What Can Evolution REALLY do? April 26, 2007 UW-Superior

1. What Can Evolution REALLY do?April 26, 2007UW-Superior Part III A sad tale of devolution and wasted potential Today’s talk: • An introduction to experimental evolution • What we thought we discovered • What we’ve learned since then

2. Bottom Line: What can evolution REALLY do?? • Not much: TWO is an evolution stopper • Less than I expected: Even ONE can be a problem, when the cell has seemingly better options. • Once you’re on a “fitness peak”, you may be stuck.

3. A comparison: What evolution has to be able to do- and one of its finest examples.

4. One Problem: How does a function evolve when multiple steps are required for an advantage? • “..A very general problem of molecular adaptive evolution: How is an advantageous phenotype selected when it requires multiple mutations, none of which are advantageous until all are present?... (This presents) a barrier that would appear to be difficult when two independent random mutations are required to improve fitness, and insuperablewhen more than two are required”. (italics added) Barry Hall, Proc. Nat. Acad. Sci. USA 88: 5882-5886, July 1991

5. Proposed Rule for Predicting What Evolution can do: If evolving a new function takes two or more steps, both or all of which must occur to produce the function IT WON’T HAPPEN

6. Testing the Rule: Give evolution a specific molecular task! 1) Find a well-studied gene- we know specific point mutations that inactivate the gene. 2) Introduce a series of point mutations into the gene- 1-4 mutations, each of which inactivates the gene. Let the gene evolve. 3) Prediction: only the mutant with one inactivating mutation will be able to regain its function through evolution.

7. Can we really test this rule? Standard answer: NO- because of ALL THE TIME NEEDED! The solution? MICROBES TO THE RESCUE!!!! WHY MICROBES????

8. ????WHY MICROBES????? • Strictly speaking, evolution doesn’t work by time, but by producing new individuals, which can take time. • It’s easy to get LOTS of individuals- easily produce a trillion overnight, and with patience, thousands of generations • Bacteria are complicated- so they have lots of functions that we can use to test evolution with

9. 60 49 234 175 Source: Hyde, et. al, 1988 The Gene of Choice: trpA of E. coli • Required for tryptophan (trp) synthesis • Mutants (trpA-) grow only when tryptophan provided • In limiting trp, evolution to trpA+ is HIGHLY selective

10. First, some technical details… The Gene is on a plasmid- pWS1 2 mutation version: pRS202-5 1 @ 49: pRS201-2 1@60: pRS213

11. What happens when you try to evolve the one with 2 mutations? • It doesn’t evolve to make tryptophan, not in: • 3600 generations • >2 trillion cells • How do we know this?

12. Testing Large Populations for Evolution- Agar Plates • Spread minimal agar plates with limiting tryptophan with RS202-5. • 2-4 X 1010 cells per plate- 20-40 Billion! • >59 plates tested, 1.1-2.2 X 1012 cells. • RS201-2 readily reverts, routinely producing >100 Trp+ colonies/plate. • No observable evidence of evolution of RS202-5.

13. Testing Multiple Generations for Evolution • If sequential events can produce a Trp+ phenotype, then multiple generations under selective conditions might result in evolution of tryptophan synthesis.

14. Each day= 6.64 generations of evolution!

16. Results of Serial Transfer • One culture lost its trpAB genes within 275 generations! Found its own fitness peak! • Two additional cultures have been tested for >544 transfers, or ~3600 generations. • No Trp+ evolution observed. • HOWEVER….

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

18. But the story gets worse… • August 2006: We make the version with a mutation at position 60 (pRS213) • The mutation DOES NOT COMPLETELY INACTIVATE THE TRP GENE!!!! Cells with this version grow slowly in the absence of tryptophan!!!

19. What this means: • We weren’t really trying to evolve a gene with two inactivating mutations! • We were trying to evolve a gene with one inactivating and one that lowered activity! • This gene should readily evolve- by going from Trp- weak Trp+ fully Trp+ • It doesn’t- not in 3600 generations, not in 60 billion cells tested.

20. But the story gets worse… • After 2,000 generations of evolution, can our evolved plasmid support the position 60 mutation, and still have it be weakly Trp+? NO! • How did we test this?

21. We do “mix the parts” “Large” SalI- HpaI fragment- after 0 and 2,000 generations of evolution. “Small” HpaI-SalI fragment- with our position 60 trpA mutation We can swap pieces, and see what happens!

22. So what happens??

23. So what’s going on???Here’s what we think • The cell is producing a useless protein- the inactive trpA- gene product- up to 15% of total protein is the useless trpA-B+gene product. • It evolves to make less of this product. • BUT- only when you make excess of this useless product, is it possible to evolve to be weakly trp+. At low levels of expression, a trpAD60N mutant would be trp-

24. The Concept of a “Fitness Peak” • Source of picture: Wikipedia

25. Some Applications • Don’t expect much help from evolution in either global warming or exotic species invasions. • Our approach to use of antibiotics has been fundamentally flawed.

26. Conclusions • A requirement for two mutations for evolution to occur remains an evolution-stopper. • Even when a single mutation (in theory) results in a fitness advantage, other mutations may place it on a fitness peak that precludes further evolution. • Evolving bacteria is a LOT of fun!

27. THANK YOU TO: UW-Superior and the Department of Biology and Earth Science for supporting my reassignment Dr. A.C. Matin & Stanford University Merck Foundation Stephanie Ebnet, Jason Uviasah, Benjamin Okemwa and Amos Tarfa