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Characterization of Bacillus thuringiensis mutants and natural isolates by molecular methods

Characterization of Bacillus thuringiensis mutants and natural isolates by molecular methods

Canadian Journal of Microbiology 43(5): 403-410

Two Bacillus thuringiensis var. kurstaki HD-1 mutants, two Bacillus thuringiensis var. israelensis HD-500 mutants, and four rice grain dust isolates were characterized using microscopic examination and protein profiles of purified crystals on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Specific detection of cryI- and cryIV-type genes was performed in a polymerase chain reaction using cryI and cryIV-specific oligonucleotide primers. The cry-type genes under study consisted of cryIA(a), cryI(A)b, cryI(A)c, cryIB, and cryIV. Presence or absence of the cryl- and cryIV-type genes was further confirmed by Southern blotting followed by hybridization with specific cryl and cryIV gene fragments. A genetically modified strain of B. thuringiensis var. kurstaki HD-1, called OZK-13 and obtained following mutagenesis with ozone, was shown to contain cryIA (a), cryIA (b), and cryIA (c) genes. Another kurstaki HD- 1 mutant, called NGK-13 and obtained following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was shown to have lost the cryIA (b) gene while retaining the cryIA (a) and cryIA (c) genes. NGI-23-1, an oligosporogenous-multicrystalliferous mutant of B. thuringiensis var. israelensis (Bti) HD-500, obtained following treatment with MNNG contained cryIV-type genes. NGI-22, an oligosporogenous-acrystalliferous mutant of Bti HD-500, contained no cryl- nor cryIV-type genes. The rice grain dust isolate BT-285 contained the cryIA(a) and cryIA(c) genes. Isolate BT-14 contained only the cryIA(c) gene, whereas isolate BT-209 contained cryIA(a), cryIA(b), and cryIB genes. Isolate BT-205 contained no cryI- nor cryIV-type genes. Bacillus thuringiensis mutants and natural isolates shown to contain cryI-type genes were tested for their insecticidal activities in a series of bioassays against Hyphantria cunea Drury (Lepidoptera: Arctiidae). All cryI-carrying strains were toxic against the insect larvae. BT-205 was also tested and exhibited no toxicity against the insect larvae.

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Accession: 002775133

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DOI: 10.1139/m97-057

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