Identification and functional analysis of two novel tpp genes in Bacillus thuringiensis HSY91

Bacillus thuringiensis (Bt) is a widely used biocontrol agent, and while research has heavily focused on the coleopteran-active binary toxins Tpp1/Tpp2, other members of the tpp family with insecticidal activity remain largely unexplored. In this study, two novel tpp-like genes, designated tpp92Aa1 and tpp92Ab1, were identified from the Bt HSY91 strain isolated from the Dongzhai Port Nature Reserve, Hainan. Phylogenetic analysis revealed a shared evolutionary origin within the Tpp family, with highly conserved C-terminal pore-forming domains but significant sequence variation in N-terminal motifs. Bioassays against larvae of Spodoptera frugiperda, Plutella xylostella, Ostrinia furnacalis, and Aedes aegypti demonstrated that both Tpp proteins only exhibited toxicity against A. aegypti larvae (Tpp92Aa1: LC₅₀ = 1.84 μg/mL; Tpp92Ab1: LC₅₀ = 1.62 μg/mL), with synergistic mosquitocidal activity observed in combination with Cyt2Aa1. Using a crystal-free Bt mutant expression system, we confirmed that Tpp92Aa1 and Tpp92Ab1 form typical spherical crystals, exhibiting robust thermal stability, resistance to trypsin digestion, and hemolytic activity. These findings enhance our understanding of Tpp protein diversity and their potential for targeted mosquito control.