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PubMed2004

Trp132, Trp154, and Trp157 are essential for folding and activity of a Cyt toxin from Bacillus thuringiensis.

Cyt2Aa2 is a cytolytic and mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. darmstadiensis. The toxin contains 3 tryptophan residues at positions 132, 154, and 157. To study the role of tryptophan on protein structure and functions, each tryptophan residue was substituted by phenylalanine and other different amino acids. Expression test in Escherichia coli showed that all mutant proteins were highly produced as inclusion bodies similar to that of the wild type. The mutant W157F showed haemolytic and mosquito larvicidal activities comparable to the wild type but the mutant W157V and all other mutants at positions 132 and 154 have completely lost these activities. Solubilization and proteinase K activation tests indicated that aromatic residue is required at position 157 and tryptophan residues at positions 132 and 154 are critical residues playing important role to maintain structure and functions of the protein and cannot be changed to any other amino acid.

Bacillus thuringiensis↗
PubMed1993

Complete nucleotide sequence and identification of a putative promoter region for the expression in Escherichia coli of the cryIA(b) gene from Bacillus thuringiensis var. aizawai HD133.

The sequence of a cry gene from Bacillus thuringiensis var. aizawai HD133 was determined. This cry gene encodes a protein of 1155 amino acids, the molecular weight of which is 130622 Da. When the nucleotide sequence of this cry gene was compared with the nucleotide sequence from B.t. var. aizawai IPL7 and var. berliner 1715, only five nucleotide changes were found. Therefore, this cry gene should be grouped into the cryIA(b) gene type. A 72 nucleotide sequence upstream of the open reading frame of the cry gene was enough to conduct the transcription of the cry gene in E. coli. Using a promoter probe vector system, it was demonstrated that any DNA fragment containing this 72 nt sequence exhibited promoter activity in vivo. It is likely that a putative promoter sequence may be present within this 72 nucleotides region for the expression of this cry gene in E. coli.

Amino Acid Sequence↗
PubMed2001

[Toxicity of isolates of Bacillus thuringiensis from Wroclaw against larvae of Aedes aegypti].

Seven field isolates of Bacillus thuringiensis from the Lower Silesia, region of Poland, the Osola plain and phylloplane niches and soil samples from the Karkonosze National Park were tested in vitro for insecticidal activity against mosquito larvae Aedes aegypti. Both the spore/crystal mixture and pured crystals from B. thuringienis strains KpC1, KpF3 and OpQ3 (belonging to the first physiological group including the subspecies japonensis, yoso, jinghongiensis ) proved to be the most active against insects (61-65% of corrected mortality). The lowest toxicity (7-28% mortality) was caused by B. thuringiensis wratislaviensis strains (PO12 and 13).

Aedes↗
PubMed1991

The C-terminal domain of the toxic fragment of a Bacillus thuringiensis crystal protein determines receptor binding.

The insecticidal crystal proteins of Bacillus thuringiensis show a high degree of specificity. In vitro binding studies with several crystal proteins demonstrated a correlation between toxicity and binding to receptors of larval midgut epithelial cells. In order to study the domain-function relationships of the toxic fragment, hybrid crystal proteins based on CryIA(b) and CryIC were constructed. Two out of 11 hybrid proteins constructed exhibited insecticidal activity. Both dispalyed an insecticidal spectrum similar to that of the parental crystal protein from which the C-terminal part of the toxic fragment originated. In addition, in vitro binding studies directly demonstrated the involvement of the C-terminal part of the toxic fragment in receptor binding. These results demonstrate that the C-terminal part of the toxic fragment determines specific receptor binding, which in turn determines, to a large extent, the insect specificity.

Amino Acid Sequence↗
PubMed2002

Active form of dipteran-specific insecticidal protein cryllA produced by Bacillus thuringiensis subsp. israelensis.

The nucleotide sequence of the cry11A gene from Bacillus thuringiensis subsp. israelensis strain HD522 was analyzed and the molecular characterization of CryllA toxin was done. The 70-kDa CryllA protoxin was processed in vitro into 36- and 32-kDa fragments by trypsin and into 34- and 32-kDa fragments by gut proteases from C. pipiens. These two processed fragments are associated together to form the heterodimer. The results of the binding assay with BBMV and the bioassay toward C. pipiens larvae suggested that the heterodimer was biologically as active as the non-digested CryllA toxin and the intramolecular cleavage did not promote the insecticidal activity. These results suggested that a probable complex of the 36- or 34-kDa and 32-kDa fragments was also one of the possible active forms of Cry11A, and that the biological functions of CryllA was not essentially affected by the intramolecular cleavage of the 70-kDa protein.

Animals↗
PubMed1999

cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori.

Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN.

Amino Acid Sequence↗
PubMed1985

Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta.

Bacillus thuringiensis subsp. kurstaki HD-73 produces a crystal protein which is lethal to many lepidopteran larvae. The gene encoding this crystal protein has been isolated from a 75-kb plasmid and engineered into a recombinant Escherichia coli plasmid for analysis. The complete nucleotide sequences of the coding region and 387-bp 5' and 376-bp 3' to the coding region have been determined. The 3537-bp of the coding region specify a protein of Mr 133 330. The full-length gene and several 3' -truncated derivatives of the gene were examined in both E. coli and in an E. coli minicell-expression system to determine if the carboxy end of the protein is essential for toxicity. The results presented here provide the primary structure of the crystal protein gene and show that the N-terminal 68-kDal peptide is toxic, but at a lower level than the full-length gene product.

Amino Acid Sequence↗
PubMed1993

The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival.

Post-exponential Bacillus thuringiensis cells produce both an endospore and a variety of intracellular inclusions. The latter are comprised of protoxins, each being specific for the larvae of certain species from at least three orders of insects. Following ingestion of spores and inclusions, toxicity results in the spores gaining access to haemolymph, a source of nutrients suitable for germination and growth. Most B. thuringiensis subspecies contain multiple, plasmid-encoded protoxin genes, often with several on the same plasmid. These genes have been manipulated in order to understand the basis of toxicity and specificity, information which is important to the use of these toxins as biological control agents. Some protoxin genes are in operons, and others are in close proximity, perhaps to enhance the chances of recombination, and some are on unstable plasmids. The arrangement of these genes is probably important for flexibility in the variety of protoxins packaged into inclusions by a particular subspecies and thus the capacity to adapt to changing populations of insects. Protoxins accumulate over a prolonged period during sporulation because of the sequential transcription from two promoters, each being dependent upon a specific sporulation sigma factor, the relative stability of the messenger RNA, and the synthesis of proteins which stabilize protoxins and perhaps facilitate inclusion assembly. During the post-exponential phase, spore and inclusion formation must be balanced so as to ensure that both are available to contribute to the survival of these bacilli.

Bacillus thuringiensis↗
PubMed1994

Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide.

Bacillus thuringiensis toxin CryIIIB2 exhibits activity against two agriculturally important pests, the Colorado potato beetle, Leptinotarsa decemlineata, and the Southern corn rootworm, Diabrotica undecimpunctata. CryIIIB2 shows significant structural similarity to Colorado potato beetle-active toxin CryIIIA, whose crystal structure has been determined elsewhere [J. Li, J. Carrol, and D. J. Ellar, Nature (London) 353:815-821, 1991]. A clone limited to the putative 7-alpha-helical bundle domain I peptide of CryIIIB2 was constructed by PCR. The truncated protein was expressed at high levels in Escherichia coli. Domain I peptide was isolated and compared with native CryIIIB2 toxin in promoting ion efflux from synthetic phospholipid vesicles and formation of ion channels in black lipid membranes. The results showed that CryIIIB2 domain I peptide is sufficient for ion channel formation and promotes ion efflux. Both native CryIIIB2 toxin and domain I peptide were inefficient channel-forming proteins that produced noisy ion channels of various conductance states. In ion efflux assays, native toxin promoted greater ion efflux from synthetic vesicles than did the truncated peptide.

Animals↗
PubMed1997

Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin.

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.

Amino Acid Sequence↗
PubMed2006

Novel preparation and characterization of the alpha4-loop-alpha5 membrane-perturbing peptide from the Bacillus thuringiensis Cry4Ba delta-endotoxin.

Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba delta-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the alpha4-alpha5 hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the alpha4-loop-alpha5 hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an alpha-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the alpha4-loop-alpha5 hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.

Amino Acid Sequence↗
PubMed1999

Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins.

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.

Animals↗
PubMed1993

Evidence that the CryIA crystal protein from Bacillus thuringiensis is associated with DNA.

Toxin generated by activation of the Bacillus thuringiensis CryIA(c) crystal protein (protoxin) with bovine trypsin was separated into two components by anion-exchange chromatography. One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (T1). Only one major protoxin component was observed, and it was found to be associated with DNA. The DNA from the T2 toxin varied in size from 100 to 300 base pairs, whereas the crystal and the solubilized protoxin contained 20-kilobase DNA as the major DNA component. DNase treatment converted the T2 toxin to the DNA-free T1 toxin. In contrast, the DNA in the crystal and the solubilized protoxin was resistant to DNase digestion and was not dissociated from the protein by 1.5 M NaCl. The protoxin and DNA appeared to elute as a complex with a molecular mass of > 2 x 10(6) Da on gel-filtration chromatography. No toxin was generated from the protoxin with trypsin after extensive digestion of the protoxin with DNase or dissociation of the DNA by succinylation of the lysine residues. It is proposed that DNA binds to the COOH-terminal half of the crystal protein and is essential for maintaining the conformational integrity required for crystal formation and generation of toxin.

Bacillus thuringiensis↗
PubMed2002

Role of helix 3 in pore formation by the Bacillus thuringiensis insecticidal toxin Cry1Aa.

Helix 3 of the Cry1Aa toxin from Bacillus thuringiensis possesses eight charged amino acids. These residues, with the exception of those involved in intramolecular salt bridges (E90, R93, E112, and R115), were mutated individually either to a neutral or to an oppositely charged amino acid. The mutated genes were expressed, and the resultant, trypsin-activated toxins were assessed for their toxicity to Manduca sexta larvae and their ability to permeabilize M. sexta larval midgut brush border membrane vesicles to KCl, sucrose, raffinose, potassium gluconate, and N-methyl-D-glucamine hydrochloride with a light-scattering assay based on osmotic swelling. Most mutants were considerably less toxic than Cry1Aa. Replacing either E101, E116, E118, or D120 by cysteine, glutamine, or lysine residues had only minor effects on the properties of the pores formed by the modified toxins. However, half of these mutants (E101C, E101Q, E101K, E116K, E118C, and D120K) had a significantly slower rate of pore formation than Cry1Aa. Mutations at R99 (R99C, R99E, and R99Y) resulted in an almost complete loss of pore-forming ability. These results are consistent with a model in which alpha-helix 3 plays an important role in the mechanism of pore formation without being directly involved in determining the properties of the pores.

Amino Acid Sequence↗
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↗
PubMed2006

Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control.

Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity against insects of different orders--Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores. Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane. Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect pests and insect vectors of human diseases.

Bacillus thuringiensis↗
PubMed1994

Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay system.

The Bacillus thuringiensis crylA(a) and crylA(c) gene specificity regions were probed by creating and testing hybrid toxins both in vivo and in vitro against cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CrylA(c) is highly active against cultured Choristoneura fumiferana cells (CF-1) whereas CrylA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis revealed that the CrylA(c) toxicity-determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258-358 and 450-510) were able to confer toxicity, although at lower levels, and one region (358-450) was present where progressive substitutions of crylA(a) with crylA(c) sequences had no effect. Exchanging the non-homologous N-terminal regions of CrylA(c) with CrylE suggested that the N-terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CrylA(b) but shows an 800-fold increase in toxicity to CF-1 cells relative to that shown by CrylA(b). Direct comparison between live Bombyx mori bioassays and a newly developed in vitro lawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity-determining region for B. mori larvae was determined to be between codons 283 and 450, although the 450-620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determining toxin specificity.

Amino Acid Sequence↗
PubMed1996

Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins.

We compared responses to six insecticidal crystal proteins from Bacillus thuringiensis by a Cry1A-resistant strain (NO-QA) and a susceptible strain (LAB-P) of the diamondback moth, Plutella xylostella. The resistant strain showed > 100-fold cross-resistance to Cry1J and to H04, a hybrid with domains I and II of Cry1Ab and domain III or Cry1C. Cross-resistance was sixfold to Cry1Bb and threefold to Cry1D. The potency of Cry1I did not differ significantly between the resistant and susceptible strains. Cry2B did not kill resistant or susceptible larvae. By combining these new data with previously published results, we classified responses to 14 insecticidal crystal proteins by strains NO-QA and LAB-P. NO-QA showed high levels of resistance to Cry1Aa, Cry1Ab, and Cry1Ac and high levels of cross-resistance to Cry1F, Cry1J, and H04. Cross-resistance was low or nil to Cry1Ba, Cry1Bb, Cry1C, Cry1D, Cry1I, and Cry2A. Cry1E and Cry2B showed little or no toxicity to susceptible or resistant larvae. In dendrograms based on levels of amino acid sequence similarity among proteins, Cry1F and Cry1J clustered together with Cry1A proteins for domain II, but not for domain I or III. High levels of cross-resistance to Cry1Ab-Cry1C hybrid H04 show that although Cry1C is toxic to NO-QA, domain III or Cry1C is not sufficient to restore toxicity when it is combined with domains I and II of Cry1Ab. Thus, diamondback moth strain NO-QA cross-resistance extends beyond the Cry1A family of proteins to at least two other families that exhibit high levels of amino sequence similarity with Cry1A in domain II (Cry1F and Cry1J) and to a protein that is identical to Cry1Ab in domain II (H04). The results of this study imply that resistance to Cry1A alters interactions between the insect and domain II.

Amino Acid Sequence↗