New functions for Cyt proteins and sporulation determinants in Bacillus thuringiensis

In this study, we first aimed at finding new insecticidal proteins and synergistic factors with activity against A. albopictus larvae. Our first goal was to characterize the individual activity of Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa against A. albopictus and find new proteins that contribute to diversifying the currently available Bt active ingredients. Interestingly, we found a new protein, Cyt1A-like, which increased the activity of Cry11Aa more than 10-fold and enabled the activity of three new Bt toxins, Cry53-like, Cry56A-like and Tpp36-like that were non-toxic on their own. These results positioned Cyt proteins, as enablers of activity for otherwise non-active crystal proteins in addition to their previously described synergistic properties. As a second objective, we focused on understanding the link between sporulation and crystal formation in Bt. In the literature, there is extensive data describing how sporulation and crystal formation are coupled. However, most of said studies usually revolve around master regulators of sporulation that are critical for the expression of crystal proteins. Here, we aimed at finding sporulation determinants that were critical for the generation of the spores but dispensable for crystal formation. Through randomized mutagenesis, we discovered an essential gene for sporulation, spoIIIAA, which did not affect the production of the crystal when interrupted. The transcriptome of the spoIIIAA mutant revealed that several sporulation genes were downregulated alongside a cell wall hydrolase. Moreover, several virulence factors were upregulated, however, the crystal protein genes had their expression levels unchanged. Finally, we reproduced the same mutation, through site directed mutagenesis, in the AM 65-52 strain. Both wild type strain and asporogenous derivative mutant retained the ability to produce crystals that were active against A. aegypti and A. albopictuslarvae. All in all, we provide novel insights into mosquitocidal crystal proteins, especially Cyt proteins and their ability to enable the activity of otherwise non-toxic proteins, and the importance of SpoIIIAA as a sporulation-specific regulator that does not affect the expression of crystal proteins.