Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.     

Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene

Summary A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato bectle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73115 dalton protein and a flat, diamond-shaped crystal composed of a protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.     

Cloning and maintenance of the housefly sodium channel gene using low copy number vector and two sequential host strains

In spite of the long history of recombinant DNA technology, some genes have not been successfully cloned in Escherichia coli. This is probably due to the toxic effects of the expressed foreign gene product on E. coli. In initial attempts to clone the full-length Vssc1 voltage-sensitive sodium channel α-subunit gene from houseflies, we used one of the most popular vectors and hosts but were unable to retrieve any intact clone. By using two vectors with different copy numbers and two alternate E. coli host strains, we found that the combined use of a low copy number vector (pALTER-1) and an E. coli host strain that suppresses plasmid replication (ABLE-K) is essential to obtain intact full-length Vssc1 clone. However, since the ABLE-K strain was not a suitable host for the long-term maintenance of Vssc1 gene due to its recombination-positive genotype, it was necessary to transfer the Vssc1 plasmid from the primary host to a secondary host with a recombination-minus genotype (Stbl2) to minimize the chances of deletion or rearrangement. We believe that this cloning strategy, with a low copy number vector and the sequential use of two E. coli strains, will be also applicable for the cloning of other toxic genes.     

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD₅₀]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD₅₀ of HD-1 with the FFD₅₀ expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.     

Synergism between Bacillus thuringiensis Spores and Toxins against Resistant and Susceptible Diamondback Moths (Plutella xylostella)

We studied the effects of combinations of Bacillus thuringiensis spores and toxins on the mortality of diamondback moth (Plutella xylostella) larvae in leaf residue bioassays. Spores of B. thuringiensis subsp. kurstaki increased the toxicity of crystals of B. thuringiensis subsp. kurstaki to both resistant and susceptible larvae. For B. thuringiensis subsp. kurstaki, resistance ratios were 1,200 for a spore-crystal mixture and 56,000 for crystals without spores. Treatment of a spore-crystal formulation of B. thuringiensis subsp. kurstaki with the antibiotic streptomycin to inhibit spore germination reduced toxicity to resistant larvae but not to susceptible larvae. In contrast, analogous experiments with B. thuringiensis subsp. aizawai revealed no significant effects of adding spores to crystals or of treating a spore-crystal formulation with streptomycin. Synergism occurred between Cry2A and B. thuringiensis subsp. kurstaki spores against susceptible larvae and between Cry1C and B. thuringiensis subsp. aizawai spores against resistant and susceptible larvae. The results show that B. thuringiensis toxins combined with spores can be toxic even though the toxins and spores have little or no independent toxicity. Results reported here and previously suggest that, for diamondback moth larvae, the extent of synergism between spores and toxins of B. thuringiensis depends on the strain of insect, the type of spore, the set of toxins, the presence of other materials such as formulation ingredients, and the concentrations of spores and toxins.     

Toxicity of Bacillus thuringiensis Spo− Cr+ Mutants for the European Corn Borer Ostrinia nubilalis

Inclusion bodies isolated from Spo⁻ Cr⁺ mutants of Bacillus thuringiensis were toxic for larvae of the European corn borer. Probit analysis revealed comparable toxicity between wild-type crystals (isolated from B. thuringiensis subsp. kurstaki) and crystals produced from two spore-free mutants of the same subspecies. Death of the larvae was due to starvation, presumably through δ-endotoxin-induced gut paralysis. Inclusion bodies pretreated with α-chymotrypsinogen were equally as toxic as native crystals for the insect larvae.     

Selection and characterization of Bacillus thuringiensis strains toxic against pyralid stored‐product pests

In this study, we present the selection and the characterization of Bacillus thuringiensis strains toxic against pyralid pests of stored products. Among 201 new B. thuringiensis strains isolated from different countries, two strains (BLB249 and BLB384) showed greater toxicity than the commercialized strain B. thuringiensis kurstaki HD1 against Ephestia kuehniella larvae. Morphological, molecular and biochemical investigations revealed that these strains were similar to HD1 but presented different cry gene content. Additional bioassays revealed that only strain BLB249 displayed higher toxicity than HD1 against Plodia interpunctella larvae. The study of Cry protoxin activation by midgut proteases of E. kuehniella and P. interpunctella larvae supported that higher toxicity of BLB249 and BLB384 strains compared to HD1 was not due to differential protoxin activation. Moreover, the toxic strains produced δ‐endotoxins and spores in similar amounts to HD1. Interestingly, the δ‐endotoxin production and the yield of BUPM26 strain were 32.87% and 35.12% greater than that of HD1. The potent insecticidal activities of BLB249 and BLB384 strains and the high level of δ‐endotoxin production by BUPM26 strain make them excellent candidates for use against E. kuehniella and P. interpunctella larvae.     

Spore Coat Protein Synergizes Bacillus thuringiensis Crystal Toxicity for the Indianmeal Moth (Plodia interpunctella)

Spores from Bacillus thuringiensis serovars kurstaki and entomocidus synergized crystal protein toxicity for larvae of the Indianmeal moth (Plodia interpunctella). Preparations of spore-crystal mixtures of either serovar were more toxic for the larvae than either purified spores or crystals alone (based on dry weight). Spores lost 53% of their toxicity for the Indianmeal moth after 2 h of UV-irradiation, but remained partially toxic (28%) even after 4 h of irradiation. Spore coat protein was toxic for the Indianmeal moth and was synergistic with B. thuringiensis serovar kurstaki HD-1 crystal protein. Enhanced toxicity of the combined spore-crystal preparation was attributed to a combination of crystal and spore coat protein, and included the effects of spore germination and resulting septicemia in the larval hemolymph. Ultraviolet irradiation of spores reduced the toxicity from septicemia but not the synergism caused by spore coat protein. The potencies of spore-crystal preparations must be carefully evaluated on the basis of contributions from all three factors. Received: 15 September 1997/Accepted: 21 October 1997     

Construction and characterisation of an antifungal recombinant Bacillus thuringiensis with an expanded host spectrum

A novel antifungal Bacillus thuringiensis strain 19–22, ssp. kurstaki (H3a3b3c), was characterised. This strain included cry1Aa, cry1Ab, cry1Ac, and cry1D, which have high insecticidal activities against lepidopteran larvae other than Spodoptera exigua. To expand the host spectrum, a cry1E gene whose product is active against S. exigua was introduced into the isolate. The transformant successfully expressed the Cry1E protein without any loss of its original antifungal activities. These results indicate that this recombinant strain exhibits dual activities and may be used as an integrated control agent to control plant diseases and insect pests.     

Screening of Brazilian Bacillus thuringiensis isolates active against Spodoptera frugiperda,Plutella xylostella and Anticarsia gemmatalis

The toxicity of a collection of 1400 isolates of Bacillus thuringiensis was assessed against the Lepidoptera Spodoptera frugiperda, Anticarsia gemmantalis and Plutella xylostella. Twenty seven isolates showed toxicity to the larvae of these insects with three isolates demonstrating significantly greater potency than the standard strain against Lepidoptera, B. thuringiensis serovar kurstaki HD1. These isolates were all found to produce bipyramidal crystals and major spore-associated protein bands of approximately 130 and 65 kDa, consistent with the detection of at least one cry1 and one cry2 family gene in each. The high level of insecticidal activity of these isolates makes them excellent candidates for further development for use in the field.     

Constructing Bacillus thuringiensis strain that co-expresses Cry2Aa and chitinase

A triple recombineering technique was used with plasmid pHT315 to produce pHTEC, a construct carrying chitinase and cry2Aa genes from Bacillus thuringiensis subsp. kurstaki 4.0718. Transformation of wild-type B. thuringiensis strain HD73 and the acrystalliferous strain Cry-B with pHTEC resulted in the recovery of recombinant strains that expressed Cry2Aa as cubic crystals in the cell pellet and soluble chitinase protein. The toxicity of HD73 (pHTEC) against Helicoverpa armigera larvae increased sevenfold when compared with HD73 (pHT315) harboring pHT315 vector. The triple recombineering protocol was optimized by comparing recombination efficacy mediated by RecE/RecT and Redα/Redβ and by using single-strand DNA as substrate.     

Recycling of ribosomal complexes stalled at the step of elongation in Escherichia coli

Translating ribosomes often stall during elongation. The stalled ribosomes are known to be recycled by tmRNA (SsrA)-mediated trans-translation. Another process that recycles the stalled ribosomes is characterized by peptidyl-tRNA release. However, the mechanism of peptidyl-tRNA release from the stalled ribosomes is not well understood. We used a defined system of an AGA-minigene containing a small open reading frame (ATG AGA AGA). Translation of the AGA-minigene mRNA is toxic to Escherichia coli because it stalls ribosomes during elongation and sequesters tRNA^Arg4 as a short-chain peptidyl-tRNA^Arg4 in the ribosomal P-site. We show that a ribosome recycling factor (RRF)-mediated process rescues the host from the AGA-minigene toxicity by releasing the peptidyl-tRNA^Arg4 from the ribosomes. The growth phenotypes of E. coli strains harboring mutant alleles of RRF and initiation factor 3 (IF3) genes and their consequences on λimmP22 phage replication upon AGA-minigene expression reveal that IF3 facilitates the RRF-mediated processing of the stalled ribosomes. Additionally, we have designed a uracil DNA glycosylase gene construct, ung-stopless, whose expression is toxic to E. coli. We show that the RRF-mediated process also alleviates the ung-stopless construct-mediated toxicity to the host by releasing the ung mRNA from the ribosomes harboring long-chain peptidyl-tRNAs.     

Cloning and Characterization of a Mosquito Larvicidal Toxin Produced During Vegetative Stage of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> 2297

The mosquitocidal toxin 1 (mtx1) gene from genomic DNA of B. sphaericus strain 2297 was cloned and expressed in E. coli. DNA sequencing analysis of the cloned gene revealed a single open reading frame encoding an 870-amino acid polypeptide. Expression level of the full-length gene in E. coli was very low even though strong promoter was used or the gene was expressed as a fusion protein. Expression level was highly improved after the putative leader sequence was deleted, and the truncated gene was expressed as a fusion protein with glutathione S-transferase (GST-tMtx1). E. coli cells expressing GST-tMtx1 was highly toxic to Culex quinquefasciatus larvae and showed lower toxicity against Anopheles dirus and Aedes aegypti larvae. Enterobacter amnigenus An11, a mosquito larval gut colonizable bacteria, transformed with the cloned gene exhibited mosquito larvicidal activity. Result suggested that there is a potential to develop this protein to be used as an alternative mosquito control agent.     

A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor ��32 of Escherichia coli

The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70''s ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ³², which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ³² facilitates RB43 bacteriophage proliferation is discussed.     

Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli

A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R · Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.     

Thioesterase II of Escherichia coli Plays an Important Role in 3-Hydroxydecanoic Acid Production

3-Hydroxydecanoic acid (3HD) was produced in Escherichia coli by mobilizing (R)-3-hydroxydecanoyl-acyl carrier protein-coenzyme A transacylase (PhaG, encoded by the phaG gene). By employing an isogenic tesB (encoding thioesterase II)-negative knockout E. coli strain, CH01, it was found that the expressions of tesB and phaG can up-regulate each other. In addition, 3HD was synthesized from glucose or fructose by recombinant E. coli harboring phaG and tesB. This study supports the hypothesis that the physiological role of thioesterase II in E. coli is to prevent the abnormal accumulation of intracellular acyl-coenzyme A.     

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.     

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC₅₀ = 0.84 mg l^?1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.