Genetic Variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) Populations from Latin America Is Associated with Variations in Susceptibility to Bacillus thuringiensis Cry Toxins

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.     

Improved production of the insecticidal CryIVD protein in Bacillus thuringiensis using cryIA(c) promoters to express the gene for an associated 20-kDa protein

Previous studies have shown that a 20-kDa protein enhances production of the insecticidal CytA and CryIVA proteins of Bacillus thuringiensis in Escherichia coli as well as CytA production and crystal formation in B. thuringiensis. To determine whether the 20-kDa protein could enhance CryIVD production, an expression vector was constructed with the 20-kDa open-reading frame under control of cryIA(c) promoters and the cryIVD gene under control of its own promoter. Acrystalliferous cells of B. thuringiensis transformed with this plasmid, designated pWF53, produced large bitrapezoidal CryIVD crystals that averaged 1.3 × 0.92 × 0.31 m, approximately fivefold larger in volume than wild-type CryIVD crystals, and 1.7 fold the volume of crystals produced using the cryIV D operon, which contains the cryIV D gene and the gene for the 20-kDa protein. These results demonstrate that the 20-kDa protein significantly improves net synthesis of CryIVD and promotes CryIVD crystal formation. Improved production of proteins as diverse as CryIVD and CytA by the 20-kDa protein indicates this protein may be useful in facilitating the production of other proteins.     

Construction of Stably Transformed Bm5 and Sf9 Cells Displaying Green Fluorescence by Using Autographa californica Nuclear Polyhedrosis Virus IE1 Gene

Transformed Bm5 or Sf9 cells displaying green fluorescence were constructed by using Autographa californica nuclear polyhedrosis virus (AcNPV) immediate early gene (ie 1). Green fluorescent protein (gfp) gene was introduced under the control of the AcNPV ie 1 promoter to yield expression plasmid pAcIE1-GFP. It was transfected into Sf9 or Bm5 cells and cell clones expressing GFP were selected by fluorescence microscopy. Genomic DNA from transformed cells was isolated and integration of AcNPV ie 1 gene harboring gfp gene was confirmed by PCR using AcNPV ie 1 gene-specific primers. The GFP was successfully expressed in the cytoplasm of insect cells transformed with pAcIEI-GFP and the expressed GFP was maintained during cell division. Furthermore, GFP expression by AcNPV ie 1 promoter in transformed cells was not interfered with viral replication. This suggests that transformed cells displaying foreign gene product by using AcNPV ie 1 promoter will be useful for the diverse applications of the insect cells.     

Expression of the mouse c-abl type IV proto-oncogene product in the insect cell baculovirus system

The cellular gene c-abl is the normal homologue of the transforming gene (v-abl) within the genome of the Abelson leukaemia virus. The cDNA sequence coding for the cellular form of the murine abl gene (c-abl type IV) has been inserted into the baculovirus transfer vector, pAc36C, so that the c-abl gene is under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells infected with the recombinant transfer vector in the presence of wild type AcNPV DNA yielded recombinant, polyhedrin negative virus that expressed moderate levesl of the c-Abl protein (representing approx. 0.5–1% of the stained cellular proteins as determined by densitometric scanning). The insect derived c-Abl protein was compared to the P210-BCR/ABL protein from K562 cells, a cell line derived from a patient with chronic myelogenous leukaemia. Antibodies raised againts synthetic peptides based on c-abl encoded peptides react with the insect derived c-Abl. In addition, the baculovirus derived c-Abl protein has a tyrosine kinase activity as demonstrated by phosphorylation of a synthetic polypeptide and also by autophosphorylation. Phosphoamino acid analysis of immunoprecipitated, autophosphorylated baculovirus derived c-Abl protein indicates that the majority of label incorporated is on the tyrosine residues. Immunofluorescence microscopy has been used to show that the majority of the c-Abl protein expressed in cells infected with recombinant virus is located in the nuclear and plasma membranes.     

Cytotoxicity Analysis of Three Bacillus thuringiensis Subsp. israelensis δ-Endotoxins towards Insect and Mammalian Cells

Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL.     

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 g 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.     

Identification of a δ-Endotoxin Gene Product Specifically Active against Spodoptera littoralis Bdv. among Proteolysed Fractions of the Insecticidal Crystals of Bacillus thuringiensis subsp. aizawai 7.29

At least three different insecticidal crystal protein genes were shown to be expressed in Bacillus thuringiensis subsp. aizawai 7.29, a strain that is potentially active against the cotton leafworm Spodoptera littoralis Bdv. Among crude K-60 fractions (60- to 70-kilodalton [kDa] molecules) that were products of proteolysed crystals containing the active domains of the protoxin molecules, we were able to distinguish several distinct components on the basis of their antigenic relationship and their larvicidal properties. A purified fraction designated SF2 was a 61-kDa component specifically active against Pieris brassicae L. and homologous to the B. thuringiensis subsp. berliner 1715 plasmid-encoded crystal protein. A second fraction designated SF1 was composed of 63- and 65-kDa polypeptides and was specifically active against S. littoralis. The SF1 fraction and particularly the 65-kDa component were not antigenically related to the 61-kDa component. The purified fractions were compared with the products of three different crystal protein genes we previously cloned from total DNA of B. thuringiensis subsp. aizawai, among them a new type of crystal protein gene encoding a protein that is specifically active against S. littoralis and other insects of the Noctuidae family. This approach led us to consider the 65-kDa component as a minimum active part of a δ-endotoxin that is encoded by this new gene. Products of the two other cloned genes can be correlated with the 61- and 63-kDa components, respectively. Thus, in B. thuringiensis subsp. aizawai 7.29, multiple δ-endotoxin genes of different structural types direct the synthesis of several δ-endotoxins with different host specificities which were identified as components of the insecticidal crystals.     

Integrative Cloning, Expression, and Stability of the cryIA(c) Gene from Bacillus thuringiensis subsp. kurstaki in a Recombinant Strain of Clavibacter xyli subsp. cynodontis

A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The cryIA(c) gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp. cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the cryIA(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The cryIA(c) gene and the tetM gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control.     

Active human erythropoietin expressed in insect cells using a baculovirus vector: A role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3′-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200000 U/mg), but was of lower M_r (23000) than human erythropoietin produced in COS cells (30000) or purified from urine (30000 to 38000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosac-charides from this M_r 23000 hormone using N-Glycanase yielded an apo-erythropoietin (M_r 18000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.     

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture

An insecticidal protein gene from Bacillus thuringiensis var. aizawal was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteloytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resist-ant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CFI ceils but not dipteran (Aedes albopictus) calls. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawal toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran ceil membranes, which may be the receptor for this toxin.     

Functional expression in insect cells of glycosylphosphatidylinositol-linked alkaline phosphatase from Aedes aegypti larval midgut: a Bacillus thuringiensis Cry4Ba toxin receptor

Bacillus thuringiensis produces insecticidal crystal (Cry) proteins which bind to cell surface receptors on the brush border membrane of susceptible midgut larvae. The toxin-receptor interaction generates pores in midgut epithelial cells resulting in cell lysis. Here, a cDNA encoding membrane-bound alkaline phosphatase from Aedes aegypti (Aa-mALP) midgut larvae, based on the sequence identity hit to Bombyx mori membrane-bound ALP, was amplified by RT-PCR and transiently expressed in Spodoptera frugiperda (Sf9) insect cells as a 58-kDa membrane-bound protein via the baculovirus expression system and confirmed by digestion with phosphatidylinositol-specific phospholipase C and LC-MS/MS analysis. Immunolocalization results showed that Cry4Ba is able to bind to only Sf9 cells-expressing Aa-mALP. Moreover, these cells were shown to undergo cell lysis in the presence of 100 ??g/ml trypsin-treated toxin. Finally, trypan blue exclusion assay also demonstrated an increase in cell death in recombinant cells treated with Cry4Ba. Overall results indicated that Aa-mALP protein was responsible for mediating Cry4Ba toxicity against Sf9 cells, suggesting its role as a receptor for Cry4Ba toxin in A. aegypti mosquito larvae.     

Introduction of a Lepidopteran-Specific Insecticidal Crystal Protein Gene of Bacillus thuringiensis subsp. kurstaki by Conjugal Transfer into a Bacillus megaterium Strain That Persists in the Cotton Phyllosphere

A lepidopteran toxin gene of the entomopathogen Bacillus thuringiensis subsp. kurstaki HD-1 was introduced into a cotton leaf-colonizing Bacillus megaterium strain, RS1, by conjugal transfer. Rifampin- and nalidixic acid-resistant colonies obtained after cell mating were screened for crystal production by microscopy. A transcipient, B. megaterium RS1-43, was selected by this procedure. Southern blot hybridization with both total DNA and HindIII-digested DNA of the transcipient showed positive signals with a cryIA-specific probe, suggesting the transfer of the lepidopteran-specific cryIA(a) gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis confirmed the presence of the 134-kDa toxic crystal protein specific to lepidopteran larvae in the transcipient. Survival studies with cultures of the transcipient at both vegetative and postvegetative growth stages on cotton, under field conditions, suggested that the bacterium persisted on the leaf surfaces for more than 28 days, with a gradual decline in the population level with time, while the donor, B. thuringiensis subsp. kurstaki, disappeared completely after 7 days following inoculation. An in situ differential crystal-staining technique indicated the production of crystals by the transcipient on cotton leaf surfaces for about 30 days. Leaf bioassays of cotton plants inoculated with a single spray of the transcipient showed 75- to 96% mortality to the first-instar larvae of Heliothis armigera up to 21 days, and this single spray conferred total protection to the plants for about 30 days by causing an antifeeding effect on the remaining larvae.     

Molecular Cloning of a New Crystal Protein Gene cry1Af1 of Bacillus thuringiensis NT0423 from Korean Sericultural Farms

A new cry1Ab-type gene encoding the 130 kDa protein of Bacillus thuringiensis NT0423 bipyramidal crystals was cloned, sequenced, and expressed in a crystal-negative B. thuringiensis host. Hybridization experiments revealed that the crystal protein gene is located on a 44 MDa plasmid of B. thuringiensis NT0423. A strong positive signal detected on the 6.6 kb HindIII fragment from B. thuringiensis NT0423 plasmid DNA was cloned and sequenced. The cry1Ab-type gene, designated cry1Af1, consisted of open reading frame of 3453 bp, encoding a protein of 1151 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 130,215 Da. With both Bt I and Br II promoter sequences were found, the B. thuringiensis NT0423 crystal protein gene promoter closely aligned with those of cry1A-type crystal protein gene. When compared with known sequences of other Cry and Cyt proteins, the Cry1Af1 protein showed maximum 93% sequence identity to Cry1Ab protein of B. thuringiensis subsp. kurstaki. The expressed Cry1Af1 protein in a crystal-negative B. thuringiensis host appears to have strong insecticidal activity against lepidopteran larvae (Plutella xylostella). Crystals containing Cry1Af1 were about six times more toxic than the wild-type crystals of B. thuringiensis NT0423. Received: 20 February 2001 / Accepted: 17 April 2001     

Insecticidal Activity of the Protein Encoded by the cryV Gene of Bacillus thuringiensis kurstaki INA-02

A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and cryV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.     

Construction of a Bioinsecticidal Strain of Pseudomonas fluorescens Active against the Sugarcane Borer, Eldana saccharina

A cryIA(c) gene was cloned from a native Bacillus thuringiensis strain showing activity against the sugarcane borer, Eldana saccharina. The sequence of the cloned gene was very similar to that of the B. thuringiensis subsp. kurstaki HD-73 cryIA(c) gene. The gene was introduced into an isolate of Pseudomonas fluorescens, capable of colonizing sugarcane, on two broad-host-range plasmids, pDER405 and pKT240, having copy numbers of 13 and 28, respectively. By using the Omegon-Km vector, the cry gene was introduced into the chromosome of P. fluorescens isolate 14. Bioassays on eldana larvae showed that the strain carrying the gene integrated into the chromosome was as toxic as one carrying it on pKT240. Glasshouse trials indicated that sugarcane treated with P. fluorescens 14::Omegon-Km-cry were more resistant to eldana damage than untreated sugarcane was.     

Recombinant Cry1Ia protein is highly toxic to cotton boll weevil (Anthonomus grandis Boheman) and fall armyworm (Spodoptera frugiperda)

Aims: To evaluate the activity of cry1Ia gene against cotton pests, Spodoptera frugiperda and Anthonomus grandis. Methods and Results: Had isolated and characterized a toxin gene from the Bacillus thuringiensis S1451 strain which have been previously shown to be toxic to S. frugiperda and A. grandis. The toxin gene (cry1Ia) was amplified by PCR, sequenced, and cloned into the genome of a baculovirus. The Cry1Ia protein was expressed in baculovirus infected insect cells, producing protein inclusions in infected cells. The Cry1Ia protein has used in bioassays against to S. frugiperda and A. grandis. Conclusions: Bioassays using the purified recombinant protein showed high toxicity to S. frugiperda and A. grandis larvae. Molecular modelling of the Cry1Ia protein translated from the DNA sequence obtained in this work, showed that this protein possibly posses a similar structure to the Cry3A protein. Ultrastructural analysis of midgut cells from A. grandis incubated with the Cry1Ia toxin, showed loss of microvilli integrity. Significance and Impact of the Study: The results indicate that the cry1Ia is a good candidate for the construction of transgenic plants resistant to these important cotton pests.     

Constitutive and tissue-specific differential expression of the cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest

 The truncated chimeric Bt gene, cryIA(b) of Bacillus thuringiensis, driven by two constitutive promoters, 35S from CaMV and Actin-1 from rice, and two tissue-specific promoters, pith tissue and pepcarboxylase (PEPC) for green tissue from maize, was introduced into several varieties of rice (indica and japonica) by microprojectile bombardment and protoplast systems. A total of 1800 putative transgenic Bt rice plants could be produced. Southern analysis revealed that more than 100 independently transformed plants could be confirmed for integration of the cryIA(b) gene. High levels of CryIA(b) proteins were obtained in the green tissue (leaves and stem) of many plants using the PEPC promoter. There was little difference in Bt protein level in leaves and stems from transgenic plants with the 35 S or Actin-1 promoter. Out of 800 Southern-positive plants that were bioassayed, 81 transgenic plants showed 100% mortality of insect larvae of the yellow stem borer (Scirpophaga incertulas). The transgene, cryIA(b), driven by different promoters showed a wide range of expression (low to high) of Bt proteins stably inherited in a number of rice varieties with enhanced yellow stem borer resistance. This first report of transgenic indica Bt rice plants with the PEPC or pith promoter either alone or in combination should provide a better strategy for providing rice plants with protection against insect pest resistance, minimizing the expression of the CryIA(b) protein in seeds and other tissues. Received: 12 November 1997 / Accepted: 25 November 1997     

A Bacillus thuringiensis S-Layer Protein Involved in Toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.     

Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product

Conditions for hyperexpression, in Escherichia coli, of the Bacillus thuringiensis var, kurstaki gene, cryIA9(c)73, encoding an insecticidal crystal protein, CryIA(c)73, were investigated by varying the promoter type, host cell, plasmid copy number, the second codon and number of terminators. The cryIA(c)73 gene was cloned into three E. coli expression vectors, pKK223-3 (Ptac promoter), pET-3a (P phi 10 promoter), and pUC19 (Ptac promoter). The level of cryIA(c)73 expression was measured by ELISA and compared to total cellular protein over growth periods of 24 and 48 h. Maximum expression levels of 284 microgram CryIA(C)73/ml (48% of cellular protein) were obtained in shake flasks with the Ptac promoter in E. coli JM103. Optimal conditions were found to be low-copy-number plasmid (pBR322 ori), 48 h of growth, in lon+ cells. A change of the gene's second codon to AAA can improve expression by two to three fold but is undetectable in the presence of a strong E. coli promoter. The cryIA(c)73 gene product, in E. coli, formed crystals with the same lattice structure as the native crystals formed in B. thuringiensis (as visualized by electron microscopy). Bioassay results (insect toxicity and specificity) of the crystal produced in E. coli were similar to that produced in B. thuringiensis.     

Construction of Novel Bacillus thuringiensis Strains with Different Insecticidal Activities by Transduction and Transformation

The shuttle vector pHT3101 and its derivative pHT408, bearing a copy of a cryIA(a) δ-endotoxin gene, were transferred into several Bacillus thuringiensis subspecies through phage CP-54Ber-mediated transduction, with frequencies ranging from 5 × 10^-8 to 2 × 10^-6 transductant per CFU, depending on the strain and on the plasmid. In Cry^- and Cry⁺ native recipients, the introduction of the cryIA(a) gene resulted in the formation of large bipyramidal crystals that were active against the insect Plutella xylostella (order Lepidoptera). In both cases, high levels of gene expression were observed. Transductants displaying a dual specificity were constructed by using as recipients the new isolates LM63 and LM79, which have larvicidal activity against insects of the order Coleoptera. It was not possible, however, to introduce pHT7911 into B. thuringiensis subsp. entomocidus, aizawai, or israelensis by transduction. However, electrotransformation was successful, and transformants expressing the toxin gene cryIIIA, carried by pHT7911, were obtained. Again, high levels of expression of the cloned gene were observed. The results indicate that CP-54Ber-mediated transduction is a useful procedure for introducing cloned crystal protein genes into various B. thuringiensis recipients and thereby creating strains with new combinations of genes. Finally it was also shown that pHT3101 is a very good expression vector for the cloned δ-endotoxin genes in the different recipients.