Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis.

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Degradation of the parasporal crystal produced by Bacillus thuringiensis var. kurstaki

Degradation products of the parasporal crystals of Bacillus thuringiensis var. kurstaki obtained by treatment with alkali, gut juice from larvae of Bombyx mori, and various plant and mammalian enzymes were compared for elution pattern, approximate molecular weight (MW), and toxicity. The results indicated that with alkaline treatment the most toxic extract was obtained with 0.05–0.1 M NaOH. Toxicity was found associated mainly with a protein peak of 230,000 MW although other toxic peaks were found in the tailing. Heat-treated midgut juice from larval B. mori gave similar results. After digestion of parasporal crystals with clarified midgut juice, five peaks causing toxicity and having MW of approximately 235,000, 67,000, 30,200, 5000, and 1000, respectively, were identified. Treatment of B. thuringiensis δ-endotoxin with α-chymotrypsin gave peaks causing mortality of approximate MW 235,000, 34,000, 5000, and 1000. Trypsin, pronase, carboxypeptidase, and enterokinase digests of the B. thuringiensis δ-endotoxin gave toxic components ranging from 235,000 to 30,000 MW. The protein protoxin molecules are digested to give small toxic subunits that may be of practical value for structural determinations and for molecular mode of action studies.     

Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus.

DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are closely related to cryI genes. Two cryV-type genes, cryV1 and cryV465, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465 protein was toxic only to Plutella xylostella.     

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.     

Simple method for the isolation of the antilepidopteran toxin from Bacillus thuringiensis subsp. kurstaki

A protein with a molecular mass of 66 kDa was isolated by a simple, rapid, and inexpensive method, using 3-N-morpholinopropanesulfonic acid, potassium thiocyanate, and dithiothreitol, from a mixture of spores, parasporal crystals, and cell debris of Bacillus thuringiensis subsp. kurstaki. The protein was active against the third instar larvae of Trichoplusia ni, was soluble in 19 mM Na2CO3, and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed as the insecticidal component of the 132-kDa protoxin of B. thuringiensis subsp. kurstaki by an enzyme-linked immunosorbent assay using antibodies prepared against the protoxin.     

Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities.

Two genes encoding insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki HD-1 were cloned and sequenced. Both genes, designated cryB1 and cryB2, encode polypeptides of 633 amino acids having a molecular mass of ca. 71 kilodaltons (kDa). Despite the fact that these two proteins display 87% identity in amino acid sequence, they exhibit different toxin specificities. The cryB1 gene product is toxic to both dipteran (Aedes aegypti) and lepidopteran (Manduca sexta) larvae, whereas the cryB2 gene product is toxic only to the latter. DNA sequence analysis indicates that cryB1 is the distal gene of an operon which is comprised of three open reading frames (designated orf1, orf2, and cryB1). The proteins encoded by cryB1 and orf2 are components of small cuboidal crystals found in several subspecies and strains of B. thuringiensis; it is not known whether the orf1 or cryB2 gene products are present in cuboidal crystals. The protein encoded by orf2 has an electrophoretic mobility corresponding to a molecular mass of ca. 50 kDa, although the gene has a coding capacity for a polypeptide of ca. 29 kDa. Examination of the deduced amino acid sequence for this protein reveals an unusual structure which may account for its aberrant electrophoretic mobility: it contains a 15-amino-acid motif repeated 11 times in tandem. Escherichia coli extracts prepared from cells expressing only orf1 and orf2 are not toxic to either test insect.     

Endogenous protease-activated 66-kDa toxin from Bacillus thuringiensis subsp. kurstaki active against Spodoptera littoralis

The anti-lepidopteran toxin from sporulated Bacillus thuringiensis subsp. kurstaki cells, generated by the proteolytic action of endogenous protease(s) on the protoxin, was purified and studied to identify the effect of such proteolysis on the biochemical nature of the toxin. The active toxin was purified employing anion-exchange chromatography to absolute homogeneity, as indicated by SDS-PAGE and Western blotting. Antisera to the purified toxin (66 kDa) crossreacted with the protoxin (132 kDa) confirming its origin from protoxin. The purified toxin with a pI of 7.95 was derived from the N-terminal region of the protoxin (pI 7.6). Circular dichroism data revealed that the toxin has significant secondary structure and it undergoes pH dependent conformational change. Unlike the toxin generated by exogenous proteases such as trypsin, etc., the endogenous protease(s) activated toxin is highly lethal to a tolerant insect variety of the lepidopteran order, Spodoptera littoralis.     

An improved enzyme-linked immunoassay for the detection and quantification of the entomocidal parasporal crystal proteins of Bacillus thuringiensis subsp. kurstaki and israelensis

An improved and simplified enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of parasporal crystalline toxins from Bacillus thuringiensis subsp. kurstaki. The improved procedure involved pretreatment of the polystyrene cuvettes with glutaraldehyde before antibody coating. A direct comparison of treated and untreated cuvettes is provided. ELISAs were then used for the analysis of the entomocidal crystalline proteins in commercial and experimental formulations of B. thuringiensis subspp. kurstaki and israelensis.     

Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua

AIMS: The objective of this work was to enhance the insecticidal activity or widen the pesticidal spectrum of a commercial Bacillus thuringiensis strain YBT1520. METHODS AND RESULTS: A vegetative insecticidal protein gene vip3Aa7, under the control of its native promoter and cry3A promoter, was subcloned into B. thuringiensis acrystalliferous BMB171 to generate BMB8901 and BMBvip respectively. It was found that the amount of Vip3Aa7 protein produced by BMBvip was 3.2-fold more than that produced by BMB8901. Therefore, the vip3Aa7 gene under the control of cry3A promoter was transformed into strain YBT1520. The toxicity of the resulting strain BMB218V against Spodoptera exigua was 10-fold more than that of YBT1520, and that the toxicity of BMB218V against Helicoverpa armigera retained the same level as that of strain YBT1520. CONCLUSIONS: Strain YBT1520 obtained high toxicity against S. exigua after it was transformed and expressed the foreign vip3Aa7 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial B. thuringiensis strain YBT1520 has high toxicity against H. armigera and Plutella xylostella, but almost no activity against S. exigua, which is a major crop pest in China. This work provides a new strategy for widening the activity spectrum of B. thuringiensis against agriculture pests.     

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)     

Characterization of the parasporal inclusion of Bacillus thuringiensis subsp. kyushuensis.

Electron microscopy of Bacillus thuringiensis subsp. kyushuensis revealed that the parasporal inclusions are composed of a homogeneous center surrounded by a thick, electron-dense coating. Antibodies directed against the 135- and 65-kilodalton B. thuringiensis subsp. israelensis peptides cross-reacted with the 70- and 26-kilodalton peptides, respectively, of B. thuringiensis subsp. kyushuensis.     

Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis.

The parasporal crystalline protoxin of Bacillus thuringiensis contains a single glycoprotein subunit that has a molecular weight of approximately 1.2 X 10(5). The carbohydrate consists of glucose (3.8%) and mannose (1.8%). At alkaline pH, the proendotoxin is apparently solubilized and activated by an autolytic mechanism involving an inherent sulfhydryl protease that renders the protoxin insecticidal. Activation generates protons, degraded polypeptides, sulfhydryl group reactivity, proteolytic activity, and insect toxicity. Chemical modification of the sulfhydryl groups inhibits the proteolytic and insecticidal activities, suggesting that cysteine residues may be present in the active site of the protein.     

Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki

AIMS: The present work aims to study a new chitinase from Bacillus thuringiensis subsp. kurstaki. METHODS AND RESULTS: BUPM255 is a chitinase-producing strain of B. thuringiensis, characterized by its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. Both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of B. thuringiensis. The identification of chitin hydrolysis products resulting from the activity, exhibited by Chi255 through heterologous expression in Escherichia coli revealed that this enzyme is a chitobiosidase. CONCLUSIONS: Another chitinase named Chi255 belonging to chitobiosidase class was evidenced in B. thuringiensis subsp. kurstaki and was shown to present several differences in its amino acid sequence with those of published ones. The functionality of Chi255 was proved by the heterologous expression of chi255 in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of the sequence of chi255 to the few sequenced B. thuringiensis chi genes might contribute to a better investigation of the chitinase 'structure-function' relation.     

昆虫中肠液性质对苏云金芽孢杆菌伴孢晶体毒力的影响

综述了昆虫中肠液性质对苏云金芽孢杆菌Bacillus thuringiensis伴孢晶体毒力的影响。中肠液的酸碱度和蛋白酶是影响伴孢晶体溶解与原毒素活化的两大因素。中肠液的酸碱度不仅影响到伴孢晶体的溶解速度,还影响到各种蛋白酶的活性表现;而蛋白酶则直接参与了原毒素的活化,其组成与活性影响着原毒素的活化速度和杀虫专一性。因中肠液蛋白水解能力过高而导致原毒素的过度降解是某些昆虫对苏云金芽孢杆菌低度敏感的主要原因,而中肠液对原毒素活化能力的降低则与昆虫抗性的形成有关。此外,中肠液的沉淀作用及其它生理生化特性也影响着原毒素毒力的正常发挥。     

Two types of entomocidal toxins in the parasporal crystals of Bacillus thuringiensis kurstaki

Two types of entomocidal proteins of Bacillus thuringiensis kurstaki were isolated from the parasporal bodies (crystals), and their structures were compared with each other in relation to the toxic activity. When the crystals were dissociated in 2% 2-mercaptoethanol at pH 10, a protein of Mr = 135,000, called delta-endotoxin, was liberated. The crystals of a strain of B. thuringiensis kurstaki, the HD-1 strain, also released another protein in small quantities. This minor component of HD-1, which had been discovered and named mosquito factor by Yamamoto and McLaughlin (T. Yamamoto and R. E. McLaughlin (1981) Biochem. Biophys. Res. Commun. 103, 414-421) because of its toxicity to mosquito larvae, could be liberated selectively from the crystals by alkali treatment without any thiol reagent at pH 11. Electron microscopic observation suggested that the bipyramidal crystal is composed of a homogeneous component, presumably the delta-endotoxin, and the mosquito factor is not within the crystal matrix. The liberated toxins, including the mosquito factor, were purified by Sephacryl S-300 column chromatography and activated by proteinases obtained from gut juice of the cabbage looper (Trichoplusia ni). The activated toxins were characterized by peptide mapping using techniques of HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping revealed that the mosquito factor is a protein distinctly different from the delta-endotoxin. Furthermore, a comparison between two strains of B. thuringiensis kurstaki indicated that minor differences in the structure of the delta-endotoxins, in particular the differences in their proteinase-resistant region, caused significant variations in their toxicity to susceptible insects.     

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.