Sulfate reducing prokaryotes in the Eastern Mediterranean A functional genomics approach

1. Sulfate reducing prokaryotes in the Eastern MediterraneanA functional genomics approach

2. Sulfate reduction: • SO42- + 8H+ +8e- S2- + 4H2O • Electron donors • Organic matter (lactate, acetate, ethanol, etc) • H2 • CH4 • Important in anoxic marine ecosystems but occurs in other ecosystems as well.

3. Sulfate reducing prokaryotes

4. Dissimilatory sulfite reductase (DSR) • Enzyme involved in sulfate reduction • Catalysis following reaction: • The gene encoding for enzyme contains conservative and variable sites • Therefore a good gene to study diversity of sulfate reducing prokaryotes in the environment

5. Deep hypersaline brines • Eastern Mediterranean contains hypersaline brines which are located at the deep-sea. • These brines are characterized by high salinity (up to 30% salt), high pressure (up to 350 bar) absence of oxygen and relatively high concentrations of sulfate and sulfide.

6. Deep hypersaline brines • 16S rDNA sequence analysis revealed many sequences related to δ-proteobacteria. • Sulfate reduction rates ranged from 8 to 80 µmol H2S day-1 in the different brines. • Conclusion: • Sulfate reduction occurs as a metabolic process in deep hypersaline brines

7. Objectives • What is the similarity of SRP communities between different sampling sites • Is their similarity between DSR sequence analysis and 16S rDNA sequence analysis. • What is the community structure of sulfate reducing prokaryotes (SRP)?

8. Mat & Meth • Study sites: • L’Atalante brine and interface • Urania brine and interface • Eastern Mediterranean deep-sea sediment three layers • α- and β-subunit of DSR gene amplified • 700 bp of α-subunit were sequenced • Amino acid alignments were created and trees were constructed using these alignments

9. Diversity and Similarity

10. Nearest relatives DSR-protein

11. Phylogenetic tree DSRa-protein

12. Phylogenetic tree δ-16S rDNA

13. δ-Proteobacterial family distribution

14. Origin of sulfate reduction

15. Percentage of clones without insertion

16. Conclusions/Discussion • All sites sampled showed diverse sulfate reducing prokaryotic communities except Urania brine. • The low diversity in Urania brine has been observed with total community structure as well. • Similarity of DSRa sequences between sites is very low thus each site studied had a unique sulfate reducing community. • There are some differences between site similarity of DSRa and δ-16S rDNA. Can be related to OTU cut-off value or that not all DSRa sequences are from δ-proteobacteria

17. Conclusions/Discussion • The obtained DSR-sequences show low similarity with GenBank sequences and represent yet-unknown DSRa genes from sulfate reducing prokaryotes. • The DSRa and 16S rDNA tree topology and family distribution were similar for AI, UB and AB. • This was not true for UI. UI DSRa sequences distantly related to Desulfotomaculum but no 16S rDNA sequences related to that cluster.

18. Conclusions/Discussion • This can be caused by • 1. These DSRa sequences are related to the δ-16S rDNA sequences but this cannot be seen because tree topologies are non congruent • 2. 16S rDNA sequences of UI related to unknown or candidate division clusters, from which metabolic capacities are unknown, are from prokaryotes with sulfate reducing capabilities.

19. Conclusions/Discussion • Allmost all DSRa sequences from deep-sea brines and interfaces contain an insertion in α-subunit. • This might indicate that sequences are from non-thermophilic sulfate reducing prokaryotes • Most DSRa sequences from intermediate layer sediment miss this insertion. • This might indicate that sequences are thermophilic sulfate reducing prokaryotes. This agrees with the thermogenic history. Why these sequences only occur at the intermediate layer is presently unknown.