Isolation and Molecular Characterization of Bacillus thuringiensis Harboring Putative ps Genes from Bangladesh

Abstract

Parasporin, another type of δ- endotoxin from Bacillus thuringiensis (Bt), renowned for its highly specific and preferential toxicity against different cancer cells, could be a potentially safe anticancer therapeutic. Considering the current cancer situation, isolation and molecular characterization of parasporin-producing Bt isolates were aimed. Hence, Bt isolates were obtained through a series of tests viz. heat treatment, acetate selection, starch hydrolysis, lecithinase activity, and microscopy for parasporal proteins. Then the biochemical properties, plasmid, parasporin gene, and δ- endotoxin protein profiles were checked. Primers were designed for ps gene detection and the molecular identification of the isolates was performed by 16S rRNA gene sequence analysis. From the tested samples (n=128), 91 Bt isolates were obtained and of them, 28 non-hemolytic Bt isolates were selected as potential parasporin producers. Plasmid profile analysis of these isolates revealed that non-hemolytic Bt isolates harbor low molecular weight plasmid (6-8 Kb) unlike the Bt with insecticidal properties. Through PCR detection, amplicons of the proteins could be obtained from indigenous Bt to desired size for ps2 and ps3 genes were yielded by two isolates namely Bt DSc5 and Bt DSf3. The crude δ- endotoxin profiles and Proteinase-K digestion generated fragments of these isolates resembled previous reports. Finally, the 16S rRNA gene sequence analysis of these two isolates followed by blastn analysis confirmed them as B. thuringiensis. It is, therefore, anticipated that effective anticancer make them useful in cancer therapy.