experimental evolution ben callahan koshlan meyer-blackwell naama pnina dekel hopkins microbiology...
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Experimental Evolution
Ben CallahanKoshlan Meyer-Blackwell
Naama Pnina Dekel
Hopkins Microbiology Course 2010
The BIG question:• What are the evolutionary consequences of
temporarily subjecting Pseudomonas fluorescens to a challenging chemical environment?
Principles:
• (Chemical) Environment Fitness• Tradeoffs• Microevolutionary processes
The challenge: Nalidixic acidNalidixic acid interferes with the ability of DNA gyrase to “nick”
the DNA. This “nicking” is necessary to relieve super-coiling which prevents DNA replication from proceeding.
Methods
2 days
20/40/60/80 μg/ml nalidixic acid
Pseudomonas fluorescens SBW25
LB
NalR
NalR
NalSNalR 1/1000
18 times ~1800 g
gyrA
-80°C
Evolution Responds
NalS-lacz+ NalR anc
NalS-lacz+ NalR der
NalS-lacz+ NalS anc
NalS-lacz+ NalR anc
Plating after inoculation and on the next day
After 1800 generations of growth in “normal” environment,we evaluate the results:
20 20 20 20 40 40 60 60 60 80 800.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
NalR Mutant NalR Mutant Derived Ancestor Derived
Antibiotic Concentration (mg/L)
Rela
tive
Fit
ness
MicroevolutionHow did selection change the (normal) fitness over the experiment?
Mutation
In E.coli:
Yoshida et al. 1998
But, In Pseudomonas fluorescens this mutation did not occur in any of the resistant strains. In fact, no mutation in gyrA was
found.
What mutational paths are available for Nalidixic resistance?
Nal resistance
Mutation
In E.coli:
Yoshida et al. 1998
But, In Pseudomonas fluorescens this mutation did not occur in any of the resistant strains. In fact, no mutation in gyrA was
found.
What mutational paths are available for Nalidixic resistance?
Nal resistance
There are multiple paths to Nalidixic acid resistance.
0 20 40 60 800
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
R² = 0.06840681715438
Nalidixic Acid Concentration
Initi
al L
oss
in F
Itnes
s
TradeoffsHow did Nal resistance impact “normal” fitness? Did [Nal] matter?
0.5 0.6 0.7 0.8 0.9 1 1.1
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5R² = 0.677470032245411
Initial Fitness
Gai
n in
Fitn
ess
Tempo and ModeInitial distance from fitness optimum affects the evolutionary “rate”?
0.5 0.6 0.7 0.8 0.9 1 1.1
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5R² = 0.677470032245411
Initial Fitness
Gai
n in
Fitn
ess
Tempo and ModeInitial distance from fitness optimum affects the evolutionary “rate”?
Nal resistance never reverted!
Fitness landscape is multidimensional Multiple paths to Nal resistance.Multiple “fixes” for Nal-resistance tradeoffs.
Suggestions• Test lower Nal concentrations– Do tradeoffs change?
• Evolve in a +Nal broth environment– Presence of Nal alters evolutionary trajectory?
Suggestions• Test lower Nal concentrations– Do tradeoffs change?
• Evolve in a +Nal broth environment– Presence of Nal alters evolutionary trajectory?
Thanks!For listening (and transferring… and counting…)