Characterization of mosquitocidal Bacillus thuringiensis strains and identification of a Cry30G variant

Mosquito-control increasingly relies on Bacillus thuringiensis based biopesticides, especially spherical crystal producing strains with diverse toxins that enhance dipteran control and may delay resistance development. Here, three such B. thuringiensis isolates were investigated for their mosquito-larvicidal potential. Strains BUPM55 and BLB516 exhibited activity against Aedes aegypti larvae, with BUPM55 showing the highest toxicity. Biochemical, molecular and bioassay analyses revealed diversity among the isolates. Phylogenetic analysis based on the gyrB gene, clustered BLB516 and BUPM55 with the sotto subspecies whereas BLB423 grouped with the kurstaki subspecies. PCR_amplification using universal cry gene primers yielded the expected fragments for BUPM55 and BLB516. BLASTx analysis of the fragments indicated the presence of cry54, cry4C/Da, cry39Ab and cry40 genes in both strains, while BLB516 additionally harbored a cry30 type gene. Crystal protein trypsin digestion profiles clearly differ between BUPM55 and BLB516 suggest functional differences in toxin composition and toxicity. PCR screening with specific primers confirmed the presence of a cry30G type gene in BUPM55, which was cloned, sequenced (GenBank Accession no. MG457160) and expressed in the B. thuringiensis strain 4Q7, producing a 78 kDa Cry30Gr_toxin. The protein showed no activity against A. aegypti larvae even at concentrations up-to 600 mg/L, but it may act synergistically with other toxins. Sequence alignment revealed 99.8% similarity to Cry30Ga2 (GenBank Accession no. ADW27188.1) from B. thuringiensis S2160-1 with two amino-acid substitutions, S321T and A569V. Cry30Ga2 was used as reference for comparative analyses. Structural_homology_modelling using Cry4Ba_structure (PDB_4MOA) as template, along with 50 ns molecular dynamics simulations, suggested an increased stability and compactness of Cry30Gr in the presence of S321T and A569V substitutions. This suggests that minor sequence variations may affect the structure function relationships and potentially influence the toxicity, pending experimental validation.