Cloning and expression of a novel toxin gene from Bacillus thuringiensis subsp. jegathesan encoding a highly mosquitocidal protein.

A gene, designated cry11B, encoding a 81,293-Da crystal protein of Bacillus thuringiensis subsp. jegathesan was cloned by using a gene-specific oligonucleotide probe. The sequence of the Cry11B protein, as deduced from the sequence of the cry11B gene, contains large regions of similarity with the Cry11A toxin (previously CryIVD) from B. thuringiensis subsp. israelensis. The Cry11B protein was immunologically related to both Cry11A and Cry4A proteins. The cry11B gene was expressed in a nontoxic strain of B. thuringiensis, in which Cry11B was produced in large amounts during sporulation and accumulated as inclusions. Purified Cry11B inclusions were highly toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi. The activity of Cry11B toxin was higher than that of Cry11A and similar to that of the native crystals from B. thuringiensis subsp. jegathesan, which contain at least seven polypeptides.     

The 60-kilodalton protein encoded by orf2 in the cry19A operon of Bacillus thuringiensis subsp. jegathesan functions like a C-terminal crystallization domain

The cry19A operon of Bacillus thuringiensis subsp. jegathesan encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the cry19A operon in an acrystalliferous strain of B. thuringiensis (4Q7) yielded one small crystal per cell, whereas no crystals were produced when cry19A or orf2 was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of the cry19A operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric cyt1A-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae of Culex quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC(95)] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC(95) = 8.2 μg/ml).     

Production of Cry11A and Cry11Ba toxins in Bacillus sphaericus confers toxicity towards Aedes aegypti and resistant Culex populations.

Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297.     

Cloning and Characterization of Two Novel Genes, cry24B and s1orf2, from a Mosquitocidal Strain of Bacillus thuringiensis serovar sotto

Two new crystal protein genes, cry24B and s1orf2, were cloned from a mosquitocidal Bacillus thuringiensis serovar sotto strain. The cry24B and s1orf2 genes encoded a 76-kDa and 62-kDa protein, respectively. The Cry24B protein retained five conserved regions commonly found in the existing Cry proteins. The amino acid sequence of the S1ORF2 had a high homology to that of the ORF2 protein of B. thuringiensis serovar jegathesan. Southern hybridization experiments with a cry24B gene-specific probe revealed that these genes are located on two large plasmids of > 100 kb. When the two genes, cry24B and s1orf2, were expressed in an acrystalliferous B. thuringiensis host, the proteins were synthesized and accumulated as inclusions. These inclusions exhibited no larvicidal activities against three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens molestus. Likewise, the inclusions contained no cytocidal activity against HeLa cells.     

Cloning and characterization of a Bacillus thuringiensis serovar higo gene encoding a novel class of the delta-endotoxin protein, Cry27A, specifically active on the Anopheles mosquito

A novel gene encoding a 98-kDa mosquitocidal delta-endotoxin protein, designated Cry27A, was cloned from a Bacillus thuringiensis serovar higo strain. The Cry27A protein contained the five sequence blocks of amino acids commonly conserved in most B. thuringiensis Cry proteins. Relatively high homologies, ranging from 43.0% to 84.4%, existed between the Cry27A protein and several established classes of mosquitocidal Cry proteins (Cry4A, Cry10A, Cry19A, Cry19B, and Cry20A) in the sequence of 51 N-terminal amino acids. The complete sequence of this protein, however, showed low levels (<40%) of amino acid identity to those of the known Cry proteins. Although the expression level of the cry27A gene was low in the transformants under the control of its own promoter, the use of the cyt1A promoter resulted in high-level expression of the gene, leading to the formation of inclusions. The expressed Cry27A protein showed larvicidal activity highly specific for Anopheles stephensi, but lacked the toxicity against Culex pipiens molestus and Aedes aegypti. The results suggest that the Cry27A protein is responsible for the Anopheles-preferential toxicity of the B. thuringiensis serovar higo strain.     

A pair of adjacent genes, cry5Ad and orf2-5Ad, encode the typical N- and C-terminal regions of a Cry5Adelta-endotoxin as two separate proteins in Bacillus thuringiensis strain L366.

A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad. cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Adelta-endotoxin. The cry5Ad sequence includes homology blocks 1-5, which are present in most B. thuringiensisdelta-endotoxins. The usual C-terminal region of a Cry5Adelta-endotoxin (including homology blocks 6-8) is encoded by orf2-5Ad. Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli, after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae (Haemonchus contortus), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.     

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.     

A pair of adjacent genes, cry5Ad and orf2-5Ad, encode the typical N- and C-terminal regions of a Cry5Aδ-endotoxin as two separate proteins in Bacillus thuringiensis strain L366

A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060 ) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad . cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Aδ-endotoxin. The cry5Ad sequence includes homology blocks 1–5, which are present in most B. thuringiensis δ-endotoxins. The usual C-terminal region of a Cry5Aδ-endotoxin (including homology blocks 6–8) is encoded by orf2-5Ad . Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli , after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae ( Haemonchus contortus ), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.     

Cloning and characterization of a novel gene cry9Ec1 encoding lepidopteran-specific parasporal inclusion protein from a Bacillus thuringiensis serovar galleriae strain

A novel delta-endotoxin gene from a lepidopteran-specific Bacillus thuringiensis serovar galleriae strain was cloned, and the full sequence of the cry gene was determined. The cloned 6.5-kb DNA fragment included the full sequence of the cry gene and three open reading frames located upstream of the cry gene. The gene, designated cry9Ec1, encodes a polypeptide of 1154 amino acid residues with a predicted molecular weight of 130 237. The deduced amino acid sequence of the Cry9Ec1 protein had the highest homology (77.7%) with the Cry9Ea1 protein when compared with existing Cry proteins. The expression, in an acrystalliferous B. thuringiensis strain, of the cry9Ec1 gene was high when controlled by the cyt1A2 promoter, leading to the formation of large spherical inclusions. The purified crystals from the recombinant strain were toxic when tested against two lepidopteran species, Bombyx mori and Plutella xylostella. However, the Cry9Ec1 protein gave no toxicity against Spodoptera litura, Spodoptera exigua, Plodia interpunctella, Helicoverpa zea, and Culex pipiens molestus.     

苏云金芽孢杆菌的cry2A芽孢期启动子和分子伴侣ORF1-ORF2对Cry11Aa蛋白表达的影响

[目的]分析苏云金芽孢杆菌的cry2A型芽孢期启动子对晶体蛋白Cry11Aa的协调作用和分子伴侣ORF1-ORF2对Cry11Aa表达的促进功能.[方法]3个包括cry11Aa编码区的重组质粒pHcy1、pHcy2和pHcy4被构建并电激转化到苏云金芽孢杆菌晶体缺陷株4Q7中,其中pHcy1质粒携带cry11Aa基因自身启动子和分子伴侣p19基因,pHcy2携带cry2A型芽孢期启动子和分子伴侣orf1-orf2基因,pHcy4质粒在pHcy1的上游插入了cry2A型芽孢期启动子和分子伴侣orf1-orf2基因.SDS-PAGE分析了Cry11Aa蛋白在各重组苏云金菌株中的表达情况,并通过生物测定确定了其对蚊虫的生物活性.[结果]SDS-PAGE结果表明,Cry11Aa蛋白在4Q7(pHcy1)和4QT(pHcy4)均获得了表达,在4Q7(pHcy2)中未检测到Cry11Aa蛋白,推测晶体蛋白Cry11A不能利用cry2A型启动子进行表达调控;Cry11Aa蛋白在等体积4Q7(pHcy4)培养液中的表达量是4Q7(pHcy1)菌株的1.25倍,暗示着分子伴侣ORF1-ORF2在某种程度上能提高Cry11Aa的蛋白表达量.4Q7(pHcy1)和4Q7(pHcy4)形成的Cry11Aa蛋白晶体的形状和大小相似,两者对致倦库蚊的生物活性没有明显差异,LC50s分别为59.33 ng/mL和66.21 ng/mL,.[结论]推测晶体蛋白Cry11A能否成功表达与其使用启动子的类型和两者的协调配合有关.分子伴侣ORF1-ORF2虽然在某种程度上能提高Cry11Aa的蛋白表达量,但对提高Cry11Aa蛋白的杀蚊毒力没有显著性帮助.     

Molecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis.

A novel mosquitocidal protein gene, cry20Aa, was cloned from Bacillus thuringiensis subsp. fukuokaensis (H-3a: 3d: 3e). The gene product, Cry20Aa, was naturally truncated and had a molecular mass of 86,138 Da. The Cry20Aa protein possessed five conserved sequence blocks, as do most other insecticidal Cry toxins. However, an amino acid comparison of Cry20Aa with other mosquitocidal toxins, including Cry4A, Cry4B, Cry10A, Cry11A, and Cry11B, demonstrated that Cry20Aa was quite different from other toxins except for the conserved blocks. The N terminus of Cry20Aa was, however, homologous to the N termini of Cry4A and Cry10A. Interestingly, an inverted repeat (IR1) sequence in the open reading frame of the cry20Aa gene caused incomplete expression of Cry20Aa. When this internal IR1 sequence was altered with no change of amino acid sequence, acrystalliferous B. thuringiensis cells transformed with cry20Aa gene dramatically produced crystal inclusions. However, the intact 86-kDa Cry20Aa protein is highly labile, and it is rapidly degraded to polypeptides of 56 and 43 kDa. To increase expression of the cry20Aa gene, the p20 chaperonelike protein and the cyt1Aa promoter were utilized. While p20 did not increase Cry20Aa expression or stability, chimeric constructs in which the cry20Aa gene was under control of the cyt1Aa promoter overexpressed the Cry20Aa protein in acrystalliferous B. thuringiensis. The expressed Cry20Aa protein showed larvicidal activity against Aedes aegypti and Culex quinquefasciatus. However, the mosquitocidal activity was low, probably due to rapid proteolysis to inactive 56- and 43-kDa proteins.     

A Bacillus thuringiensis subsp. israelensis gene encoding a 125-kilodalton larvicidal polypeptide is associated with inverted repeat sequences.

A gene encoding a 125-kilodalton (kDa) mosquitocidal delta-endotoxin was cloned from the 72-MDa resident plasmid of Bacillus thuringiensis subsp. israelensis. This gene is similar in its 3' region to the gene encoding the 135-kDa protein previously cloned (C. Bourgouin, A. Klier, and G. Rapoport, Mol. Gen. Genet. 205:390-397, 1986). Escherichia coli recombinant clones harboring the 125-kDa gene were toxic to larvae of the three mosquito species Aedes aegypti, Anopheles stephensi, and Culex pipiens. In addition, the B. thuringiensis subsp. israelensis DNA fragment carrying the 125-kDa protein gene contains two sets of inverted repeat sequences, identified either by the S1 nuclease method or by electron microscopic observation. The structural organization of inverted repeat sequences and of the 125-kDa gene was analyzed and suggests that this B. thuringiensis subsp. israelensis delta-endotoxin gene is located within a transposable element.     

Deletion by in vivo recombination shows that the 28-kilodalton cytolytic polypeptide from Bacillus thuringiensis subsp. israelensis is not essential for mosquitocidal activity.

The cytA gene encoding the 28-kDa polypeptide of Bacillus thuringiensis subsp. israelensis crystals was disrupted in the 72-MDa resident plasmid by in vivo recombination, thus indicating that homologous recombination occurs in B. thuringiensis. The absence of the 28-kDa protein in B. thuringiensis did not affect the crystallization of the other toxic components of the parasporal body (68-, 125-, and 135-kDa polypeptides). The absence of the 28-kDa protein abolished the hemolytic activity of B. thuringiensis subsp. israelensis crystals. However, the mosquitocidal activity of the 28-kDa protein-free crystals did not differ significantly from that of the wild-type crystals when tested on Aedes aegypti and Culex pipiens larvae. The 28-kDa protein contributed slightly to the toxicity to Anopheles stephensi larvae. This indicates that the 28-kDa protein is not essential for mosquitocidal activity, at least against the three species tested.     

Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control.     

苏云金杆菌辅助蛋白P19对杀虫晶体蛋白Cry11Aa表达的影响

苏云金杆菌以色列亚种的p19基因、cry11Aa基因和p20基因位于同一操纵子上,据推测辅助蛋白P19可能与Cry11Aa蛋白的晶体化相关。本研究利用穿梭载体pHT3101构建了两个重组质粒pHcy1和pHcy3,两质粒均携带cry11Aa基因,但后者完全缺失了cry11Aa基因上游的p19基因。将重组质粒电激转化至苏云金杆菌无晶体突变株4Q7中进行蛋白表达,SDS-PAGE结果表明在4Q7(pHcy1)和4Q7(pHcy3)中均能检测到正常表达的Cry11Aa蛋白,但单位体积培养液的Cry11Aa蛋白在辅助蛋白P19存在时的表达量明显高于其单独表达的表达量;透射电镜观察显示两菌株中的Cry11Aa蛋白形成了大小相近、形状相似的双梯形晶体;另外,生物测定结果表明重组菌株4Q7(pHcy1)和4Q7(pHcy3)对三龄致倦库蚊的杀虫活性没有显著性差异。该现象说明辅助蛋白P19的缺失对Cry11Aa蛋白的晶体形成和杀蚊活性没有影响,但P19作为分子伴侣在一定程度上帮助提高了Cry11Aa蛋白的表达水平。     

Role of the CryIVD polypeptide in the overall toxicity of Bacillus thuringiensis subsp. israelensis.

The gene encoding the CryIVD protein of B. thuringiensis subsp. israelensis crystals was disrupted by in vivo recombination. The toxicity of the CryIVD protein-free inclusions was similar to that of the wild-type crystals on Anopheles stephensi larvae but was half the wild-type toxicity on Culex pipiens and Aedes aegypti larvae.     

Cloning and expression of two crystal protein genes, cry30Ba1 and cry44Aa1, obtained from a highly mosquitocidal strain, Bacillus thuringiensis subsp. entomocidus INA288

Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.     

Characterisation of the cry54Ab1 operon of Bacillus thuringiensis subsp. sichuansis strain MC28

A novel cry gene operon harbouring two open reading frames (ORFs) was discovered in a large plasmid of Bacillus thuringiensis (Bt) subsp. sichuansis MC28 (pMC189). The first ORF encoded 743 amino acids. It had a deduced molecular mass of 84.5 kDa and 73.2% identity with Cry54Aa1 protein as indicated by amino acid homology. This cry gene was designated as cry54Ab1 by the Bt Toxin Nomenclature Committee. The second ORF encoded 503 amino acids and had a deduced molecular mass of 58.2 kDa. It is here called as orf2. The whole cry54Ab1 operon, cry54Ab1 and orf2 were digested with BamHI/SalI and inserted into a shuttle vector, pSTK. The recombinant plasmids were transferred into an acrystalliferous mutant Bt HD73^?. Parasporal inclusions were observed only under expression of the whole cry54Ab1 operon. The bioassay experiments showed that expressed products of the cry54Ab1 and the whole cry54Ab1 operon were all toxic to Culex quinquefasciatus (Diptera).     

Construction and characterization of a recombinant Bacillus thuringiensis subsp. israelensis strain that produces Cry11B

The mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis (Bti) produces four major endotoxin proteins, Cry4A, Cry4B, Cry11A, and Cyt1A, and has toxicity in the range of many synthetic chemical insecticides. Cry11B, which occurs naturally in B. thuringiensis subsp. jegathesan, is a close relative of Cry11A, but is approximately 10-fold as toxic to Culex quinquefasciatus. To determine whether the addition of Cry11B to Bti would improve its toxicity, we produced this protein in Bti. High levels of Cry11B synthesis were obtained by expression of the cry11B gene under the control of cyt1A promoters and the STAB-SD sequence. This construct was cloned into the shuttle vector pHT3101, yielding the derivative plasmid pPFT11Bs, which was then transformed by electroporation into acrystalliferous (4Q7) and crystalliferous (IPS-82) strains of Bti. Synthesis of Cry11B in Bti 4Q7 produced crystals approximately 50% larger than those produced with its natural promoters without STAB-SD. However, less Cry11B was produced per unit culture medium with this construct than with the wild-type construct, apparently because the latter construct produced more cells per unit medium. Nevertheless, the Bti IPS-82 strain that produced Cry11B with pPFT11Bs was twice as toxic as the parental IPS-82 strain (LC(50) = 1.4 ng/ml versus 3.3 ng/ml, respectively) to fourth instars of C. quinquefasciatus. Against fourth instars of Aedes aegypti, no statistically significant difference between parental Bti IPS-82 (LC(50) = 4.7 ng/ml) and the Bti IPS-82 recombinant producing Cry11B (LC(50) = 3.5 ng/ml) was found in toxicity.     

Active form of dipteran-specific insecticidal protein cryllA produced by Bacillus thuringiensis subsp. israelensis

The nucleotide sequence of the cry11A gene from Bacillus thuringiensis subsp. israelensis strain HD522 was analyzed and the molecular characterization of CryllA toxin was done. The 70-kDa CryllA protoxin was processed in vitro into 36- and 32-kDa fragments by trypsin and into 34- and 32-kDa fragments by gut proteases from C. pipiens. These two processed fragments are associated together to form the heterodimer. The results of the binding assay with BBMV and the bioassay toward C. pipiens larvae suggested that the heterodimer was biologically as active as the non-digested CryllA toxin and the intramolecular cleavage did not promote the insecticidal activity. These results suggested that a probable complex of the 36- or 34-kDa and 32-kDa fragments was also one of the possible active forms of Cry11A, and that the biological functions of CryllA was not essentially affected by the intramolecular cleavage of the 70-kDa protein.