Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases

The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect-specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines.     

Activated insecticidal crystal proteins from Bacillus thuringiensis serovars killed adult house flies

Insecticidal crystal proteins (ICP) from Bacillus thuringiensis serovar kurstaki HD-1 and HD-73 were activated by immobilized trypsin or chymotrypsin. The activated toxins (10 μ g or more) as well as unactivated ICP killed adult house flies but not larvae. Bacillus thuringiensis strain son diego did not kill house flies. In this experimental system, the average life span of the adult house fly was 8 days and the activated toxins reduced it to 2 days. The unactivated insecticidal crystal protein also reduced it to 4 days at the same concentration as the activated toxin.     

Genomic amplification and expression of delta-endotoxin fragment of Bacillus thuringiensis.

delta-Endotoxin gene of Bacillus thuringiensis HD-1 var kurstaki codes for the insecticidal crystal protein (ICP) specific for lepidopteran insects. Since the N-terminal half of the toxin is sufficient both for insect specificity and toxicity, the coding sequence of this part of the gene CryIA(b) was amplified by PCR and cloned in pUC19. As there was no expression of immunologically detectable delta-endotoxin in this clone in E. coli, the amplified ICP gene was transferred to an expression vector pGEx2T. Restriction mapping and immunoblotting confirmed the presence and expression of the CryIA(b) gene. This insert should be suitable for expression in plant system if it is mobilized into a plant binary vector.     

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains.

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.     

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.     

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.     

Differential specificity of two insecticidal toxins from Bacillus thuringiensis var. aizawai

Bacillus thuringiensis var. aizawai HD-249 produces more than one protein of 130-135 kD in its insecticidal crystal delta-endotoxin. We describe an indirect method of assessing the relative contribution to toxicity of two of these protoxins using monospecific antibodies directed against their active proteolytic products. Our results show that one toxin is active against Spodoptera frugiperda but not Choristoneura fumiferana cells in vitro, while the other lyses C. fumiferana but not S. frugiperda cells. There is no indication of synergism between these toxins in vitro.     

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.     

Comparison of the in vivo and in vitro activity of the delta-endotoxin of Bacillus thuringiensis var. morrisoni (HD-12) and two of its constituent proteins after cloning and expression in Escherichia coli

The insecticidal crystal delta-endotoxin of Bacillus thuringiensis var. morrisoni HD-12 contains at least five polypeptides in the range 126-140 kDa. Immune blotting revealed that individual proteins in this complex share homology with a range of other B. thuringiensis delta-endotoxins. In vivo the native HD-12 crystal killed a lepidopteran larva (Pieris brassicae) and a dipteran larva (Anopheles gambiae), but not the related dipteran Aedes aegypti. In vitro the solubilized activated crystal lysed Choristoneura fumiferana cells (lepidopteran) and dipteran cells derived from Anopheles gambiae and Culex quinquefasciatus but not those from Aedes aegypti. An intragenic probe derived from a B. thuringiensis var. sotto lepidoptera-specific delta-endotoxin gene hybridized with one of six plasmids extracted from HD-12. When cloned into pUC18 two HindIII fragments from this plasmid (pEG1 and pEG2) were shown to encode polypeptides cross-reacting with HD-12 antiserum. Escherichia coli lysates containing pEG2 were toxic in vivo to lepidoptera and diptera larvae and in vitro to a broader range of insect cell lines than the native crystal. E. coli cells containing pEG3, a subclone derived from pEG1, synthesised large amounts of a 140-kDa protein in the cytoplasm as inclusion bodies. The cytotoxicity of the protein encoded by pEG3 was restricted to C. fumiferana and A. gambiae cell lines.     

The inactivation of Bacillus thuringiensis subsp. israelensis toxin by mosquito larvae proteases liberated into the medium

Crystal serine-proteases of B. thuringiensis subsp. israelensis were able to process the 28,000-dalton protein during crystal solubilization. On the other hand, solubilized crystal proteins were degraded during the larvicidal bioassay by the action of serine-proteases liberated by mosquito larvae into the medium, with loss of toxicity. However, proteins in intact crystals were protected from the action of these proteases. This resistance to degradation of crystals partly explains the observation that they are more toxic than solubilized crystal proteins.     

The parasporal inclusion of Bacillus thuringiensis subsp. shandongiensis: characterization and screening for insecticidal activity.

The parasporal body of Bacillus thuringiensis subsp. shandongiensis was characterized in terms of its structure, protein composition, and toxicological properties against several types of insects. The crystals of B. thuringiensis shandongiensis appear to consist of a major protein of 144 kDa present in an spherical inclusion, as determined by transmission electron microscopy, titration curve analysis, and SDS-PAGE of the solubilized crystals. A second protein of ca. 60 kDa is present in trace amounts and appears to be associated with a small bar-shaped inclusion. The 144-kDa protein has been characterized by isoelectric point determination, N-terminal amino acid sequence analysis, amino acid analysis, and immunological cross reactivity. Its N-terminal amino acid sequence differed from that of other B. thuringiensis crystal proteins. The 144-kDa protein was not immunologically related to the crystal proteins of two toxic serovars (B. thuringiensis israelensis and B. thuringiensis kurstaki HD-1) and one nontoxic serovar (B. thuringiensis indiana), as shown in immunoblots probed with antiserum raised against the 144-kDa B. thuringiensis shandongiensis protein, the B. thuringiensis israelensis crystal proteins, and the trypsin resistant fragment of B. thuringiensis kurstaki P1 proteins. In contrast to most B. thuringiensis serovars, B. thuringiensis shandongiensis crystals did not dissolve at pH 12. Solubilization was achieved in sodium bicarbonate at pH 8.3 and in the presence of 25 mM dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego

A gene from Bacillus thuringiensis subsp. san diego that is responsible for a delta-endotoxin active against Colorado potato beetle and some other Coleoptera was sequenced and shown to have surprising regional homology with both lepidopteran and dipteran active delta-endotoxins from other strains of B. thuringiensis. Unlike the lepidopteran active toxins from B. thuringiensis subsp. kurstaki that exist as approx. 130-kDa protoxins and form bipyramidal crystalline inclusions, the coleopteran toxic protein forms a square-shaped crystal composed of an approx. 65-kDa protein. Comparisons of the gene sequences encoding the active portions of these protoxins indicate conservation of N-terminal hydrophilic and hydrophobic regions, and suggest a distant ancestral origin for these insecticidal proteins.     

球形芽孢杆菌Mtx1蛋白和苏云金杆菌Cyt1Aa晶体蛋白的协同作用

采用常规的生物测定方法确定了纯化的球形芽孢杆菌(Bacillus sphaericus)的缺失信号肽的97kDa营养期杀蚊毒素(Mosquitocidal toxin 1,Mtx1)蛋白和苏云金芽孢杆菌(Bacillus thuringiensis)27.3kDa的Cyt1Aa晶体蛋白对致倦库蚊(Culex quinquefasciatus)幼虫的杀虫活性。结果表明Mtx1和Cyt1Aa不同比例的混合物对致倦库蚊的毒力比单独毒素蛋白高,经统计分析表明两毒素蛋白对目标蚊幼虫具有明显的协同作用。在LC98处理浓度下,Mtx1和Cyt1Aa按3∶1混合的混合物LT50值比单独Mtx1的提前了6.36h。表明Cyt1Aa和Mtx1对致倦库蚊具有协同毒杀作用,提高对目标蚊虫的毒力、缩短半致死时间。该结果为深入研究Mtx1和Cyt1Aa的杀蚊作用方式奠定了基础,同时为其在蚊虫防治中的应用提供了新的思路和方法。     

Location of the dipteran specificity region in a lepidopteran-dipteran crystal protein from Bacillus thuringiensis.

Two highly related crystal protein genes from Bacillus thuringiensis subsp. kurstaki HD-1, designated cryIIA and cryIIB (previously named cryB1 and cryB2, respectively), were used to study host range specificity. Their respective gene products are 87% identical but exhibit different toxicity spectra; CryIIA is toxic to both mosquito and tobacco hornworm larvae, whereas CryIIB is toxic only to the latter. Hybrids of the cryIIA and cryIIB genes were generated, and their resultant gene products were assayed for toxicity. A short segment of CryIIA corresponding to residues 307 through 382 was shown to be sufficient for altering host range specificity-i.e., when this region replaced the corresponding segment of CryIIB, the resulting hybrid protein acquired toxicity against mosquitoes. The CryIIA and CryIIB polypeptides differ by only 18 amino acids in this region, indicating that very few amino acid changes can have a substantial effect on the toxicity spectra of these proteins.     

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.     

Decreased toxicity of Bacillus thuringiensis subsp. israelensis to mosquito larvae after contact with leaf litter

Bacillus thuringiensis subsp. israelensis is a bacterium producing crystals containing Cry and Cyt proteins, which are toxic for mosquito larvae. Nothing is known about the interaction between crystal toxins and decaying leaf litter, which is a major component of several mosquito breeding sites and represents an important food source. In the present work, we investigated the behavior of B. thuringiensis subsp. israelensis toxic crystals sprayed on leaf litter. In the presence of leaf litter, a 60% decrease in the amount of Cyt toxin detectable by immunology (enzyme-linked immunosorbent assays [ELISAs]) was observed, while the respective proportions of Cry toxins were not affected. The toxicity of Cry toxins toward Aedes aegypti larvae was not affected by leaf litter, while the synergistic effect of Cyt toxins on all B. thuringiensis subsp. israelensis Cry toxins was decreased by about 20% when mixed with leaf litter. The toxicity of two commercial B. thuringiensis subsp. israelensis strains (VectoBac WG and VectoBac 12AS) and a laboratory-produced B. thuringiensis subsp. israelensis strain decreased by about 70% when mixed with leaf litter. Taken together, these results suggest that Cyt toxins interact with leaf litter, resulting in a decreased toxicity of B. thuringiensis subsp. israelensis in litter-rich environments and thereby dramatically reducing the efficiency of mosquitocidal treatments.     

Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains.

Parasporal crystals of Bacillus thuringiensis subspp. kurstaki, tolworthi, alesti, berliner, and israelensis were compared by electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, immunological analysis, and insecticidal activity. Spore coats also were compared by polyacrylamide gel electrophoresis. B. thuringiensis subsp. israelensis crystals were lethally toxic to mosquito larvae and nontoxic to tobacco hornworm larvae. Conversely, crystals from the other subspecies killed tobacco hornworm larvae but were ineffective against mosquitoes. Crystalline inclusion bodies of all subspecies contained a protoxic subunit that had an apparent molecular weight of approximately 1.34 X 10(5). However, polyacrylamide gel electrophoretic patterns of solubilized crystals revealed a small-molecular-weight component (apparent molecular weight, 26,000) in B. thuringiensis subsp. israelensis that was absent in the other subspecies. Also, differences were noted in amino acid composition and tryptic peptide fingerprints. Crystal proteins were found in spore coats of all subspecies. The results suggest that insecticidal specificity is due to unique polypeptide toxins.     

苏云金杆菌伴孢晶体的蛋白质和抗蛋白酶多肽及其毒力特性

以SDS-聚丙烯酰胺凝胶电泳分析了19种苏云金杆菌伴孢晶体的蛋白质和抗胰蛋白酶多肽。以鳞翅目的家蚕和双翅目的致倦库蚊进行了毒力测定。根据蛋白质和抗酶多肽的特性,19种晶体可分为7个类型。晶体蛋白质的组成、溶解特性及抗酶多肽的性质与其对两种昆虫的毒力特性密切相关。含分子量为130—138 kD(千道尔顿)或130—138 kD及60一65kD蛋白质,抗蛋白酶多肽(PRP)为68—75kD的晶体,对家蚕高毒,但绝大部分对库蚊无毒或微毒。含3种以上蛋白质(15一138kD),抗酶多肽为35—65kD的晶体,对库蚊有强烈毒性,对家蚕则无毒。缺少135kD蛋白质的两种晶体对家蚕低毒。HD一282和L—14晶体的P1蛋白质中同时含有杀鳞翅目和杀蚊毒素。讨论了伴孢晶体的蛋白质结构及结构与毒力之间的关系、晶体蛋白质及毒性肽的性质在菌株定向筛选和遗传工程研究中的意义。     

Integration of the delta-endotoxin gene of Bacillus thuringiensis into the chromosome of root-colonizing strains of pseudomonads using Tn5

The delta-endotoxin gene (tox) from Bacillus thuringiensis subsp. kurstaki HD-1 was cloned into Tn5 and the resulting Tn5-tox element transposed from a vector plasmid into the chromosome of six corn-root-colonizing strains of Pseudomonas fluorescens and Agrobacterium radiobacter. Chromosomal integration of the tox gene maximized stability and minimized the potential for horizontal transfer of the tox gene to other bacterial species. Expression of the tox gene was demonstrated by Western blot analysis and by toxicity against larvae of the tobacco hornworm (Manduca sexta). The method described illustrates how a given gene can be stably integrated into the chromosome of diverse bacterial species.     

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.