1. Advancing Science with DNA Sequence Ecology of activated sludge Victor Kunin

2. Advancing Science with DNA Sequence Phil Hugen- holtz Victor Kunin Falk Warnecke Suzan Yilmaz Hector Garcia Martin Natalia Ivanova Trina McMahon (UW) Linda Blackall (UQ) Project powered by DOE

3. Advancing Science with DNA Sequence Metagenomes in pipeline Wastewater sludge (EBPR) Termite gut Guerrero Negro Hypersaline mat Antarctic subglacial lake Vostok

4. Advancing Science with DNA Sequence Ecology? population structure predator-pray interactions biogeography

5. Advancing Science with DNA Sequence Microbial ecology Local population structure Global metapopulation structure –Biogeography –Dispersal mechanisms –Survival strategies of the species Phage-host interactions –Can we link an unculturable bacterium to its phage by (meta)genomic sequence? Ecosystem resilience

6. Advancing Science with DNA Sequence Excessive P affects water quality and ecosystem balance through eutrophication. Enhanced Biological Phosphorus Removal (EBPR)

7. Advancing Science with DNA Sequence P is the key pollutant. P limitations in released water very effective Enhanced Biological Phosphorus Removal (EBPR) N,CN,C N,C,P

8. Advancing Science with DNA Sequence Brisbane Madison US and Australian (OZ) samples 8 L 2 L * *

9. Advancing Science with DNA Sequence Community composition Rhodothermus Cytophaga Chryseobacterium Flavobacteriales Clostridium Prosthecobacter Chlorobium Rhodospirillum Caulobacter Rhizobiales Thiothrix Xanthomonadales Acidovorax Dechloromonas Rhodocyclus 0.10 Bacteroidetes Firmicutes Verruco- microbia Chlorobi Proteobacteria Candidatus Accumulibacter phosphatis    US Oz 16S rDNA What organisms live there?

10. Advancing Science with DNA Sequence Bacterial biogeography Everything is everywhere and the environment selects Microbial species are ubiquitous Beijerinck, 1913 Microbial populations have astronomical sizes Microbes travel well 10 18 microbes are estimated to cross continents per year by airlift Identical 16S rRNA molecules are found as far apart as polar oceans Undetected biogeography - poor methods Travel takes time, methods are insufficient to detect divergence Geographical isolation exists in hot spring bacteria Whitaker et al, 2003, Papke et al, 2003 What is the biogeography of Accumulibacter?

11. Advancing Science with DNA Sequence Any geographic isolation of strains? Geographic isolation Oz US Yes No US Oz US OZ

12. Advancing Science with DNA Sequence How do they move? CAP is globally dispersed Never observed outside of sludge How do they move?

13. Advancing Science with DNA Sequence Metabolic reconstruction of CAP ->Looks like it can grow in C,N,P-limited habitats ->oligotrophic ancestry or current lifestyle? N2 fixing genes CO2 fixing genes High affinity P transporters Flagellar genes

14. Advancing Science with DNA Sequence Looking for environmental reservoirs Contra Costa wastewater treatment plant * * * * * * PCR detection using: 16S rRNA gene Polyphosphokinase gene

15. Advancing Science with DNA Sequence Previous map CAP environmental reservoirs * * Aquatic samples: mostly yes Terrestrial samples: mostly no Metapopulation

16. Advancing Science with DNA Sequence In-strain variation dominant strain minor Dominant strain is near clonal

17. Advancing Science with DNA Sequence Monoculture Population structure –One clonal strain dominates –No homologous recombination The bulk of cells in the system are identical Vulnerable ecosystem! Explains the crashes of the system –Waste water engineers: bad influent –No bad influent in bioreactors, still crashes Kill the winner

18. Advancing Science with DNA Sequence Genomic imprints of virus-host interactions >95% nt identity across most of the genome Major differences include: * EPS gene cassettes defence against predation important for settling in EBPR * CRISPR elements

19. Advancing Science with DNA Sequence CRISPR elements 1 GTTTGCCGCCGTGATGGCGGCTTAGAAATCGATGATCATCGACAGGAGCAGATCGCCCAC 61 GTTTGCCGCCGTGATGGCGGCTTAGAAACAGTGTCGCTCAGTCCGCCGACCAGATTCTTC 121 GTTTGCCGCCGTGATGGCGGCTTAGAAATCAACCGGAATCGCGTCTGCTTGTTCGAGGTC 181 GTTTGCCGCCGTGATGGCGGCTTAGAAATCGACGTGCCGAGCGACGACAGTTGCGATGCG 241 GTTTGCCGCCGTGATGGCGGCTTAGAAAACATCGTGGCGCGCCTTGATGAGCGCCTGCTC 301 GTTTGCCGCCGTGATGGCGGCTTAGAAATGCGCAGGCACCGCAGCGCCCAGGCCACCGAC 361 GTTTGCCGCCGTGATGGCGGCTTAGAAAGTGCAGGGCGAGGCGGCACGTGAATATCCCGA 421 GTTTGCCGCCGTGATGGCGGCTTAGAAAACGAATCTGGTCTGGCCCAGGCTGCAAGTCCT 481 GTTTGCCGCCGTGATGGCGGCTTAGAAATATCATGACCACCAATCGGTATACATGATCCT CRISPR - a mechanism to keep the record of and destroy invasive elements EPS - mechanical defence against direct contact with phages Viral influence: Genomic - CRISPR/EPS Population structure - non-recombining, clonal dominant strains

20. Advancing Science with DNA Sequence Phage metagenome Virion phages metagenome was sequenced –Loads of phages –Some phages have deep coverage - high abundance –CRISPR spacers hit phages 8 in US sludge 2 in OZ sludge –Some phage contigs are hit by multiple bacterial CRISPR spacers –Linking unculturable host and its phage While dispersal of the host is global, adaptation to phages is local

21. Advancing Science with DNA Sequence Expression studies An array with viral and microbial genes was prepared The expression was monitored for ~3 months Any active viruses? Phages apply a constant pressure on the bacterial community

22. Advancing Science with DNA Sequence Low complexity engineered systems Most cells in ecosystem are virtually clonal, non- recombining cells Yogurt production EPS cassettes & CRISPR are exchanged in yogurt-producing strains of Streptococcus thermophilus Internal instability due to phages - common to all low- complexity engineered systems?

23. Advancing Science with DNA Sequence Conclusions Ecogenomics Community & population structure –Domination of a clonal asexual strain Global population structure –Global dispersal –Dispersal strategies & lifestyle Phage pressure –Linking a unculturable host to its phage –Constant phage expression Ecosystem vulnerability –Skewed strain abundance –Potential stabilization / diagnostics / cure of failing plants –May be common to many low-complexity engineered systems

24. Advancing Science with DNA Sequence Acknowledgments Falk Warnecke Hector Garcia Martin Philip Hugenholtz Linda Blackall Shaomei He S. Brook Peterson Katherine D. McMahon Matthew Haynes Forest Rohwer