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

2. 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

3. The challenge: Nalidixic acid Nalidixic 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.

4. Methods 2 days 20/40/60/80 μg/ml nalidixic acid Pseudomonas fluorescens SBW25 LB NalR NalS NalR 1/1000 18 times ~1800 g gyrA -80°C

5. 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:

6. Microevolution How did selection change the (normal) fitness over the experiment?

7. 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

8. 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. There are multiple paths to Nalidixic acid resistance.

9. Tradeoffs How did Nal resistance impact “normal” fitness? Did [Nal] matter?

10. Tempo and Mode Initial distance from fitness optimum affects the evolutionary “rate”?

11. Tempo and Mode Initial distance from fitness optimum affects the evolutionary “rate”? Nal resistance never reverted!

12. Fitness landscape is multidimensional Multiple paths to Nal resistance. Multiple “fixes” for Nal-resistance tradeoffs.

13. Suggestions Test lower Nal concentrations – Do tradeoffs change? Evolve in a +Nal broth environment – Presence of Nal alters evolutionary trajectory?

14. 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…)