Identification and Characterization of Three Previously Undescribed Crystal Proteins from Bacillus thuringiensis subsp. jegathesan

Abstract

The total protoxin complement in the parasporal body of mosquitocidal strain, Bacillus thuringiensis subsp. jegathesan 367, 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 as cry30Ca-gap-orf2 and cry60Ba-gap-cry60Aa. The cry30Ca and a downstream orf2 gene were oriented in the same direction and separated by 114 bp, and cry60Ba was located 156 bp upstream from and in the same orientation to cry60Aa. The three new protoxin genes were cloned from B. thuringiensis subsp. jegathesan and expressed in an acrystalliferous strain under the control of cyt1A gene promoters and the STAB-SD stabilizer sequence. Recombinant strain containing only cry30Ca did not produce visible inclusion under microscope observation, while that containing both cry30Ca and orf2 could 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 of Culex quinquefasciatus, Cry60Aa and Cry60Ba alone or together had estimated 50% lethal concentrations of 2.9 to 7.9 g/ml; however, Cry30Ca with or without ORF2 was not toxic to this mosquito.

In Vivo Crystallization of Three-Domain Cry Toxins

“…Figure 1 shows the configuration of various mosquitocidal Bt toxin genes including cry10A [16], cry19A [17,18], cry24B [19], cry30C [20], cry39A [21], cry40A (GenBank accession No. AB074414), cry44A [22], and cry59A [23].…”

“…The length of the intergenic regions is 48 bp, 145 bp, and 114 bp in cry10A, cry19A, and cry30C, respectively [16,18,20]. There is no homology between these regions and any known transposon or insertion element sequence that could explain the gene separation.…”

“…However, factors responsible for this arrangement are still unknown. It may have evolved through the insertion of a DNA fragment into a gene that previously encoded a 135 kDa protein [17,20]. …”

Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae

“…Found after Bti, Btjeg is another B. thuringiensis strain highly toxic against mosquito larvae with parasporal crystal components distinct from those of Bti. Parasporal crystals produced by Btjeg contain 8 protoxins that include Cry11Ba, Cry19Aa, Cry24Aa, Cry25Aa, Cyt2Bb, as well as the 3 newly described Cry30Ca, Cry60Aa, and Cry60Ba protoxins (Sun et al, 2013). The 81 kDa Cry11Ba of Btjeg has the highest toxicity of known Cry proteins to Culex, Anopheles, and Aedes species (Delecluse et al, 1995).…”

Chitosan/DsiRNA nanoparticle targeting identifies AgCad1 cadherin in Anopheles gambiae larvae as an in vivo receptor of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan

“…jegathesan (Btjeg) (Selena et al, 1995). The crystals of Btjeg have eight insecticidal proteins (Sun et al, 2013), of which Cry11Ba has been studied in the most detail as Cry11Ba is the single most active Cry protein across the three major genera of disease-vectoring mosquitoes.…”