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PubMed1981

[Extracellular serine proteinase of Bacillus thuringiensis].

Pure extracellular serine proteinase has been isolated from a broth filtrate of Bacillus thuringiensis, strain 69-6R by fractionation with ammonium sulfate and affinity chromatography on Sepharose 4B derivatives containing p-(omega-aminomethyl)-phenylboronic acid and cyclopeptide bacillichin as ligands. The enzyme is completely inactivated by phenylmethylsolfonyl fluoride, a specific reagent for serine proteinases, has the molecular weight of 29 000 and pI of 8.4, reveals maximal activity and stability at pH 8.5 and is inactivated at pH values below 4 and above 10 and at temperatures above 60 degrees. The enzyme hydrolyzed azokasein, bovine serum albumin and synthetic chromogenic peptide substrates, e.g. benzyloxycarbonyl-L-alanyl-L-alanyl-L-leucyl p-nitroanilide and possesses the esterolytic activity. In terms of its physico-chemical characteristics, interaction with specific inhibitors and substrates, extracellular serine proteinase from Bacillus thuringiensis can be related to subtilisins. However, its amino acid composition-Lys16, His4, Arg8, Asx28, Thr16, Ser18, Glx29, Pro12, Gly32, Ala31, Val19, Met5, Ile12, Leu18, Tyr11, Phe10, Trp4 appears to be an intermediate between that of subtilisins and intracellular serine proteinases of Bacilli.

Amino Acids↗
PubMed2006

Characterization of a cry4Ba-type gene of Bacillus thuringiensis israelensis and evidence of the synergistic larvicidal activity of its encoded protein with Cry2A delta-endotoxin of B. thuringiensis kurstaki on Culex pipiens (common house mosquito).

A newly isolated strain of Bacillus thuringiensis, named BUPM97, was identified as affiliated to the israelensis subspecies. This strain was selected for its insecticidal activity against larvae of several dipteran insects, such as the common house mosquito (Culex pipiens). A 5 kb EcoRI fragment, containing a cry4Ba-type gene, named cry4BLB, was cloned from BUPM97. The sequencing of this gene revealed an open reading frame of 3411 bp encoding a protein of 1136 amino acid residues. Similarity analysis of both nucleotide and amino acid sequences revealed that cry4BLB presents several differences from the other previously described cry4Ba-type genes. Particular attention was paid to a two-amino-acid substitution located in domain III of the N-terminal moiety of this protein, which is very important for both toxicity and specificity of the toxin. The transfer of cry4BLB to an acrystalliferous B. thuringiensis kurstaki strain, HD1CryB, showed that it was expressed, resulting in the production of the typical parasporal crystal inclusions. On the other hand, its transfer to another B. thuringiensis kurstaki strain synthesizing Cry2A endotoxin known to be weakly toxic to the dipteran insect C. pipiens, resulted in the clear increase in the insecticidal activity of the transformed strain. Hence, in the present paper, we demonstrate for the first time that Cry2A and Cry4B act synergistically towards C. pipiens.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of the gene encoding novel delta-endotoxin from Bacillus thuringiensis serovar japonensis strain Buibui specific to scarabaeid beetles.

A new isolate of Bacillus thuringiensis serovar japonensis strain Buibui, which was specific to scarab beetles (M. Ohba et al., Lett. Appl. Microbiol. 14:54, 1992), was shown to have a 130-kDa insecticidal crystal protein (ICP) (H. Hori et al., J. Appl. Bacteriol. 76:307, 1994). ClaI restriction enzyme fragments of total cell DNA of the isolate were cloned into E. coli (Sato et al., Curr. Microbiol. 28:15, 1994). Whole 3480-bp nucleotide sequence of the gene encoding 130-kDa ICP was determined, and the molecular weight of the ICP was estimated to be 130,424. The strongly conserved five blocks that occur in almost all ICP genes of B. thuringiensis were detected in the ORF with the same order and almost the same intervals as elsewhere. The amino acid sequence homologies of the whole ICP or N-terminus half portion to that of the CryIIIA, B, C, D, and CryV were about 35%.

Amino Acid Sequence↗
PubMed1998

Characterization of mosquito larvicidal parasporal inclusions of a Bacillus thuringiensis serovar higo strain.

The parasporal inclusion proteins of the type strain of Bacillus thuringiensis serovar higo (H44), that have moderate mosquitocidal activity, were characterized. The purified parasporal inclusions, spherical in shape, were examined for activity against the two mosquito species, Culex pipiens molestus and Anopheles stephensi and the moth-fly, Telmatoscopus albipunctatus. The LC50 values of the inclusion for the two mosquitoes were 3.41 and 0.15 microgram.ml-1, respectively. No mortality was shown for T. albipunctatus larvae by the inclusions at concentrations up to 1 mg ml-1. Solubilized parasporal inclusions exhibited no haemolytic activity against sheep erythrocytes. Parasporal inclusions consisted of eight proteins with molecular masses of 98, 91, 71, 63, 59, 50, 44 and 27 kDa. Of these, the 50 and 44 kDa proteins were the major components. Analysis with immunoblotting revealed that, among several inclusion proteins of B. thuringiensis serovar israelensis, only two proteins of 130 kDa and 110 kDa reacted weakly with antibodies against higo proteins. N-terminal amino acid sequences of the 98, 91, and 71 kDa proteins showed 85-100% identity to those of the two established Cry protein classes, Cry4A and Cry10A.

Amino Acid Sequence↗
PubMedDate not supplied

Enhanced cytotoxicity caused by increased DNA strand breakage resulting from synergistic potentiation of bleomycin with Bacillus thuringiensis subsp. israelensis delta-endotoxin.

We previously reported that a 25 kDa Bacillus thuringiensis subsp. israelensis (BTI) delta-endotoxin potentiates the cytotoxicity of bleomycin (BLM) in cultured cells. In order to clarify the mechanism involved, we examined the induction of DNA strand breakage by BLM in the presence of BTI toxin. Results showed that the increase in strand breakage resulted in enhanced cytotoxicity through the synergistic potentiation of BLM with BTI toxin.

Animals↗
PubMed1996

[Thermodynamic analysis of domain organization of Bacillus thuringiensis toxins].

The structure of delta-endotoxins CryIA(c) and CryIIIA from Bacillus thuringiensis was studied by differential scanning microcalorimerty. The analysis of molecular melting showed that the N- and C-terminal halves of the CryIA(c) protoxin from B. thuringiensis subspecies kurstaki HD-73 are thermodynamically independent subunits, with the C-terminal fragment being denatured at a much lower temperature than the N-terminal fragment. The tertiary structure of the N-terminal fragment undergoes no changes during the protoxin-toxin transition. The melting of the native structure of CryIA(c) at pH 9.7-11.0 suggests that it consists of two domains. In CryIIIA from B. thuringiensis subspecies tenebrionis, the transition from the native to denatured state under alkaline conditions (pH 9.7-11.0) proceeds by the "two-state" principle; i.e., the protein melts as one cooperative domain. The melting of the CryIIIA toxin at pH 2.2-3.5 is described by two transitions overlapping by temperatures, indicating the presence of two domains.

Bacillus thuringiensis↗
PubMed1995

In vitro binding of Bacillus thuringiensis var. israelensis individual toxins to midgut cells of Anopheles gambiae larvae (Diptera: Culicidae).

Midguts from Anopheles gambiae fourth instars were dissected and processed for immuno-light microscopy. Cloned insecticidal crystal proteins (ICPs) from Bacillus thuringiensis var. israelensis (Bti) were individually expressed in crystal-negative strains of Bacillus thuringiensis. Tissue sections of A. gambiae were incubated in vitro with each solubilized and trypsin-activated ICP. Immunodetection of CryIVA, CryIVB, CryIVD and CytA toxins on sections was performed using purified rabbit IgG directed against Bti ICPs, in combination with an anti-rabbit IgG/peroxidase. CryIVA, CryIVB, CryIVD and CytA toxins were detected on the apical brush border of midgut cells, in the gastric caecae and posterior stomach. CytA was also detected, to a lesser extent, on microvilli of anterior stomach cells.

Animals↗
PubMed1997

A high homology exists in N-terminal amino acid sequences of delta-endotoxins between Lepidoptera-specific and Coleoptera-specific Bacillus thuringiensis strains.

The 130 kDa parasporal inclusion proteins of the Lepidoptera-specific Bacillus thuringiensis reference strains for four H serovars were examined for sequences of 14 N-terminal amino acids. The four sequences fell into a single category, MNRNNQNEYEVIDA, and were dissimilar to any of the established sequences for Lepidoptera- and/or Diptera-killing crystal proteins. They were highly related to the reported sequence of the 130 kDa protein of the strain Buibui (serovar japonensis), which is specific for scarabaeid coleopterans.

Amino Acid Sequence↗
PubMed1992

Characterization and properties of a novel plasmid vector for Bacillus thuringiensis displaying compatibility with host plasmids.

A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker gene from Bacillus subtilis, was constructed for use in B.t. Unlike other vectors which have been reported to be acceptable for B.t., this new B.t. vector was stably maintained in the absence of Er and did not displace host plasmids, some of which carry crystal protein-encoding genes (cry genes). The compatibility of this B.t. vector with native plasmids is highly desirable when introducing new cry genes into a wild-type B.t. strain. When a cryIIIA gene of B.t. tenebrionis was cloned in this vector and introduced into B.t. kurstaki (kur) HD119, cryIIIA was highly expressed without affecting the level of expression of native cry genes. The stability of this vector and its compatibility with native B.t. plasmids were achieved by subcloning only nucleotide sequences required for the vector to replicate in B.t. The origin of replication was first cloned on a 9.6-kb Bg/II fragment from a 75-kb plasmid of B.t. kur HD73 and then localized to a 2.4-kb region within the 9.6-kb fragment. Sequencing of the 2.4-kb region revealed the presence of an open reading frame (ORF), encoding a putative 312-amino acid (aa) protein. The deduced aa sequence of the ORF showed no homology to any published aa sequences. Deletion analysis indicated that the B.t. vector required at least the ORF and up to 300 bp surrounding the ORF, in order to replicate.

Amino Acid Sequence↗
PubMed1998

Bacillus thuringiensis and its pesticidal crystal proteins.

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Amino Acid Sequence↗
PubMed1995

Structure, function and engineering of Bacillus thuringiensis toxins.

Nature has provided potent insecticidal toxins as fermentation products of many Bacillus thuringiensis strains. Elucidation of structure-function relationships for this class of natural toxins is in its early stages. Both direct experimentation and application of theoretical structure-function principles emerging from the rapidly growing field of protein structure are accelerating understanding of these toxins. Coupled with the increasing demand for biologically sound pesticides, the benefits from engineering nature's toxins for improved performance set the stage for exciting, fast growth in discovery, characterization and commercialization of new active ingredients for biopesticides and transgenic plants.

Animals↗
PubMed1999

Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains.

An extended PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to lepidopteran, coleopteran, and dipteran pests (Ben-Dov et al., Appl. Environ. Microbiol. 63:4883-4890, 1997). To optimize identification of all reported cry genes, this methodology needs a complete PCR set of primers. In the study reported here, a set of universal (Un9) and specific primers for multiplex rapid screening for all four known genes from the cry9 group was designed. PCR analyses were performed for cry9 genes on 16 standard strains and 215 field isolates of B. thuringiensis. Among the standard strains, only B. thuringiensis subsp. aizawai HD-133, which harbors cry1 and cry2 genes, was positive with Un9 but negative to all four specific primers for cry9 genes. DNA of 22 field-collected isolates was also found to be positive with Un9. These isolates were classified into three cry9 profiles using specific primers; all of them harbor cry1 and cry2. This newly designed set of primers complements the existing PCR methodology for most currently known cry genes.

Bacillus thuringiensis↗
PubMed1997

Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis.

An extended multiplex PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial stages of large-scale screening of cry genes by pinpointing isolates that contain specific genes or unique combinations of interest with potential insecticidal activities, thus facilitating subsequent toxicity assays. Five pairs of universal primers were designed to probe the highly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 genes. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty-two distinct cry-type profiles were identified from 126 field-collected B. thuringiensis strains. Several of them were found to be different from all published profiles. Some of the field-collected strains, but none of the 16 standard strains, were positive for cry2Ac. Three standard and 38 field-collected strains were positive by universal primers but negative by specific primers for all five known genes of cry7 and cry8. These field-collected strains seem to contain a new gene or genes that seem promising for biological control of insects and management of resistance.

Animals↗
PubMed1990

Construction of genetically engineered baculovirus insecticides containing the Bacillus thuringiensis subsp. kurstaki HD-73 delta endotoxin.

The delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki HD-73 was inserted into Autographa californica nuclear polyhedrosis virus (AcMNPV) using two transfer vector systems. In the first, the delta-endotoxin gene was placed under the control of the polyhedrin gene promoter in lieu of the polyhedrin coding sequences, thus deriving a polyhedrin-negative virus. In the second, it was inserted under the control of a copy of the AcMNPV p10 promoter positioned upstream of the polyhedrin gene to produce a polyhedrin-positive virus. Analysis of infected cell extracts showed that the delta-endotoxin was expressed in insect cells as 130K, 62K and 44K proteins, with peak syntheses at 18 h post-infection. Each of these products reacted with antisera specific for the complete protoxin and the cleaved, active form. When extracts from the cells infected with the polyhedrin-negative virus were fed to Trichoplusia ni larvae, feeding by the insects was inhibited and deaths occurred that were inconsistent with virus infection. This effect was also observed after the inoculum had been treated with detergents to inactivate virus particles prior to feeding to the larvae. These data indicate that the expression of the B. thuringiensis delta-endotoxin gene by a baculovirus in insect cells produces material with insecticidal activity. The biological activities of the two recombinant viruses were assessed in conventional bioassay tests by feeding virus particles or occlusion bodies to the insects. The polyhedrin-negative virus preparation appeared to be contaminated with endotoxin which inhibited feeding of the insects and prevented determination of the LD50 value. The polyhedrin-positive virus had an LD50 value about twofold higher than that of unmodified AcMNPV. The significance of these data for the genetic engineering of virus insecticides is discussed.

Animals↗
PubMed1970

Urea-mercaptoethanol-soluble protein from spores of Bacillus thuringiensis and other species.

Treatment with urea-mercaptoethanol of purified spores of Bacillus thuringiensis, other Bacillus species, and Clostridium roseum solubilizes a protein fraction between 5 and 12% of the dry weight of the spores. This fraction behaves identically to the crystal protein of B. thuringiensis on acrylamide-gel electrophoresis. The protein from all of the Bacillus species shows partial homology with crystal protein, using the Ouchterlony immunodiffusion technique. A further fraction, similar in amount, can be removed from spores of B. thuringiensis by the addition of sodium lauryl sulfate to the urea-mercaptoethanol. Spores of B. thuringiensis extracted in these ways show no difference when compared to untreated spores with respect to viability or resistance to heat and ultraviolet-irradiation. The extracted spores do show differences in their germination requirements and their susceptibility to phase-darkening by lysozyme. It is concluded that an urea-mercaptoethanol-soluble protein or class of protein is a widespread component of bacterial spores, possibly located in the spore coat, and that this protein may be related to the crystal protein of B. thuringiensis.

Amino Acids↗
PubMed1994

Ligand blot identification of a Manduca sexta midgut binding protein specific to three Bacillus thuringiensis CryIA-type ICPs.

The CryIA(a), CryIA(b) and CryIA(c) Bacillus thuringiensis insecticidal crystal proteins (ICPs) were used in ligand-blot experiments to detect specific binding proteins in brush-border membrane vesicles (BBMV) of Manduca sexta. We identified a protein which binds these three CryIA-type ICPs. The apparent molecular mass of the protein, estimated on SDS-PAGE, was 210 kDa as was the CryIA(b) binding protein previously described by Vadlamudi and col. We have also demonstrated, in ligand blot experiments, that CryIA(a) and CryIA(c) compete with CryIA(b) for binding this 210 kDa protein. Properties of the binding molecule can be correlated with knowledge previously acquired through radiolabelled binding experiments.

Animals↗
PubMed1991

Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution.

The structure of the delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis that is specifically toxic to Coleoptera insects (beetle toxin) has been determined at 2.5 A resolution. It comprises three domains which are, from the N- to C-termini, a seven-helix bundle, a three-sheet domain, and a beta sandwich. The core of the molecule encompassing all the domain interfaces is built from conserved sequence segments of the active delta-endotoxins. Therefore the structure represents the general fold of this family of insecticidal proteins. The bundle of long, hydrophobic and amphipathic helices is equipped for pore formation in the insect membrane, and regions of the three-sheet domain are probably responsible for receptor binding.

Amino Acid Sequence↗
PubMed1989

Evidence for two different types of insecticidal P2 toxins with dual specificity in Bacillus thuringiensis subspecies.

Analysis of polypeptides in the crystalline delta-endotoxins from different Bacillus thuringiensis strains revealed two antigenically similar forms of the P2 protein which differed in molecular mass, peptide profile, and amino acid sequence. Purified preparations of the two forms displayed the characteristic dual toxicity of the P2 protein towards members of the orders Lepidoptera and Diptera in vivo but differed markedly in potency for the insects tested. Both species of the P2 protoxin, solubilized and activated by sequential proteolysis with insect gut extract and alpha-chymotrypsin, retained activity in vivo and in vitro, despite the removal of 144 residues from the N terminus. For the low-molecular-mass form, the dual insecticidal activity was reproducible in the in vitro assays.

Amino Acid Sequence↗