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.     

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).     

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

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-μm 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 N-terminal 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.The subspecies Bacillus thuringiensis subsp. israelensis (serotype H-14) was discovered by Goldberg and Margalit in 1977 (11). To date, its insecticidal potential has not been overcome by any other bacterium (or any biological control agent) as an effective control measure against mosquito and blackfly larvae (8). Recently, its toxicity spectrum has been expanded to a coleopteran pest, the coffee berry borer (Hypothenemus hampei) (23), indicating that this strain may have potential versatility. Also, the so-called pBtoxis megaplasmid harbored in this strain, containing all the endotoxin-encoding genes found in its parasporal crystal, including cry4A, cry4B, cry10A, cry11A, and cyt1A, was recently sequenced (1). Among many other interesting aspects of this serotype, the occurrence of this mosquitocidal arsenal in one strain and their synergistic interaction make this bacterium scientifically and technologically attractive.The parasporal crystal of B. thuringiensis subsp. israelensis contains large amounts of Cry4A, Cry4B, Cry11A, and Cyt1A toxins (14), and consequently, most of the knowledge about the toxicity of this strain has been focused on these proteins, acting either as a complex (31) or tested separately (6). Although the cry10Aa gene was originally cloned in 1986 (known then as cryIVC) (30), to date, little is known about cry10Aa and the protein it encodes, mostly due to its very low levels of expression (10) in B. thuringiensis subsp. israelensis. Interestingly, cry10Aa is an operon as it includes two open reading frames (ORFs), previously reported as pBt047 and pBt048 (hereafter referred to only as ORF1 and ORF2, respectively), separated by a 48-bp untranslated gap (1). ORF1 contains the complete δ-endotoxin sequence (active toxin), with a coding capacity for a 78-kDa protein. Interestingly, ORF2 shows high identity with the coding sequence of the C-terminal half of Cry4-type proteins, with a coding capacity for a 56-kDa protein. Therefore, it is believed that a putative ancestral cry10Aa gene is similar in size to the cry4-type genes (ca. 4 kbp), but either a small sequence had been inserted in the middle of the coding sequence or site mutations produced end codons (two end codons flank the gap) in this region (1).Previous attempts to clone and express the cry10Aa gene included ORF1 and only part of ORF2 (7, 10, 30). This was a reasonable strategy, as most of the so-called “complete” protoxins are partially digested to become active toxins (δ-endotoxins) (28), and ORF1 included the complete sequence to code the Cry10Aa δ-endotoxin. However, in all these cases, the expression levels were very low, and no parasporal body was formed. Similar results were obtained when the promoter was changed and a stabilizing sequence was added to the construction (13). The low expression levels achieved in these cases led to conclusions that assumed low toxic levels of Cry10Aa when tested against mosquito larvae (30). In spite of the low toxicity of Cry10Aa found against mosquito larvae, a synergistic effect was reported between Cry10Aa and Cry4Ba toxins in Culex (7). Obtaining high levels of expression and crystallization of Cry10Aa are required to properly characterize and understand the toxic spectrum of this protein.In this report, we show the formation of parasporal bodies of Cry10Aa, achieved by cloning the whole Cry10Aa operon under the control of the cyt1A promoter and the STAB-SD sequence. We also show that Cry10Aa is as toxic as most of the other B. thuringiensis subsp. israelensis toxins acting separately, and in synergism with the Cyt1A toxin.     

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.     

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。     

Characterization of Cry34/Cry35 binary insecticidal proteins from diverse Bacillus thuringiensis strain collections

Bacillus thuringiensis crystal proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm, Diabrotica virgifera virgifera LeConte. We surveyed 6,499 B. thuringiensis isolates by hybridization for sequences related to cry35A genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34 cry34 genes and 16 cry35 genes were also obtained from cloning, PCR analysis, and DNA sequencing. These included representatives of all known Cry34A, Cry34B, Cry35A, and Cry35B classes, as well as a novel Cry34A/Cry35A-like pair. Bioassay analysis indicated that cry35-hybridizing strains not producing a ca. 14-kDa protein, indicative of Cry34, were not active on corn rootworms, and that the previously identified Cry34A/Cry35A pairs were more active than the Cry34B/Cry35B pairs. The cry35-hybridizing B. thuringiensis strains were found in locales and materials typical for other B. thuringiensis strains. Comparison of the sequences with the geographic origins of the strains showed that identical, or nearly identical, sequences were found in strains from both Australasia and the Americas. Sequence similarity searches revealed that Cry34 proteins are similar to predicted proteins in Photorhabdus luminescens and Dictyostelium discoidium, and that Cry35Ab1 contains a segment similar to beta-trefoil domains that may be a binding motif. The binary Cry34/Cry35 B. thuringiensis crystal proteins thus appear closely related to each other, are environmentally ubiquitous, and share sequence similarities consistent with activity through membrane disruption in target organisms.     

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.     

Contribution of the 65-kilodalton protein encoded by the cloned gene cry19A to the mosquitocidal activity of Bacillus thuringiensis subsp. jegathesan.

Two new crystal protein genes, cry19A and orf2, isolated from Bacillus thuringiensis subsp. jegathesan were cloned and characterized. The cry19A gene encodes a 74.7-kDa protein, and the orf2 gene encodes a 60-kDa protein. Cry19A contains the five conserved blocks present in most B. thuringiensis delta-endotoxins. The ORF2 amino acid sequence is similar to that of the carboxy terminus of Cry4 proteins. The cry 19A gene was expressed independently or in combination with orf2 in a crystal-negative B. thuringiensis host. The proteins accumulated as inclusions. Purified inclusions containing either Cry19A alone or Cry19A and ORF2 together were toxic to Anopheles stephensi and Culex pipiens mosquito larvae. They were more toxic to C. pipiens than to A. stephensi. However, inclusions containing Cry19A and ORF2 together were more toxic than inclusions of Cry19A alone but less toxic than the wild-type inclusions of B. thuringiensis subsp. jegathesan.     

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.     

Co-Expression of the Mosquitocidal Toxins Cyt1Aa and Cry11Aa from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">israelensis</Emphasis> in <Emphasis Type="Italic">Asticcacaulis excentricus</Emphasis>

The cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti), whose product synergizes other mosquitocidal toxins, and functions as a repressor of resistance developed by mosquitoes against Bacilli insecticides, was introduced into the aquatic Gram-negative bacterium Asticcacaulis excentricus alongside the cry11Aa gene. The genes were introduced as an operon, but although mRNA was detected for both genes, no Cyt1Aa toxin was detected. Both proteins were expressed using a construct in which a promoter was inserted upstream of each gene. Recombinant A. excentricus expressing both toxins was found to be approximately twice as toxic to third instar larvae of Culex quinquefasciatus as transformants expressing just Cry11Aa.     

Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth larvae resistant to Cry1A or Cry1C

A synthetic Bacillus thuringiensis (Bt) cry1C gene was introduced into broccoli (Brassica oleracea ssp. italica) by Agrobacterium-mediated transformation. Twenty-one Cry1C transgenic plants were regenerated from 400 hypocotyl and petiole explants. Variable amounts of stable steady- state cry1C mRNA accumulated in different transgenic plants. Cry1C protein (up to 0.4% of total soluble protein) was produced in correlation with the cry1C mRNA levels. Leaf section and whole-plant bioassays were done using diamondback moth (DBM) larvae from lines susceptible to Bt or resistant to Cry1A or Cry1C proteins (Cry1AR or Cry1CR, respectively). Plants with high levels of Cry1C protein caused rapid and complete mortality of all three types of DBM larvae with no defoliation. Plants with lower levels of Cry1C protein showed an increasing differential between control of susceptible of Cry1AR DBM. This study demonstrated that high production of Cry1C protein can protect transgenic broccoli not only from susceptible or Cry1AR DBM larvae but also from DBM selected for moderate levels of resistance of Cry1C. The Cry1C- transgenic broccoli were also resistant to two other lepidopteran pests of crucifers (cabbage looper and imported cabbage worm). These plants will be useful in studies of resistance management strategies involving multiple transgenes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Functional analysis of two processed fragments of Bacillus thuringiensis Cry11A toxin

The 70-kDa protoxin of Cry11A, a dipteran-specific insecticidal protein, was processed by trypsin into 36- and 32-kDa fragments. To investigate the potent function of the two processed fragments, a GST (Glutathione-S-transferase) fusion protein of each polypeptide was constructed. While neither the 36- nor the 32-kDa fragment was toxic to Culex pipiens larvae, coexpression of the two fragments restored the insecticidal activity. Furthermore, the coprecipitation experiment demonstrated that the 36-kDa fragment was associated with the 32-kDa fragment. It was, therefore, shown that the coexistence of the two processed fragments of Cry11A was essential for the toxicity. The mutant of the 36-kDa fragment lacking the region from Gly(257) to Arg(360) bound to the 32-kDa fragment but the coexpression with the 32-kDa fragment resulted in no toxicity, suggesting that this region was involved in insecticidal activity.     

Efficient expression of vip184DeltaP gene under the control of promoters plus Shine-Dalgarno (SD) sequences of cry genes from Bacillus thuringiensis

AIMS: To compare vip184DeltaP gene expression time course and Vip184 protein yield under the control of promoters and Shine-Dalgarno (SD) sequences of vip184, cry3A and cry1A gene from Bacillus thuringiensis respectively. METHODS AND RESULTS: Derived from the shuttle vector pHT3101, recombinant plasmids pHPT3, pHTP3A(Delta)P and pHTP1A(Delta)P were constructed with the native vip184 gene and the vip184(Delta)P gene, either under the control of promoters and SD sequences of cry3A or cry1A genes. When the above plasmids were transformed into an acrystalliferous B. thuringiensis strain Cry(-)B, their expression time course were consistent with those of vip184, cry3A and cry1A gene respectively. The maximum yields of Vip184 protein were increased when under the control of promoters plus SD sequences of cry3A and cry1A gene. CONCLUSIONS: The results showed that both cry3A and cry1A promoter/SD sequence combinations were able to enhance synthesis of Vip184 and change its expression time course. SIGNIFICANCE AND IMPACT OF THE STUDY: Both cry3A and cry1A promoter/SD systems offer a method for improving the expression efficacy of the vip184 gene in B. thuringiensis and it is possible to co-express the vip184 gene and cry genes and accumulate Vip184 in the form of inclusion bodies by these systems in order to construct novel useful B. thuringiensis engineered strains.     

Toxic activity of Bacillus Thuringiensis isolates to Aedes Aegypti (L.) (Diptera: Culicidae) larvae

Aedes aegypti (L.), the main vector of dengue fever in Brazil, has been controlled with the use of massive chemical products, contributing to the development of resistance and decreasing the insect control efficiency. The control of dipterans with bioinsecticides based on Bacillus thuringiensis has been satisfactory, due to the production of insecticidal proteins denominated Cry (crystal), Cyt (cytolytic) toxins and Chi (chitinase), and to the synergistic effects among them. The present work aimed to select B. thuringiensis isolates efficient against A. aegypti larvae. A bacterial collection containing 1,073 isolates of B. thuringiensis, obtained from different locations of Brazilian territory, had the DNA isolated and submitted to PCR amplifications using specific primers for cry4Aa, cry4Ba, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa and chi genes. For the LC50 and LC90 determination, the entomopathogenic isolates were evaluated by selective and quantitative bioassays. Only 45 isolates (4.2%) presented amplicons for the cry and cyt genes. The chi gene sequence was detected in 25 (54.3%) of those isolates. From the 45 isolates submitted to the selective bioassays, 13 caused 100% mortality of A. aegypti larvae. The identification of cry, cyt and chi genes of B. thuringiensis and the toxicity analysis on A. aegypti led to the selection of a set of isolates that have the potential to be used in the formulation of new bioinsecticides.