Large crystal toxin formation in chromosomally engineered Bacillus thuringiensis subsp. aizawai due to σE accumulation.

Seven distinct Bacillus thuringiensis subsp. aizawai integrants were constructed that carried the chitinase (chiBlA) gene from B. licheniformis under the control of the cry11Aa promoter and terminator with and without p19 and p20 genes. The toxicity of B. thuringiensis subsp. aizawai integrants against second-instar Spodoptera litura larvae was increased 1.8- to 4.6-fold compared to that of the wild-type strain (BTA1). Surprisingly, the enhanced toxicity in some strains of B. thuringiensis subsp. aizawai integrants (BtaP19CS, BtaP19CSter, and BtaCAT) correlated with an increase in toxin formation. To investigate the role of these genes in toxin production, the expression profiles of the toxin genes, cry1Aa and chiBlA, as well as their transcriptional regulators (sigK and sigE), were analyzed by quantitative real-time RT-PCR (qPCR) from BTA1, BtaP19CS, and BtaCAT. Expression levels of cry1Aa in these two integrants increased about 2- to 3-fold compared to those of BTA1. The expression of the transcription factor sigK also was prolonged in the integrants compared to that of the wild type; however, sigE expression was unchanged. Western blot analysis of σ(E) and σ(K) showed the prolonged accumulation of σ(E) in the integrants compared to that of BTA1, resulting in the increased synthesis of pro-σ(K) up to T(17) after the onset of sporulation in both BtaP19CS and BtaCAT compared to that of T(13) in BTA1. The results from qPCR indicate clearly that the cry1Aa promoter activity was influenced most strongly by σ(E), whereas cry11Aa depended mostly on σ(K). These results on large-crystal toxin formation with enhanced toxicity should provide useful information for the generation of strains with improved insecticidal activity.