Bacillus thuringiensis strain M15, a novel autoagglutinable, nonserotypeable strain-cloning and characterization of a novel cry31A-type crystal protein gene, cry31Aa2, and two new insertion sequences, IS231M and -N

The synthetic organic pesticides have caused considerable damage on the environment and human health due to their accumulation in the ecosystem and the contamination of the food chain in the environment. Several resistances to synthetic insecticides have appeared in some insect pest populations. For these reasons, there is a need for the development of biological control agents as an alternative for the management of insect pests.; Bacillus thuringiensis is the leading biocontrol agent and has been used as a biorational pesticide in insect pest control. B. thuringiensis is a Gram-positive, spore-forming bacterium that is commonly found in natural environments. During the stationary phase of its growth cycle, B. thuringiensis produces a parasporal inclusion that exhibits specific insecticidal activity against certain insect species among Lepidoptera, Diptera, and Coleoptera. The parasporal inclusions are made of proteins. These are encoded by toxin genes. Our research project was to isolate and characterize a new B. thuringiensis strain from the non-insect, two-spotted spider mite (Tetranychus urticae Koch; Arthropoda: Arachnida: Tetranychidae). A novel crystal protein gene was cloned and sequenced. It was designated cry31Aa2 by the B. thuringiensis Pesticide Crystal Protein Nomenclature Committee. It was expressed in an acrystalliferous B. thuringiensis strain. The parasporal inclusion protein expressed was isolated and confirmed to be composed of single major polypeptide of 83-kDa (Chapter 1).; Insertion sequences and transposons have been found in the vicinity of cry genes. We have cloned and characterized two new insertion sequences of the IS231 family, IS231M (Chapter 2) and N (Chapter 3). Although analyses of their DNA sequences revealed high homology to the IS231 family, their structural maps were different. IS231M has two overlapping open reading frames, ORF1 and ORF2, that could encode polypeptides of 334 and 143 amino acids, respectively, while IS231N contains three open reading frames (ORFs) that could code for polypeptides of 329 (ORF1), 118 (ORF2) and 17 (ORF3) amino acids, respectively. IS231N shows 99% nucleotide identity to IS231M, but shares only 83% amino acid identity because of nucleotide substitutions and additions. Whether IS231M and -N are functional transposable elements remains to be determined.