Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis , and Cry10Aa-Cyt1Aa Synergism

Abstract

Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-m amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by Nterminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anopheles albimanus

“…In contrast, LC 50 values of Cyt toxins were 60-1245 ng per ml when tested alone against Ae. aegypti and C. quinquefasciatus (Crickmore et al, 1995;Hernández-Soto et al, 2009;Pérez et al, 2005;Promdonkoy et al, 2005;Tabashnik, 1992;Wu et al, 1994). The results here imply that even though Cyt1Aa has no toxicity by itself against An.…”

“…israelensis has been demonstrated against the disease-transmitting mosquitoes Aedes aegypti and Culex quinquefasciatus (Crickmore et al, 1995;Hernández-Soto et al, 2009;Pérez et al, 2005;Promdonkoy et al, 2005;Tabashnik, 1992;Wu and Chang, 1985;Wu et al, 1994). However, synergism between Cry and Cyt toxins has not been examined previously in Anopheles mosquitoes that transmit malaria, which causes approximately one million deaths annually (Greenwood et al, 2008).…”

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

“…jegathesan 367, and their encoding genes were found to be separated by a 114-bp interval. The similar gene structure (ORF1-gap-ORF2), in which a typical 130-kDa toxin gene was split into two separate ORFs, has been found in some other instances, such as cry10A (24), cry19A (13,25), cry39A (26), cry40A (GenBank accession no. AB074414), and cry44A (8).…”

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].…”