1. Siobhán O’Brien, Adela Luján & Angus Buckling College of Life and Environmental Sciences, University of Exeter Cornwall Campus, UK Email: s.obrien@exeter.ac.uk Cooperative heavy-metal detoxification by P. aeruginosa

2. S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

3. Braud et al. 2010. Environ Microbiol. 2: 419 – 425 West SA & Buckling A. 2003 Proc.R.Soc.Lond.B 270:37 – 44. Griffin AS et al. Nature. 430 :1024 – 1027 West SA et al 2007 Annu. Rev. Ecol. Evol.Syst.38:53 – 77 Fe2+ Siderophore production: A model system for studying cooperation S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

4. Our Questions Can siderophore production in response to copper toxicity be a cooperative trait? Can toxic copper environments select for rapid evolution of cheats? How is cheat fitness affected by the soil microbial community? Part 1Part 3Part 2 S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

5. There is a benefit to the recipient Do cooperators outcompete cheats when grown alone? The behaviour itself is costly Do cheats outcompete cooperators in competition? How do we determine if a behaviour is altruistic? WT Ch WT Ch S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

6. ChWt1:1 Copper Sulfate Cooperator Cheat (X12) 6mM CuSO 4 1.Is heavy metal detoxification by siderophores a cooperative trait? Strains: Wt: PAO1 Ch:PAO1 ΔPvdΔPch S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

7. 1.Is heavy metal detoxification by siderophores a cooperative trait? Cheat relative fitness ~ metal treatment: conditions: F 1,89 =207.89, p < 0.001) 1.Do cooperators do better than cheats when grown alone in toxic environments? ✔ 1.Do cheats outcompete cooperators in competition in toxic environments? ✔ O’Brien, Hodgson & Buckling. Social Evolution of toxic Metal Bioremediation in P. aeruginosa. 2014.Proc.R.Soc.Lond.B 281(1787) S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

8. Experimental evolution (approx. 105 generations) WT Copper Iron X12 Frequency of cheats Total siderophore (pyoverdine) production 1.Is heavy metal detoxification by siderophores a cooperative trait? S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

9. *** 1.Is heavy metal detoxification by siderophores a cooperative trait? Conclusion: Copper detoxification is a social trait that actively selects for the evolution of social “cheats”. Conclusion: Copper detoxification is a social trait that actively selects for the evolution of social “cheats”. O’Brien, Hodgson & Buckling. Social Evolution of toxic Metal Bioremediation in P. aeruginosa. 2014.Proc.R.Soc.Lond.B 281(1787) S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

10. Our Questions Can siderophore production in response to copper toxicity be a cooperative trait? Can toxic copper environments select for rapid evolution of cheats? How is cheat fitness affected by the soil microbial community? Part 1Part 3Part 2 S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

11. S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014 How is cheat fitness affected by the presence of the soil microbial community? Cheat (species 2) Cooperator (species 1)

12. How is cheat fitness affected by the presence of the soil microbial community? S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

13. (cheat fitness~ copper*community, p=0.03). Error bars represent +/-SE (cheat fitness~ copper, p=0.01). Error bars represent +/- SE Monoculture Co-culture Cheat relative fitness (w) How is cheat fitness affected by the presence of the soil microbial community? S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014 -Comm. + Comm. - Comm. + Comm. +Cu

14. Summary Heavy metal detoxification by bacterial siderophores is a cooperative behaviour, that selects for rapid evolution of social ‘cheats’ In toxic copper soil, cheat fitness is increased in the presence of the community, but in monoculture conditions only. Cheats cannot ‘invade’ populations of cooperators in soil as they can in synthetic media. S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

15. Thanks for listening! Angus Buckling Dave Hodgson Adela Luján Florian Bayer Elze Hesse Eleanor Van Veen S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

18. Cu 2+ Cheat Cheat (species 2) Cooperator How is cheat fitness affected by the presence of the soil microbial community? S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

19. (cheat fitness~ copper*community, p=0.03). Error bars represent +/-SE (cheat fitness~ copper, p=0.01). Error bars represent +/- SE Monoculture Co-culture Iron-limitedToxic copper - community<1*** + community=1 Iron-limitedToxic copper - community<1*=1 + community=1 Cheat relative fitness (w) How is cheat fitness affected by the presence of the soil microbial community?

20. Mineral/protein Fe 3 +   transportation Fe 2 +  Siderophore production: A model system for studying cooperation Fe 3 + siderophore producer Fe 2+ West SA, Buckling A. 2003 Cooperation, virulence and siderophore production in bacterial parasites. Proc.R.Soc.Lond.B 270:37 – 44. Griffin AS, West SA, Buckling A. 2004 Cooperation and competition in pathogenic bacteria. Nature. 430 :1024 – 1027 West SA, Diggle SP, Buckling A, Gardner A, Griffin AS. 2007 The social lives of microbes. Annu. Rev. Ecol.Evol.Syst.38:53 – 77. S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014

21. Cu 2+  ✗ Siderophores bind other heavy metals too… …but don’t take them into the cell siderophore producer Braud A, Geoffroy V, Hoegy F, Mislin GLA, Schalk IJ. 2010 Presence of the siderophores pyoverdine and pyochelin in the extracellular medium reduces toxic metal accumulation in Pseudomonas aeruginosa and increases bacterial metal tolerance. Environ Microbiol. 2: 419 – 425 S.OBrien@exeter.ac.uk Microbial communities- Cambridge 28 October 2014