Expression of the mosquitocidal cryIVB gene under the control of different promoters in Bacillus sphaericus 2362 and acrystalliferous Bacillus thuringiensis subsp. israelensis c4Q2-72

The 3.7 kb XbaI fragment harbouring the cryIVB gene which encoded a 130 kDa mosquitocidal toxin protein from Bacillus thuringiensis subsp. israelensis (B.t.i.) was placed downstream to the cat-86 gene promoter (Pcat-86, spore stage specific expression) or bgaB gene promoter (PbgaB, vegetative stage specific expression). The constructs were subcloned into pBC16 to obtain pBTC3 and pBTC6, respectively. Both plasmids and the other construct, pBTC1 were successfully transferred into B. thuringiensis subsp. israelensis c4Q2-72 and B. sphaericus 2362. Western blot analysis showed that PbgaB in front of PcryIVB could enable cells to produce a 130 kDa protein from the vegetative stage (4 h) whereas those with Pcat-86 could not. The positive detection of 130 kDa crystal protein during the vegetative stage (4 h) by Western blot analysis indicated the vegetative-stage-specific expression of PbgaB, while the 130 kDa crystal protein produced from cryIVB gene under control of Pcat-86 was detected only at 48 h. The strong activity of PbgaB, together with PcryIVB within pBTC6 in both bacterial hosts was also shown by the toxicity assay against Aedes aegypti larvae (B.t.i. c4Q2-72, 5.6 ± 3.6 × 102 c.f.u./ml; B. sphaericus 2362, 5.4 ± 2.5 × 102 c.f.u./ml) which were 100-fold and 10-fold more toxic to such larvae when compared with pBTC3 (Pcat-86 together with PcryIVB) and pBTC1 (contained only its self promoter) in the same bacterial host strains, respectively. The plasmid pBTC6 is not stable in either Bacillus host.