Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins

Abstract

Two homologous genes encoding 130-kilodalton (kDa) mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis have been cloned and expressed in Escherichia coli or Bacillus subtilis or both. One of these genes, pPC130, was expressed as a lacZ transcriptional fusion in E. coli at a level sufficient to produce phase-bright inclusions, which were purified and shown to be toxic to Aedes aegypti larvae. The second gene, pCH130, was expressed at a low level in recombinant E. coli cells and was therefore cloned in B. subtilis as a transcriptional fusion of the promoter sequences corresponding to a B. thuringiensis subsp. israelensis 27-kDa delta-endotoxin (E. S. Ward, A. R. Ridley, D. J. Ellar, and J. A. Todd, J. Mol. Biol. 191:13-22, 1986) and the structural gene. Recombinant B. subtilis cells produced phase-bright inclusions during late sporulation; these were partially purified and shown to be toxic to A. aegypti larvae at an LC50 (concentration required to cause 50% mortality of larvae after 24 h of assay) which is significantly lower than that of the pPC130 protein. Neither 130-kDa protein was hemolytic under the assay conditions. Comparison of the nucleotide sequences of these two genes indicates that they share a high degree of homology in the C-terminal portions, but relatively little similarity in the N termini. In addition, significant homologies were found between the pCH130 gene and the HD-1 Dipel gene of B. thuringiensis subsp. kurstaki (H. E. Schnepf, H. C. Wong, and H. R. Whiteley, J. Biol. Chem. 260:6264-6272, 1985).