Large Crystal Toxin Formation in Chromosomally Engineered Bacillus thuringiensis subsp. aizawai Due to σEAccumulation
ABSTRACTSeven distinctBacillus thuringiensissubsp.aizawaiintegrants were constructed that carried the chitinase (chiBlA) gene fromB. licheniformisunder the control of thecry11Aapromoter and terminator with and withoutp19andp20genes. The toxicity ofB. thuringiensissubsp.aizawaiintegrants against second-instarSpodoptera lituralarvae was increased 1.8- to 4.6-fold compared to that of the wild-type strain (BTA1). Surprisingly, the enhanced toxicity in some strains ofB. thuringiensissubsp.aizawaiintegrants (BtaP19CS,BtaP19CSter, andBtaCAT) correlated with an increase in toxin formation. To investigate the role of these genes in toxin production, the expression profiles of the toxin genes,cry1AaandchiBlA, as well as their transcriptional regulators (sigKandsigE), were analyzed by quantitative real-time RT-PCR (qPCR) from BTA1,BtaP19CS, andBtaCAT. Expression levels ofcry1Aain these two integrants increased about 2- to 3-fold compared to those of BTA1. The expression of the transcription factorsigKalso was prolonged in the integrants compared to that of the wild type; however,sigEexpression was unchanged. Western blot analysis of σEand σKshowed the prolonged accumulation of σEin the integrants compared to that of BTA1, resulting in the increased synthesis of pro-σKup toT17after the onset of sporulation in bothBtaP19CS andBtaCAT compared to that ofT13in BTA1. The results from qPCR indicate clearly that thecry1Aapromoter activity was influenced most strongly by σE, whereascry11Aadepended mostly on σK. These results on large-crystal toxin formation with enhanced toxicity should provide useful information for the generation of strains with improved insecticidal activity.