Diversity of Cry48/Tpp49 binary toxins from Lysinibacillus sphaericus and differential activities of their pairwise cross combinations with BinA/BinB against Culex quinquefasciatus larvae.

BACKGROUND: The mosquitocidal activity of Lysinibacillus sphaericus primarily results from the binary toxin BinA/BinB, encoded by the binA/binB operon within the 30-kb mobile genetic element GI8. Promising for alleviating BinA/BinB resistance, the two-component toxins Cry48Aa1/Tpp49Aa1 are of prime interest, but their very low expression levels in wild-type isolates have so far hampered their use in mosquito biocontrol. RESULTS: Besides the reported cry48Aa1/tpp49Aa1, this study identified two new sets of cry48/tpp49 variants neighboring the GI8-like region(s) in L. sphaericus. The Cry48 variants could be expressed in BMB171, an acrystalliferous isolate of Bacillus thuringiensis sv. israelensis, but not in L. sphaericus; whereas the Tpp49 variants were expressed in both B. thuringiensis and L. sphaericus, except that the expression of tpp49Ab2 was not observed in L. sphaericus G725 which also produces BinA/BinB. Interestingly, Cry48 or Tpp49 alone have no mosquitocidal activity against Culex quinquefasciatus larvae, whereas all the tested Cry48/Tpp49 combinations, at an equal molar ratio, displayed comparable toxicity to the BinA/BinB combination. Remarkably also, the combination of BinA - Tpp49Ab2, and BinB with Tpp49Aa1 exhibited mosquitocidal activity which is likely to be related to the structural similarities between BinB and Tpp49Ab2, and between BinA and Tpp49Aa1. CONCLUSION: The study identified novel cry48/tpp49 variants and proposes their evolutionary origin through GI8 duplication. The Cry48 variants were successfully expressed in B. thuringiensis sv. israelensis but not in L. sphaericus. The Tpp49 variants exhibited functional compatibility in hybrid combinations with Bin, suggesting conserved structural features that mediate cross-toxin synergy. © 2026 Society of Chemical Industry.