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Catalytic Efficiencies of Enolase from Fast- and SlowKilling Genotypes of Paenibacillus larvae Bryan Lehner and Ross Koning Department of Biology, Eastern Connecticut State University Background 4 1 Paenibacillus larvae causes the honeybee disease American Foulbrood. Honeybee 2 6.9 KB kcat/Km= 670 Larva Infected larva Produce spores Kill larvae Grow from sugar Genotype ERIC I ERIC II, III, IV 1.3 KB 3 Ingested by larvae Summary Germinate Larval-level virulence Kills larvae in 12 days Kills larvae in 6 days kcat/Km= 2300 -1 -1 M s 5 5.6 KB Paenibacillus kills honeybee larvae to reproduce. -1 -1 M s Colony-level virulence More virulent Less virulent ERIC I enolase has a 3.4- fold lower catalytic efficiency than ERIC III due to a single amino acid mutation preceding the enolase active site Experimental 1. Clone and transform ERIC I and ERIC III enolase into E. coli Enolase is a multifunctional protein implicated in 2. Isolate enolase clones Paenibacillus virulence and a central glycolytic enzyme 3. Express and purify ERIC I and ERIC III enolase protein Enolase 2-Phosphoglycerate Phosphoenolpyruvate 4. Compare ERIC I and ERIC III enolase catalytic efficiency with enzyme kinetics 5. Compare DNA and protein sequences ERIC I enolase Gly Ala substitution ERIC I kills larvae slower Lower Catalytic efficiency of ERIC I enolase ERIC I escapes nurse bee removal ERIC I Paenibacillus metabolizes sugar slower ERIC I produces more spores in the hive ERIC I Paenibacillus grows slower ERIC I is more virulent to colony References Genersch, E. (2010) American Foulbrood in honeybees and its causative agent, Paenibacillus larvae. Journal of Invertebrate Pathology 103, S10-S19. Antúnez, K., Anido, M., Arredondo, D., Evans, J., and Zunino, P. (2010) Paenibacillus larvae enolase as a virulence factor in honeybee larvae infection. Veterinary Microbiology 147, 83-89. Johnson, K.A., and Goody, R.S. (2011) The original Michaelis constant: Translation of the 1913 Michaelis-Menten paper (Die kinetik der ivertinwirkung). Biochemistry 4, 8264-8269.
