What Can Evolution REALLY Do?

How Microbes Can Help Us Find the Answer

May 10, 2008

Ralph Seelke, U. Wisconsin-Superior

Where We’re Going

• Confessions of an experimentalist who loves making (bacterial) mutants

• What evolution has been able to accomplish

• What it has NOT been able to accomplish

• Some conclusions

Two Warnings

1) Do not expect to be overwhelmed.

2) Some of this will get complicated!

Four Take-Homes Lessons

1) We really CAN test evolution2) Two is an evolution stopper3) Even doing one thing at a time can be a

problem for evolution- a gene can “evolve to become unevolveable”

4) God can even use dull, plodding people for his glory

Experimental Evolution?

???

Source: Ronald Pine, http://home.honolulu.hawaii.edu/~pine/book1-2.html

We Can Do This “Absurd” Experiment With Microbes!For Evolution to Occur You Need

A LARGE Population and/or

MANY Generations

A Trait That Can Evolve

!!!!BACTERIA!!!!

• Up to 4 TRILLION in a 1 Gal Milk Jug!

• Thousands of Generations in a Year!

• COMPLEX Traits!

• When they evolve, we can FIND THEM!

Each Transfer Produces 6.6 generations of evolution!46 generations per week

Almost 400 per monthOver 2,400 in a year24,000 in ten years!

Inoculate flask

10 ml broth

Grow overnight->Grow overnight->

Transfer 0.1 ml to new flask Transfer 0.1 ml to new flask

You have just produced 6.6 generations of Evolution!

We can produce thousands of generations in a year!

We can FIND evolution Because

When the microbe EVOLVESit

GROWSor

GROWS BETTER!!!!

Evolution Before your very eyes!

MORE Evolution Before your very eyes!

About 3 mm

My Question:

Can evolution do two things at once?• Can a microbe evolve when two mutational

events BOTH have to happen for evolution to occur?

• Not just my pet question! • Acknowledged by evolutionists • The fundamental problem of irreducible

complexity- 2 or more components, all required for a function, and all required for any function.

Why Should Requiring Two Changes Make Evolution Difficult?

• Mutation rates are typically one in 100 million; a teaspoonful of bacteria would have over about 50 mutants!

• If you need TWO mutations, then the chances of BOTH mutations occurring is 1 in 10,000 trillion!

• Now you would need a population of 10,000 liters to produce the mutant!

Is the Need for Two Independent Mutations REALLY an Evolution-

Stopper?

Studies with the trpA Gene of Escherichia coli

Testing the Two Mutation Rule

• Find a well-studied gene, with known mutations that inactivate it.

• Introduce 1,2,3, or four inactivating mutations

• Let the gene evolve under highly selective conditions

49

175

234

Source: Hyde, et. al, 1988

60

The Gene of Choice: trpA (tryptophan synthase A)

Mutation 1

Mutation 2

Results So Far:If Evolution Requires Two or More Independent Mutations

NOTHING HAPPENS

Testing Large Populations of RS202-5 (two

mutations) for Evolution:

• Test in liquid culture: about 1 trillion cells tested without evidence of evolution.

• Test on agar plates: ~1.2-2.4 trillion cells tested without evidence of evolution.

• RS201-2 (mutation one) routinely produced 10-20 Trp+ colonies/plate, 104/ml Trp+ cells/ml in liquid culture.

• No evidence of evolution of RS202-5

Results of Serial Transfer

• One culture lost its trpAB genes within 275 generations.

• Two additional cultures have been tested for ~870 transfers, or about 5,700 generations

• No Trp+ evolution observed. The dead trpA gene after 2,000 generations is identical to the original, dead gene.

• HOWEVER….

The cultures have evolved to be able to grow better in the tryptophan-limited

medium

Increased Fitness of RS202-5 in Low-Trp Medium

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 500 1000 1500 2000

Generations of Evolution

Gro

wth

Rat

e,

Gen

erat

ion

s/h

r

CONCLUSION:TWO is an evolution stopper

Important, but somewhat boring

Warning: This Next Part May be Hard!

Then the story got more interesting- and more complicated

DeadTrpA

We thought that BOTH mutations inactivated trpA,

but only Mutation 1 did; Mutation 2 only weakened

the gene

When we made a version with just Mutation two- the gene was not

completely dead!

weakTrpA

Why was this a big deal? It meant that our gene with two mutations should have evolved!

M1, M2Dead TrpA M1, M2

Weak TrpA

Mutation

Selection because of fitness advantage

M1, M2Strong TrpA

Population of fully Trp+ cells

THIS DOES NOT HAPPEN!!

Evolution not only can’t do two things at a time, in this case it can’t do one thing at a time!

So why didn’t our gene evolve??

Maybe, while it was evolving, the expression of the weak trpA gene was lowered, so that now the trpA gene with just M2 was now DEAD. This would mean that EVOLUTION MADE IT UNEVOLVEABLE

How can we find out if it “evolved to become unevolveable”?

We did “part swapping” experiments! We took the large part (red) from our original plasmid, combined it with trpA-M2; the gene was weakly trpA. When we took the same piece was from a plasmid that had evolved, the trpA-M2 gene was DEAD!

Original piece- trpA-M2 gene WEAK

Evolved piece- trpA-M2 gene DEAD

What did this mean? Most likely, a mutation had occurred, outside the trpA-M2 gene, that had turned the weakly trpA gene OFF.

Mutation somewhere in red, not in the trpA gene! Evolution in red switched off the trpA gene in blue. That somewhere was a single base change at 1584.

1584

A case of evolution preventing evolution

M1, M2DeadTrpA M1, M2

Weak TrpA

Mutation

Selection because of fitness advantage

M1, M2Strong TrpA

Population of fully Trp+ cells

M1, M2Dead TrpA

Mutation lowers trpA expression, increases fitness

M1, M2STILL Dead TrpA

Reversion of M1 no longer selective; both must now revert for a Trp+ cell

Change at 1584 ALWAYS happens

This path NEVER happens

The Concept of a “Fitness Peak”

How is evolution like a guy?

1) It has trouble doing two things at once

2) Even when it only has one thing to do, it sometimes gets sidetracked

Acknowledgements

• Merck Foundation

• Biologic Institute

• UW-Superior

• A.C. Matin Lab and Stanford University

• NUMEROUS undergraduate students!– Pravien Abeywickrema,Kayo Sakaguchi

– Robert Jennings, Ranjuna Weerasekera

– Lynn Meyers, Sarah Rahn, Stephanie Ebnet, Benjamin Okemwa