Cloning and expression of mosquitocidal endotoxin gene crylVB from Bacillus thuringiensis var israelensis in the obligate methylotroph Methylobacillus flagellatum

The crylVB gene from a new isolate of Bacillus thuringiensis var israelensis was cloned and sequenced. Two nucleotide replacements resulted in changing Asp³⁸⁵-Thr³⁸⁶ to Glu³⁸⁵-Ser³⁸⁶ were found in comparison with the previously sequenced crylVB gene. Two genetic constructions were designed for expression of crylVB in the obligate methylotroph Methylobacillus flagellatum. In the first construction, crylVB was cloned under the strong inducible lac promoter and contained original ribosome binding site and 150 bp of 5' transcribed but untranslated region. In the second construct, the first five codons of the lacZ gene were fused to the second codon of the crylVB gene. Both E. coli and M. flagellatum harboring both constructs were toxic to insect larvae of Anopheles stephensi and Aedes aegypti. However, the toxicity of the methylotroph was about 450 times less. This study is the first attempt to use methylotrophs as an insecticidal endotoxin producer.