Download Biofilms are diverse

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
Our
interests
How do effects of beneficial mutations
How do symbionts become
depend upon (and influence)
their genetic and ecological context?
(distribution of mutational effects)
How does replication timing
influence evolutionary rates
throughout genomes?
mutualists or pathogens? Why
are some more prone than
others?
How does (bacterial) diversity
evolve, persist, and influence
Population structure of potentially pathogenic
Vibrio, andfunction?
the oyster microbiome,
community
in New Hampshire’s Great Bay Estuary
?
slow
faster
fastest
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.
A biofilm is a tangled bank
?
Planktonic growth
Are changes adaptive and heritable?
What mutations?
Are population dynamics exceptional?
How do mutants function?
Persist? Interact? Converge?
Sustained biofilm
“small colony variants”
Adaptive radiation or phenotypic plasticity?
It may matter for treatment
Adaptive radiation
• Diversity breeds true
• Goal: eliminate the
keystone species in the
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)
How does biodiversity affect
productivity?
Competition
for shared
resources
Emergent properties
of diverse
communities
(Selection)
(Complementarity)
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)
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
lacZ-
lacZ+
6 Biofilm
Temperature: 37C
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
S. Poltak
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%
t = 1000
Ruffled (R)
10-15%
Wrinkly (W)
~5%
t = 150
300
450
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
Biofilm diversity is synergistic
Productivity
Biofilm production
Fitness
Antibiotic resistance
Observed productivity >> expected from sum of
parts
How does biodiversity affect
productivity?
8.E+08
7.E+08
Productivity (cfu/ml)
Mutualism
W
S
6.E+08
Sum
5.E+08
4.E+08
3.E+08
2.E+08
1.E+08
(Selection)
(Complementarity)
0.E+00
0
0.5
Frequency of S
What mechanisms would explain increased
productivity?
1
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
200
180
160
Growth (AUC)
140
120
100
S
80
R
60
W
S
40
20
W
0
WT
S
R
W
Supernatant Producer
R
B1
..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?
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. 807–8
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)
Frequency of major adaptive mutations in the community
a'
g
a
b
c
d
e
h
f’’
f
e’
f’
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? (D yciR + 94 genes)
g. increased c-di-GMP (wspD) + altered
signaling
d. increased polysaccharide (manC)
h. D 45 genes. ??
N actually increases
Despite large selective advantage of these
mutations, their rise was slowed by
clonal interference
Haplotype
s in Metagenome
s in Niche
s 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
Different alleles,
different effects and interactions
HPLC-MS
Thanks to Chris Waters @
Despite convergence in some
adaptive 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-evolved
Pseudomonas?
• High diversity
• Less parallelism
• Each biofilm population
becomes a mutator
Pseudomonas community fitness (competitive ability) also req
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 Burkhol
reveals niche complementarity and synergy
Conclusions
1. 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.
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
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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 in
the evolving population B1
metagenome
Allelic diversity persists
throughout and no allele
fixes.
This diversity (clonal
interference) likely fuels
adaptation
Selection favored changes
in genes affecting
 oxidative stress
resistance
 cyclic-di-GMP
 exopolysaccharide
 affinity for long transcripts
by RNAp?
 stability of some mRNAs?
altered central metabolism