1. Sequencing the World of Possibilities for Energy & Environment

2. Metagenomics with spatial resolution When doing metagenomics, we take a sample from a community and homogenise The structure and complexity of a community are ignored What about layers in microbial communities?

3. Sequencing the World of Possibilities for Energy & Environment Microelectrode measurements Depth mm O2H2SO2H2S µM Introduction Hypersaline mat, Guerrero Negro, Baja California –~6cm thick The most diverse community on Earth –Probably :) –Tens of thousands of microbial species –Dozens of novel phyla Steep physico/chemical gradients with depth –O 2, H 2 S, light Highly stratified (layered) community –Cyanobacteria on top –Cloroflexi, proteobacteria, bacteroides below

4. Sequencing the World of Possibilities for Energy & Environment

5. Sequenced 10 layers of the mat Samples are taken: 1mm top layers, 3mm lower layers A parallel project is aimed to identify the diversity, deep sequencing of 16S is under way in the Norm Pace’ lab. Sequencing, from each layer: –~13,000 reads (between 11,574 and 15,663) –Assembly gives contigs less than 10kb, all analysis done with reads –~13,600 proteins (between 12,012 and 15,984) –Loaded into IMG/M` Sequencing strategy

6. Sequencing the World of Possibilities for Energy & Environment The questions How does the mat live? –Metabolism, interactions, chemotaxis Are there gradients observable from the data? –Species –Protein families –Functional families –Other characteristics? What is special about the mat as compared to other environments? –Adaptations similar across many layers? –Adaptations that cross phylogenetic boundaries? Stress management –Salinity –Heat

7. Sequencing the World of Possibilities for Energy & Environment Blast

8. Sequencing the World of Possibilities for Energy & Environment Marker genes

9. Sequencing the World of Possibilities for Energy & Environment 16S clone library

10. Sequencing the World of Possibilities for Energy & Environment Species content - the discrepancies 16S clone library Blast Marker genes

11. Sequencing the World of Possibilities for Energy & Environment Functional content

12. Sequencing the World of Possibilities for Energy & Environment

13. Depth mm O2H2SO2H2S µM

14. Sequencing the World of Possibilities for Energy & Environment

18. Unifying features? Layers are different –Phylogenetically –Functionally Anything common to all layers –Hypersalinity is common –Adaptations to hypersalinity? –K/Na exchange, increasing intracellular K In Archaea Salinibacter Ruber In the mat?

19. Sequencing the World of Possibilities for Energy & Environment Isoelectric profiles

20. Sequencing the World of Possibilities for Energy & Environment Amino acid usage

21. Sequencing the World of Possibilities for Energy & Environment

22. Conclusions Gradients found of –Phylogenetic groups –Protein families –Functional families Adaptations to salinity –Isoelectric point –Amino acid usage

23. Sequencing the World of Possibilities for Energy & Environment Acknowledgments Jeroen Raes Kirk Harris Natalia Ivanova John Spear Christian Von Mering Brad Bebout Peer Bork Norman Pace Philip Hugenholtz