Characterization of CwlC, an autolysin, and its role in mother cell lysis of Bacillus thuringiensis subsp. israelensis.

Bacillus thuringiensis subsp. israelensis (Bti) has been proven to efficiently control mosquitoes, of which many species are important vectors of human disease. The larvicidal action is attributed to the parasporal crystals formed in the sporulating cells and released upon cell autolysis. In this study, a sporulation-specific cwlC gene that encodes an N-acetylmuramoyl-L -alanine amidase was characterized in Bti strain Bt-59. CwlC was the only cell wall hydrolase in Bti found to contain both MurNAc-LAA and Amidase02_C domains. A recombinant CwlC-His protein was able to digest the Bacillus cell wall. Deletion of the cwlC gene delayed Bti mother cell lysis without impacting vegetative growth or insecticidal efficacy. Transcriptional analyses indicated that cwlC was expressed at the late sporulation stage and was controlled by SigK. Two other cell wall hydrolase genes, cwlB and cwlE, with high expression levels at T14 in Bt-59, were also identified. Like cwlC, cwlB expression was controlled by SigK; in contrast, cwlE was found not to be under the control of this sigma factor and unlike the other two, its gene was found to be plasmid encoded.

Temperature-dependent development of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) and its larval parasitoid, Habrobracon hebetor (Say) (Hymenoptera: Braconidae): implications for species interactions.

Habrobracon hebetor (Say) is a parasitoid of various Lepidoptera including Helicoverpa armigera (Hübner), a key pest of different crops and vegetables. The development of both H. armigera and H. hebetor were simultaneously evaluated against a wide range of constant temperatures (10, 15, 17.5, 20, 25, 27.5, 30, 35, 37.5 and 40 °C). Helicoverpa armigera completed its development from egg to adult within a temperature range of 17.5-37.5 °C and H. hebetor completed its life cycle from egg to adult within a temperature range of 15-40 °C. Based on the Ikemoto and Takai model the developmental threshold (To) and thermal constant (K) to complete the immature stages, of H. armigera were calculated as 11.6 °C and 513.6 DD, respectively, and 13 °C and 148 DD, respectively, for H. hebetor. Analytis/Briere-2 and Analytis/Briere-1 were adjudged the best non-linear models for prediction of phenology of H. armigera and H. hebetor, respectively and enabled estimation of the optimum (Topt) and maximum temperature (Tmax) for development with values of 34.8, 38.7, 36.3, and 43 °C for host and the parasitoid, respectively. Parasitisation by H. hebetor was maximal at 25 °C but occurred even at 40 °C. This study suggests although high temperature is limiting to insects, our estimates of the upper thermal limits for both species are higher than previously estimated. Some biological control of H. armigera by H. hebetor may persist in tropical areas, even with increasing temperatures due to climate change.

Novel genetic factors involved in resistance to Bacillus thuringiensis in Plutella xylostella.

The widespread and sustainable exploitation of the entomopathogen Bacillus thuringiensis (Bt) in pest control is threatened by the evolution of resistance. Although resistance is often associated with loss of binding of the Bt toxins to the insect midgut cells, other factors have been implicated. Here we used suppressive subtractive hybridization and gene expression suppression to identify additional molecular components involved in Bt-resistance in Plutella xylostella. We isolated transcripts from genes that were differentially expressed in the midgut of larvae from a resistant population, following ingestion of a Bt kurstaki HD1 strain-based commercial formulation (DiPel), and compared with a genetically similar susceptible population. Quantitative real-time polymerase-chain reaction (RT-PCR) analysis confirmed the differential basal expression of a subset of these genes. Gene expression suppression of three of these genes (P. xylostella cyclin-dependent kinase 5 regulatory subunit associated protein 1-like 1, stromal cell-derived factor 2-like 1 and hatching enzyme-like 1) significantly increased the pathogenicity of HD1 to the resistant population. In an attempt to link the multitude of factors reportedly influencing resistance to Bt with the well-characterized loss of toxin binding, we also considered Bt-resistance models in P. xylostella and other insects.

Beyond the spore--past and future developments of Bacillus thuringiensis as a biopesticide.

Formulated and sporulated cultures of Bacillus thuringiensis (Bt) are widely used as foliar sprays as part of integrated pest management strategies against insect pests of agricultural crops. Although in several cases the presence of the spore has been shown to improve the activity of the product, other Bt-based insecticides have been developed in which the spore is absent. The most notable of these are transgenic plants expressing just the insect toxin gene from the bacterium. This paper will discuss these developments, and the advantages and disadvantages of having the spore present.

Cyt1Aa from Bacillus thuringiensis subsp. israelensis is toxic to the diamondback moth, Plutella xylostella, and synergizes the activity of Cry1Ac towards a resistant strain.

The Bacillus thuringiensis subsp. israelensis cytolytic protein Cyt1Aa was found to be toxic to an insecticide-susceptible laboratory population of Plutella xylostella. Cry1Ac-resistant populations of P. xylostella showed various degrees of resistance to Cyt1Aa. Cyt1Aa/Cry1Ac mixtures showed a marked level of synergism in the Cry1Ac-resistant populations.

Expression and crystallization of an N-terminally activated form of the Bacillus thuringiensis Cry1Ca toxin.

When the active form of the Bacillus thuringiensis delta-endotoxin Cry1Ca was expressed in E. coli severe growth retardation was observed. The absence of a short peptide from the N-terminus of the protoxin was responsible for this effect. The introduction of a mutation at an amino acid previously reported as being involved in the initial stages of pore formation within the natural insect target partially abolished the growth retardation effect. We suggest that removal of the N-terminal peptide is a necessary step in toxin activation, the presence of this peptide preventing proper interaction of the toxin with the target membrane. Expression of the truncated toxin in Bacillus thuringiensis also prevented the formation of Cry1Ca crystals.

Susceptibility of a field-derived, Bacillus thuringiensis-resistant strain of diamondback moth to in vitro-activated Cry1Ac toxin.

Resistant and susceptible populations of the diamondback moth (Plutella xylostella) were tested with crystalline, solubilized, and partially and fully activated forms of the Bacillus thuringiensis Cry1Ac delta-endotoxin. Fully activated toxin greatly reduced the resistance ratio (ratio of the 50% lethal concentration for the resistant population to that for the susceptible population) of the resistant population, suggesting that a defect in toxin activation is a major resistance mechanism.

Cross-resistance and stability of resistance to Bacillus thuringiensis toxin Cry1C in diamondback moth.

We tested toxins of Bacillus thuringiensis against larvae from susceptible, Cry1C-resistant, and Cry1A-resistant strains of diamondback moth (Plutella xylostella). The Cry1C-resistant strain, which was derived from a field population that had evolved resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai, was selected repeatedly with Cry1C in the laboratory. The Cry1C-resistant strain had strong cross-resistance to Cry1Ab, Cry1Ac, and Cry1F, low to moderate cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to Cry1Bb, Cry1Ja, and Cry2A. Resistance to Cry1C declined when selection was relaxed. Together with previously reported data, the new data on the cross-resistance of a Cry1C-resistant strain reported here suggest that resistance to Cry1A and Cry1C toxins confers little or no cross-resistance to Cry1Bb, Cry2Aa, or Cry9Ca. Therefore, these toxins might be useful in rotations or combinations with Cry1A and Cry1C toxins. Cry9Ca was much more potent than Cry1Bb or Cry2Aa and thus might be especially useful against diamondback moth.

Cellular localization and characterization of the Bacillus thuringiensis Orf2 crystallization factor.

In vivo crystallization of the Bacillus thuringiensis insecticidal protein toxin Cry2Aa has been studied in the presence and absence of its associated crystallization factor Orf2. When expressed in the same cell, both proteins were localized to the cuboidal cytoplasmic crystal. When expressed in the absence of Cry2Aa, Orf2 was randomly distributed throughout the cytoplasm. Cry2Aa when expressed in the absence of Orf2 did not form visible crystals but could be recovered from the insoluble fraction of the cell lysate and solubilized at high pH. Purified Orf2 was found to be soluble over a wide range of pH although it could be co-precipitated in the presence of Cry2Aa, suggesting a direct interaction between the two.

How Bacillus thuringiensis has evolved specific toxins to colonize the insect world.

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. Together the subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. This is mainly determined by the arsenal of crystal proteins that the bacterium produces during sporulation. Here we describe the properties of these toxin proteins and the current knowledge of the basis for their specificity. Assessment of phylogenetic relationships of the three domains of the active toxin and experimental results indicate how sequence divergence in combination with domain swapping by homologous recombination might have caused this extensive range of specificities.

Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.

The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.

Bacillus thuringiensis and its pesticidal crystal proteins.

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N.

A 120 kDa glycoprotein in the larval midgut membrane of the lepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin, has been purified by a combination of protoxin affinity chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin in the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.

Use of an operon fusion to induce expression and crystallisation of a Bacillus thuringiensis delta-endotoxin encoded by a cryptic gene.

A delta-endotoxin gene previously cloned from Bacillus thuringiensis subsp. galleriae has been shown by a combination of restriction mapping and DNA sequence analysis to be a cryIIB clone; in common with other cryIIB genes it was found to lack a functional promoter. Addition of a promoter resulted in expression of the gene in Bacillus thuringiensis but did not result in the formation of the crystalline inclusions normally associated with such toxins. Inclusion formation was only observed when the gene was incorporated into an operon containing a gene known to be involved in the crystallisation of another delta-endotoxin.

Effects on toxicity of eliminating a cleavage site in a predicted interhelical loop in Bacillus thuringiensis CryIVB delta-endotoxin.

When activated by treatment with mosquito (Aedes aegypti) gut extract, the Bacillus thuringiensis CryIVB delta-endotoxin lysed A. aegypti cells in vitro. SDS-PAGE and N-terminal sequence determination showed that in addition to removal of the C-terminal half of the molecule, the activated toxin had undergone proteolytic cleavage at two internal regions producing 47-48-kDa and 16-18-kDa polypeptides. Aligning the CryIVB protein sequence with the crystallographic structure of the CryIIIA toxin suggested that one set of cleavages occurred in a region before the start of the N-terminal helical bundle and the second cleavage site occurred in a predicted loop between helices 5 and 6 in the bundle at arginine-203. To investigate the suggestion by Li et al. that interhelical proteolysis is important in the cytolytic mechanism of these toxins, arginine-203 was substituted by alanine. The mutated toxin now resisted proteolysis at this position and showed a marked decrease in cytolysis in vitro but an increase in larvicidal activity.

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo.

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.

Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis.

The Bacillus thuringiensis cryIIA delta-endotoxin gene is found as the third-gene in a three-gene operon, with a sporulation-dependent promoter lying upstream of the first gene, orf1. We show here that the polypeptide product of the middle gene (orf2) is required for efficient expression of the toxin gene. In the absence of a functional ORF2 polypeptide the toxin does not form the crystalline inclusions characteristic of other known Bacillus thuringiensis toxins. We discuss the importance of this finding with respect to the possible role of chaperonins in the crystallization of these proteins.

Cytotoxicity of a cloned Bacillus thuringiensis subsp. israelensis CryIVB toxin to an Aedes aegypti cell line.

A cloned CryIVB toxin was purified from a cured strain of Bacillus thuringiensis (BT) containing the cryIVB gene on the recombinant plasmid Cam135. Solubilized protoxin was treated with Aedes gut extract or trypsin for varying times and tested for toxicity in vitro on three dipteran and one lepidopteran cell line. Treatment with the Aedes extract but not trypsin, produced an active toxin which lysed only Aedes aegypti cells out of those tested. This activation was time-dependent reaching a maximum after 6 h. Both the Aedes extract-treated and trypsin-treated toxin killed A. aegypti larvae, but this toxicity declined rapidly with increasing time of exposure to the proteolytic preparations.

The construction of Bacillus thuringiensis strains expressing novel entomocidal delta-endotoxin combinations.

Using our recently reported method of electroporation to transform Bacillus thuringiensis [Bone & Ellar (1989) FEMS Microbiol. Lett. 58, 171-178], cloned B. thuringiensis entomocidal delta-endotoxin genes have been introduced into several native B. thuringiensis strains. In many cases the resulting transformants expressed both their native toxins and the cloned toxin, producing strains with broader toxicity spectra. The introduction of the var. tenebrionis toxin gene into B. thuringiensis var. israelensis resulted in a strain with activity against Pieris brassicae (cabbage white butterfly), an activity which neither parent strain possesses. We discuss further the possibility of synergism and also the problems associated with introducing cloned DNA by this method.