Transcript Catalytic Efficiencies of Fast and Slow Killing

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.