Abstract
Bacillus thuringiensis (Bt) is a delta-endotoxins producing bacterium that inhibits the digestive process of many insect orders such as; lepidopteran, dipteran and coleopteran. A comparative study between sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and DNA hybridization was performed on 25 chosen different strains of B. thuringiensis. Based on patterns from these molecular tools, the 25 isolates where grouped based on insect killing capability. Soil samples from Tennessee were collected and analyzed for the presence of B. thuringiensis.Fifty six (56) of more than one hundred isolates collected were initially processed using the sodium acetate and a crystal protein staining procedure. Proteinaceous and genomic profiles were gathered for 25 isolates which were named Bt1 thru Bt25. DNA was extracted, quantified, amplified, cloned and sequenced. Alignment studies were performed on the sequenced products. This sequencing data helped decipher which of the Bt isolates belonged to the resembling parent category. The spore/crystal mixtures produced during sporulation were harvested by centrifugation at 10,000 g at 4°C. The spores and crystals were then separated using a discontinuous sucrose gradient with ultracentrifugation. The separation was confirmed by polarized microscopy. The crystal proteins were quantified and then separated using sodium dodecyl sulphate-polyacrylamide gel electrophresis (SDS-PAGE). A correlation was achieved between genomic and proteomic profiles which directly helped in grouping the 25 isolates. The compilation of data suggest that 14 of the 25 isolates resembledBacillus thuringiensis subspecies kurstaki, four of the 25 isolates resembled B. thuringiensis subspecies israelensis while just one of the 25 isolates resembled B. thuringiensis subspecies tenebrionis. The overwhelming majority of the Bt isolates collected in our study resembled B. thuringiensis subspecies kurstaki. This can bedue to the over use of genetically modified Bt corn in the late 1980’s which was subsequently dispersed into the environment. Key words: Bacillus thuringiensis, Nashville Tennessee area, bacterium, SDS-PAGE
Highlights
Bacillus thuringiensis (Bt) is one of over 20 species of soil growing, spore-forming, Gram positive, aerobic Bacilli that exist
Bacillus thuringiensis (Bt) is a delta-endotoxins producing bacterium that inhibits the digestive process of many insect orders such as; lepidopteran, dipteran and coleopteran
A comparative study between sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and DNA hybridization was performed on 25 chosen different strains of B. thuringiensis
Summary
Bacillus thuringiensis (Bt) is one of over 20 species of soil growing, spore-forming, Gram positive, aerobic Bacilli that exist. A few important species of the genera Bacilli are; Bacillus subtilis, a source of industrial enzymes; B. thuringiensis,a known insecticidal bacterium; Bacillus cereus, the cause of gastrointestinal discomfort and Abbreviations: IEBC, International Entomopathogenic Bacillus Centre; Btk, B. thuringiensis subspecies kurstaki; Bti, B. thuringiensis subspecies israelensis; Cry, crystal protein; Cyt, cytolytic proteins; VIP, vegetative insecticidal proteins; PCR, polymerase chain reaction; NCBI, National Center of Biotechnological Investigation; GM, genetically modified. The International Entomopathogenic Bacillus Centre (IEBC) in the early 1990’s reported that there were 3,493 B. thuringiensis strains grouped into 69 serotypes based on their flagellar H antigens. The two most discussed of these serotypes are B. thuringiensis subspecies kurstaki (Btk) and B. thuringiensis subspecies israelensis (Bti). These serotypes are important because they produce -endotoxins that have insecticidal capabilities. Btk is used for the control of lepidopteran larvae while Bti is used to control dipteran larvae
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