Ecological succession in long-term experimentally evolved ... › 2010 › 02 ›...
Transcript of Ecological succession in long-term experimentally evolved ... › 2010 › 02 ›...
Ecological succession in long-term experimentally evolved biofilms produces
synergistic communities
Steffen Poltak Cooper Laboratory
Biofilms
Doctorspiller.com Meruseonline.com
Biofilm Development
Cystic fibrosis biofilm infections
Burkholderia cenocepacia
Pseudomonas aeruginosa
cdc.org
S. Poltak
S. Poltak
Important questions about chronic infection variants?
• Do variants serve different functions or occupy separate niches within the biofilm?
• Are variants more fit than their ancestor?
• Do variants cooperate or compete?
• How does diversity affect persistence and virulence?
How do we model biofilm population evolution?
Biofilm experimental evolution
6 Populations 6 Populations
Burkholderia cenocepacia HI2424
Environmental isolate and dominant CF epidemic strain (48 U.S. states)
Carbon source: Galactose (most abundant sugar in mucus)
Temperature: 37 C
Conditions: 18 x 150mm test tubes; rollerdrum 50 rpm
Serial transfer: Every 24 hours; ~1500 generations; 9 generations/day
Does selection for biofilm formation produce diverse populations?
Biofilm mutants are diverse and ecologically distinct
Bead competition
All biofilm populations undergo the same pattern of diversification
“Smooth” / “Studded” (S) ~88%
Ruffled (R) ~9%
Wrinkly (W) ~2%
750 generations
What is the cause of this diversity? and why does it persist?
Adaptive Radiation
• The evolution of ecological and phenotypic diversity within a rapidly multiplying lineage.
• Typically follows the colonization of a new environment or the establishment of a “key innovation,” which opens new ecological niches and/or new evolutionary paths
Adaptive radiation is an extension of speciation to larger temporal and spatial scales. • Driven by ecological factors and
subject to certain initial conditions
1. Adaptation to pre-existing niches
2. Adaptation to newly constructed niches (Succession)
Origin of diversity
Maintenance of diversity
1. Facilitation (mutualism?)
2. Tolerance (commensalism?)
Based on Odum 1969 and Connell and Slatyer 1977
What are the benefits of diverse biofilm populations?
Niche construction: Food and Space
• Architectural support
1. Increased surface area 2. Binding strength of cells
• Cross-feeding
1. Secondary metabolites “One man’s trash is another man’s treasure”
Image: P. Stewart, Nature reviews microbiology 2008
Mutants segregate the biofilm structure and increase binding surface area for others
Confocal microscopy of population B1, S=blue, R = green, W = red
How does biodiversity affect productivity?
Tolerance Inhibition
Facilitation
Observed productivity >> expected from sum of parts
Is synergy caused by cross-feeding or spatial partitioning?
Morphotypes construct metabolic niches by conditioning media
Numerical value = Benefit of growing in genotype supernatant
(AUC in Supernatant / AUC in Galactose M9)
We see synergy, so who is facilitating who?
Starting Density of Resident Starting Density of Resident
Ab
so
lute
fitn
ess (
M)
Ab
so
lute
fitn
ess (
M)
COMPETITION FACILITATION
Resident
Invader : Constant starting density Troy Day
3-way density dependence competitions of morphotypes
Is biofilm diversification driven by competition, facilitation, or both…
‐5
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Fitness
Studdedstar9ngfrequency
Sfitness
Rfitness
Wfitness
Log.(Sfitness)
Poly.(Rfitness)
Poly.(Wfitness)
Studded = Resident
Ruffled spreader = Invader
Wrinkly = Invader
S facilitates fitness of R and W in mixed culture
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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Fitness
Ruffledspreaderstar9ngfrequency
Sfitness
Rfitness
Wfitness
Poly.(Sfitness)
Poly.(Rfitness)
Poly.(Wfitness)
Studded = Resident
Ruffled spreader = Invader
Wrinkly = Invader
R facilitates S and W through biotic structure and cross-feeding
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0
5
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0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
Wrinklystar9ngfrequency
Sfitness
Rfitness
Wfitness
Poly.(Sfitness)
Poly.(Rfitness)
Poly.(Wfitness)
Studded = Resident
Ruffled spreader = Invader
Wrinkly = Invader
W facilitates R and S mainly through biotic structure
An ecological model of biofilm diversity and of the evolution of a stable community
Benefit of cross feeding
Req
uire
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t for
spa
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S
R
W
Model of experimental biofilm succession
• At time=0 only ancestor exists • All niches open for colonization • No competition Blue
After 150 generations
S outcompetes ancestor by better use of food and space and is fed by the ancestor
Blue
Yellow
• Studded established in population
Origin: Niche construction?
After 300 generations
W and R invade because they are better competitors for space and better consumers but benefit S by creating a biotic surface for binding.
Blue
Yellow
Green
Red
• Studded thriving • Ancestor going extinct • RS and W established
Origin: Niche construction?
What are the genetic mechanisms underlying biofilm
adaptation?
We resequenced Studded, Ruffled Spreader, and Wrinkly from generation 1500 via Illumina sequencing
# Annotation of mutated gene in B. cenocepacia HI2424
Studded (n=7)
Ruffled (n=9)
Wrinkly (n=5)
Implication P. aeruginosa homolog
m1 Deletion of 95 genes, first is yciR, diguanylate cyclase and phosphodiesterase domains
X X X Alters cyclic-di-GMP concentration and promotes biofilm production
†
m2 Bacterioferritin X X X Iron acquisition †
m3 2-oxoglutarate dehydrogenase E2 subunit
X X X Central metabolism
m4 Premature stop in mannose-1-phosphate guanylyltransferase, manC
X X Polysaccharide/capsule biosynthesis algA†
m5 Phenylacetate-coA oxygenase, paaI X Acetate metabolism; part of ferritin superfamily †
m6 Deletion of 46 genes X Unknown
m7 GC, 90 bp 5' of operon containing putative aminoglycoside phosphotransferase and glycosyltransferase
X Possible altered expression of glycoproteins rhlB, rhamnolipid biosynthesis†
m8 cheW X Scaffold protein in Wsp pathway, affects cyclic-di-GMP concentration
wspD†
m9 Cation/multidrug efflux pump X Multidrug resistance associated with biofilms mexD†
m10 mltA, membrane-bound lytic murein transglycosylase
X Growth/maintenance of peptidoglycan (58)
m11 Receiver domain of methyl-accepting chemotaxis sensory transducer
X Regulatory, wrinkly phenotype, adherence wspA†
Conclusions • Selection for biofilm formation in Burkholderia
results in rapid and successive diversification.
• Diverse biofilms produce synergistic communities.
• Evolved morphotypes exhibit frequency dependence thereby influencing productivity.
• Biofilm evolution model produces mutations that parallel in vivo mutations.
Further work
• Identification of the cross-feeding metabolites
• Assess the stability of the community to disturbance
• Examine the influence of community synergy on antibiotic resistance
Acknowledgments
• Cooper Lab • O’Toole Lab: Dartmouth • Tim Cooper: University of Houston • Mike Travisano: Minnesota • Mark Townley-CSLM • Nancy Cherim-SEM • Grad Students, Friends, Family
• Turner Lab
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WT S450 R450 W450
Yield
Fitness
Bea
d Yi
eld
CFU
/ml A
bsolute fitness (M)
Efficiency of bead colonization
# Annotation of mutated gene in B. cenocepacia HI2424
Studded (n=7)
Ruffled (n=9)
Wrinkly (n=5)
Implication P. aeruginosa homolog
m1 Deletion of 95 genes, first is yciR, diguanylate cyclase and phosphodiesterase domains
X X X Alters cyclic-di-GMP concentration and promotes biofilm production
†
m2 GA, 37bp 5’ of bacterioferritin X X X Iron acquisition †
m3 R304S in 2-oxoglutarate dehydrogenase E2 subunit
X X X Central metabolism
m4 Deletion @ 771A. Premature stop in mannose-1-phosphate guanylyltransferase, manC
X X Polysaccharide/capsule biosynthesis algA†
m5 A31S, phenylacetate-coA oxygenase, paaI
X Metabolism; iron acquisition, part of ferritin superfamily
†
m6 Deletion of 46 genes X Unknown
m7 GC, 90 bp 5' of operon containing putative aminoglycoside phosphotransferase and glycosyltransferase
X Possible altered expression of glycoproteins rhlB, rhamnolipid biosynthesis†
m8 L34P in cheW X Scaffold protein in Wsp pathway, affects cyclic-di-GMP concentration
wspD†
m9 V618A, cation/multidrug efflux pump X Multidrug resistance associated with biofilms mexD†
m10 GA 18 bp 5’ of mltA, membrane-bound lytic murein transglycosylase
X Growth/maintenance of peptidoglycan (58)
m11 A406V in receiver domain of methyl-accepting chemotaxis sensory transducer
X Regulatory, wrinkly phenotype, adherence wspA†