Detection of N-Acyl Homoserine Lactone -Degrading Enzyme and Insecticidal Protein Genes in Bacillus thuringiensis
Bacillus thuringiensis (Bt) is a Gram positive, endospore-forming, soil bacterium. It is the most widely used bio-pesticide worldwide, making it the sole source of toxin genes for the development of insect-resistant plants. Bt produces a factor that interferes with quorum sensing among gram-negative organisms. This lactone-degrading enzyme is produced from the aiiA gene. In addition, Bt strains are reported to produce the Vip3A protein, which is an insecticidal protein with high toxicity to lepidopteran insects. Thus, the purpose of this study was to assess the presence of the aiiA and Vip3A genes in strains of Bt. Multiple strands of Bacillus thuringiensis obtained from our stock of Middle Tennessee isolates were tested for the presence of the aiiA and Vip3A genes. Two reference strains, Bacillus thuringiensis subsp. Israelensis (I) and Bacillus thuringiensis kurstaki (K) and 72 isolates were used in PCR amplifications with specific primers for the genes. The PCR products were sequenced, the sequences were subjected to BLAST analysis, and the sequences were aligned to assess relationships. Results showed that a total of 28 strains of the 74 studied contained the aiiA gene only, 3 contained the Vip3A gene only, and 16 strains contained both genes. Interestingly, several strains (27) did not yield an amplified product for either gene. The BLAST analysis showed that the strains that yielded a PCR product to the aiiA gene were members of the B. cereus and B. thuringiensis lactonase family. In addition, alignment analysis of these genes showed the Bt strains could be grouped into parent clusters with multiple sub clusters. The BLAST and alignment analyses of the Vip3A genes showed that a less specific primer set was needed to assess the distribution of the Vip genes in the Bt strains. Only minimal relations were seen between the Vip3A genes, and BoxShade display showed differences between the Vip3A genes from the Bt strains. It was concluded that nearly 60% of the Bt strains studied carry the aiiA gene and only 24% carry the Vip3A gene. There was greater variability between the strains carrying the Vip3A gene than those with the aiiA gene. Further studies are needed to assess the relationship between Bt strains carrying the Vip gene, and studies of the specific lactonase activity of Bt strains is needed to further assess the diversity of this activity in these organisms.
Kesha R Bailey,
"Detection of N-Acyl Homoserine Lactone -Degrading Enzyme and Insecticidal Protein Genes in Bacillus thuringiensis"
ETD Collection for Tennessee State University.