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

The cryIVB gene from a new isolate of Bacillus thuringiensis var israelensi s was cloned and sequenced. Two nucleotide replacements resulted in changin g Asp(385)-Thr(386) to Glu(385)-Ser(386) were found in comparison with the previously sequenced cryIVB gene. Two genetic constructions were designed f or expression of cryIVB in the obligate methylotroph Methylobacillus flagel latum, In the first construction, cryIVB was cloned under the strong induci ble 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 cryIVB gene. Both E. coli and M. flagellatum harboring both constructs were toxic to insect larvae of Anopheles stephensi and Aedes aegypti. However, the tox icity of the methylotroph was about 450 times less. This study is the first attempt to use methylotrophs as an insecticidal endotoxin producer.