The amino acid sequences of all Bacillus thuringiensis (B.t.) delta- endotoxins deduced from gene sequences share certain conserved regions which very likely reflect a similar mode of action. Inclusions containing the delta-endotoxins (or protoxins) are solubilized in insect larval guts and converted to toxins which bind to specific cell membrane receptors. The toxins then insert into the membrane to form or alter cation pores. The sequenced toxins are active on larvae from different insect orders including Lepidoptera, Coleoptera and Diptera. Among the target insects are those which damage trees and crops as well as vectors for pathogens. In fact, WHO is employing a B.t. isolate in Africa for vector control. Since there is likely to be a common mode of action, one protoxin gene has been selected for extensive mutagenic analysis in order to assess the contribution of various portions of the protoxin molecule to processing, specificity and toxicity. Both site-directed and mutagenic oligonucleotide techniques will be used to alter specific residues or regions of the gene. The mutated genes will be cloned back into B.t. so that pure inclusions may be isolated for bioassays and for in vitro vesicle binding and transport experiments. An understanding of the contribution of various conserved regions to these processes should be helpful in the design of new toxins, for elucidating the mode of action and for understanding the basis for resistance. Most B.t. isolates contain multiple protoxin genes which are expressed to different extents. In general, protoxin genes are on large plasmids so differential expression could be influenced by gene copy number, plasmid stability and the availability of transcription factors. These particular parameters will be examined in at least two isolates employing gene-specific probes to measure protoxin gene content., steady state mRNA levels and the presence or absence of a particular protoxin-encoding plasmid. Understanding the regulation of protoxin synthesis would be important for the evaluation of the efficacy of new isolates, for constructing new strains by mating and/or transformation and for determining the stability of certain genes in a particular isolate.
Showing the most recent 10 out of 12 publications
