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PubMed1996

Expression of 135-kDa insecticidal protein gene from Bacillus thuringiensis in the yeast Saccharomyces cerevisiae.

Bacillus thuringiensis subsp. aizawai produces 130-kDa and 135-kDa (CryIA(a)) insecticidal proteins. When Saccharomyces cerevisiae was transformed by the vector carrying a cryIA(a) gene, the gene expression could not be observed. When the 5'-upstream region from the initiation codon was removed using a synthetic oligonucleotide, the CryIA(a) protein was successfully synthesized in yeast. The yeast extract containing CryIA(a) protein had insecticidal activity against Plutella xylostella larvae.

Bacillus thuringiensis↗
PubMed1987

Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli.

A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein.

Amino Acid Sequence↗
PubMed2000

Feeding behavior of bollworm and tobacco budworm (Lepidoptera: Noctuidae) larvae in mixed stands of nontransgenic and transgenic cotton expressing an insecticidal protein.

Feeding behavior of third-instar bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), was observed in pure and mixed stands of nontransgenic and transgenic cotton (BTK), Gossypium hirsutum L., expressing an insecticidal protein CryIA(c) from a bacterium, Bacillus thuringiensis Berliner subsp. kurstaki. Five plant stands composed of BTK and non-BTK plants were evaluated; two pure stands and three mixed stands. Percentage ratios of BTK to non-BTK plants in the stands were 100:0, 75:25, 50:50, 25:75 and 0:100, respectively. In all stands with BTK plants, fewer bollworm and tobacco budworm larvae were found on BTK plants than non-BTK plants 24 h after infestation with third instars. At 48 h, significantly fewer tobacco budworm larvae, but not fewer bollworm larvae, were found on BTK plants. However, the number of larvae of either insect did not increase on non-BTK plants compared with the initial infestation density of three larvae per plant. The number of obacco budworm injured flower buds, and capsules was lower in all plant stands containing BTK plants compared with the pure stand of non-BTK at 48 h after infestation. Higher numbers of larvae on non-BTK plants were possibly the result of larval intoxication, reduced feeding, and increased plant abandonment and death on BTK plants rather than a classical feeding preference. Unexpectedly, the number of flower buds and capsules injured by bollworm and tobacco budworm when averaged per plant for all plants in a stand, differed little among the 75:25, 50:50 and 25:75 plant mixtures. These data suggest that larvae of both species frequently moved among plants, feeding indiscriminately on BTK and non-BTK plants.

Animals↗
PubMed2005

Insertion behavior of the Bacillus thuringiensis Cry4Ba insecticidal protein into lipid monolayers.

Toxicity mechanisms of Bacillus thuringiensis Cry insecticidal proteins involve membrane insertion and lytic pore formation in lipid bilayers of the target larval midgut cell membranes. The B. thuringiensis Cry4Ba mosquito-larvicidal protein has been shown to be capable of permeabilizing liposome vesicles and of forming ion channels in planar lipid bilayers. Here, the membrane interaction of the 65-kDa activated Cry4Ba protein with the lipid monolayers, comprising dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and cholesterol (Chol), was studied using Langmuir-Blodgett technique. The interactions of the Cry4Ba protein with the lipid monolayers were measured from the surface pressure versus area isotherms of the protein-lipid monolayers. The increase in the mean molecular area was demonstrated as an incorporation of the protein into lipid monolayers. The insertion of the Cry4Ba protein was monitored by measuring as an increase of the surface pressure at constant molecular area. For a given monolayer, the membrane insertion of the Cry4Ba reduced as the initial surface pressure increased. The Cry4Ba protein showed a strong preference of an insertion towards a Chol monolayer. In addition, the mixed monolayers of Chol showed an enhanced effect on the insertion kinetics of Cry4Ba into lipid films, suggesting its involvement in the modulation of the protein insertion. These findings provide the first evidence that the Cry4Ba protein is capable of inserting itself into lipid monolayers, depending on the packing density of the monolayers. Our results also indicate that only a limited part of the protein is likely to be involved in the insertion.

Animals↗
PubMed2003

The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin.

The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent.

Animals↗
PubMed1996

Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.

A novel vegetative insecticidal gene, vip3A(a), whose gene product shows activity against lepidopteran insect larvae including black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), tobacco budworm (Heliothis virescens), and corn earworm (Helicoverpa zea) has been isolated from Bacillus thuringiensis strain AB88. VIP3-insecticidal gene homologues have been detected in approximately 15% of Bacillus strains analyzed. The sequence of the vip3A(b) gene, a homologue of vip3A(a) isolated from B. thuringiensis strain AB424 is also reported. Vip3A(a) and (b) proteins confer upon Escherichia coli insecticidal activity against the lepidopteran insect larvae mentioned above. The sequence of the gene predicts a 791-amino acid (88.5 kDa) protein that contains no homology with known proteins. Vip3A insecticidal proteins are secreted without N-terminal processing. Unlike the B. thuringiensis 5-endotoxins, whose expression is restricted to sporulation, Vip3A insecticidal proteins are expressed in the vegetative stage of growth starting at mid-log phase as well as during sporulation. Vip3A represents a novel class of proteins insecticidal to lepidopteran insect larvae.

Amino Acid Sequence↗
PubMed2000

[Expression and synergism of two cry insecticidal protein genes in Pseudomonas fluorescens].

Several engineered Pseudomonas fluorescens(Pf) strains were constructed mainly based on a Pseudomonas plasmid pJMS6 alpha-lac and two insecticidal crystal protein genes of Bacillus thuringiensis, cry1Ac and cry2Aa, and the host Pf strain, P303, which was with highly antifungal activity to some plant disease fungi and colonizing ability on a wide range of plants. The DNA introduction was confirmed by PCR-RFLP and Southern blot. The 132 kD insecticidal protein was detected in IPP101 and IPP202 by SDS-PAGE and rhombic insecticidal protein crystals of them were observed through electron microscope, also indicating that cry1Ac gene was highly expressed. The results of insecticidal bioassay indicated that IPP101 was more toxic than IPP201, and IPP202 was the most toxic among the 3 strains. LC50 to the neonates of cotton boll worm(Helicoverpa armigera) were 0.02604, 0.00812 and 0.00186 mL/g feed, consecutively. In IPP202, two gene products showed significant synergism, with the co-toxicity coefficient of 332.8.

Animals↗
PubMed1998

Bacillus thuringiensis insecticidal proteins: molecular mode of action.

Growing interest in biorational pesticides has placed the Bacillus thuringiensis insecticidal crystal proteins at the forefront of pesticides for plant genetic engineering. The development of improvement pesticides, both in enhanced activity and broader host range, depends on an understanding of its mechanism of action. This review presents a complete overview of the bacterium and the group of insecticidal proteins known as Cry proteins or delta-endotoxins. The molecular mode of action is described in detail, including the mapping of receptor binding sites by site-directed mutagenesis, the known receptors, and the ion-channel activity of the toxins.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of Bacillus thuringiensis var. tenebrionis insecticidal proteins produced in E. coli.

Native and single amino acid variants of the Bacillus thuringiensis var. tenebrionis insecticidal proteins were expressed in Escherichia coli, purified and examined for biological and biochemical properties. A novel, pH dependent, preferential precipitation method was implemented to purify Escherichia coli produced Bacillus thuringiensis var. tenebrionis proteins, which are active against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Cysteine residues of the native Bacillus thuringiensis var. tenebrionis protein were replaced by serine residues by site-directed mutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionis proteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteine residue (amino acid 540) caused complete inactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidal activities comparable to each other and to solubilized crystals from the original strain.

Animals↗
PubMed1997

Identification of a second transcriptional start site for the insecticidal protein gene cryIVA of Bacillus thuringiensis subsp. israelensis.

Expression of cryIVA, one of the insecticidal protein genes of B. thuringiensis subsp. israelensis, is regulated at the transcriptional level. The cryIVA gene is specifically transcribed during the stationary phase of this bacterium. As shown in our previous report [Yoshisue et al. (1993a)], the transcription from the -364 position of the cryIVA gene is conducted by the major promoter P1 that is functional during middle stages of the stationary phase of B. thuringiensis. In the present study, we have identified a second transcriptional start point P2 for the cryIVA gene in addition to P1, the major transcriptional start point. The transcription from P2 of the cryIVA gene occurred later than that from P1, during later stages of stationary phase of B. thuringiensis subsp. israelensis. The -10 and -35 nt sequences upstream from P2 of cryIVA are similar to those of the omega 28-specific promoters of B. thuringiensis genes and of the omega K-specific promoters of B. subtilis genes. It is most likely that the region upstream from P2 of cryIVA contains the nt sequences that determine the omega 28-specific promoter, the second one, for the cryIVA gene.

Amino Acid Sequence↗
PubMed1990

Construction of chimeric insecticidal proteins between the 130-kDa and 135-kDa proteins of Bacillus thuringiensis subsp. aizawai for analysis of structure-function relationship.

Eight chimeric insecticidal protein (IP) genes were constructed between the 130-kDa and 135-kDa IP genes of Bacillus thuringiensis subsp. aizawai, and expressed in Escherichia coli JM103 cells. The characterization of the produced chimeric IPs indicated that the variable region (VR1) in the amino-terminal half of the IPs is responsible for the insecticidal activity against larvae of Spodoptera litura and Plutella xylostella. The carboxy-terminal half of VR1 was important for the formation of the 60-kDa active fragment in the gut juice of S. litura larvae. Also, combination of the other two variable regions (VR2 and VR3), which were in the central and carboxy-terminal portions of the IPs, appeared to be related to the solubility of the IPs in the gut juice.

Animals↗
PubMedDate not supplied

Studies on the growth metabolism of Bacillus thuringiensis and its vegetative insecticidal protein engineered strains by microcalorimetry.

The metabolic power-times curves of Bacillus thuringiensis and its vegetative insecticidal protein engineered strains were determined at 30 degrees C by using a thermal activity monitor air Isothermal Microcalorimeter, ampoule method. From the power-times curves, the maximum power (Pmax) in the log phase, the growth rate constant (k), the generation times (tG), the time of the maximum power (tmax), the heat effects (Qlog) for log phase, and the total heat effect in 45 h (Qtotal) of B. thuringiensis strains can be obtained. The results indicate that their power-times curves are different. The relationship between their metabolic power-times curves and character of bacteria metabolism, and thermokinetics and gene expression were analyzed and discussed. The character of the bacteria power-times curves reflected the physiologic character of gene expression. The microcalorimetric method proved to be a reliable and sensitive tool for the assessment of the growth metabolism, the heat output in bacteria and its engineered strains. The determination of the thermokinetic character is beneficial to the control of fermentation.

Animals↗
PubMed1995

Enhanced production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional crystal protein gene in their chromosomes.

A two-step procedure was used to place a cryIC crystal protein gene from Bacillus thuringiensis subsp. aizawai into the chromosomes of two B. thuringiensis subsp. kurstaki strains containing multiple crystal protein genes. The B. thuringiensis aizawai cryIC gene, which encodes an insecticidal protein highly specific to Spodoptera exigua (beet armyworm), has not been found in any B. thuringiensis subsp. kurstaki strains. The cryIC gene was cloned into an integration vector which contained a B. thuringiensis chromosomal fragment encoding a phosphatidylinositol-specific phospholipase C, allowing the B. thuringiensis subsp. aizawai cryIC to be targeted to the homologous region of the B. thuringiensis subsp. kurstaki chromosome. First, to minimize the possibility of homologous recombination between cryIC and the resident crystal protein genes, B. thuringiensis subsp. kurstaki HD73, which contained only one crystal gene, was chosen as a recipient and transformed by electroporation. Second, a generalized transducing bacteriophage, CP-51, was used to transfer the integrated cryIC gene from HD73 to two other B. thuringiensis subsp. kurstaki stains. The integrated cryIC gene was expressed at a significant level in all three host strains, and the expression of cryIC did not appear to reduce the expression of the endogenous crystal protein genes. Because of the newly acquired ability to produce the CryIC protein, the recombinant strains showed a higher level of activity against S. exigua than did the parent strains. This two-step procedure should therefore be generally useful for the introduction of an additional crystal protein gene into B. thuringiensis strains which have multiple crystal protein genes and which show a low level of transformation efficiency.

Animals↗
PubMed2005

Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis.

Twenty-four serovars of Bacillus thuringiensis (Bt) were screened by polymerase chain reaction to detect the presence of vegetative insecticidal protein gene (vip)-like sequences by using vip3Aa1-specific primers. vip-like gene sequences were identified in eight serovars. These genes were cloned and sequenced. The deduced amino acid sequence of the vip3Aa14 gene from Bacillus thuringiensis tolworthi showed considerable differences as compared to those of Vips reported so far. The vip3Aa14 gene from Bt tolwarthi was expressed in Escherichia coli using expression vector pET29a. The expressed Vip3Aa14 protein was found in cytosolic supernatant as well as pellet fraction, but the protein was more abundant in the cytosolic supernatant fraction. Both full-length and truncated (devoid of signal sequence) Vips were highly toxic to the larvae of Spodoptera litura and Plutella xylostella. Truncation of Vip3Aa14 protein at N-terminus did not affect its insecticidal activity.

Animals↗
PubMed1996

Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins by site-directed mutagenesis--a minireview.

The current model of the mechanism of action of several Bacillus thuringiensis insecticidal crystal proteins (Cry) is reviewed and tested by site-directed mutagenesis experiments. Amino acid (aa) residues were substituted in each of the three domains of Cry toxins and the effects on toxin stability, binding to receptors, irreversible insertion into the membrane, and ion channel activity were examined. Mutant proteins with aa altered on the putative membrane-proximal surface of domain I are affected in insertion into the membrane and toxicity, but not in binding to the receptor. Alterations in the putative receptor-binding loops of domain II show an effect on the initial (reversible) binding to the receptor when certain aa are altered, while affecting irreversible binding when other aa are altered. Mutant proteins with aa altered in a conserved track of aa of domain III have altered ion channel properties, as measured by the voltage clamping of insect midguts and the K+ permeability of brush border membrane vesicles. In summary, domain I is involved in insertion into the membrane and affects ion channel function, domain II is involved in receptor binding and insertion into the membrane, and domain III is involved ion channel function, receptor binding, and insertion into the membrane.

Bacillus thuringiensis↗
PubMed2006

Vegetative insecticidal protein (Vip1Ac) of Bacillus thuringiensis HD201: evidence for oligomer and channel formation.

The binding component (Vip1Ac) of the ADP-ribosylating vegetative insecticidal protein (Vip) of Bacillus thuringiensis HD201 was isolated from the supernatant of cell cultures. Vip1Ac protein solubilized at room temperature ran as oligomers on SDS-PAGE. These oligomers were not resistant to heating. Mass spectroscopic analysis of this high molecular mass band identified it as Vip1Ac. The protein formed in artificial lipid bilayer membranes channels with two conductance states of about 350 and 700 pS in 1 M KCl. The channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero-current membrane potential measurements showed that the Vip1Ac channel has a slightly higher permeability for chloride than for potassium ions. Asymmetric addition of Vip1Ac to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. The functional role of Vip1Ac and its relationship to other ADP-ribosylating toxins are discussed.

Adenosine Diphosphate Ribose↗
PubMed2006

Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua.

AIMS: The objective of this work was to enhance the insecticidal activity or widen the pesticidal spectrum of a commercial Bacillus thuringiensis strain YBT1520. METHODS AND RESULTS: A vegetative insecticidal protein gene vip3Aa7, under the control of its native promoter and cry3A promoter, was subcloned into B. thuringiensis acrystalliferous BMB171 to generate BMB8901 and BMBvip respectively. It was found that the amount of Vip3Aa7 protein produced by BMBvip was 3.2-fold more than that produced by BMB8901. Therefore, the vip3Aa7 gene under the control of cry3A promoter was transformed into strain YBT1520. The toxicity of the resulting strain BMB218V against Spodoptera exigua was 10-fold more than that of YBT1520, and that the toxicity of BMB218V against Helicoverpa armigera retained the same level as that of strain YBT1520. CONCLUSIONS: Strain YBT1520 obtained high toxicity against S. exigua after it was transformed and expressed the foreign vip3Aa7 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial B. thuringiensis strain YBT1520 has high toxicity against H. armigera and Plutella xylostella, but almost no activity against S. exigua, which is a major crop pest in China. This work provides a new strategy for widening the activity spectrum of B. thuringiensis against agriculture pests.

Animals↗
PubMed1992

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco.

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 microgram ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.

Bacillus thuringiensis↗