Toxicity and synergism in transgenic Escherichia coli expressing four genes from Bacillus thuringiensis subsp. israelensis

The genes cyt1Aa and p20 , encoding, respectively, cytolytic and accessory proteins of Bacillus thuringiensis subsp. israelensis , were introduced into previously constructed clones expressing cry4Aa and cry11Aa in Escherichia coli ( Ben-Dov et al ., 1995 ). Fifteen clones with all possible combinations of the four genes were obtained and found to express the genes included. Two new combinations, pVE4-ADRC and pVE4-ARC, expressing cyt1Aa , p20 and cry4Aa , with or without cry11Aa , respectively, were more toxic than their counterparts without cyt1Aa . They displayed the highest toxicity against Aedes aegypti larvae ever reached in transgenic bacteria. Five out of the six clones (except pVE4-DC) containing cry4Aa or cry11Aa (with or without p20 ) displayed varying levels of synergism with cyt1Aa : they are 1.5-to 34-fold more toxic than the respective clones without cyt1Aa against exposed larvae. Their lethal times also decreased (they kill larvae quicker), more so at higher cell concentrations. These clones are anticipated to dramatically reduce the likelihood of resistant development in the target organisms ( Wirth et al ., 1997 ).     

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。     

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.     

Enduring toxicity of transgenic Anabaena PCC 7120 expressing mosquito larvicidal genes from Bacillus thuringiensis ssp. israelensis

Persistence of biological control agents against mosquito larvae was tested under simulated field conditions. Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing cry4Aa, cry11Aa and p20 from Bacillus thuringiensis ssp. israelensis was greater than B. thuringiensis ssp. israelensis primary powder (fun 89C06D) or wettable powder (WP) (Bactimos products) when either mixed with silt or exposed to sunlight outdoors. Reduction of Bactimos primary powder toxicity was at least 10-fold higher than Anabaena's after mixing with silt. In outdoors experiments, Bactimos WP remained toxic (over 30% mortality of 3rd instar Aedes aegypti larvae) for 2-4 days only, while transgenic Anabaena's toxicity endured 8-21 days.     

cry4Aa、cry4Ba和cry11Aa基因在苏云金杆菌无晶体型菌株中的表达

利用穿梭载体pBU4,将苏云金杆菌以色列亚种（Bti)的cry4Aa、cry4Ba和cry11Aa基因分别转入Bti无晶体突变株4Q7中,获得了转化菌株Bt-B601、Bt-B611和Bt-B640。SDS－PAGE结果显示：cry4Aa、cry4Ba和cry11Aa蛋白均分别获得了表达。透射电镜下观察,转化菌 有产生球形或菱形伴胞晶体。转化菌株对敏感和抗性致倦库蚊及白纹伊蚊幼虫的生物测定结果显示：cry4Aa、cry4Ba和cry11Aa蛋白对库蚊和伊蚊的毒力较低,二元毒素抗性库蚊幼虫对Bti杀蚊毒素蛋白无明显的交叉抗性。     

A plasmid encoding a combination of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus confers toxicity against a broad range of mosquito larvae when expressed in Gram-negative bacteria

A recombinant plasmid harboring cry4A, cry4B and cry11A from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus has been constructed. The three cry genes were placed under the control of the cry4B promoter whereas the binary toxin gene was controlled by its native promoter. The expression of toxins in Escherichia coli harboring the resulting plasmid, p4BDA-5142, was investigated. Cry4B expression was highest compared to other toxins. Although the level of toxin expression was low compared with E. coli expressing single toxins, the recombinant E. coli strain harboring p4BDA-5142 exhibited broad range mosquito-larvicidal activity against all Aedes, Culex and Anopheles larvae. This work has shown that the development of the recombinant plasmid can be used to broaden the host range spectrum of the appropriate bacterial host for mosquito control.     

Diversity of Bacillus thuringiensis strains from Latin America with insecticidal activity against different mosquito species

The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented. Characterization was based on their insecticidal activity against Aedes aegypti, Culex quinquefasciatus, and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for cry and cyt genes encoding proteins active against mosquitoes (cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40). Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A. aegypti and C. quinquefasciatus larvae than B. thuringiensis subsp. israelensis and displayed high similarities with the B. thuringiensis subsp. israelensis used in this study with regard to protein and plasmid profiles and the presence of cry genes. Strain 147-8906 has activity against A. aegypti similar to that of B. thuringiensis subsp. israelensis but has different protein and plasmid profiles. This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions. Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene.     

Co-expression of Bacillus thuringiensis Cry4Ba and Cyt2Aa2 in Escherichia coli revealed high synergism against Aedes aegypti and Culex quinquefasciatus larvae

Cry4Ba is a delta-endotoxin produced by Bacillus thuringiensis subsp. israelensis and Cyt2Aa2 is a cytolytic delta-endotoxin produced by B. thuringiensis subsp. darmstadiensis. Cry4Ba produced in Escherichia coli was toxic to Aedes aegypti larvae (LC(50)=140 ng ml(-1)) but virtually inactive to Culex quinquefasciatus larvae. Cyt2Aa2 expressed in E. coli exhibited moderate activity against A. aegypti and C. quinquefasciatus larvae with LC(50) values of 350 and 250 ng ml(-1), respectively. Co-expression of both toxins in E. coli dramatically increased toxicity to both A. aegypti andC. quinquefasciatus larvae (LC(50)=7 and 20 ng ml(-1), respectively). This is the first report to demonstrate that Cry4Ba and Cyt2Aa2 have high synergistic activity against C. quinquefasciatus larvae.     

苏云金杆菌以色列亚种杀蚊蛋白Cyt1Aa在离中不粘柄菌中的表达

在蚊幼虫生活水域里的离中不粘柄菌(Asticcacaulis excentricus,Ae)中已成功表达苏云金芽孢杆菌以色列亚种(Bacillus thuringiensis subsp.israelensis,Bti)杀蚊蛋白基因cry11Aa的基础上,将另一Bti杀蚊蛋白基因cyt1Aa转化入Ae中表达。构建并转化了分别单独含有cyt1Aa基因、及同时含有cry11Aa基因的表达质粒pSODCyt20和pSODCryCyt20,蛋白免疫杂交检测相应的Ae重组子分别表达产生了Cyt1Aa和Cry11Aa蛋白。为了探究Ae(pSODCryCyt20)重组子不能表达cyt1Aa的原因,提取了重组子总RNA、并与同是革兰氏染色阴性的大肠杆菌的总RNA比较,结果显示两者RNA系统显著不同,推测Ae中多个外源基因的表达,可能要求每个基因必需一个启动子。     

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.     

Variations in the mosquito larvicidal activities of toxins from Bacillus thuringiensis ssp. israelensis

Comparing activities of purified toxins from Bacillus thuringiensis ssp. israelensis against larvae of seven mosquito species (vectors of tropical diseases) that belong to three genera, gleaned from the literature, disclosed highly significant variations in the levels of LC₅₀ as well as in the hierarchy of susceptibilities. Similar toxicity comparisons were performed between nine transgenic Gram-negative species, four of which are cyanobacterial, expressing various combinations of cry genes, cyt1Aa and p20 , against larvae of four mosquito species as potential agents for biological control. Reasons for inconsistencies are listed and discussed. Standard conditions for toxin isolation and presentation to larvae are sought. A set of lyophilized powders prepared identically from six Escherichia coli clones expressing combinations of four genes displayed toxicities against larvae of three mosquito species, with levels that differed between them but with identical hierarchy.     

Cyt1A of Bacillus thuringiensis delays evolution of resistance to Cry11A in the mosquito Culex quinquefasciatus

Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.     

球形芽孢杆菌和苏云金芽孢杆菌以色列亚种杀蚊毒素间的协同作用

本研究测定了分别表达苏云金芽孢杆菌Cry4Aa、Cry4Ba、Cry11Aa、Cyt1Aa和球形芽孢杆菌二元毒素Bin的转化菌株Bt B60 1、Bt B611、Bt B640、Bt U 30和Bt CW 3全发酵培养物两两或两两以上不同组合对抗性库蚊的毒力 ,分析了杀蚊毒素间的协同作用。结果表明 ,Bin和Cry4Aa、Bin和Cry 4Ba间有明显的协同作用 ,此外 ,Cry4Aa和Cry4Ba、Cry4Aa和Cry11Aa、Cyt1Aa和Cry4Aa之间也有明显的协同作用     

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.     

Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain.

A gene designated cyt1Ab1, encoding a 27,490-Da protein, was isolated from Bacillus thuringiensis subsp. medellin (H30 serotype) by using an oligonucleotide probe corresponding to the cyt1Aa1 gene. The sequence of the Cyt1Ab1 protein, as deduced from the sequence of the cyt1Ab1 gene, was 86% identical to that of the Cyt1Aa1 protein and 32% identical to that of the Cyt2Aa1 protein from B. thuringiensis subsp. kyushuensis. The cyt1Ab1 gene was flanked upstream by a p21 gene, in the same orientation, encoding a 21,370-Da protein that showed 84% similarity to the putative chaperone P20 protein from B. thuringiensis subsp. israelensis and downstream, on the opposite strand, by a sequence showing 85% identity to the IS240A insertion sequence. The cyt1Ab1 gene was expressed at a high level in a nontoxic strain of B. thuringiensis subsp. israelensis in which large inclusions of the Cyt1Ab1 protein were produced. Purified Cyt1Ab1 crystals were as hemolytic as those of the Cyt1Aa1 protein and were twice as hemolytic as those from the wild-type strain. Mosquitocidal activity toward Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae was assayed. The toxicity of the Cyt1Ab1 protein was slightly lower than that of the Cyt1Aa1 protein for all three mosquito species, and Cyt1Ab1 was 150, 300, and 800 times less active toward Culex, Anopheles, and Aedes larvae, respectively, than were the native crystals from B. thuringiensis subsp. medellin.     

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.     

Cytolytic toxin Cyt1Aa of Bacillus thuringiensis synergizes the mosquitocidal toxin Mtx1 of Bacillus sphaericus

Using the shuttle vector pBU4, the mosquitocidal toxin gene mtx1 from Bacillus sphaericus strain SSII-1 was introduced into an acrystalliferous strain of B. thuringiensis both individually and in combination with the accessory protein gene p20 and the cytolytic protein gene cyt1Aa from B. thuringiensis subsp. israelensis. Bioassay results indicated that the recombinants B-pMT4(Mtx1) and B-pMT9(Mtx1), both individually containing mtx1, had moderate toxicities to binary toxin susceptible and binary toxin resistant Culex quinquefasciatus larvae during the vegetative growth stage, but that their toxicities declined rapidly during the sporulation phase. The LC50 values were 2.5 and 4.8 mg/ml respectively, against 3-4 instar susceptible and resistant larvae for the final sporulated cultures of recombinants B-pMT9(Mtx1), and little toxicity was detected for B-pMT4(Mtx1). Meanwhile, the recombinant B-pMPX2(Mtx1+Cyt1Aa) expressing Mtx1, P20 alone, and Cyt1Aa in combination had stable toxicities during both the vegetative phase and the sporulation phase, with a LC50 ranging from 0.45-0.58 mg/ml. Furthermore, expression of Cyt1Aa appeared to enhance the activity of Mtx1 to target mosquito larvae, suggesting a synergism between Cyt1Aa and Mtx1 toxins.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression.

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.