NOVEL CLONING AND TRANSFER SYSTEMS FOR BACILLUS THURINGIENSIS INSECTICIDAL GENES

THE PRINCIPAL OBJECTIVES OF THIS PROPOSAL ARE (1) TO EVALUATE A NOVEL BACILLUS HOST-VECTOR SYSTEM FOR THE CLONINGAND EXPRESSION OF GENES SPECIFYING MICROBIAL INSECTICIDAL ACTIVITIES; AND (2) TO DETERMINE THE FEASIBILITY OF AN INTERGENIC CONJUGAL TRANSFER SYSTEM FOR MOBILIZING RECOMBINANT PLASMIDS BETWEEN A VARIETY OF GRAM-POSITIVE HOSTS. A DELTA-ENDOTOXIN GENE FROM A PROTOTYPE STRAIN OF BACILLUS THURINGEIENSIS VAR. KURSTAKI (HD-73) WILL BE CLONED INTO THEPLASMID VECTOR PBD9, AND TRANSFORMED INTO A PLASMID-FREE STRAIN OF B. MEGATERIUM. RECOMBINANT CLONES CONTAINING THE TOXIN GENE WILL BE IDENTIFIED EITHER BY MICROSCOPIC EXAMINATION OF SPORULATED COLONIES FOR VISIBLE CRYSTALS OR BY COLONY HYBRIDIZATION. THESE RECOMBINANT PLASMIDS WILL BEEVALUATED FOR TRANSFERABILITY BETWEEN VARIOUS STRAINS OF BACILLUS AND STREPTOCOCCUS, USING WELL-CHARACTERIZED TRANSMISSIBLE PLASMIDS AS THE MOBILIZING PLASMID. THESE APPROACHES WILL ULTIMATELY ENABLE THE CONSTRUCTION OF NOVEL COMBINATIONS OF INSECTICIDAL GENES IN HOST BACTERIA THAT AREMAXIMIZED FOR FERMENTATION YIELDS.