Showing papers on "Bacillus thuringiensis published in 1988"

Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

Abstract: Binding studies were performed with two 125I-labeled Bacillus thuringiensis delta-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One delta-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other delta-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis delta-endotoxins active against M. sexta compete for binding of 125I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles.

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro.

Abstract: Summary: Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.

Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene

Abstract: A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato bectle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73115 dalton protein and a flat, diamond-shaped crystal composed of a protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.

Monoclonal Antibody Analysis and Insecticidal Spectrum of Three Types of Lepidopteran-Specific Insecticidal Crystal Proteins of Bacillus thuringiensis

Abstract: We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135- and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity.

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

Abstract: 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.

Characterization of the Coleopteran–Specific Protein Gene of Bacillus thuringiensis Var. tenebrionis

Abstract: The gene encoding the coleopteran–specific insect control protein from Bacillus thuringiensis var. tenebrionis was isolated and characterized. When expressed in either E. coli or P. fluorescens the gene directs the production of biologically active insect control protein that is lethal to Colorado potato beetle. The gene contains two functional translational initiation codons in the same reading frame leading to the production of both the full length protein and an N–terminal truncated form. The gene has been engineered to produce each of these two proteins separately, and each is active against Colorado potato beetle. Native crystals from B.t. var. tenebrionis were found to contain five distinct proteins derived from this single gene either as primary translation products or the result of proteolysis. Comparison of this gene to a lepidopteran–specific B.t. gene revealed only limited regions of homology.

Multiplicity of delta-endotoxin genes with different insecticidal specificities in Bacillus thuringiensis aizawai 7.29.

Abstract: Summary The hypothesis according to which multiple and different 5-endotoxin genes could determine the host-range specificity of the lepidopteran strains of Bacillus thuringiensis is being checked in the case of strains aizawai 7.29 (serotype 7) and entomocidus 601 (serotype 6). From these strains, several crystal protein genes, belonging to different structural types, have been isolated. One of the cloned genes that is not present in strain entomocidus 601 is duplicated in strain aizawai 7.29. This gene belongs to a previously characterized type of crystal protein gene and encodes a protein preferentially active against Pieris brassicae. Two other genes, of presumed chromosomal location, are present In both strains and each displays a unique physical map. In both strains the two genes are in close proximity and in the same orientation. The first, which belongs to a new type of crystal protein gene, encodes a 130–140 kD protein that is not significantly active against the two insect species tested. The other new type of crystal protein gene directs the synthesis of a polypeptide preferentially active against Spodoptera littoralis.

Insect-resistant plants

Abstract: A method for producing genetically transformed plants exhibiting toxicity to Coleopteran insects is disclosed. In another aspect, the present invention embraces chimeric plant genes, genetically transformed cells and differentiated plants which exhibit toxicity to Coleopteran insects. In yet another aspect, the present invention embraces bacterial cells and plant transformation vectors comprising a chimeric plant gene encoding a Coleopteran toxin protein of Bacillus thuringiensis.

Novel isolates of bacillus thuringiensis having activity against nematodes

Abstract: The invention concerns novel isolates of Bacillus thuringiensis (B.t.) which contains a toxin(s) which is active against adult nematode worms and larvae. This B.t. toxin(s) can be used to treat animals and plants hosting susceptible nematodes.

Starch-Encapsulated Bacillus thuringiensis: A Potential New Method for Increasing Environmental Stability of Entomopathogens

Abstract: Bacillus thuringiensis Berliner was encapsulated within a starch matrix and assayed for biological activity against neonate and second-instar larvae of the European corn borer, Ostrinia nubilalis (Hubner). When larvae ingested encapsulated B. thuringiensis , they digested the starch matrix and released into their guts B. thuringiensis crystals and spores, which initiated infection. Nearly 100% mortality occurred at all dosages and concentrations tested whenever the starch granules were hydrated and high relative humidity conditions (>80%) were maintained during the 24-h exposure period of the assay. Encapsulated B. thuringiensis stored in the laboratory for 4 mo exhibited no detectable decrease in pathological activity.

Nucleotide sequence of crystal protein gene isolated from B.thuringiensis subspecies entomocidus 60.5 coding for a toxin highly active against Spodoptera species

Abstract: The isolation of a toxin gene from B. thuringiensis subsp. entomocidus 60.5. which did not belong to one of three welldescribed gene families (1), was described (2). Its gene product is highly active against Spodoptera species. Here the complete nucleotide sequence of this gene (BTVI) encoding a protein with a calculated weight of 134.740 kDa is presented. Extensive homology with the 4.5 kb-class gene isolated from B. thuringiensis subsp. kurstaki HD-1 Dipel (3), was found downstream from the proteolytic cleavage site.

Novel coleopteran-active bacillus thuringiensis isolate

Abstract: The subject invention concerns a novel and useful insecticide with activity against insect pests of the order Lepidoptera. Pests in the order Lepidoptera do heavy damage to crops, e.g., cabbage and broccoli. The insecticide of the subject invention is a novel B. thuringiensis microbe referred to as B.t. PS85A1, or mutants thereof. Specifically disclosed is an asporogenous mutant designated B.t. PS85A1-168. The spores or crystals of B.t. PS85A1, or the crystals of B.t. PS85A1-168, are useful to control lepidopteran pests in various environments.

Novel bacillus thuringiensis isolate

Abstract: The subject invention concerns a novel and useful insecticide with activity against insect pests of the order Coleoptera and the order Lepidoptera. Pests in the order Coleoptera do heavy damage to crops, e.g., corn. The insecticide of the subject invention is a novel B. thuringiensis microbe referred to as B.t. MT 104, or mutants thereof. The spores or crystals of this microbe are useful to control coleopteran and lepidopteran pests in various environments.

Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera.

Abstract: The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus thuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LC50 value of 43 ng/ml to third-instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68-78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N-terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera-specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C-terminal half of the proteins. The N-terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological cross-reaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect-host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes.

Sequence of the Bacillus thuringiensis phosphatidylinositol specific phospholipase C

Abstract: A number of eukaryotic proteins have been shown to be anchored to membranes by covalent linkage to a phosphatidylinositol molecule(l). Bacterial phospholipases have been instrumental in the identification of proteins which are anchored by this mechanism. To increase the availability of this enzyme, and to possibly understand its mechanism of action, we have isolated the gene for the phosphatidylinositol specific phospholipase C of B. thuringiensis. Oligonucleotide probes based on the amino acid sequence (2) of the purified enzyme were used to isolate clones in pBR322. The sequence was determined by the dideoxy sequencing method(3). The amino acids corresponding to the determined amino acid sequence are underlined. When either E. coli OTB. subtilis was transformed with this gene on a plasmid, enzymatic activity could be detected in amounts at least 500-fold higher than nontransformed cultures. No obvious sequence similarities were found in the Dayhoff protein sequence data base, nor were there similarities to the recently reported sequence of a mammalian phosphoinositide-specific phospholipase C (4).

Three trophic level interactions: allelochemicals, Manduca sexta (L.) , and Bacillus thuringiensis var. kurstaki Berliner

Abstract: The role of plant allelochemicals in reducing the ability of Bacillus thuringiensis var. kurstaki Berliner to kill larvae of Manduca sexta (L.) was studied by comparing the effects of the alkaloid nicotine and the flavonoid rutin on the toxicity of B. thuringiensis. Concentrations of nicotine and rutin reflecting levels found in tobacco and tomato plants (0, 0.2, 0.4, 0.6, 0.8, 1.0% wet weight) were incorporated into synthetic diet containing 0.001% B. thuringiensis and B. thuringiensis -free diet. Survivorship of M. sexta larvae increased at higher levels of nicotine (0.6, 0.8, 1.0% wet weight), but higher concentrations of rutin did not increase larval survivorship. Experiments on colony growth of B. thuringiensis on nutrient agar containing nicotine or rutin (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9% wet weight) demonstrated that nicotine reduced colony growth to a greater extent than rutin. Consequently, plant allelochemicals potentially are an important factor in reducing larval mortality caused by B. thuringiensis on certain crops. Specialist herbivores that can tolerate high concentrations of allelochemicals may gain protection from pathogens by feeding on plants or plant parts with higher levels of toxins.

Nucleotide sequence of an additional crystal protein gene cloned from Bacillus thuringiensis subsp. thuringiensis

Abstract: The nucleotide sequences of 3 homologous crystal protein genes (cry A1,A2,A3) from Bacillus thuringiensis subsp. kurstaki and subsp. thuringiensis have been reported previously (1, 2, 3, respectively). We report the nucleotide sequence of an additional crystal protein gene {cry hi) from B. thuringiensis subsp. thuringiensis strain HD2 that differs markedly in the aminoterminal region from the 3 homologous genes and includes a TTG translational initiation codon.

Coleopteran active microorganisms, related insecticide compositions and methods for their production and use.

Abstract: This invention relates to Bacillus thuringiensis strains that have insecticidal activity against lepidopteran and coleopteran insects, the coleopteran-active endotoxin being produced by an acquired plasmid. This invention also relates to the crystalline protein toxin useful as a biological insecticide against Coleoptera which toxin is produced by the strain of Bacillus thuringiensis. This invention also relates to the expression in various microorganisms of the gene, known as cryC, which codes for this toxin, and for related novel insecticide compositions and methods for their use.

Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins.

Abstract: Two homologous genes encoding 130-kilodalton (kDa) mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis have been cloned and expressed in Escherichia coli or Bacillus subtilis or both. One of these genes, pPC130, was expressed as a lacZ transcriptional fusion in E. coli at a level sufficient to produce phase-bright inclusions, which were purified and shown to be toxic to Aedes aegypti larvae. The second gene, pCH130, was expressed at a low level in recombinant E. coli cells and was therefore cloned in B. subtilis as a transcriptional fusion of the promoter sequences corresponding to a B. thuringiensis subsp. israelensis 27-kDa delta-endotoxin (E. S. Ward, A. R. Ridley, D. J. Ellar, and J. A. Todd, J. Mol. Biol. 191:13-22, 1986) and the structural gene. Recombinant B. subtilis cells produced phase-bright inclusions during late sporulation; these were partially purified and shown to be toxic to A. aegypti larvae at an LC50 (concentration required to cause 50% mortality of larvae after 24 h of assay) which is significantly lower than that of the pPC130 protein. Neither 130-kDa protein was hemolytic under the assay conditions. Comparison of the nucleotide sequences of these two genes indicates that they share a high degree of homology in the C-terminal portions, but relatively little similarity in the N termini. In addition, significant homologies were found between the pCH130 gene and the HD-1 Dipel gene of B. thuringiensis subsp. kurstaki (H. E. Schnepf, H. C. Wong, and H. R. Whiteley, J. Biol. Chem. 260:6264-6272, 1985).

A Bacillus thuringiensis subsp. israelensis gene encoding a 125-kilodalton larvicidal polypeptide is associated with inverted repeat sequences.

Abstract: A gene encoding a 125-kilodalton (kDa) mosquitocidal delta-endotoxin was cloned from the 72-MDa resident plasmid of Bacillus thuringiensis subsp. israelensis. This gene is similar in its 3' region to the gene encoding the 135-kDa protein previously cloned (C. Bourgouin, A. Klier, and G. Rapoport, Mol. Gen. Genet. 205:390-397, 1986). Escherichia coli recombinant clones harboring the 125-kDa gene were toxic to larvae of the three mosquito species Aedes aegypti, Anopheles stephensi, and Culex pipiens. In addition, the B. thuringiensis subsp. israelensis DNA fragment carrying the 125-kDa protein gene contains two sets of inverted repeat sequences, identified either by the S1 nuclease method or by electron microscopic observation. The structural organization of inverted repeat sequences and of the 125-kDa gene was analyzed and suggests that this B. thuringiensis subsp. israelensis delta-endotoxin gene is located within a transposable element.

Genes from Bacillus thuringiensis entomocidus 60.5 coding for insect-specific crystal proteins

Abstract: Five crystal protein genes have been isolated from DNA of Bacillus thuringiensis entomocidus 60.5, an isolate selected for its high toxicity against Spodoptera littoralis and Spodoptera exigua. Two of these genes belong to a family of well-described crystal protein genes. The toxic properties of the corresponding proteins are similar to those of isolate kurstaki HD1. The other three genes belong to gene families not described before. One of these genes codes for a protein product exhibiting a high degree of specificity towards Spodoptera species, explaining the high toxicity of isolate entomocidus 60.5 against these species. This gene product is much less toxic against larvae of Heliothis virescens and Pieris brassicae. Its coding sequence is separated from a supposed fourth crystal protein gene by a stretch of DNA of 3 kb. The crystal protein encoded by the fifth gene is mainly active against P. brassicae. Homology between the crystal protein genes is limited to the central region of the coding sequences, including the proteolytic cleavage site, except for the first two genes between which homology is extensive.

Chimeric bacillus thuringiensis crystal protein gene comprising hd-73 and berliner 1715 toxin genes, transformed and expressed in pseudomonas fluorescens

Abstract: A novel hybrid B.t. toxin gene toxic to lepidopteran insects has been cloned. The DNA encoding the B.t. toxin can be used to transform various prokaryotic and eukaryotic microbes to express the B.t. toxin. These recombinant microbes can be used to control lepidopteran insects in various environments.

Effect of culture conditions on growth and sporulation of Bacillus thuringiensis subsp. israelensis and development of media for production of the protein crystal endotoxin

Abstract: The influence of medium composition on the inoculum and production stages of theBacillus thuringiensis subsp.israelensis bioinsecticide fermentation was investigated. Media which inhibited sporulation were selected for inoculum development stages. Bioinsecticide production media were designed to produce high cell counts and >90% sporulation in a 48h fermentation. Maximum insecticidal activity occurred at the point of maximum bacterial cell lysis/spore release. A process involving two inoculum stages and a 48h production stage in a 40 l fermenter yielded a viable cell count of 6.5 x 109/ml with greater than 95% sporulation. Good correlation existed between spore counts and bioinsecticide activity.

Barium and calcium block Bacillus thuringiensis subspecies Kurstaki delta-endotoxin inhibition of potassium current across isolated midgut of larval Manduca sexta

Abstract: Ba2+ and Ca2+ prevent and reverse the Btk delta-endotoxin inhibition of the short-circuit current in isolated lepidopteran midgut. These findings support the K+ pump-leak steady-state model for midgut K+ homeostasis and the K+ channel mechanism of Bt toxin action. They provide a new tool with which to study the interactions between Bt toxin and midgut cell membranes.

Cloning and Nucleotide Sequences of the Two 130kDa Insecticidal Protein Genes of Bacillus thuringiensis var. israelensis

Abstract: Two types of 130kDa insecticidal protein genes isolated from large plasmid DNAs of Bacillus thuringiensis var. israelensis HD522 were cloned in an Escherichia coli plasmid vector. Analysis of the nucleotide sequences revealed that the two genes encoded a 127, 500-dalton protein (ISRH3) consisting of 1, 135 amino acids and a 134, 400-dalton protein (ISRH4) consisting of 1, 180 amino acids. The two insecticidal proteins were identical in a region of the C-terminal 467 amino acids.