pH-Controlled Bacillus thuringiensis Cry1Ac Protoxin Loading and Release from Polyelectrolyte Microcapsules

Crystal proteins synthesized by Bacillus thuringiensis (Bt) have been used as biopesticides because of their toxicity to the insect larval hosts. To protect the proteins from environmental stress to extend their activity, we have developed a new microcapsule formulation. Poly (acrylic acid) (PAH) and poly (styrene sulfonate) (PSS) were fabricated through layer-by-layer self-assembly based on a CaCO₃ core. Cry1Ac protoxins were loaded into microcapsules through layer-by-layer self-assembly at low pH, and the encapsulated product was stored in water at 4°C. Scanning electron microscopy (SEM) was used to observe the morphology of the capsules. To confirm the successful encapsulation, the loading results were observed with a confocal laser scattering microscope (CLSM), using fluorescein-labeled Cry1Ac protoxin (FITC-Cry1Ac). The protoxins were released from the capsule under the alkaline condition corresponding to the midgut of certain insects, a condition which seldom exists elsewhere in the environment. The following bioassay experiment demonstrated that the microcapsules with Cry1Ac protoxins displayed approximately equivalent insecticidal activity to the Asian corn borer compared with free Cry1Ac protoxins, and empty capsules proved to have no effect on insects. Further result also indicated that the formulation could keep stable under the condition of heat and desiccation. These results suggest that this formulation provides a promising methodology that protects protoxins from the environment and releases them specifically in the target insects’ midgut, which has shown potential as biopesticide in the field.     

Shared Binding Sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A Toxins

Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.     

Efficient Production of Bacillus thuringiensis Cry1AMod Toxins under Regulation of cry3Aa Promoter and Single Cysteine Mutations in the Protoxin Region

Bacillus thuringiensis Cry1AbMod toxins are engineered versions of Cry1Ab that lack the amino-terminal end, including domain I helix α-1 and part of helix α-2. This deletion improves oligomerization of these toxins in solution in the absence of cadherin receptor and counters resistance to Cry1A toxins in different lepidopteran insects, suggesting that oligomerization plays a major role in their toxicity. However, Cry1AbMod toxins are toxic to Escherichia coli cells, since the cry1A promoter that drives its expression in B. thuringiensis has readthrough expression activity in E. coli, making difficult the construction of these CryMod toxins. In this work, we show that Cry1AbMod and Cry1AcMod toxins can be cloned efficiently under regulation of the cry3A promoter region to drive its expression in B. thuringiensis without expression in E. coli cells. However, p3A-Cry1Ab(c)Mod construction promotes the formation of Cry1AMod crystals in B. thuringiensis cells that were not soluble at pH 10.5 and showed no toxicity to Plutella xylostella larvae. Cysteine residues in the protoxin carboxyl-terminal end of Cry1A toxins have been shown to be involved in disulfide bond formation, which is important for crystallization. Six individual cysteine substitutions for serine residues were constructed in the carboxyl-terminal protoxin end of the p3A-Cry1AbMod construct and one in the carboxyl-terminal protoxin end of p3A-Cry1AcMod. Interestingly, p3A-Cry1AbMod C654S and C729S and p3A-Cry1AcMod C730S recover crystal solubility at pH 10.5 and toxicity to P. xylostella. These results show that combining the cry3A promoter expression system with single cysteine mutations is a useful system for efficient expression of Cry1AMod toxins in B. thuringiensis.     

Resistance of Trichoplusia ni Populations Selected by Bacillus thuringiensis Sprays to Cotton Plants Expressing Pyramided Bacillus thuringiensis Toxins Cry1Ac and Cry2Ab

Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops.     

苏云金芽孢杆菌伴孢晶体20kbDNA中cry1Ac基因的克隆、高效表达和生物活性研究

已经证实苏云金芽孢杆菌(Bacillus thuringiensis,Bt)伴孢晶体结合20kb DNA,但其序列特异性及作用有待进一步研究阐明.研究了选择性溶解Bt 4.0718菌株Cry1类原毒素所形成的菱形伴孢晶体,从中抽提出与其结合的20kb DNA.经Nde Ⅰ酶切消化后亚克隆构建文库,通过PCR-RFLP及测序筛选出含cry1Ac基因的转化子.然后设计引物PCR扩增出cry1Ac基因的ORF并与pET30a连接,转化E.coli BL21(DE3),高效表达了141kD蛋白.表达蛋白占总蛋白量的50%以上,且90%以上以包涵体形式存在.利用穿梭载体pHT304构建表达质粒pHTX42,电转化Bt无晶体突变株XBU001,获得重组菌株HTX42,经SDS-PAGE分析,cry1Ac基因得到强表达,蛋白质定量分析显示目的蛋白量占总蛋白量的79.28%,且其在细胞中累积达细胞干重的64.13%,比文献报道的25%左右高了1倍以上.原子力显微镜(Atomic Force Microscopy,AFM)检测显示,目的基因在大肠杆菌(E.coli)中表达的包涵体呈不规则形状且较小,而在无晶体突变株中表达的晶体呈典型菱形晶体,大小约为1.2 μm×2.0 μm.生测结果显示,包涵体与晶体对小菜蛾(Plutella xylostella)幼虫均有高效杀虫活性.本研究为构建高效杀虫工程菌及进一步阐明Bt伴孢晶体中20kb DNA分子的来源、结构和功能奠定了重要的基础.     

Lipid-induced Pore Formation of the Bacillus thuringiensis Cry1Aa Insecticidal Toxin

After activation, Bacillus thuringiensis (Bt) insecticidal toxin forms pores in larval midgut epithelial cell membranes, leading to host death. Although the crystal structure of the soluble form of Cry1Aa has been determined, the conformation of the pores and the mechanism of toxin interaction with and insertion into membranes are still not clear. Here we show that Cry1Aa spontaneously inserts into lipid mono- and bilayer membranes of appropriate compositions. Fourier Transform InfraRed spectroscopy (FTIR) indicates that insertion is accompanied by conformational changes characterized mainly by an unfolding of the β-sheet domains. Moreover, Atomic Force Microscopy (AFM) imaging strongly suggests that the pores are composed of four subunits surrounding a 1.5 nm diameter central depression. Received: 14 July 2000/Revised: 28 December 2000     

Common, but Complex, Mode of Resistance of Plutella xylostella to Bacillus thuringiensis Toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.     

Aggregation of Bacillus thuringiensis Cry1A Toxins upon Binding to Target Insect Larval Midgut Vesicles

During sporulation, Bacillus thuringiensis produces crystalline inclusions comprised of a mixture of δ-endotoxins. Following ingestion by insect larvae, these inclusion proteins are solubilized, and the protoxins are converted to toxins. These bind specifically to receptors on the surfaces of midgut apical cells and are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible oligomerization to form a channel have been examined. When bound to vesicles from the midguts of Manduca sexta larvae, the Cry1Ac toxin was largely resistant to digestion with protease K. Only about 60 amino acids were removed from the Cry1Ac amino terminus, which included primarily helix α1. Following incubation of the Cry1Ab or Cry1Ac toxins with vesicles, the preparations were solubilized by relatively mild conditions, and the toxin antigens were analyzed by immunoblotting. In both cases, most of the toxin formed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates did not include the toxin receptor aminopeptidase N, but interactions with other vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (α5) and known to insert into the membrane were tested. Active toxins with other mutations in this helix did form oligomers. There was one exception; a very active helix α5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices α2 and α3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the Heliothis virescens midgut than with those from the M. sexta midgut, which correlated with observed differences in toxicity. Tight binding of virtually the entire toxin molecule to the membrane and the subsequent oligomerization are both important steps in toxicity.     

Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.     

Common Receptor for Bacillus thuringiensis Toxins Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera, Helicoverpa zea, and Spodoptera exigua

Binding studies using ¹²⁵I-Cry1Ac and biotinylated Cry1Fa toxins indicate the occurrence of a common receptor for Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera, Helicoverpa zea, and Spodoptera exigua. Our results, along with previous binding data and the observed cases of cross-resistance, suggest that this pattern seems to be widespread among lepidopteran species.     

Domain I plays an important role in the crystallization of Cry3A in Bacillus thuringiensis

The insecticidal bacterium Bacillus thuringiensis synthesizes endotoxin Cry proteins of two size classes, 135 and 70 kDa, and both form crystalline inclusions in cells after synthesis. Crystallization of 135-kDa proteins is due to intermolecular attraction of regions in the C-terminal half of the molecule, and the N-terminal half fails to crystallize when synthesized in vivo. Alternatively, endotoxins of the 70-kDa class such as Cry2A and Cry3A, which correspond to the N-terminal half of 135-kDa molecules, crystallize readily after synthesis. Cry molecules of this size class consist of three principal domains, but the domains responsible for crystallization are not known. To identify these domains, chimeric proteins were constructed in which Cry3A Domains I or III, or I and III were substituted for the corresponding domains in truncated Cry1C molecules. Cry1C molecules with only Cry3A Domain III did not crystallize, whereas when Cry3A Domains I and III, or Domain I alone, were substituted, large inclusions were obtained. Except for the chimera consisting of Cry3A Domains I and III and Cry1C Domain II, most chimeras were not as stable as wild-type Cry3A or truncated Cry1C. These results show that Cry3A Domain I plays an important role in its crystallization in vivo.     

Effect of Insect Larval Midgut Proteases on the Activity of Bacillus thuringiensis Cry Toxins

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.     

Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.     

Lack of Cross-Resistance to Cry19A from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) Resistant to Cry Toxins from Bacillus thuringiensis subsp. israelensis

Culex quinquefasciatus mosquitoes with high levels of resistance to single or multiple toxins from Bacillus thuringiensis subsp. israelensis were tested for cross-resistance to the Bacillus thuringiensis subsp. jegathesan polypeptide Cry19A. No cross-resistance was detected in mosquitoes that had been selected with the Cry11A, Cry4A and Cry4B, or Cry4A, Cry4B, Cry11A, and CytA toxins. A low but statistically significant level of cross-resistance, three to fourfold, was detected in the colony selected with Cry4A, Cry4B, and Cry11A. This cross-resistance was similar to that previously detected with B. thuringiensis subsp. jegathesan in the same colony. These data help explain the toxicity of B. thuringiensis subsp. jegathesan against the resistant colonies and indicate that the Cry19A polypeptide might be useful in managing resistance and/or as a component of synthetic combinations of mosquitocidal toxins.     

Formation of Macromolecule Complex with Bacillus thuringiensis Cry1A Toxins and Chlorophyllide Binding 252-kDa Lipocalin-Like Protein Locating on Bombyx mori Midgut Membrane

P252, a 252-kDa Bombyx mori protein located on the larval midgut membrane, has been shown to bind strongly with Bacillus thuringiensis Cry1A toxins (Hossain et al. Appl Environ Microbiol 70:4604-4612, 2004). P252 was also shown to bind chlorophyllide (Chlide) to form red fluorescence-emitting complex Bm252RFP with significant antimicrobial activity (Pandian et al. Appl Environ Microbiol 74:1324-1331, 2008). In this article, we show that Cry1A toxin bound with Bm252RFP and Bm252RFP-Cry1A macrocomplex, with both antimicrobial and insecticidal activities, was formed. The insecticidal activity of Bm252RFP-Cry1Ab was reduced from an LD?? of 1.62 to 5.05 μg, but Bm252RFP-Cry1Aa and Bm252RFP-Cry1Ac did not show such reduction. On the other hand, the antimicrobial activity of Bm252RFP-Cry1Ab was shown to retain almost the same activity as Bm252RFP, while the other two complexes lost around 30% activity. The intensity of photo absorbance and fluorescence emission of Bm252RFP-Cry1Ab were significantly reduced compared to those of the other two complexes. Circular dichroism showed that the contents of Cry1Ab α-helix was significantly decreased in Bm252RFP-Cry1Ab but not in the other two toxins. These data suggested that the reduction of contents of α-helix in Cry1Ab affected the insecticidal activity of the macrocomplex but did not alter the antimicrobial moiety in the macrocomplex of Bm252RFP-Cry1Ab.     

Effect of Bacillus thuringiensis Cry1 Toxins in Insect Hemolymph and Their Neurotoxicity in Brain Cells of Lymantria dispar

Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 μg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 μg/0.2 g fresh wt). Cry1E and Cry1Ac (5 μg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to N. bullata larvae upon injection. Injection of 5 μg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 μg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.     

Cross-Resistance and Stability of Resistance to Bacillus thuringiensis Toxin Cry1C in Diamondback Moth

We tested toxins of Bacillus thuringiensis against larvae from susceptible, Cry1C-resistant, and Cry1A-resistant strains of diamondback moth (Plutella xylostella). The Cry1C-resistant strain, which was derived from a field population that had evolved resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai, was selected repeatedly with Cry1C in the laboratory. The Cry1C-resistant strain had strong cross-resistance to Cry1Ab, Cry1Ac, and Cry1F, low to moderate cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to Cry1Bb, Cry1Ja, and Cry2A. Resistance to Cry1C declined when selection was relaxed. Together with previously reported data, the new data on the cross-resistance of a Cry1C-resistant strain reported here suggest that resistance to Cry1A and Cry1C toxins confers little or no cross-resistance to Cry1Bb, Cry2Aa, or Cry9Ca. Therefore, these toxins might be useful in rotations or combinations with Cry1A and Cry1C toxins. Cry9Ca was much more potent than Cry1Bb or Cry2Aa and thus might be especially useful against diamondback moth.