1. Catalytic efficiencies of enolase from fast- and slow-killing genotypes of Paenibacillus larvae Bryan Lehner Department of Biochemistry ECSU

2. Paenibacillus larvae causes American Foulbrood Honeybee Healthy Larva Infected with Paenibacillus Brood comb

3. Paenibacillus larvae kills honeybee larvae to reproduce Produce spores Ingested by larvae Germinate Grow from sugar Kill larvae 1 2 3 4 5

4. Virulence varies with genotype Slow-KillingFast-Killing 1st Genotype 2 nd genotype 3 rd genotype 4 th genotype

5. What is the biochemical difference between fast- and slow-killing genotypes? Enolase is expressed in multiple places Enolase toxic to honeybee larvae … but enolase is also an enzyme

6. Yes enolase might aid in tissue degradation, but if enolase is expressed in multiple places, maybe its enzymatic function affects virulence.

7. Enzymes are biological catalysts

8. Fast vs. Slow genotypes Is there is difference in catalytic efficiency of enolase? Is there a difference in amino acid sequence of enolase?

9. Cloned enolase DNA Fast enolase Slow enolase Methods

10. Expressed and purified enolase protein Fast enolase Slow enolase

11. Enolase converts 2-PGA to PEP 2-phosphoglycerate phosphoenolpyruvate

12. k cat / K m = 2300 M -1 s -1 k cat / K m = 670 M -1 s -1 Catalytic Efficiency Slow- Killing Genotype Fast- Killing Genotype

13. …traced to a single amino acid mutation… Fast-Killing genotype Slow-Killing genotype DNA

14. …that precedes the active site! Protein

15. Fast-Killing Genotype = Higher Catalytic Efficiency Slow-Killing Genotype = Lower Catalytic Efficiency Paenibacillus virulence is correlated to the catalytic efficiency of enolase Produce spores Ingested by larvae Germinate Grow from sugar Kill larvae Enolase affects  Results Paenibacillus Life Cycle

16. 6 million 2.5 million United States 1940- 2010 * National Agricultural Statistics Service

17. Amino acid mutation Lower Catalytic efficiency of enolase Metabolize sugar slower Grow Slower Kill larvae slower Escape detected by nurse bees Produce more spores in the hive More virulent to honeybee colonies Conclusion

18. Acknowledgements Honors Program CT Agricultural Experiment Station Biology Department Dr. Ross Koning

19. Honeybee populations have declined since the 1940’s * National Agricultural Statistics Service

20. Enzyme Kinetics

22. Acid–Base Catalysis