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PubMed1996

Digestion of delta-endotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to CryIC.

The present study describes the correlation between gut protease activity of lepidopteran larvae of different instars, the inactivation of Bacillus thuringiensis delta-endotoxins in crystalline and noncrystalline forms, and the reduced susceptibility of advanced larval instars of Spodoptera littoralis to the toxin. The original assembly of delta-endotoxins in a crystal structure is essential for causing efficient larval mortality. Denaturation and renaturation (D/R) of delta-endotoxin crystals increased the vulnerability of the toxin molecules to proteolysis, reduced their capability to kill neonate larvae of S. littoralis, but sustained most of their larval growth-inhibition activity. E. coli-produced CryIC delta-endotoxin applied as a fraction of inclusion bodies exerted a growth inhibition effect, similar to the molecules released from the crystals by denaturation and subsequent renaturation. Incubation of CryIC with gut juice of 1st or 2nd instar larvae, left part of the CryIC toxin intact, while the toxin was completely degraded when incubated with gut juice of 5th instar larvae. The degradation rate was consistent with the increase of protease specific activity of the gut juice during larval development. This increase in toxin degradation may account for the loss of sensitivity of 5th instar larvae to CryIC. Specific protease inhibitors such as PMSF and Leupeptin were shown to inhibit gut proteases activity in all instar larvae, while, 1,10 phenanthroline, TLCK and TPCK were effective only in young instar larvae. The differential effect of protease inhibitors on proteases obtained from different larval instars indicated that gut juice protease profiles change with larval age. The observed quantitative and qualitative differences in degradation of delta-endotoxin by larval gut proteases that occur during larval maturation may account for the difference in susceptibility to the delta-endotoxin. This finding should be taken into consideration when designing strategies for the development of transgenic crops expressing delta-endotoxins as potent insecticidal proteins.

Animals↗
PubMed2001

Structural and functional studies of alpha-helix 5 region from Bacillus thuringiensis Cry1Ab delta-endotoxin.

The crystal insecticidal proteins from Bacillus thuringiensis are modular proteins comprised of three domains connected by single linkers. Domain I is a seven alpha-helix bundle, which has been involved in membrane insertion and pore formation activity. Site-directed mutagenesis has contributed to identify regions that might play an important role in the structure of the pore-forming domain within the membrane. There are several evidences that support that the hairpin alpha4-alpha5 inserts into the membrane in an antiparallel manner, while other helices lie on the membrane surface. We hypothesized that highly conserved residues of alpha5 could play an important role in toxin insertion, oligomerization and/or pore formation. A total of 15 Cry1Ab mutants located in six conserved residues of Cry1Ab, Y153, Y161, H168, R173, W182 and G183, were isolated. Eleven mutants were located within helix alpha5, one mutant was located in the loop alpha4-alpha5 and three mutants, W182P, W182I and G183C, were located in the loop alpha5-alpha6. Their effect on binding, K(+) permeability and toxicity against Manduca sexta larvae was analyzed and compared. The results provide direct evidence that some residues located within alpha5 have an important role in stability of the toxin within the insect gut, while some others also have an important role in pore formation. The results also provide evidence that conserved residues within helix alpha5 are not involved in oligomer formation since mutations in these residues are able to make pores in vitro.

Amino Acid Sequence↗
PubMed2004

Composition of grain and forage from corn rootworm-protected corn event MON 863 is equivalent to that of conventional corn (Zea mays l.).

Insect-protected corn hybrids containing event MON 863 protect corn plants against feeding damage from corn rootworm (Diabrotica), a major North American insect pest. Corn event MON 863 contains a gene that expresses an amino acid sequence variant of the wild-type Cry3Bb1 insecticidal protein from Bacillus thuringiensis. The purpose of this study was to compare the composition of corn containing event MON 863 with that of conventional nontransgenic corn. Compositional analyses were conducted to measure proximates, fiber, amino acids, fatty acids, minerals, folic acid, thiamin, riboflavin, vitamin E, antinutrients, and certain secondary metabolites in grain and proximates and fiber content in forage collected from a total of eight field sites in the U.S. and Argentina. Compositional analyses demonstrated that the grain and forage of event MON 863 are comparable in their nutritional content to the control corn hybrid and conventional corn. These comparisons, together with the history of the safe use of corn as a common component of animal feed and human food, support the conclusion that corn event MON 863 is compositionally equivalent to, and as safe and nutritious as, conventional corn hybrids grown commercially today.

Amino Acids↗
PubMed1989

Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var. tenebrionis.

Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin.

Amino Acid Sequence↗
PubMed2005

Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants.

Transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) were grown on over 13 million ha in the United States and 22.4 million ha worldwide in 2004. Preventing or slowing the evolution of resistance by insects ("resistance management") is critical for the sustainable use of Bt crops. Plants containing two dissimilar Bt toxin genes in the same plant ("pyramided") have the potential to delay insect resistance. However, the advantage of pyramided Bt plants for resistance management may be compromised if they share similar toxins with single-gene plants that are deployed simultaneously. We tested this hypothesis using a unique model system composed of broccoli plants transformed to express different Cry toxins (Cry1Ac, Cry1C, or both) and a synthetic population of the diamondback moth (Plutella xylostella) carrying genes for resistance to Cry1Ac and Cry1C at frequencies of approximately 0.10 and 0.34, respectively. After 24-26 generations of selection in the greenhouse, the concurrent use of one- and two-gene plants resulted in control failure of both types of Bt plants. When only two-gene plants were used in the selection, no or few insects survived on one- or two-gene Bt plants, indicating that concurrent use of transgenic plants expressing a single and two Bt genes will select for resistance to two-gene plants more rapidly than the use of two-gene plants alone. The results of this experiment agree with the predictions of a Mendelian deterministic simulation model and have important implications for the regulation and deployment of pyramided Bt plants.

Adaptation, Biological↗
PubMed2005

Disruption of a cadherin gene associated with resistance to Cry1Ac {delta}-endotoxin of Bacillus thuringiensis in Helicoverpa armigera.

A laboratory strain (GY) of Helicoverpa armigera (Hubner) was established from surviving larvae collected from transgenic cotton expressing a Bacillus thuringiensis var. kurstaki insecticidal protein (Bt cotton) in Gaoyang County, Hebei Province, People's Republic of China, in 2001. The GYBT strain was derived from the GY strain through 28 generations of selection with activated Cry1Ac delivered by diet surface contamination. When resistance to Cry1Ac in the GYBT strain increased to 564-fold after selection, we detected high levels of cross-resistance to Cry1Aa (103-fold) and Cry1Ab (>46-fold) in the GYBT strain with reference to those in the GY strain. The GYBT strain had a low level of cross-resistance to B. thuringiensis var. kurstaki formulation (Btk) (5-fold) and no cross-resistance to Cry2Aa (1.4-fold). Genetic analysis showed that Cry1Ac resistance in the GYBT strain was controlled by one autosomal and incompletely recessive gene. The cross-resistance pattern and inheritance mode suggest that the Cry1Ac resistance in the GYBT strain of H. armigera belongs to "mode 1," the most common type of lepidopteran resistance to B. thuringiensis toxins. A cadherin gene was cloned and sequenced from both the GY and GYBT strains. Disruption of the cadherin gene by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. Tight linkage between Cry1Ac resistance and the cadherin locus was observed in a backcross analysis. Together with previous evidence found with Heliothis virescens and Pectinophora gossypiella, our results confirmed that the cadherin gene is a preferred target for developing DNA-based monitoring of B. thuringiensis resistance in field populations of lepidopteran pests.

Amino Acid Sequence↗
PubMed2005

Evolutionary trade-offs of insect resistance to Bacillus thuringiensis crops: fitness cost affecting paternity.

Evolution of resistance to insecticides provides a useful model for examining fitness trade-offs associated with adaptation to stress. Here, we examined male reproductive costs in pink bollworm (Pectinophora gossypiella) resistant to an insecticidal protein of Bacillus thuringiensis (Bt) produced by transgenic cotton, using contrasts between two pairs of related susceptible and resistant strains. Without competition for access to females, no costs affecting reproductive success of resistant males were observed. Resistant and susceptible males had similar mating frequency and fertility. Additionally, fecundity of females mated to resistant and susceptible males was comparable. In competition for matings with virgin females, resistant and susceptible males had comparable success in one strain, whereas susceptible males tended to mate more often than resistant males in the other. However, irrespective of strain origin, resistant males that mated first sired significantly less offspring than susceptible males that mated first. The reduced first-male paternity in resistant males may involve reduced sperm precedence caused by mutations in a cadherin gene linked with resistance to Bt cotton.

Analysis of Variance↗
PubMed1991

Functional domains of Bacillus thuringiensis insecticidal crystal proteins. Refinement of Heliothis virescens and Trichoplusia ni specificity domains on CryIA(c).

Insecticidal crystal proteins (delta-endotoxins), CryIA(a) and CryIA(c), from Bacillus thuringiensis are 82% homologous. Despite this homology, CryIA(c) was determined to have 10-fold more insecticidal activity toward Heliothis virescens and Trichoplusia ni than CryIA(a). Reciprocal recombinations between these two genes were performed by the homolog-scanning technique. The resultant mutants had different segments of their primary sequences exchanged. Bioassays with toxin proteins from these mutants revealed that amino acids 335-450 on CryIA(c) are associated with the activity against T. ni, whereas amino acids 335-615 on the same toxin are required to exchange full H. virescens specificity. One chimeric protein toxin, involving residues 450-612 from CryIA(c), demonstrated 30 times more activity against H. virescens than the native parental toxin, indicating that this region plays an important role in H. virescens specificity. The structural integrity of mutant toxin proteins was assessed by treatment with bovine trypsin. All actively toxic proteins formed a 65-kDA trypsin-resistant active toxic core, similar to the parental CryIA(c) toxin, indicating that toxin protein structure was not altered significantly. Contrarily, certain inactive mutant proteins were susceptible to complete protease hydrolysis, indicating that their lack of toxicity may have been due to structural alterations.

Bacillus thuringiensis↗
PubMed1991

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.

Amino Acid Sequence↗
PubMed1996

Effect of insecticidal crystal proteins of Bacillus thuringiensis on human erythrocytes in vitro.

Effect of intact and alkali solubilized insecticidal crystal protein (ICP) preparations from a mutant strain of B. thuringiensis var. israelensis (VCRC MB24) and the wild type strain (VCRC B17) in vitro on human erythrocytes with respect to lipid peroxidation, osmofragility and membrane bound enzymes was determined. The alkali solubilized ICPs of both B. thuringiensis strains caused increased lipid peroxidation, decreased resistance to hypotonic lysis and reduction in the activity of membrane bound enzymes. On the contrary, the intact ICPs did not produce any such adverse effect on RBCs under the same experimental conditions. It is suggested that the ICPs are safe when they are intact when compared with solubilized ones.

Bacillus thuringiensis↗
PubMed1989

Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities.

Two genes encoding insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki HD-1 were cloned and sequenced. Both genes, designated cryB1 and cryB2, encode polypeptides of 633 amino acids having a molecular mass of ca. 71 kilodaltons (kDa). Despite the fact that these two proteins display 87% identity in amino acid sequence, they exhibit different toxin specificities. The cryB1 gene product is toxic to both dipteran (Aedes aegypti) and lepidopteran (Manduca sexta) larvae, whereas the cryB2 gene product is toxic only to the latter. DNA sequence analysis indicates that cryB1 is the distal gene of an operon which is comprised of three open reading frames (designated orf1, orf2, and cryB1). The proteins encoded by cryB1 and orf2 are components of small cuboidal crystals found in several subspecies and strains of B. thuringiensis; it is not known whether the orf1 or cryB2 gene products are present in cuboidal crystals. The protein encoded by orf2 has an electrophoretic mobility corresponding to a molecular mass of ca. 50 kDa, although the gene has a coding capacity for a polypeptide of ca. 29 kDa. Examination of the deduced amino acid sequence for this protein reveals an unusual structure which may account for its aberrant electrophoretic mobility: it contains a 15-amino-acid motif repeated 11 times in tandem. Escherichia coli extracts prepared from cells expressing only orf1 and orf2 are not toxic to either test insect.

Aedes↗
PubMed2005

Identification of a promoter for the vegetative insecticidal protein-encoding gene vip3LB from Bacillus thuringiensis.

The expression of vip3LB, one of the vegetative insecticidal protein-encoding genes of Bacillus thuringiensis, was studied at the transcriptional level. By primer extension analysis, we have identified, for the first time, the transcription start point of vip3-type gene. Upstream from vip3LB transcription start point, was found a nucleotide sequence partially homologous to the consensus sequence for the E sigma(E) holoenzyme of B. subtilis. Thus, it was strongly suggested that the identified vip3 promoter was under the control of sigma(35)-like enzyme, the B. thuringiensis homolog of sigma(E). The transcriptional activity from the promoter was detected at the vegetative stage of growth starting at mid-log phase as well as during the middle stage of sporulation.

Bacillus thuringiensis↗
PubMed1997

Cloning of the nprA gene for neutral protease A of Bacillus thuringiensis and effect of in vivo deletion of nprA on insecticidal crystal protein.

The nprA gene, encoding Bacillus thuringiensis neutral protease A, was cloned by the use of gene-specific oligonucleotides. The size of neutral protease A deduced from the nprA sequence was 566 amino acids (60,982 Da). The cloned nprA gene was partially deleted in vitro, and the deleted allele, designated nprA3, was used to construct an nprA3 strain (neutral protease A-deficient strain) of B. thuringiensis. Growth and sporulation of the nprA3 strain were similar to those of an isogenic nprA+ strain, although the extracellular proteolytic activity of the nprA3 strain was significantly less than that of the nprA+ strain. The nprA3 strain produced insecticidal crystal proteins that were more stable than those of the isogenic nprA+ strain after solubilization in vitro, and sporulated cultures of the nprA3 strain contained higher concentrations of full-length insecticidal crystal proteins than did those of its isogenic counterpart. The absence of neutral protease A did not affect the insecticidal activity of a lepidopteran-specific crystal protein of B. thuringiensis. These results indicate that crystal protein stability and yield may be improved by deletion of specific proteases from B. thuringiensis.

Amino Acid Sequence↗
PubMed1991

Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts.

We have examined expression of several insecticidal crystal protein (ICP) genes of Bacillus thuringiensis in transgenic tobacco plants and electroporated carrot protoplasts. We determined that low levels of lepidopteran toxin cryIA(b) ICP gene expression in plants and electroporated carrot cells is due to RNA instability. We used a series of 3' deleted by cryIA(b) constructs directed by the cauliflower mosaic virus 35S promoter to demonstrate that this instability is minimally contained in the first 579 bases of the gene in both systems. This instability may result from 5'----3' as well as 3'----5' RNA metabolism. The coleopteran toxic cryIIIA gene was also examined in electroporated carrot cells, and found to be poorly expressed. A model for improvement of ICP RNA stability in plants is presented.

Bacillus thuringiensis↗
PubMed1999

Insecticidal activity of transgenic tobacco plants expressing both Bt and CpTI genes on cotton bollworm (Helicoverpa armigera).

Transgenic tobacco plants expressing both Bacillus thuringiensis (Bt) insecticidal protein and cowpea trypsin inhibitor (CpTI) genes was used to evaluate the insecticidal activity on the cotton bollworm (Helicoverpa armigera) compared with transgenic tobacco with Bt insecticidal protein gene alone. Mortality of first to third instar larvae fed on transgenic two genes tobacco for three days was significantly higher than that fed on transgenic Bt tobacco. First to fourth instar larvae fed on transgenic two genes tobacco continuously could not survive until pupation. Second instar larvae were fed on transgenic tobacco plants for three days and then were transferred to an artificial diet. The efficacy of transgenic two genes tobacco on mortality, larvae weight, percentage of pupation, and time to pupation were significantly higher than that of transgenic tobacco with Bt insecticidal protein gene alone. The results with transgenic tobacco as model plant were valuable for other crops both for the enhancement of insecticidal efficacy and for the delay of insect adaptation to transgenic Bt crops.

Animals↗
PubMed1987

Cloning and expression in Escherichia coli of an insecticidal crystal protein gene from Bacillus thuringiensis var. aizawai HD-133.

Using a gene probe derived from the cloned var. sotto insecticidal crystal protein (ICP) gene, we have cloned a Bacillus thuringiensis var. aizawai HD-133 ICP gene in Escherichia coli. The gene encodes a polypeptide that is toxic to Lepidoptera in vivo and in vitro. The protein is expressed at a level sufficient to produce phase-bright inclusions in recombinant E. coli strains, and these inclusions can be partially purified using discontinuous sucrose density gradients. Immunoblotting shows that the inclusions contain a 135 kDa polypeptide which reacts strongly with antiserum raised against the B. thuringiensis var. kurstaki HD-1 P1 polypeptide.

Bacillus thuringiensis↗
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↗
PubMed2026

Paradigm shift for cry gene expression in Bacillus thuringiensis.

In most Bacillus thuringiensis strains, the cry genes are transcribed by RNA polymerases containing sporulation-sigma factors E or K, leading to the formation of an insecticidal crystal within the mother cell along spore development. The kurstaki HD1 strain, a parent of commercial strains, also releases the insecticidal proteins Cry1I and Vip3A in the extracellular medium. vip3A expression is activated by the transcriptional regulator VipR at the onset of the stationary phase. Here, we expanded the VipR regulon in strain HD1 by identifying the VipR-binding box upstream from the cry2Aa, cry2Ab, and cry1Ia genes, and conducting transcription assays. Unexpectedly, a VipR box was located in the promoter of a putative N-acetylmuramoyl-l-alanine amidase (ami) gene upstream from cry1Ac in strain kurstaki HD73, closely related to the HD1 but devoid of vipR. Introduction of vipR in this strain led to the expression of the ami-cry1Ac operon, resulting in an early and increased production of Cry1Ac. We demonstrated that Cry1Ac was also produced in a VipR-dependent manner in an HD73 ∆spo0A mutant. Similarly, an HD1 ∆spo0A strain produces all the insecticidal proteins encoded in its genome, including cry2Ab, previously considered unexpressed. A genomic analysis also revealed the presence of putative VipR-binding sequences in lepidopteran-active strains, upstream from cry genes such as cry1E, cry1F, cry9D, and cry9E. Overall, our results break the dogma on the regulation of cry1A and cry2A genes and provide evidence of sporulation-independent Cry toxin production in biopesticidal Bt strains.IMPORTANCEBacillus thuringiensis is a remarkably efficient entomopathogen due to its ability to produce various insecticidal proteins, such as Cry or Vip. This property has made it a highly effective biopesticide used worldwide. Our work modifies the paradigm of cry1 and cry2 genes being regulated solely by sporulation-specific sigma factors and thus exclusively expressed during this process. Indeed, we demonstrated that the VipR regulator controls the transcription of vip3Aa, cry2Aa, cry2Ab, cry1Ia, and the ami-cry1A operons encoded by a strain closely related to that of commercial biopesticides and specifically turns on their expression from the onset of the stationary phase, leading to the production of insecticidal crystals independently of sporulation. By providing new knowledge on the regulation of insecticidal protein genes, these findings bring new insight for the genetic improvement of Bt strains used as commercial biopesticides.

Bacillus thuringiensis↗