Tube worm clump at the sampling site. Tevnia jerichonana and Riftia pachyptila
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specimens at Tica vent, 9°50’N. The picture was taken on January 11th 2008 directly before
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collection of the tube worms. The long red plumes of the larger Riftia specimens in the center
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of the clump are more distant from the diffuse flow source at the seafloor than the plumes of
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the smaller Tevnia specimens.
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Figure S2: Single gene comparison. 24 key genes encoding major metabolic enzymes
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involved in sulfide oxidation, carbon fixation, nitrogen metabolism and oxidative stress
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response from the Riftia 1 symbiont, the Riftia 2 symbiont, and the Tevnia symbiont
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metagenome were subjected to a mutual alignment and compared to the respective Riftia
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symbiont sequences published previously (Robidart et al., 2008) using the Geneious ProTM
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tool. The green bar indicates the nucleotide consensus sequence (shown on top), i. e. the
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homogeneity of the four symbiont metagenomes. It is interrupted in differing (non-
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homologous) DNA regions. Protein coding sequences (CDS) of the respective symbionts are
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indicated by a yellow bar and specified by their GenBank accession numbers on the left.
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Translations (above the DNA sequences) indicate whether nucleotide deviations impact the
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amino acid sequences.
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Figure S3: ITS and 16S rRNA alignment. ITS and 16S rRNA sequences of the three
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metagenomes sequenced in this study (Riftia 1 symbiont, Riftia 2 symbiont, Tevnia symbiont)
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were compared to each other and to the sequences previously published by Robidart et al.
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(2008) using Geneious ProTM. Additionally, ITS sequences of two individual Riftia symbiont
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phylotypes described by Harmer et al. (2008) and of seven Riftia and Tevnia symbiont
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phylotypes as published by Di Meo et al. (2000) were included in the alignment. The 16S
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rRNA alignment also includes two 16S rRNA sequences published by Di Meo et al. (2000).
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GenBank accession numbers of publically accessible sequences are indicated on the left. The
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pink bar characterizes tRNA sequences located within the ITS sequence, whereas the rRNA
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sequence is shown as a red bar. The consensus sequence (homogeneity of the compared
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genes) is highlighted in green.
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