ABSTRACTThe total protoxin complement in the parasporal body of mosquitocidal strain,Bacillus thuringiensissubsp.jegathesan367, was determined by use of a polyacrylamide gel block coupled to mass spectrometry. A total of eight protoxins were identified from this strain, including five reported protoxins (Cry11Ba, Cry19Aa, Cry24Aa, Cry25Aa, and Cyt2Bb), as well as three previously undescribed (Cry30Ca, Cry60Aa, and Cry60Ba) in this isolate. It was interesting that the encoding genes of three new protoxins existed ascry30Ca-gap-orf2andcry60Ba-gap-cry60Aa. Thecry30Caand a downstreamorf2gene were oriented in the same direction and separated by 114 bp, andcry60Bawas located 156 bp upstream from and in the same orientation tocry60Aa. The three new protoxin genes were cloned fromB. thuringiensissubsp.jegathesanand expressed in an acrystalliferous strain under the control ofcyt1Agene promoters and the STAB-SD stabilizer sequence. Recombinant strain containing onlycry30Cadid not produce visible inclusion under microscope observation, while that containing bothcry30Caandorf2could produce large inclusions. Cry60Aa and Cry60Ba synthesized either alone or together in the acrystalliferous host could yield large inclusions. In bioassays using the fourth-instar larvae ofCulex quinquefasciatus, Cry60Aa and Cry60Ba alone or together had estimated 50% lethal concentrations of 2.9 to 7.9 μg/ml; however, Cry30Ca with or without ORF2 was not toxic to this mosquito.
Identification and characterization of the new Bacillus thuringiensis serovars pirenaica (serotype H57) and iberica (serotype H59)
