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.     

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.     

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture

An insecticidal protein gene from Bacillus thuringiensis var. aizawai was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteolytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resistant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CF1 cells but not dipteran (Aedes albopictus) cells. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawai toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran cell membranes, which may be the receptor for this toxin.     

Characterization of the Bacillus thuringiensis strains isolated from Taiwan

Over 100 Bacillus thuringiensis (Bt) isolates which produced phase bright inclusions have been isolated from soil samples from different areas in Taiwan. Three types of crystal proteins were visualized by phase contrast microscopy. Among these isolates, only 14 different types of plasmid profiles have been observed. They all possess a variety of plasmids ranging from a few kb to around 250 kb in size. With respect to the crystal protein profiles, the plasmid profiles, and the shapes of crystal proteins, we found that the majority of our isolates (87%) were different from most of the known Bt strains. Our other two types of isolates (10 and 3%) resembled Bt var. kurstaki HD1 and Bt var. israelensis, respectively. Most of our isolates were active against Bombyx mori (Lepidoptera) and Aedes aegypti (Diptera). Most interestingly, two of our isolates, Nos. 82 and 96, were found highly toxic to Heliothis virescens, even compared with the standard strain, Bt var. kurstaki HD1. Using insecticidal crystal protein (ICP) gene probe from Bt var. aizawai HD-133 to probe the total DNA of our isolates, we observed that at least one plasmid from each of the tested strains reacted with the probe. A 10 kb plasmid from some of our isolates hybridized with the probe. This probably is the first evidence demonstrating that the ICP gene sequence can be found in a low molecular weight plasmid.     

Insecticidal activity of the CryIIA protein from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki expressed in Escherichia coli and Bacillus thuringiensis and in a leaf-colonizing strain of Bacillus cereus.

A 4.0-kb BamHI-HindIII fragment encoding the cryIIA operon from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki was cloned into Escherichia coli. The nucleotide sequence of the 2.2-kb AccI-HindIII fragment containing the NRD-12 cryIIA gene was identical to the HD-1 and HD-263 cryIIA gene sequences. Expression of cryIIA and subsequent purification of CryIIA inclusion bodies resulted in a protein with insecticidal activity against Heliothis virescens, Trichoplusia ni, and Culex quinquefasciatus but not Spodoptera exigua. The 4.0-kb BamII-HindIII fragment encoding the cryIIA operon was inserted into the B. thuringiensis-E. coli shuttle vector pHT3101 (pMAU1). pMAU1 was used to transform an acrystalliferous HD-1 strain of B. thuringiensis subsp. kurstaki and a leaf-colonizing strain of B. cereus (BT-8) by using electroporation. Spore-crystal mixtures from both transformed strains were toxic to H. virescens and T. ni but not Helicoverpa zea or S. exigua.     

The hypervariable region in the genes coding for entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhd1 gene of subsp. kurstaki HD1

One of the genes for the entomophatogenic crystal protein of Bacillus thuringiensis (subsp. kurstaki strain HD1) has been cloned in Escherichia coli, and its nucleotide sequence determined completely. The gene is contained within a 4360-bp-long HpaI-PstI DNA restriction fragment and codes for a polypeptide of 1,155 amino acid residues. The protoxin protein has a predicted Mr of 130,625. The E. coli-derived protoxin gene product is biologically active against Heliothis virescens larvae in a biotest assay. Extensive computer comparisons with other published B. thuringiensis subsp. kurstaki strains HD1, HD73, and B. thuringiensis subsp. sotto gene sequences reveal hypervariable regions in the first half of the protoxin coding sequence. These regions are responsible for the biological activity of the protein product of the cloned gene, and may explain the different biological activities of these different protoxins.     

Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki

The gene encoding the 66-kDa entomocidal protein (P2 protein or mosquito factor) from Bacillus thuringiensis var. kurstaki has been isolated by the use of a 62-mer oligonucleotide probe that encoded 21 amino acids of the P2 protein NH2 terminus. The DNA sequence of the gene, designated cryBI, was unique from the published sequences of other B. thuringiensis genes. However, the amino acid sequence of the P2 protein, as deduced from the DNA sequence of the cryBI gene, was found to contain a sequence of 100 amino acids having 37% homology to a group of B. thuringiensis entomocidal proteins, the P1 proteins. Late stationary phase Bacillus megaterium cells harboring the cloned B. thuringiensis cryBI gene contained large aggregates of the P2 protein, and the cells were highly toxic to both lepidopteran and dipteran larvae. In contrast, Escherichia coli cells harboring the cloned cryBI gene contained very low levels of the P2 protein. DNA blot hybridization experiments showed that certain B. thuringiensis strains contained at least one cryBI-related DNA sequence in addition to the cryBI gene itself.     

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.     

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.     

Nucleotide sequence coding for the insecticidal fragment of the Bacillus thuringiensis crystal protein

The insecticidal crystal protein (ICP) gene, icp, from a 68-kb plasmid derived from Bacillus thuringiensis subsp. sotto was cloned in Escherichia coli. The icp expression in E. coli cells was confirmed by both immunological and insect-toxicity assays of the cell extract. The entire icp gene resides in the 6.6-kb PstI fragment, which codes for a 144-kDal peptide identical to the intact ICP, as determined by its size and reaction with anti-ICP antibody. Deletion analysis further revealed that the 2.8-kb region within the 6.6-kb PstI fragment codes for ICP. Analysis of the nucleotide sequence indicated that a peptide of 934 amino acid residues truncated at the C-terminal end is encoded by this 2.8-kb fragment. A unique feature of this truncated ICP is the abundance of cysteine and lysine residues within its C-terminal region.     

Isolation of a strain of Bacillus thuringiensis ssp. kurstaki HD-1 encoding δ-endotoxin Cry1E

A strain of Bacillus thuringiensis, STB-1, toxic against Spodoptera exigua , was isolated. Bacillus thuringiensis STB-1 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis ssp. kurstaki . The plasmid and protein profiles of B. thuringiensis STB-1 were compared with those of its reference strains, ssp. kurstaki and ssp. kenyae . To verifiy the gene type of B. thuringiensis STB-1, PCR analysis was performedwith Spodoptera -specific cry gene primers. The result showed that B. thuringiensis STB-1, unlike its reference strains, had cry1Aa , cry1Ab , cry1Ac and cry1E , suggesting that B. thuringiensis STB-1 was a unique strain with respect to gene type. In addition, B. thuringiensis STB-1 showed a high level of toxicity against both S. exigua and Bombyx mori , whereas B. thuringiensis ssp. kurstaki HD-1 or ssp. kenyae showed a high level of toxicity against only Bombyx mori or S. exigua , respectively.     

Cloning and characterization of a novel gene cry9Ec1 encoding lepidopteran-specific parasporal inclusion protein from a Bacillus thuringiensis serovar galleriae strain

A novel delta-endotoxin gene from a lepidopteran-specific Bacillus thuringiensis serovar galleriae strain was cloned, and the full sequence of the cry gene was determined. The cloned 6.5-kb DNA fragment included the full sequence of the cry gene and three open reading frames located upstream of the cry gene. The gene, designated cry9Ec1, encodes a polypeptide of 1154 amino acid residues with a predicted molecular weight of 130 237. The deduced amino acid sequence of the Cry9Ec1 protein had the highest homology (77.7%) with the Cry9Ea1 protein when compared with existing Cry proteins. The expression, in an acrystalliferous B. thuringiensis strain, of the cry9Ec1 gene was high when controlled by the cyt1A2 promoter, leading to the formation of large spherical inclusions. The purified crystals from the recombinant strain were toxic when tested against two lepidopteran species, Bombyx mori and Plutella xylostella. However, the Cry9Ec1 protein gave no toxicity against Spodoptera litura, Spodoptera exigua, Plodia interpunctella, Helicoverpa zea, and Culex pipiens molestus.     

Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715

A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution. Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus.     

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture

An insecticidal protein gene from Bacillus thuringiensis var. aizawal was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteloytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resist-ant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CFI ceils but not dipteran (Aedes albopictus) calls. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawal toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran ceil membranes, which may be the receptor for this toxin.     

Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. israelensis.

A gene encoding a 72,357-dalton (Da) crystal protein of Bacillus thuringiensis var. israelensis was isolated from a native 75-MDa plasmid by the use of a gene-specific oligonucleotide probe. Bacillus megaterium cells harboring the cloned gene (cryD) produced significant amounts of the 72-kDa protein (CryD), and the cells were highly toxic to mosquito larvae. In contrast, cryD-containing Escherichia coli cells did not produce detectable levels of the 72-kDa CryD protein. The sequence of the CryD protein, as deduced from the sequence of the cryD gene, was found to contain regions of homology with two previously described B. thuringiensis crystal proteins: a 73-kDa coleopteran-toxic protein and a 66-kDa lepidopteran- and dipteran-toxic protein of B. thuringiensis subsp. kurstaki. A second gene encoding the B. thuringiensis subsp. israelensis 28-kDa crystal protein was located approximately 1.5 kilobases upstream from and in the opposite orientation to the cryD gene.