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PubMed1995

Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis.

Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the hornworm. BT-R1 shares sequence similarity with the cadherin superfamily of proteins.

Amino Acid Sequence↗

PubMed2005

Cry3Aa11: a new Cry3Aa delta-endotoxin from a local isolate of Bacillus thuringiensis.

A local isolate of Bacillus thuringiensis Mm2 had insecticidal activity against the larvae of Melolontha melolontha, Agelastica alni, Leptinotarsa decemlineata and Amphimallon solstitiale and produced a 65 kDa protein. SDS-PAGE profile of B. thuringiensis Mm2 was compared with those of 29 different Cry3Aa producers which verified Cry3Aa biosynthesis by the isolate. The cry3Aa gene of Mm2 was cloned, sequenced and the deduced amino acid sequence was compared with the cry3Aa sequences of ten different quaternary ranks. Its identity to these sequences ranged between 97.4% and 99.2%. The gene was next cloned into E. coli-Bacillus shuttle vector pNW33N and expressed at a low level in B. subtilis 168.

Amino Acid Sequence↗

PubMed1987

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains.

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.

Bacillus thuringiensis↗

PubMed1985

Nematoda: susceptibility of the egg to Bacillus thuringiensis toxins.

Crystalline toxins from Bacillus thuringiensis israelensis and B.t. kurstaki were lethal in vitro to eggs of the nematode Trichostrongylus colubriformis. The LD50 values for the two toxins were 0.38 ng and 37.5 micrograms total protein/ml, respectively. After 1 week at ambient temperature, the LD50 of B.t. kurstaki decreased to less than 4 micrograms/ml. Toxin from B.t. israelensis had no effect within 48 hr on survival of adult nematodes or on their feeding in vitro. Third-stage larvae of T. colubriformis were also unaffected by B.t. israelensis toxin. Exposure of third-stage larvae of Nippostrongylus brasiliensis to 1.1 micrograms total protein/ml of B.t. israelensis for 4 hr had no effect on their infectivity in mice, based on recovery of helminths at 7 days after infection. Similar exposure of 5-day-old N. brasiliensis and subsequent transfer into the intestine of mice gave recoveries that were similar to the untreated control. Thirty strains of B. thuringiensis caused mortality in nematode eggs, but over a 77,000-fold range of activity was found, based on the LD50 values. Toxin from B.t. israelensis was lethal to eggs of six zooparasitic and one free-living species of nematode, but the LD50 values varied 28-fold. Addition of B.t. israelensis to feces that contained eggs of T. colubriformis reduced subsequent recovery of larvae, with an LD50 of 260 ng/g of feces.

Animals↗

PubMed1989

Location of the Bombyx mori specificity domain on a Bacillus thuringiensis delta-endotoxin protein.

Bacillus thuringiensis produces different types of insecticidal crystal proteins (ICPs) or delta-endotoxins. In an effort to identify the insect specificity of ICP toxins, two icp genes were cloned into the Escherichia coli expression vector pKK223-3, and bioassays were performed with purified crystals. The type A protein [from an icpA1, or 4.5-kilobase (kb) gene, from B. thuringiensis var. kurstaki HD-1] was found to be 400 times more active against Bombyx mori than type C protein (from an icpC73, or 6.6-kb gene, from B. thuringiensis var. kurstaki HD-244). The type C protein was 9 times more active against Trichoplusia ni than the type A protein, while both have similar activity against Manduca sexta. To locate the specificity domain of the type A protein for B. mori, site-directed mutagenesis was used to introduce or remove restriction enzyme sites, facilitating the exchange of regions of the two genes. The hybrid genes were overexpressed, and purified ICP was used in bioassays. The B. mori specificity domain for the ICP A toxin is located in the amino-terminal portion of the hypervariable region between amino acids 332 and 450.

Animals↗

PubMed1999

The cadherin-like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin.

The Bacillus thuringiensis CryIAa toxin binds a cadherin-like protein (BtR175) on the brush-border membranes of the Bombyx mori midgut columnar cells, which are the targets. By introducing the BtR175 gene with a baculovirus, Spodoptera frugiperda Sf9 cells expressed BtR175 protein on the cell membrane and became susceptible to the CryIAa toxin. The toxin bound the cadherin repeat adjacent to the membrane and made a pore that passed inorganic ions, causing the cell to swell and burst. This was not observed with a BtR175 variant lacking the toxin-binding site. This in vitro experiment mimicked the specific insecticidal action of the toxin in vivo well.

Bacillus thuringiensis↗

PubMed1998

Cloning, sequencing, and expression of the Bombyx mori receptor for Bacillus thuringiensis insecticidal CryIA(a) toxin.

Bacillus thuringiensis strains produce insect-specific Bt toxins. Bt CryIA(a) toxin binds to a 175-kDa glycoprotein (BtR175) on the microvillus membranes of columnar cells in the Bombyx mori midgut and causes lysis of the cells. BtR175 was purified, and its cDNA was cloned. The cDNA encodes a newly identified 193.3-kDa preproprotein form of BtR175 that includes nine extracellular cadherin repeats, a 23.5-kDa membrane-proximal domain, a membrane-spanning region, and a 13.6-kDa cytoplasmic domain. Spodoptera frugiperda cells transfected with a recombinant baculovirus DNA carrying the cDNA produced a 175-kDa protein that reacted with anti-BtR antibodies and the Bt CryIA(a) toxin.

Amino Acid Sequence↗

PubMed1986

Biological control of mosquitoes by the larvicidal activity of Bacillus thuringiensis var. israelensis delta endotoxin.

Bacillus thuringiensis var. israelensis (B.t.i.) is a promising, safe toxic agent for control of mosquitoes, but the rapid disappearance of its toxicity makes its use in practice economically unattractive. The lack of evidence for B.t.i. multiplication in water also makes the natural ecology of B.t.i. puzzling. The observation that mosquito larvae readily cannibalize carcasses of B.t.i.-killed larvae, and that the carcasses become toxic to scavenging larvae provides a possible solution to this puzzle. Several experimental techniques have been developed to study these phenomena (e.g., quantitative determination of spore numbers despite aggregation, protocols for following toxicity development, etc.). Results suggest a cycle involving larval poisoning and death due to delta-endotoxin of ca. 1,000 spores, germination, vegetative growth and sporulation (with toxin production) of B.t.i. after multiplication to several million in the carcass. Implications of these results for the ecology of B.t.i., and for practical applications in mosquito control, are discussed.

Animals↗

PubMed1998

Purification and partial amino acid sequences of the binding protein from Bombyx mori for CryIAa delta-endotoxin of Bacillus thuringiensis.

The binding protein for Bacillus thuringiensis delta-endotoxin, CryIAa, from the brush border membrane of the midgut of Bombyx mori was purified by the dot blot method and delta-endotoxin affinity chromatography. The binding protein was purified to 235-fold enrichment from cholic acid extracts of brush border membranes from B. mori midgut by activated CryIAa-affinity chromatography and DEAE ion-exchange chromatography. The purified binding protein showed a single band of 180 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and this band specifically reacted to 125I-labeled CryIAa on Immobilon membrane. The affinity of the binding protein for CryIAa was equivalent to that of the brush border membrane vesicles and solubilized membrane proteins. Partial amino acid sequences of the binding protein showed sequence similarity to the cadherin-like binding protein for CryIAb from Manduca sexta, but not for CryIAc binding protein from M. sexta and Heliothis virescens.

Amino Acid Sequence↗

PubMed1997

Bacillus thuringiensis: from biodiversity to biotechnology.

Bacillus thuringiensis is a Gram-positive bacterium, widely used in agriculture as a biological pesticide. The biocidal activity mainly resides in a parasporal protein inclusion body, or crystal. The inclusion is composed of one or more types of delta-endotoxins (Cry and Cyt proteins). Cry proteins are selectively toxic to different species from several invertebrate phyla: arthropods (mainly insects), nematodes, flatworms and protozoa. The mode of action of the insecticidal proteins is still a matter of investigation; generally, the active toxin is supposed to bind specific membrane receptors on the insect midgut brush-border epithelium, leading to intestinal cell lysis and subsequent insect death by starvation or septicemia. The toxin-encoding cry genes have been extensively studied and expressed in a large number of prokaryotic and eukaryotic organisms. The expression of such genes in transgenic plants has provided a powerful alternative for crop protection.

Bacillus thuringiensis↗

PubMed1991

Expression of the cryIB crystal protein gene of Bacillus thuringiensis.

The cryIB gene of Bacillus thuringiensis subsp. thuringiensis HD-2 codes for a Mr 139492 protein that is lethal to certain lepidopteran larvae. We used primer extension to map transcriptional initiation sites and found that cryIB was transcribed from two sites that are activated at different times during sporulation. The presumed promoter regions for the two start sites are very similar to the two promoters preceding the cryIA (a) gene, and the in vivo transcriptional start sites were found to be identical. Variable amounts of the full-length cryIB protein were detected by immunoblotting of extracts of recombinant cells of Escherichia coli; larger amounts were found when the TTG translational start codon was changed to ATG and when an htpR- strain of E. coli was used as the recipient for transformation. When expressed in E. coli, the cryIB protein was found to be toxic to the larvae of Artogeia rapae (LC50 of 58 ng/cm2) and exhibited little toxicity to the larvae of Manduca sexta (LC50 greater than 5000 ng/cm2).

Animals↗

PubMed1995

Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin.

The Bacillus thuringiensis CryIA(c) insecticidal delta-endotoxin binds to a 120-kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect Manduca sexta. This glycoprotein has recently been purified and identified as aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were isolated, and their combined 3095-nucleotide sequence contains an open reading frame encoding a 990-residue pre-pro-protein. The N-terminal amino acid sequence derived from the glycoprotein is present in the open reading frame, immediately following a predicted cleavable signal peptide and a pro-peptide. There are four potential N-linked glycosylation sites. The C-terminal sequence contains a possible glycosylphosphatidylinositol (GPI) anchor signal peptide, which suggests that, unlike most other characterized aminopeptidases, the lepidopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by phosphatidylinositol-specific phospholipase C. The deduced amino acid sequence shows significant similarity to the zinc-dependent aminopeptidase gene family, particularly in the region surrounding the consensus zinc-binding motif characteristic of these enzymes.

Amino Acid Sequence↗

PubMed1999

Analysis of mutations in the pore-forming region essential for insecticidal activity of a Bacillus thuringiensis delta-endotoxin.

The Bacillus thuringiensis insecticidal delta-endotoxins have a three-domain structure, with the seven amphipathic helices which comprise domain I being essential for toxicity. To better define the function of these helices in membrane insertion and toxicity, either site-directed or random mutagenesis of two regions was performed. Thirty-nucleotide segments in the B. thuringiensis cry1Ac1 gene, encoding parts of helix alpha4 and the loop connecting helices alpha4 and alpha5, were randomly mutagenized. This hydrophobic region of the toxin probably inserts into the membrane as a hairpin. Site-directed mutations were also created in specific surface residues of helix alpha3 in order to increase its hydrophobicity. Among 12 random mutations in helix alpha4, 5 resulted in the total loss of toxicity for Manduca sexta and Heliothis virescens, another caused a significant increase in toxicity, and one resulted in decreased toxicity. None of the nontoxic mutants was altered in toxin stability, binding of toxin to a membrane protein, or the ability of the toxin to aggregate in the membrane. Mutations in the loop connecting helices alpha4 and alpha5 did not affect toxicity, nor did mutations in alpha3, which should have enhanced the hydrophobic properties of this helix. In contrast to mutations in helix alpha5, those in helix alpha4 which inactivated the toxin did not affect its capacity to oligomerize in the membrane. Despite the formation of oligomers, there was no ion flow as measured by light scattering. Helix alpha5 is important for oligomerization and perhaps has other functions, whereas helix alpha4 must have a more direct role in establishing the properties of the channel.

Animals↗

PubMed2006

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Colorado potato beetle-active Bacillus thuringiensis delta-endotoxins.

Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry1-hybrid SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square or rectangular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions. Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude activity against CPB, but that interfering with this crystal formation can increase the activity.

Amino Acid Sequence↗

PubMed2000

Role of proteolysis in determining potency of Bacillus thuringiensis Cry1Ac delta-endotoxin.

Bacillus thuringiensis protein delta-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac delta-endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of approximately 56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of approximately 58, approximately 40, and approximately 20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.

Amino Acid Sequence↗

PubMed2005

Fingerprinting of Bacillus thuringiensis type strains and isolates by using Bacillus cereus group-specific repetitive extragenic palindromic sequence-based PCR analysis.

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native beta-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.

Bacillus cereus↗

PubMed1981

[Instability of the auxotrophic markers in Bacillus thuringiensis].

Auxotrophic mutants of Bacillus thuringiensis have been isolated and compared with the mutants of Bacillus subtilis. It has been found that after spore formation and a prolonged storage, the considerable part of cells in a population of some strains display a genetically unstable additional requirement for arginine with high frequency. The correlation between tetracycline sensitivity and unstable requirement for arginine and other growth factors has been found. The possible reasons for instability of genetic markers in Bac. thuringiensis are discussed.

Arginine↗

PubMedDate not supplied

[Antibiotic sensitivity of Bacillus thuringiensis var. galleriae strains].

Bacillus thuringiensis strains forming colonies of the S and R morphology were found to be susceptible to streptomycin, chloramphenicol, rifampicin, neomycin, lincomycin, monomycin, kanamycin, and resistant to ampicillin and polymyxin. The S strains were shown to be susceptible to tetracycline (Tets), whereas the R strains were either susceptible (Tets) or resistant (Tetr). The population of Tetr strains was heterogeneous in its resistance to tetracycline (the frequency of Tetr cells was from 10(-4) to 1). The incubation with a subinhibiting concentration of tetracycline (2 micrograms/ml) caused "induction", which decreased in the presence of chloramphenicol, in the Tetr strains. No "induction" was detected in Tets strains. No significant differences were found in the plasmid composition of the Tets and Tetr strains, and no correlation was established between the presence of plasmids and the resistance to tetracycline. It may be concluded therefore that determinants for the resistance to tetracycline are located in the chromosomes. Possible factors causing the resistance to tetracycline in B. thuringiensis are discussed.

Anti-Bacterial Agents↗