Cross-resistance spectra of Culex quinquefasciatus resistant to mosquitocidal toxins of Bacillus thuringiensis towards recombinant Escherichia coli expressing genes from B. thuringiensis ssp. israelensis

Sixteen Escherichia coli clones were assayed against susceptible and Bacillus thuringiensis-resistant Culex quinquefasciatus larvae. The clones expressed different combinations of four genes from Bacillus thuringiensis ssp. israelensis; three genes encoded mosquitocidal toxins (Cry11Aa, Cry4Aa and Cyt1Aa) and the fourth encoded an accessory protein (P20). The cross-resistance spectra of the mosquitoes were similar to the profiles for recombinant B. thuringiensis strains expressing B. thuringiensis toxin genes, but with varied toxicity levels. The toxicity of the recombinants towards resistant mosquito larvae was improved when p20 and cyt1Aa were expressed in combination with cry4Aa and/or cry11Aa. Recombinant pVE4-ADRC, expressing cry4Aa, cry11Aa, p20 and cyt1Aa, was the most active against the resistant Culex, and resistance levels did not exceed fourfold. These results indicate that B. thuringiensis ssp. israelensis genes expressed in a heterologous host such as E. coli can be effective against susceptible and B. thuringiensis-resistant larvae and suppress resistance.     

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.     

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.     

Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis.

Mosquitocidal Bacillus thuringiensis strains show as a common feature the presence of toxic proteins with cytolytic and hemolytic activities, Cyt1Aa1 being the characteristic cytolytic toxin of Bacillus thuringiensis subsp. israelensis. We have detected the presence of another cyt gene in this subspecies, highly homologous to cyt2An1, coding for the 29-kDa cytolytic toxin from B. thuringiensis subsp. kyushuensis. This gene, designated cyt2Ba1, maps upstream of cry4B coding for the 130-kDa crystal toxin, on the 72-MDa plasmid of strain 4Q2-72. Sequence analysis revealed, as a remarkable feature, a 5' mRNA stabilizing region similar to those described for some cry genes. PCR amplification and Southern analysis confirmed the presence of this gene in other mosquitocidal subspecies. Interestingly, anticoleopteran B. thuringiensis subsp. tenebrionis belonging to the morrisoni serovar also showed this gene. On the other hand, negative results were obtained with the anti-lepidopteran strains B. thuringiensis subsp. kurstaki HD-1 and subsp. aizawai HD-137. Western analysis failed to reveal Cyt2A-related polypeptides in B. thuringiensis subsp. israelensis 4Q2-72. However, B. thuringiensis subsp. israelensis 1884 and B. thuringiensis subsp. tenebrionis did show cross-reactive products, although in very small amounts.     

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.     

Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins.

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.     

Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.     

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 ).     

Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan.

A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.     

Medium for the production of primary powder of Bacillus thuringiensis subsp. israelensis

Five media, formulated from the seeds of five legume varieties, dried cow blood, and mineral salts, were assessed for the growth and production of insecticidal properties of Bacillus thuringiensis subsp. israelensis. Bacterial powders prepared from the broth cultures were assayed against the larvae of Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A standard primary powder of B. thuringiensis subsp. israelensis (IPS78) was included in the assay for comparison. Good growth was obtained in all the media, and all powders were effective against the three types of mosquito larvae. The powder containing ground seeds of Voandzeia subterranean was the most effective and compared favorably with the standard (IPS78). The concentrations required to kill 50% of the larvae of Aedes aegypti, C. quinquefasciatus, and Anopheles gambiae were 1.13 X 10(-2) +/- 1.79 X 10(-3), 1.83 X 10(-2) +/- 2.55 X 10(-3), and 2.25 X 10(-2) +/- 1.88 X 10(-3) micrograms/ml, respectively. This investigation shows that the medium containing V. subterranean can be used for the production of B. thuringiensis subsp. israelensis primary powder.     

Medium for the production of primary powder of Bacillus thuringiensis subsp. israelensis.

Five media, formulated from the seeds of five legume varieties, dried cow blood, and mineral salts, were assessed for the growth and production of insecticidal properties of Bacillus thuringiensis subsp. israelensis. Bacterial powders prepared from the broth cultures were assayed against the larvae of Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A standard primary powder of B. thuringiensis subsp. israelensis (IPS78) was included in the assay for comparison. Good growth was obtained in all the media, and all powders were effective against the three types of mosquito larvae. The powder containing ground seeds of Voandzeia subterranean was the most effective and compared favorably with the standard (IPS78). The concentrations required to kill 50% of the larvae of Aedes aegypti, C. quinquefasciatus, and Anopheles gambiae were 1.13 X 10(-2) +/- 1.79 X 10(-3), 1.83 X 10(-2) +/- 2.55 X 10(-3), and 2.25 X 10(-2) +/- 1.88 X 10(-3) micrograms/ml, respectively. This investigation shows that the medium containing V. subterranean can be used for the production of B. thuringiensis subsp. israelensis primary powder.     

The Bacillus thuringiensis cyt genes for hemolytic endotoxins constitute a gene family

In the same way that cry genes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, the cyt genes for hemolytic toxins seem to compose another set of highly related genes in Bacillus thuringiensis. Although the occurrence of Cyt hemolytic factors in B. thuringiensis has been typically associated with mosquitocidal strains, we have recently shown that cyt genes are also present in strains with different pathotypes; this is the case for the morrisoni subspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae. In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such as B. thuringiensis subsp. israelensis and subsp. morrisoni PG14, and that this could also be the case for many others. In fact, PCR screening and Western blot analysis of 50 B. thuringiensis strains revealed that cyt2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges. Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains. These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of many B. thuringiensis strains.     

Characterization of Cyt2Bc toxin from Bacillus thuringiensis subsp. medellin

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.     

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.     

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.     

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.     

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.     

Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects

AIM: To evaluate the genetic and molecular diversity and insecticidal activity of Bacillus thuringiensis isolates from all the natural regions of Colombia. METHODS AND RESULTS: A total of 445 isolates from a collection of B. thuringiensis were characterized. The parasporal crystal morphology that was most abundant was bipyramidal (60%). Almost 10% of the isolates were toxic to Spodoptera frugiperda and 5.6% against Culex quinquefasciatus larvae. cry gene content determined by PCR indicated that 10.6% of the isolates contained cry1 genes and 1.1% contained cry2, cry4 or cry11 genes. Protein content of the parasporal crystal was determined by SDS-PAGE; 25 and 18 different protein profiles were found in isolates active against S. frugiperda and C. quinquefasciatus, respectively. CONCLUSIONS: Bacillus thuringiensis presents great genetic and molecular diversity even in isolates from the same soil sample. Moreover, the diversity and activity of the isolates might have a relationship with the geographical origin of the samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here indicate that some of the B. thuringiensis isolates characterized in this study are potential control agents that could be used in programmes against mosquitoes and S. frugiperda.     

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。