Experimental Evolution Ben Callahan Koshlan Meyer-Blackwell Naama Pnina Dekel Hopkins Microbiology Course 2010.

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…)