Abstract
The genes encoding the d-endotoxins of Bacillus thuringiensis are located on plasmids ranging in size from 45 to 1000 kb. Plasmid size and variety are diagnostic features for characterizing subspecies of this aerobic spore-forming crystalliferous entomopathogen. Two of 25 B. thuringiensis isolates obtained from Middle Tennessee were characterized into subspecies on the basis of size, number, and varieties of plasmids they host using pulse field gel electrophoresis (PFGE). By using specific pulse angle (120°), field strength (5 V/cm), pulse time (26 h), and agarose concentration (1.2%) these DNA molecules were separated from other genomic DNA. The purified DNA product the agarose gel was tested for homology to 49 d-endotoxin gene using PCR.. The PFGE data for 5 of the 25 isolates collected in Tennessee showed distinct banding patterns. Two of the isolates had only 1 band whereas the others had more than 4 ranging from 45 to 1000 kb in size. Isolate 6 and 10 both yielded one 49 kb fragment that contained the cry1Agene. Key words: Bacillus thuringiensis, d-endotoxin, pulsed-field gel electrophoresis, plasmid.
Highlights
Bacillus thuringiensis (Bt) is one of more than 20 species of soil growing Bacilli
The most discussed of the other species are Bacillus subtilis, a source of industrial enzymes, B. thuringiensis, in a lot of cases is confused with Bacillus cereus, and Bacillus anthracis, the causative agent of anthrax
Of the thousands of colonies formed on the sodium acetate plates 25 isolates had the characteristics of B. thuringiensis based on crystal protein production as shown by the crystal stain used in these studies
Summary
Bacillus thuringiensis (Bt) is one of more than 20 species of soil growing Bacilli. This bacterium is a gram positive, aerobic, spore-forming organism. The most discussed of the other species are Bacillus subtilis, a source of industrial enzymes, B. thuringiensis, in a lot of cases is confused with Bacillus cereus, and Bacillus anthracis, the causative agent of anthrax. All of these different Bacilli are mostly found in the soil, whereas B. thuringiensis has been sited as been found in living and dead insects, insect feces, granaries, and on the surface of plants (Ohba and Aizawa, 1978). On January 1999 the International Entomopathogenic Bacillus Centre (IEBC) suggested that there are currently 3,493 B. thuringiensis strains grouped into 69 serotypes based on their flagellar H antigens. More recent studies suggest that these strains are 82 serotypes (Lecadet and Frachon, 1999)
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