What Can Evolution REALLY Do? How Microbes Can Help Us Find the Answer May 10, 2008

1. What Can Evolution REALLY Do?How Microbes Can Help Us Find the AnswerMay 10, 2008 Ralph Seelke, U. Wisconsin-Superior

2. 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

3. Two Warnings 1) Do not expect to be overwhelmed. 2) Some of this will get complicated!

4. Four Take-Homes Lessons • We really CAN test evolution • Two is an evolution stopper • Even doing one thing at a time can be a problem for evolution- a gene can “evolve to become unevolveable” • God can even use dull, plodding people for his glory

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

6. 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!!!!

7. 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!

8. Transfer 0.1 ml to new flask Transfer 0.1 ml to new flask Inoculate flask Grow overnight-> Grow overnight-> 10 ml broth You have just produced 6.6 generations of Evolution! We can produce thousands of generations in a year! Each Transfer Produces 6.6 generations of evolution! 46 generations per week Almost 400 per month Over 2,400 in a year 24,000 in ten years!

9. We can FIND evolution BecauseWhen the microbe EVOLVESitGROWSor GROWS BETTER!!!!

10. Evolution Before your very eyes!

11. About 3 mm MORE Evolution Before your very eyes!

12. 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.

13. 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!

14. Is the Need for Two Independent Mutations REALLY an Evolution-Stopper?Studies with the trpA Gene of Escherichia coli

15. 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

16. 60 49 234 175 Source: Hyde, et. al, 1988 The Gene of Choice: trpA (tryptophan synthase A) Mutation 2 Mutation 1

17. Results So Far:If Evolution Requires Two or More Independent MutationsNOTHING HAPPENS

18. 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

19. 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….

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

21. CONCLUSION:TWO is an evolution stopperImportant, but somewhat boring

22. Warning: This Next Part May be Hard!

23. Then the story got more interesting- and more complicated DeadTrpA

24. 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

25. Why was this a big deal? It meant that our gene with two mutations should have evolved! Selection because of fitness advantage Mutation M1, M2 Dead TrpA M1, M2 Weak TrpA 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! M1, M2 Strong TrpA Population of fully Trp+ cells

26. 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

27. 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! Evolved piece- trpA-M2 gene DEAD Original piece- trpA-M2 gene WEAK

28. 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

29. A case of evolution preventing evolution This path NEVER happens Selection because of fitness advantage Mutation M1, M2 DeadTrpA M1, M2 Weak TrpA Change at 1584 ALWAYS happens M1, M2 Dead TrpA M1, M2 Strong TrpA M1, M2 STILL Dead TrpA Mutation lowers trpA expression, increases fitness Population of fully Trp+ cells Reversion of M1 no longer selective; both must now revert for a Trp+ cell

30. The Concept of a “Fitness Peak”

31. 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

32. 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