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Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus
W P Donovan1, C Dankocsik, M P Gilbert
1Ecogen Inc., Langhorne, Pennsylvania 19047.
Abstract:
A gene encoding a 72,357-dalton (Da) crystal protein of Bacillus thuringiensis var. israelensis was isolated from a native 75-MDa plasmid by the use of a gene-specific oligonucleotide probe. Bacillus megaterium cells harboring the cloned gene (cryD) produced significant amounts of the 72-kDa protein (CryD), and the cells were highly toxic to mosquito larvae. In contrast, cryD-containing Escherichia coli cells did not produce detectable levels of the 72-kDa CryD protein. The sequence of the CryD protein, as deduced from the sequence of the cryD gene, was found to contain regions of homology with two previously described B. thuringiensis crystal proteins: a 73-kDa coleopteran-toxic protein and a 66-kDa lepidopteran- and dipteran-toxic protein of B. thuringiensis subsp. kurstaki. A second gene encoding the B. thuringiensis subsp. israelensis 28-kDa crystal protein was located approximately 1.5 kilobases upstream from and in the opposite orientation to the cryD gene.
Insights
Researchers isolated the cryD gene from Bacillus thuringiensis var. israelensis, producing a toxic 72-kDa crystal protein (CryD) in Bacillus megaterium. This protein showed toxicity to mosquito larvae, aiding in pest control research.
Area of Science:
- Molecular Biology
- Microbiology
- Insect Toxicology
Background:
- Bacillus thuringiensis produces crystal proteins toxic to specific insect orders.
- The genetic basis for the toxicity of Bacillus thuringiensis var. israelensis against mosquito larvae was not fully elucidated.
- Crystal protein genes (cry genes) are often located on plasmids within Bacillus thuringiensis.
Purpose of the Study:
- To isolate and characterize the gene encoding the 72-kDa crystal protein (CryD) from Bacillus thuringiensis var. israelensis.
- To assess the expression and toxicity of the CryD protein in heterologous hosts.
- To investigate the genetic organization of cryD in relation to other crystal protein genes.
Main Methods:
- Gene isolation using a gene-specific oligonucleotide probe.
- Cloning of the cryD gene into an expression vector.
- Heterologous expression of the cryD gene in Bacillus megaterium and Escherichia coli.
- Protein characterization and toxicity assays against mosquito larvae.
- DNA sequencing and sequence homology analysis.
Main Results:
- The cryD gene encoding the 72-kDa CryD protein was successfully isolated from a 75-MDa plasmid.
- Expression of cryD in Bacillus megaterium resulted in significant CryD production and high toxicity to mosquito larvae.
- Escherichia coli failed to produce detectable levels of the CryD protein.
- The CryD protein sequence exhibited homology to other known Bacillus thuringiensis crystal proteins.
- A second gene for a 28-kDa crystal protein was identified upstream of cryD.
Conclusions:
- The isolated cryD gene is responsible for the production of the 72-kDa crystal protein in Bacillus thuringiensis var. israelensis.
- Heterologous expression in Bacillus megaterium confirms the toxicity of the CryD protein to mosquito larvae.
- The CryD protein shares structural similarities with other insecticidal crystal proteins, suggesting a conserved mechanism of action.
- The genetic organization provides insights into the regulation and evolution of crystal protein genes in Bacillus thuringiensis.