Mode of Action of Bipyramidal delta-Endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1

The mode of action of the toxic fragment (P-59) derived from bipyramidal-shaped delta-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 on the silkworm Bombyx mori was investigated. An enzyme-linked immunosorbent assay showed that there was no translocation of P-59 from the gut lumen to the hemocoel. When membrane vesicles prepared from silkworm midgut were incubated with P-59, normally smooth surface of vesicles became rough, and patch formation was observed on the surface. Vesicles treated with P-59 tended to agglutinate. The vesicle-denaturing activity of a 130,000-dalton subunit protein of bipyramidal toxin was enhanced by treatment with a gut juice protease of the silkworm. P-59 did not cause any uncoupling effect on mitochondria of the silkworm midgut. These results suggest that the attacking site of this toxin is not the mitochondrion but the cell membrane of the susceptible cell.     

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD₅₀]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD₅₀ of HD-1 with the FFD₅₀ expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.     

Mode of Action of Bipyramidal δ-Endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1

The mode of action of the toxic fragment (P-59) derived from bipyramidal-shaped δ-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 on the silkworm Bombyx mori was investigated. An enzyme-linked immunosorbent assay showed that there was no translocation of P-59 from the gut lumen to the hemocoel. When membrane vesicles prepared from silkworm midgut were incubated with P-59, normally smooth surface of vesicles became rough, and patch formation was observed on the surface. Vesicles treated with P-59 tended to agglutinate. The vesicle-denaturing activity of a 130,000-dalton subunit protein of bipyramidal toxin was enhanced by treatment with a gut juice protease of the silkworm. P-59 did not cause any uncoupling effect on mitochondria of the silkworm midgut. These results suggest that the attacking site of this toxin is not the mitochondrion but the cell membrane of the susceptible cell.     

Comparative toxicity of the HD-1 and NRD-12 strains of Bacillus thuringiensis subsp. kurstaki to defoliating forest Lepidoptera.

Insecticidal activities of sporulated cultures of the HD-1 and NRD-12 strains of Bacillus thuringiensis subsp. kurstaki were compared against four species of defoliating forest lepidopterans in diet-incorporation assays. There was no difference in LC50 between the two strains to larvae of spruce budworm (Choristoneura fumiferana), gypsy moth (Lymantria dispar), eastern hemlock looper (Lambdina fiscellaria fiscellaria), and whitemarked tussock moth (Orgyia leucostigma), whether expressed as total alkaline soluble protein, activated toxin protein, or International Units as determined by bioassay against Trichoplusia ni. Both strains were consistently more toxic than HD-1-S-1980 when compared on the basis of alkali-soluble protein, but not on the basis of activated toxin or International Units. Hybridization of genomic DNA after restriction with HindIII revealed the presence of all three cryIA toxin genes in each of the isolates used in this study, including HD-1-S-1980, which was previously reported to have lost the cryIA(b) gene.     

Purification of Vip3Aa from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera)

We developed a protocol for obtaining high yields (10-15 mg per 1100 ml of culture supernatant) of highly purified (up to 95%) Vip3Aa protein from HD-1 cultures. The protocol is based on acetone precipitation of supernatant protein, followed by HPLC fractionation (DEAE-5PW column) and several concentration steps. Our protocol resulted in higher yields and purity of Vip3Aa than a previously published method [Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., Koziel, M.G., 1996. Vip3A, a 353 novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of 354 activities against lepidopteran insects. Proc. Nat. Acad. Sci. USA 93, 5389-5394.]. This was achieved by using acetone rather than ammonium sulfate for precipitation of proteins from culture supernatants, and a shallow rather than a steep NaCl gradient for elution of the toxin, and by conducting all the purification steps at low temperature to prevent toxin degradation. In bioassays of the purified protein, Choristoneura fumiferana and Lymantria dispar larvae were less susceptible than Spodopteraexigua (10- and ∼100-fold, respectively). A B. thuringiensis var. kurstaki strain HD-1 from which the vip3Aa gene had been deleted (EG12414) showed reduced toxicity to S. exigua relative to the unmodified parental strain (EG2001), but not to L. dispar or C. fumiferana. We interpret these results as indicating that the Vip3Aa toxin does not contribute measurably to pathogenicity of HD-1 in these species.     

Secondary structure of the entomocidal toxin fromBacillus thuringiensis subsp.kurstaki HD-73

The secondary structure of the toxin fromBacillus thuringiensis subsp.kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33–40% α-helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32–40% β-sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both α-helical and β-sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.     

Cellular Fatty Acid Analysis of Isolates of Bacillus thuringiensis Serovar kurstaki,Strain HD-1

Whole-cell cellular fatty acid (CFA) composition was utilized to determine if Bacillus thuringiensis serovar kurstaki (HD-1) larvicides produced by different manufacturers could be distinguished from each other and to determine whether these larvicides were distinguishable from isolates deposited in public collections. This study analyzed Biobit, Dipel, Foray, Thuricide, and the non HD-1 larvicides Delfin and Javelin, as well as the 1971 and 1980 Standards of HD-1. Isolates of HD-1 deposited in the collections of the American Type Culture Collection, Bacillus Genetic Stock Center, Pasteur Institute, and United States Department of Agriculture (USDA) were also analyzed. The data were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages (UPGMA). Samples that linked at a Euclidean distance ≤2.0 units were considered to belong to the same fatty acid strain. Isolates of HD-1 from commercial products and deposits of HD-1 in the public collections and Standards were polytypic; 22 separate fatty acid strains were identified in the 1971 and 1980 Standards and 35 fatty acid strains were identified in the public collections. The type strain for Btk contained multiple fatty acid strains; three fatty acid strains were present in both the Bacillus Genetic Stock Center and the Pasteur Institute collections. In contrast, the type strain for serovar kurstaki in the USDA collection (HD-73) was monotypic and its fatty acid strain did not occur in other collections. We could distinguish between HD-1 and non-HD-1 larvicides using CFA composition. We conclude that CFA analysis may be used to identify commercial products.     

Secondary structure of the entomocidal toxin from Bacillus thuringiensis subsp. kurstaki HD-73

The secondary structure of the toxin fromBacillus thuringiensis subsp.kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33–40% -helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32–40% -sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both -helical and -sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.     

固定化果胶酶澄清果汁的条件及效果

利用固定化果胶酶对四种不同果汁澄清条件及效果进行研究,结果表明,固定化果胶酶澄清四种不同果汁的效果明显,其中澄清桔汁的果胶酶重复使用20次以上,酶活力及透光率仍可维持在80%以上,其最适反应条件是:果汁浓度50%;pH 3.0~3.5;温度45~50℃;反应时间2小时;酶量每毫升果汁0.05 g固定化果胶酶;澄清时间20小时。     

Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes

Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp. sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity. Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae. In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE). Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity. Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands. Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact. The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity. However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests.     

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.     

pH对苏云金杆菌晶体致病性的影响

报道了以苏云金杆菌5个菌株的晶体,经粘虫和黄粉虫幼虫的肠液在pH为6．4、7,4、8．4和9．4的缓冲液中（28℃）消化6小时后,其上清液（消化部分）和沉淀（未消化部分）对三龄末粘虫和黄粉虫幼虫的毒力。结果表明,457株、HD－1珠和10－4－13株的上清波和沉淀对粘虫均有不同程度的毒效,T84-1,株和T2株的上清液和沉淀对粘虫均无毒效;而对黄粉虫幼虫,以pH9．4时的上清液和沉淀均无毒效,在pH6．4和7．4时晶体被消化得极少,pH8．4时能被消化但较pH9．4时少。证明了pH对晶体的消化起重要作用,也表明了晶体之间在结构上存在着差异。     

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.     

The Bacillus thuringiensis Cry1Aa toxin: effects of trypsin and chymotrypsin site mutations on toxicity and stability

The objective of the present work was to create an active Cry1Aa toxin showing enhanced resistance to degradation by spruce budworm (Choristoneura fumiferana) midgut proteases by mutating potential chymotrypsin and trypsin sites. Fourteen Cry1Aa mutants were created in an Escherichia coli-Bacillus shuttle vector and expressed in a crystal minus Bacillus thuringiensis host. Using spruce budworm gut juice, commercial bovine trypsin and chymotrypsin we performed protease resistance assays with Cry1Aa wild type and mutant toxins. Although many mutants showed little or no change, several mutants showed a > 2-fold increase (R543S, R566G, and F570S) up to a > 4-fold increase in toxicity (F576S), in bioassay studies against C. fumiferana. The in vitro protease resistance assay results indicated a possible involvement of other gut juice components in toxin overdigestion.     

Dissolution and Degradation of Bacillus thuringiensis delta-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori

The dissolution and degradation of dagger-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease.     

Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.     

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin

BUPM95 is a Bacillusthuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22 kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62 kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65 kDa compared to the 55 and 100 kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.     

Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 protoxins and sporulated cultures of Bacillus thuringiensis subsp. kurstaki HD-1 and NRD-12.

The toxicities to neonate Spodoptera exigua and Trichoplusia ni of lyophilized powders obtained from sporulated liquid cultures (referred to as sporulated cultures) and Escherichia coli-expressed P1 [cryIA(a) cryIA(b) cryIA(c)] protoxins from three-gene strains of NRD-12 and HD-1 of Bacillus thuringiensis subsp. kurstaki were determined by using diet incorporation bioassays. Although sporulated cultures from both strains were more toxic to T. ni than S. exigua, there were no differences in toxicity between NRD-12 and HD-1. Toxicities of the three individual P1 protoxins against S. exigua varied by at least fivefold, with the cryIA(b) protein being the most toxic. These same protoxins varied in toxicity against T. ni by at least 16-fold, with the cryIA(c) protein being the most toxic. However, when tested against either S. exigua or T. ni, there were no differences in toxicity between an NRD-12 P1 protoxin and the corresponding HD-1 P1 protoxin. Comparing the toxicities of individual protoxins with that of sporulated cultures demonstrates that no individual protoxin was as toxic to S. exigua as the sporulated cultures. However, this same comparison against T. ni shows that both the cryIA(b) and cryIA(c) proteins are at least as toxic as the sporulated cultures. Results from this study suggest that NRD-12 is not more toxic to S. exigua than HD-1, that different protein types have variable host activity, and that other B. thuringiensis components are not required for T. ni toxicity but that other components such as spores might be required for S. exigua toxicity.     

Cloning and expression in Escherichia coli of an insecticidal crystal protein gene from Bacillus thuringiensis var. aizawai HD-133

Using a gene probe derived from the cloned var. sotto insecticidal crystal protein (ICP) gene, we have cloned a Bacillus thuringiensis var. aizawai HD-133 ICP gene in Escherichia coli. The gene encodes a polypeptide that is toxic to Lepidoptera in vivo and in vitro. The protein is expressed at a level sufficient to produce phase-bright inclusions in recombinant E. coli strains, and these inclusions can be partially purified using discontinuous sucrose density gradients. Immunoblotting shows that the inclusions contain a 135 kDa polypeptide which reacts strongly with antiserum raised against the B. thuringiensis var. kurstaki HD-1 P1 polypeptide.     

Dissolution and Degradation of Bacillus thuringiensis δ-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori

The dissolution and degradation of †-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease.