a tangled bank: laboratory biofilm evolution mimics the ecology of chronic infections
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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. - PowerPoint PPT PresentationTRANSCRIPT
A tangled bank: laboratory biofilm evolution mimics the ecology of chronic
infectionsKey contributorsChuck TraverseSteffen PoltakCrystal EllisKenny FlynnRachel StaplesLeslie Mayo-SmithLaura BentonThomas JohnsonWendy Carlson
http://cooperlab.micropopbio.org University of New Hampshire
Our interests
How do symbionts become mutualists or pathogens? Why are some more prone than others?
How does (bacterial) diversity evolve, persist, and influence community function?
How does replication timing influence evolutionary rates throughout genomes?
?slow
faster
fastest
How do effects of beneficial mutationsdepend upon (and influence) their genetic and ecological context?(distribution of mutational effects)
Population structure of potentially pathogenic Vibrio, and the oyster microbiome, in New Hampshire’s Great Bay Estuary
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.
“small colony variants”
Planktonic growth Sustained biofilm
?Are changes adaptive and heritable?
What mutations?
Are population dynamics exceptional?
How do mutants function?
Persist? Interact? Converge?
A biofilm is a tangled bank
Adaptive radiation or phenotypic plasticity?
It may matter for treatmentAdaptive radiation
• Diversity breeds true• Goal: eliminate the keystone species
in the community
Phenotypic plasticity
• Diversity reverts to WT• Goal: disable the switching
mechanism
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)
(Selection) (Complementarity)
How does biodiversity affect productivity?
Emergent propertiesof diversecommunities
Competitionfor sharedresources
Experimental evolution is a method of simplifying complex processes to study mechanisms of adaptation
Natural biofilm life cycle Model biofilm life cycle
M9 minimal salts +galactose (the primary sugar in mucus)
Selection for reversible stickiness
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
6 Planktonic 6 Biofilm
S. Poltak
lacZ-
lacZ+
Temperature: 37C Conditions: 18x150mm test tubes in a rollerdrum(7mm polystyrene beads)Serial Transfer: every 24 hrs. for 6 months or ~1000 generations*
S. Poltak
Biofilm evolved Planktonic evolved
Fitness: relative colonization efficiency or relative realized growth versus the competitor (ancestor)
All biofilm populations undergo the same pattern of
diversification“Smooth” / “Studded” (S) majority >70%
Ruffled (R) 10-15%
Wrinkly (W)~5%
t = 150 300 450
t = 1000
Morphs inhabit different niches and
exhibit different functions
WT Smooth Rough Wrinkly
Biofilm + ++ +++ ++++
Growth rate
+++ ++ ++ +
Motility +++ + - -Nematode
killing++++ +++ ++ ++
S R W
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?
Biofilm diversity is synergistic
ProductivityBiofilm production
FitnessAntibiotic resistance
Observed productivity >> expected from sum of parts
(Selection) (Complementarity)
How does biodiversity affect productivity?
What mechanisms would explain increased productivity?
Mutualism
Mutants segregate the biofilm structure and increase binding surface area for
others
Confocal microscopy of population B1, S=blue, R = green, W = red
Morphotypes cross-feed one another
SS
RRWW
..and grow optimally when confined to a single bead or slide
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
From evolutionary ecology to medical microbiology
What mutations, what functions,
and what relevance?
How does diversity relate to infections?
Chantratita et al. JOURNAL OF BACTERIOLOGY, Feb. 2007, p. 807–817
B. pseudomallei from human blood and sputum samples.
Morphs vary in lethality in BALB/c mice and A549 human epithelial cells
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)
N actually increases
ab
c d e
f’
g
h
e’f
f’’
a'
a. increased c-di-GMP (yciR SNP) e. increased Fe3+ storage (bfr promoter)
b. central metabolism (2-oxog) f. increased c-di-GMP (wspA or wspE)
c. increased c-di-GMP and altered RNA stability? (yciR + 94 genes)
g. increased c-di-GMP (wspD) + altered signaling
d. increased polysaccharide (manC) h. 45 genes. ??
Frequency of major adaptive mutations in the community
Despite large selective advantage of these mutations, their rise was slowed
by clonal interference
Haplotypes in
Metagenome s in Niches vs
ancestor
M1/M2 0.053 0.066 0.450
+M3 0.009 0.029 0.401
+M4/M5 0.008 0.030 0.512
+M6 0.015 0.035 0.568
+M7 0.014 0.015 0.519
Predicts fixation in ~60 generations
Extent of parallelism among bead-evolved populations?Convergence with chronic
infections?
Adaptation and ecological specificity occur by altered regulation of
cyclic-di-GMP
Thanks to Chris Waters @ MSU
HPLC-MSDifferent alleles, different effects and interactions
Despite convergence in someadaptive mutations:
1.Each community evolved a unique pattern of assembly
2. Each community is synergistic
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.
If experimental evolution of Burkholderia in biofilms favors
mutations found in Pseudomonas from infections…
…what happens to biofilm-evolvedPseudomonas?
• High diversity• Less parallelism• Each biofilm population
becomes a mutator
Pseudomonas community fitness (competitive ability) also requires diversity. No cheaters found.The community is more invasible when certain types are lacking
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
Preliminary experimentation mixing mutants of Pseudomonas and Burkholderiareveals niche complementarity and synergy
Conclusions1. Similar mutations in Burkholderia, a -Proteobacteria, in
vitro and in Pseudomonas, a -Proteobacteria, in vivo suggests that biofilm adaptation may follow a common program in a wide range of organisms and environments.
2. This model enables experiments in vitro that could shed light on chronic biofilm-related infections.
3. Productivity can be enhanced by diversity if colonists construct new, vacant niches:there is strength in numbers in the tangled bank of biofilms.
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
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