Fig. S1. Summarized taxonomic composition of three 16S rRNA gene libraries.

Fig. S2. Rarefaction curves of dsrAB, aprA and gammaproteobacterial 16S rRNA gene sequences recovered from Janssand sediment. A coverage of 79% for the 16S rRNA, 78% for dsrAB and 67% for the aprA gene library was calculated according to C = 1 − n/N (Good, 1953) indicating the extent to which the actual species richness is reflected in the libraries.

Fig. S3. Agar enrichment tubes with opposing gradients of oxygen and sulfide. A–C. Tubes inoculated with surface sediment after 2 weeks of incubation in the dark; note the layer of sulfur precipitates few millimetres below the agar surface. D. Control tube, not inoculated.

Fig. S4. Consensus tree based on maximum-likelihood (RAxML) of AprA amino acid sequences recovered from the intertidal sediments of Janssand. Single OTUs are represented by selected clones (‘Wadden Sea sediment clone’), ‘n’ equals numbers of sequences per OTU. Gammaproteobacterial OTUs that affiliated with known sulfur oxidizers are shown in bold. The sequence cluster related to endosymbionts of Oligobrachia spp. is highlighted. Circles indicate lineages with > 70% (closed) and > 50% (open) RAxML bootstrap support. The bar indicates 10% sequence divergence. Unc, uncultured organisms; Sym, symbionts; Tht, Thiotrichaceae; Chr, Chromatiaceae; Ect, Ectothiorhodospiraceae; SAR, SAR11 clade.

Fig. S5. A. Geographic locations where sequences or organisms targeted by probes WS-Gam209 (▲), WS-Gam446 (●) and WS-Gam1030 (■) have been detected in the SILVA SSU Reference database release 100 (green) and by CARD-FISH (red) respectively. In silty intertidal sediments of a second site in the Wadden Sea (Sylt 55°1′31.584″N, 8°25′54.1194″E) single cells were detected by probes WS-Gam1030 and WS-Gam446. Similarly, populations WS-Gam446, WS-Gam1030 and WS-Gam209 were in detected in abundances comparable to those at Janssand site in non-tidal, sandy sediment from the German North Sea (Helgoland Island, 54.182917°N, 7.902617°E). Here, counts averaged 1.2%, 2.3% and 2.4% of all cells respectively. Moreover, the groups were present in sediment from Hydrate Ridge, Cascadia Margin (Knittel et al., 2003). In addition, probe WS-Gam446 hybridized to cells from the Logatchev hydrothermal vent field (site Anyas Garden, 14°45′174″N, 44°58′768″ W) and arctic coastal sediment from Svalbard (Ravenschlag et al., 2001). B. Epifluorescence microscopy images of the widely distributed WS-Gam446 group at site Logatchev Hydrothermal vent field (I), Hydrate Ridge (II) and Svalbard (II). Green: Alexa 488-conferred probe signal; blue: DAPI-stained DNA.

Fig. S6. Epifluorescence microscopy images of 14CO2-incorporating cells revealed by microautoradiography combined with FISH. Green (Alexa 488): fluorescence-conferred signal of probes targeting different gammaproteobacterial populations; red (Alexa 594): Bacteria (probe EUBI-III); blue, DAPI-stained DNA; black, precipitation of silver grains indicating substrate incorporation. Arrows indicate 14CO2-positive/incorporating cells. (A–C) Probe WS-Gam209-targeted cells related to symbionts of Oligobrachia spp.; (D) probe WS-Gam1030-targeted cells related to Thioalkalivibrio thiocyanodenitrificans; (E and F) probe GAM42a-targeted Gammaproteobacteria displaying no significant 14CO2 uptake; (G and H) dead control without silver grain precipitation; (I–M) probes GAM42a (Gammaproteobacteria) and EUBI-III (Bacteria). The scale bars correspond to 5 μm.

Table S1. Taxonomic affiliation of 16S rRNA sequences related to known sulfur-oxidizing bacteria.

Table S2. Primer sets use for PCR amplification of the dsrAB gene.

Table S3. Oligonucleotides and clones applied for probe optimization.

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