Characterization of a cry4Ba-type gene of Bacillus thuringiensis israelensis and evidence of the synergistic larvicidal activity of its encoded protein with Cry2A delta-endotoxin of B. thuringiensis kurstaki on Culex pipiens (common house mosquito).

A newly isolated strain of Bacillus thuringiensis, named BUPM97, was identified as affiliated to the israelensis subspecies. This strain was selected for its insecticidal activity against larvae of several dipteran insects, such as the common house mosquito (Culex pipiens). A 5 kb EcoRI fragment, containing a cry4Ba-type gene, named cry4BLB, was cloned from BUPM97. The sequencing of this gene revealed an open reading frame of 3411 bp encoding a protein of 1136 amino acid residues. Similarity analysis of both nucleotide and amino acid sequences revealed that cry4BLB presents several differences from the other previously described cry4Ba-type genes. Particular attention was paid to a two-amino-acid substitution located in domain III of the N-terminal moiety of this protein, which is very important for both toxicity and specificity of the toxin. The transfer of cry4BLB to an acrystalliferous B. thuringiensis kurstaki strain, HD1CryB, showed that it was expressed, resulting in the production of the typical parasporal crystal inclusions. On the other hand, its transfer to another B. thuringiensis kurstaki strain synthesizing Cry2A endotoxin known to be weakly toxic to the dipteran insect C. pipiens, resulted in the clear increase in the insecticidal activity of the transformed strain. Hence, in the present paper, we demonstrate for the first time that Cry2A and Cry4B act synergistically towards C. pipiens.