ecological succession in long-term experimentally evolved ... › 2010 › 02 ›...
TRANSCRIPT
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Ecological succession in long-term experimentally evolved biofilms produces
synergistic communities
Steffen Poltak Cooper Laboratory
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Biofilms
Doctorspiller.com Meruseonline.com
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Biofilm Development
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Cystic fibrosis biofilm infections
Burkholderia cenocepacia
Pseudomonas aeruginosa
cdc.org
S. Poltak
S. Poltak
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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?
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How do we model biofilm population evolution?
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Biofilm experimental evolution
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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
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Does selection for biofilm formation produce diverse populations?
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Biofilm mutants are diverse and ecologically distinct
Bead competition
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All biofilm populations undergo the same pattern of diversification
“Smooth” / “Studded” (S) ~88%
Ruffled (R) ~9%
Wrinkly (W) ~2%
750 generations
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What is the cause of this diversity? and why does it persist?
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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
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Adaptive radiation is an extension of speciation to larger temporal and spatial scales. • Driven by ecological factors and
subject to certain initial conditions
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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
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What are the benefits of diverse biofilm populations?
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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
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Mutants segregate the biofilm structure and increase binding surface area for others
Confocal microscopy of population B1, S=blue, R = green, W = red
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How does biodiversity affect productivity?
Tolerance Inhibition
Facilitation
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Observed productivity >> expected from sum of parts
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Is synergy caused by cross-feeding or spatial partitioning?
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Morphotypes construct metabolic niches by conditioning media
Numerical value = Benefit of growing in genotype supernatant
(AUC in Supernatant / AUC in Galactose M9)
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We see synergy, so who is facilitating who?
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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
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3-way density dependence competitions of morphotypes
Is biofilm diversification driven by competition, facilitation, or both…
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‐5
0
5
10
15
20
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
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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‐5
0
5
10
15
20
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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‐5
0
5
10
15
20
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
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An ecological model of biofilm diversity and of the evolution of a stable community
Benefit of cross feeding
Req
uire
men
t for
spa
ce
S
R
W
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Model of experimental biofilm succession
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• At time=0 only ancestor exists • All niches open for colonization • No competition Blue
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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?
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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?
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What are the genetic mechanisms underlying biofilm
adaptation?
We resequenced Studded, Ruffled Spreader, and Wrinkly from generation 1500 via Illumina sequencing
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# 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†
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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.
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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
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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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0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0.00E+00
1.00E+08
2.00E+08
3.00E+08
4.00E+08
5.00E+08
6.00E+08
WT S450 R450 W450
Yield
Fitness
Bea
d Yi
eld
CFU
/ml A
bsolute fitness (M)
Efficiency of bead colonization
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# 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†