A tangled bank: laboratory biofilm evolution mimics the ecology of chronic infections

1. A tangled bank: laboratory biofilm evolution mimics the ecology of chronic infections Key contributors Chuck Traverse Steffen Poltak Crystal Ellis Kenny Flynn Rachel Staples Leslie Mayo-Smith Laura Benton Thomas Johnson Wendy Carlson http://cooperlab.micropopbio.org University of New Hampshire

2. Our interests How do effects of beneficial mutationsdepend upon (and influence) their genetic and ecological context? (distribution of mutational effects) How do symbionts become mutualists or pathogens? Why are some more prone than others? How does replication timing influence evolutionary rates throughout genomes? How does (bacterial) diversity evolve, persist, and influence community function? Population structure of potentially pathogenic Vibrio, and the oyster microbiome, in New Hampshire’s Great Bay Estuary ? slow faster fastest

3. Darwin’s Tangled Bank "It is interesting to contemplate a tangled bank, clothed with many plants of many kinds, with birds singing on the bushes, with various insects flitting about, and with worms crawling through the damp earth, and to reflect that these elaborately constructed forms, so different from each other, and dependent upon each other in so complex a manner, have all been produced by laws acting around us.

4. A biofilm is a tangled bank ? Are changes adaptive and heritable? What mutations? Are population dynamics exceptional? How do mutants function? Persist? Interact? Converge? Planktonic growth Sustained biofilm “small colony variants”

5. Adaptive radiation or phenotypic plasticity? It may matter for treatment Adaptive radiation Phenotypic plasticity Diversity reverts to WT Goal: disable the switching mechanism • Diversity breeds true • Goal: eliminate the keystone species in the community

6. Why biofilms may become diverse • Environmental structure (space) alone • Allows multiple lineages to persist • Local ecological interactions facilitated by structure • New variants may evolve in response to biotic inputs (niche construction)

7. How does biodiversity affect productivity? Competitionfor shared resources Emergent properties of diverse communities (Selection) (Complementarity)

8. Experimental evolution is a method of simplifying complex processes to study mechanisms of adaptation Natural biofilm life cycle Model biofilm life cycle Selection for reversible stickiness M9 minimal salts +galactose (the primary sugar in mucus)

9. Our model organism: Burkholderia cenocepacia str. HI2424 - soil isolate of the PHDC epidemic strain type - species is the most threatening to persons with cystic fibrosis (CF) - phenotypically plastic biofilm former Temperature: 37C Conditions: 18x150mm test tubes in a rollerdrum(7mm polystyrene beads)Serial Transfer: every 24 hrs. for 6 months or ~1000 generations* 6 Planktonic 6 Biofilm lacZ- lacZ+ S. Poltak

10. Biofilm evolved Planktonic evolved S. Poltak Fitness: relative colonization efficiency or relative realized growth versus the competitor (ancestor)

11. All biofilm populations undergo the same pattern of diversification “Smooth” / “Studded” (S) majority >70% Ruffled (R) 10-15% Wrinkly (W)~5% t = 1000 t = 150 300 450

12. Morphs inhabit different niches and exhibit different functions S R W

13. How is biofilm diversity maintained? 1. Ability to invade when rare • Residents facilitate the growth of invaders when at high density 2. Niche complementarity • Different mutants of the same type are functionally equivalent in mixture …but how does diversity influence community function?

14. Biofilm diversity is synergistic Productivity Biofilm production Fitness Antibiotic resistance

15. Observed productivity >> expected from sum of parts

16. How does biodiversity affect productivity? Mutualism What mechanisms would explain increased productivity? (Selection) (Complementarity)

17. Mutants segregate the biofilm structure and increase binding surface area for others Confocal microscopy of population B1, S=blue, R = green, W = red

18. Morphotypes cross-feed one another S W R ..and grow optimally when confined to a single bead or slide

19. Coevolution in the biofilm: good fences make good neighbors • Early populations benefit less from diversity because of greater competition between morphs • Character displacement minimizes the cost of competition over time, such that all morphs benefit from mixture • The S ecotype experiences competition from biofilm specialists early, but evolves a net benefit from mixture PhD thesis of Crystal Ellis

20. From evolutionary ecology to medical microbiology What mutations, what functions, and what relevance?

21. How does diversity relate to infections? Morphs vary in lethality in BALB/c mice and A549 human epithelial cells B. pseudomallei from human blood and sputum samples. Chantratita et al. JOURNAL OF BACTERIOLOGY, Feb. 2007, p. 807–817

23. What are the genetic mechanisms underlying biofilm adaptation? We sequenced Single clones of S, R, W from generations 300 and 1000 Metagenomes from 300, 500, and 1000 generations (by Illumina) Mutated loci identified from above in 10 random clones of S,R,W from 500 and 1000, to build haplotypes (by conventional methods)

24. Frequency of major adaptive mutations in the community a' g b a c d e h f’’ f’ e’ f N actually increases

25. Despite large selective advantage of these mutations, their rise was slowed by clonal interference Predicts fixation in ~60 generations

27. Extent of parallelism among bead-evolved populations?Convergence with chronic infections?

28. Adaptation and ecological specificity occur by altered regulation of cyclic-di-GMP HPLC-MS Different alleles, different effects and interactions Thanks to Chris Waters @ MSU

29. Despite convergence in someadaptive mutations: • Each community evolved a • unique pattern of assembly • 2. Each community is synergistic

30. Recurrent evolution and a revolution • Ecotypes are genetically distinct and persist by both sequential and recurrent evolution • Suggests strong niche-specific selection and high mutation supply • Biofilm adaptation occurs by : • Altered cyclic-di-GMP regulation, leading to higher concentrations • polysaccharide biosynthesis • Tit-for-tat competition for limiting iron • Affinity for ‘slow-turnover’ transcripts by RNAp ? (after Palsson et al.) • metabolic efficiency, particularly through TCA cycle • In the structured biofilm environment, multiple contending lineages persist for long periods without fixation or loss • Demonstrates role of structure, enhances potential for coevolution • A globally adaptive mutation affecting iron metabolism remodels the community. Biofilm-specific ecotypes re-evolve on this background.

31. If experimental evolution of Burkholderia in biofilms favors mutations found in Pseudomonas from infections……what happens to biofilm-evolvedPseudomonas?

32. High diversity • Less parallelism • Each biofilm populationbecomes a mutator

33. Pseudomonas community fitness (competitive ability) also requires diversity. No cheaters found. The community is more invasible when certain types are lacking

34. Temporal dynamics of PA biofilm assembly reveal competition and facilitation

35. c-di-GMP degradation by a PDE expressed by one mutant (1/7) decreases community fitness

36. Preliminary experimentation mixing mutants of Pseudomonas and Burkholderiareveals niche complementarity and synergy

37. Conclusions • Similar mutations in Burkholderia, a b-Proteobacteria, in vitro and in Pseudomonas, a g-Proteobacteria, in vivo suggests that biofilm adaptation may follow a common program in a wide range of organisms and environments. • This model enables experiments in vitro that could shed light on chronic biofilm-related infections. • Productivity can be enhanced by diversity if colonists construct new, vacant niches:there is strength in numbers in the tangled bank of biofilms.

38. Thanks • My team • Whistler laboratory, UNH • T. Cooper, Houston; W. Sung, H. Zhang, UNH • G. O’Toole group, Dartmouth • Chris Waters, MSU • NIH, NSF • DOE/JGI Community Sequencing Award and analyst W. Schackwitz

39. Mutations identified inthe evolving population B1metagenome • Allelic diversity persiststhroughout and no allelefixes. • This diversity (clonal interference) likely fuels adaptation • Selection favored changesin genes affecting • oxidative stress resistance • cyclic-di-GMP • exopolysaccharide • affinity for long transcripts by RNAp? • stability of some mRNAs? • altered central metabolism