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PubMed1996

Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: the cryV-encoded protein is expressed early in stationary phase.

A CryV-type protein (CGCryV) has been isolated from supernatant fluids of Bacillus thuringiensis AB88 cultures. Previous reports have suggested the cryptic nature of the cryV-type genes on the basis of the absence of CryV-type proteins in parasporal crystals. The CryV-type protein reported here is expressed early in stationary phase, and evidence indicates that it is an exported protein. Analysis of the deduced protein sequence from this gene reveals the presence of an N-terminal domain that likely acts as a signal peptide. The CGCryV protein is the first reported case of a delta-endotoxin being a secreted protein, which may influence the biological relevance of these proteins.

Amino Acid Sequence↗
PubMed2002

[Transfer of insecticidal protein gene from Bacillus thuringiensis into conventional maize inbred-line mediated by Agrobacterium tumefaciens].

Excellent inbred-lines of maize,340 and 4112, which were used largely in hybridized combination were transformed with Agrobacterium tumefaciens. The immature embryos and their original calli were infected by A.tumefaciens LBA4404 containing plasmid pGBIL04. After 3 days of co-cultivation, the immature embryos and calli were continuously selected on the medium containing phosphinothricin (PPT) for 3 generations, then plants were regenerated. It was proved by PCR analysis that the target Bt gene had been integrated into the genome of regenerated plants. The results showed that fresh original calli from the immature embryos after pre-culture were suitable acceptors. The results also showed that it could increase the frequency of selection by properly lowering the co-culture temperature to 22 degrees .

English Abstract↗
PubMed1993

[Nucleotide sequence of the toxic domain of an insecticidal protein gene from B. thuringiensis subsp. kurstaki HD-1].

Two crystal protein genes, the 5.3kb and 6.6kb class respectively, from Bacillus thuringiensis subsp. kurstaki HD-1 (B. t HD-1) had been cloned previously. Based on the classification system of Hofte and Whiteley, these two genes should belong to Cry I A (b) and Cry I A (c) gene type respectively. The nucleotide sequence of the toxic domain of this Cry I A (c) gene from B. t kurstaki HD-1 is firstly reported here and compared with that of Cry I A (c) gene from B. t HD-73 and Cry I A (b) gene from B. t HD-1.

Amino Acid Sequence↗
PubMed2002

Interaction of gene-cloned and insect cell-expressed aminopeptidase N of Spodoptera litura with insecticidal crystal protein Cry1C.

Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.

Amino Acid Sequence↗
PubMed2004

Characterization of Cry34Ab1 and Cry35Ab1 insecticidal crystal proteins expressed in transgenic corn plants and Pseudomonas fluorescens.

Cry34Ab1 and Cry35Ab1 proteins, identified from Bacillus thuringiensis strain PS149B1, act together to control corn rootworms. Transgenic corn lines coexpressing the two proteins were developed to protect corn against rootworm damage. Large quantities of the two proteins were needed to conduct studies required for assessing the safety of this transgenic corn crop. Because it was technically infeasible to obtain sufficient quantities of high purity Cry34Ab1 and Cry35Ab1 proteins from the transgenic corn plants, the proteins were produced using a recombinant Pseudomonas fluorescens (Pf) production system. The two proteins from both the transgenic corn and the Pf were purified and characterized. The proteins from each host had the expected molecular mass and were immunoreactive to specific antibodies in enzyme-linked immunosorbent assay and Western blot analysis. Data from N-terminal sequencing, tryptic peptide mass fingerprinting, internal peptide sequencing, and biological activity provided direct evidence that the Cry34Ab1 and Cry35Ab1 proteins produced in Pf and transgenic corn were, respectively, comparable or equivalent molecules. In addition, neither protein had detectable glycosylation regardless of the host.

Amino Acid Sequence↗
PubMed2005

Characterization of a novel vip3-type gene from Bacillus thuringiensis and evidence of its presence on a large plasmid.

A novel vip3-type gene named vip3LB has been isolated from Bacillus thuringiensis strain BUPM95. The corresponding secreted vegetative insecticidal protein was active against the lepidopteran insect Ephestia kuehniella. The vip3LB gene was shown, for the first time, to be carried by the large plasmid containing the cry1Ia genes of B. thuringiensis. The nucleotide sequence predicted a protein of 789 amino acids residues with a calculated molecular mass of 88.5kDa. Both nucleotide and amino acid sequences similarity analysis revealed that vip3LB is a new vip3-type gene, presenting several differences with the other vip3-type genes. Heterologous expression of the vip3LB under the control of the strong promoter P(BAD) was performed in Escherichia coli and the produced protein conferred insecticidal activity against Ephestia kuehniella. This novel vegetative insecticidal protein Vip3LB could be a very useful biological control agent.

Animals↗
PubMed1989

Molecular cloning and expression of Bacillus thuringiensis subsp. galleriae insecticidal crystal protein genes in Escherichia coli.

The location of the toxin gene of B. thuringiensis subsp. galleriae (H5ab) on the Mr-130Md plasmid is determined by molecular cloning. Double digestion fragments (BamHI and SalI) and PstI restriction fragments as well, from the 130 Md plasmid, of B. thuringiensis subsp. galleriae, are ligated with the cloning vector pAT 153 respectively and transformed into E. coli strain HB 101. Out of 208 transformants, three colonies (FG2, FG9, FG19) give positive hybridization reaction using the HD-1 delta-endotoxin gene as a probe. They are presumed to contain the delta-endotoxin gene of B. thuringiensis subsp. galleriae. Western blot assays indicate that Mr-130 kDal and 68 kDal, crystal proteins produced by clone FG2 react with anticrystal protein antibody. The protein extracts of clone FG2 are lethal to Ostrinia furnacalis (Guenee). This is the first report with regard to the cloning and expression of the B. thuringiensis subsp. galleriae (H5ab) delta-endotoxin gene.

Bacillus thuringiensis↗
PubMed1998

Role of DNA in the activation of the Cry1A insecticidal crystal protein from Bacillus thuringiensis.

The Cry1A insecticidal crystal protein (protoxin) from six subspecies of Bacillus thuringiensis as well as the Cry1Aa, Cry1Ab, and Cry1Ac proteins cloned in Escherichia coli was found to contain 20-kilobase pair DNA. Only the N-terminal toxic moiety of the protoxin was found to interact with the DNA. Analysis of the crystal gave approximately 3 base pairs of DNA per molecule of protoxin, indicating that only a small region of the N-terminal toxic moiety interacts with the DNA. It is proposed that the DNA-protoxin complex is virus-like in structure with a central DNA core surrounded by protein interacting with the DNA with the peripheral ends of the C-terminal region extending outward. It is shown that this structure accounts for the unusual proteolysis observed in the generation of toxin in which it appears that peptides are removed by obligatory sequential cleavages starting from the C terminus of the protoxin. Activation of the protoxin by spruce budworm (Choristoneura fumiferana) gut juice is shown to proceed through intermediates consisting of protein-DNA complexes. Larval trypsin initially converts the 20-kilobase pair DNA-protoxin complex to a 20-kilobase pair DNA-toxin complex, which is subsequently converted to a 100-base pair DNA-toxin complex by a gut nuclease and ultimately to the DNA-free toxin.

Animals↗
PubMed1995

Regulation of insecticidal crystal protein production in Bacillus thuringiensis.

The production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis normally coincides with sporulation, resulting in the appearance of parasporal crystalline inclusions within the mother cell. In most instances, the temporal and spatial regulation of ICP gene expression is determined at the transcriptional level by mother-cell-specific sigma factors that share homology with sigma E and sigma K from Bacillus subtilis. The cryIII ICP genes are a notable exception; these genes are transcribed from sigma A-like promoters during vegetative growth, are induced or derepressed at the onset of stationary phase, and are overexpressed in sporulation mutants of B. thuringiensis blocked in the phosphorylation of Spo0A, a key regulator of sporulation initiation. Transcription alone, however, cannot account for the impressive ability of this bacterium to accumulate insecticidal proteins. A variety of post-transcriptional and post-translational mechanisms also contribute to the efficient production of ICPs in B. thuringiensis, thus making this bacterium a cost-effective biological control agent.

Amino Acid Sequence↗
PubMed2004

Cryptic endotoxic nature of Bacillus thuringiensis Cry1Ab insecticidal crystal protein.

Cry1Ab is one of the most studied insecticidal proteins produced by Bacillus thuringiensis during sporulation. Structurally, this protoxin has been divided in two domains: the N-terminal toxin core and the C-terminal portion. Although many studies have addressed the biochemical characteristics of the active toxin that corresponds to the N-terminal portion, there are just few reports studying the importance of the C-terminal part of the protoxin. Herein, we show that Cry1Ab protoxin has a unique natural cryptic endotoxic property that is evident when their halves are expressed individually. This toxic effect of the separate protoxin domains was found against its original host B. thuringiensis, as well as to two other bacteria, Escherichia coli and Agrobacterium tumefaciens. Interestingly, either the fusion of the C-terminal portion with the insecticidal domain-III or the whole N-terminal region reduced or neutralized such a toxic effect, while a non-Cry1A peptide such as maltose binding protein did not neutralize the toxic effect. Furthermore, the C-terminal domain, in addition to being essential for crystal formation and solubility, plays a crucial role in neutralizing the toxicity caused by a separate expression of the insecticidal domain much like a dot/anti-dot system.

Agrobacterium tumefaciens↗
PubMed2006

[Influence of accessory protein P19 from Bacillus thuringiensis on insecticidal crystal protein Cry11Aa].

p19 gene, cry11Aa gene and p20 gene from Bacillus thuringienesis subsp. israelensis are organized as a single operon. It is reported that P20 polypeptide is not required for high-level expression of Cry11Aa and crystal formation in B. thuringiensis. It is deduced that P19 might relate to Cry11Aa crystallization. In this study, two recombinant plasmids pHcy1 and pHcy3 containing cryllAa gene were constructed, the latter absent from p19 gene encoding a possible accessory protein between cry11Aa promoter and cry11Aa gene. The recombinant plasmids were introduced into an acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis. SDS-PAGE showed that Cry11Aa protein per unit of culture medium had a higher expression level in 4Q7(pHcy1) with p19 and cry11Aa genes than in 4Q7(pHcy3) with only cry11Aa gene. Both two B. thuringiensis strains formed Cry11Aa crystals in a similar size and shape during sporulation. Toxicity bioassay showed 4Q7 (pHcy1) and 4Q7 (pHcy3) exhibited a comparable mosquito-larvicidal activity against 3rd-instar Culex quinquefasciatus. It indicated that accessory protein P19 did not have an effect on cry11Aa crystallization and high mosquitocidal toxicity. However, it could enhance Cry11Aa expression amount to a certain extent.

Animals↗
PubMed1993

Biotinylation of Bacillus thuringiensis Insecticidal Crystal Proteins.

Biotinylation of Bacillus thuringiensis insecticidal crystal proteins (ICPs) was evaluated for its potential use in an alternative ICP screening method and in the characterization of ICP receptors. In vivo biological activity of CryIA(b), as inferred from bioassays with Manduca sexta and Ostrinia nubilalis and from histopathological effects on O. nubilalis midgut cells induced by force feeding, was not affected by biotinylation at moderate biotinylation ratios. A competitive radioreceptor assay showed that there was only a minor reduction in binding affinity of biotin-labeled CryIA(b) for M. sexta brush border membrane vesicles. On midgut tissue sections, the binding pattern along the midgut epithelium and the staining intensity of biotinylated ICPs detected with streptavidin-enzyme conjugate were virtually identical to the binding pattern and staining intensity of native CryIA(b) detected with antibodies. The specificity of biotinylated ICP binding to larval midgut tissue was demonstrated by performing homologous competition experiments. The relationship between different ICP receptor types in Plutella xylostella, as inferred from radioligand binding studies, was confirmed by the results of heterologous competition experiments performed with biotinylated and native ICPs.

Journal Article↗
PubMed2002

Rapid degradation of the Cry1F insecticidal crystal protein in soil.

The gene for the core Cry1F insecticidal crystal protein (ICP) from Bacillus thuringiensis Berliner (Bt) has been incorporated into the genome of maize plants, Zea mays L. Plants expressing this ICP are protected from attack by various Lepidopteran pests including the European corn borer, Ostrinia nubilalis (Hübner). The stability of the Cry1F ICP in soil was assessed in a laboratory study designed to determine the persistence of the active protein residue in soil over time, using insect bioassay as the analytical quantification method. The GI(50) (concentration estimated to inhibit growth by 50%) rose at each consecutive incubation interval, indicating a consistent decline in Cry1F activity over time. The residue data were poorly described by a first-order model when fit to either the full data or a truncated data set where the last interval (28 days) was excluded. Data were well described by a shift-log model, and this model predicted DT(50) (time until 50% decay) and DT(90) (time until 90% decay) values of 0.6 and 6.9 days, respectively. This rapid degradation rate was consistent with other Bt proteins evaluated in our laboratory.

Animals↗
PubMed2000

Transcription of the insecticidal crystal protein genes of Bacillus thuringiensis.

Production of a large amount of insecticidal crystal proteins encoded on large plasmids is largely dependent upon the mother cell, Bacillus thuringiensis (B. thuringiensis, also Bt), specific transcription systems attributable to sporulation. In the middle stages of sporulation, cry4A is most actively transcribed from the promoter cry4A-P1. The proximal transcriptional start point of cry4A, which is under the control of the promoter P1, is used in Bacillus subtilis (B. subtilis) in the middle stage of sporulation. The nucleotide sequence that determines the cry4A-P1 promoter is homologous to the consensus sequence for the promoter of sigma E-specific genes in B. subtilis, and to those promoters of the insecticidal protein genes that are efficiently transcribed in vitro with the RNA polymerase E sigma 35 isolated from B. thuringiensis. The sigma factor sigma 35 of B. thuringiensis is highly homologous and functionally equivalent to sigma E of B. subtilis. These results suggest that the cry4A transcription from P1 is under the control of sigma E in B. subtilis, and under the control of sigma 35 in B. thuringiensis.

Amino Acid Sequence↗
PubMed1990

A Translation Fusion Product of Two Different Insecticidal Crystal Protein Genes of Bacillus thuringiensis Exhibits an Enlarged Insecticidal Spectrum.

Two truncated Bacillus thuringiensis crystal protein genes, belonging to the classes cryIA(b) and cryIC and both coding for insecticidal N-terminal fragments of the corresponding crystal proteins, were translationally fused. Expression of the gene fusion in Escherichia coli showed a biologically active protein with a toxicity spectrum that overlapped those of both contributing crystal proteins.

Journal Article↗
PubMedDate not supplied

[The construction and use of inosine-containing primers for searching for and identifying the genes of insecticidal crystal proteins in Bacillus thuringiensis].

The specific to 3 types of Cry genes primers containing inosine were constructed to find crystal insecticidal protein Bacillus thuringiensis genes using PCR. A number of new B. thuringiensis strains isolated in Ukraine were investigated using these PCR primers. As a results, Cry genes were found, some of them were identified and demonstrated high homology to Cry1Ba2 and Cry1Bc genes.

Animals↗
PubMed1998

Primary cause of mortality in the armyworm larvae simultaneously parasitized by parasitic wasp and infected with bacteria.

Parasitoid wasps never kill their hosts before the wasp larvae emerge from the host. However, almost 100% of the host armyworm larvae Pseudaletia separata die within 2-3 days by parasitization with the wasp Cotesia kariyai or by injection of polydnavirus, the wasp symbiont virus, when they are simultaneously infected by the pathogenic bacterium Serratia marcescens. The present study was conducted to elucidate the crucial factor causing this larval mortality. An insecticidal protein has been shown to exist in the hemolymph of dying host larvae; it has been purified by procedures consisting of reverse-phase column extraction, gel filtration and ion-exchange column chromatography. The purified protein showed a strong insecticidal effect with a median lethal dosage (LD50) of 13 pmol/larva and was estimated to have a molecular mass of 57 kDa. The amino acid sequence of the insecticidal protein was partially characterized and used for isolation and sequencing of the genomic DNA. The deduced amino acid sequence for this protein revealed striking similarity with the metalloprotease of S. marcescens enterobacter.

Amino Acid Sequence↗