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

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

‐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

‐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

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

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

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

# 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†