Heterologous expression of green fluorescent protein in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees.

AIMS: We aimed at expressing heterologous proteins in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees, as a prerequisite for future studies on the molecular pathogenesis of P. larvae infections. METHODS AND RESULTS: For this purpose, we established a protocol for the transformation of the plasmid pAD43-25 carrying a functional GFP gene sequence (gfpmut3a) into different P. larvae strains representing the two most relevant P. larvae genotypes ERIC I and ERIC II. We determined the optimal field strength for electroporation and the optimal regeneration time after transformation. Stable GFP expression could be detected in the mutants during their entire life cycles and even after sporulation and re-germination. CONCLUSIONS: This method is suitable not only for the expression of GFP in P. larvae but also for the expression of heterologous proteins or GFP-tagged proteins in P. larvae. Mutants can be used for infection assays because GFP expression remained stable after sporulation and re-germination. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides the first true molecular tool for P. larvae and, therefore, is an immense advancement from what we had previously at our hands for the study of P. larvae pathogenesis.

Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin.

The sesquiterpenoid artemisinin, isolated these from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight.