An extensive characterization study of different Bacillus thuringiensis strains collected from the Nashville Tennessee area

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 resembled Bacillus 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 be due to the over use of genetically modified Bt corn in the late 1980’s which was subsequently dispersed into the environment.

Keywords: Bacillus thuringiensis, Nashville Tennessee area, bacterium, SDS-PAGE.

African Journal of Biotechnology Vol. 12(30), pp. 4827-4835