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