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PubMed2002

Physical mapping of the Bacillus thuringiensis subsp. kurstaki and alesti chromosomes.

Two strains of the well-known insect pathogen and biopesticide, Bacillus thuringiensis (Bt), belonging to subspecies alesti (strain Bt5) and kurstaki (strain Bt213), were chosen for genetic characterization. The two strains belong to different serotypes and are currently classified into different subspecies, although their insecticidal activity is similar. Physical maps were constructed of Bt alesti and Bt kurstaki using Pulsed Field Gel Electrophoreses (PFGE), and the map positions of several genes were determined. The 5.5 Mb combined genetic and physical chromosome maps of the two strains were found to be indistinguishable, and the only differences detected between the strains were of extrachromosomal origin. A cryIA toxin gene probe hybridised to a chromosome fragment and to two extrachromosomal elements in both strains, migrating as 100 kb and 350 kb, respectively. In addition a cry hybridizing extrachromosomal element migrating as 80 kb was present only in Bt alesti. Both strains were also found to contain sequences hybridizing to an enterotoxin (hbla) gene probe. Such sequences were positioned on the 350 kb extrachromosomal element, as well as on the chromosome.

Bacillus thuringiensis↗
PubMed2003

Ex vivo cytotoxicity of the Bacillus thuringiensis Cry4B delta-endotoxin to isolated midguts of Aedes aegypti larvae.

The pathological effect of the Bacillus thuringiensis Cry delta- endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.

Aedes↗
PubMed1999

Integrative model for binding of Bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth (Plutella xylostella).

Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.

Animals↗
PubMed2001

Crystallization of the Bacillus thuringiensis toxin Cry1Ac and its complex with the receptor ligand N-acetyl-D-galactosamine.

Cry1Ac from Bacillus thuringiensis ssp. kurstaki HD-73 is a pore-forming protein specifically toxic to lepidopteran insect larvae. It binds to the cell-surface receptor aminopeptidase N in Manduca sexta midgut via the sugar N-acetyl-D-galactosamine (GalNAc). By using 1,3-diaminopropane (DAP) as the buffer throughout protoxin activation and chromatography on Q-Sepharose at pH 10.3, trypsin-activated Cry1Ac has been purified in a monomeric state, which was crucial to obtaining single crystals of Cry1Ac and of the Cry1Ac-GalNAc complex. Crystals of Cry1Ac alone are triclinic, with unit-cell parameters a = 51.78, b = 113.23, c = 123.41 A, alpha = 113.11, beta = 91.49, gamma = 100.46 degrees; those of the Cry1Ac-GalNAc complex show P2(1) symmetry, with unit-cell parameters a = 121.36, b = 51.19, c = 210.56 A, beta = 105.75 degrees. Data sets collected to 2.36 and 2.95 A resolution, respectively, show that both crystal forms contain four molecules of the 66 kDa toxin in the asymmetric unit and have related packing arrangements. The deaggregating effect of DAP may be explained by its capacity for bivalent hydrogen bonding and hydrophobic interactions at protein interfaces.

Acetylgalactosamine↗
PubMed1990

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.

Bacillus thuringiensis↗
PubMed2004

Comparative production of Helicoverpa zea (Lepidoptera: Noctuidae) from transgenic cotton expressing either one or two Bacillus thuringiensis proteins with and without insecticide oversprays.

Transgenic cotton, Gossypium hirsutum (L.), expressing either one or two Bacillus thuringiensis ssp. kurstaki Berliner (Bt) proteins was compared with the conventional sister line in field experiments with regard to production of bollworm, Helicoverpa zea (Boddie), and bolls damaged by bollworm. The relative numbers of bollworms that developed on Bollgard (Monsanto Co., St. Louis, MO), Bollgard II (Monsanto Co.), and conventional cotton were estimated under nontreated conditions in 2000 and both insecticide-treated and nontreated conditions in 2001-2002 in North Carolina tests. Averaged across seven field studies under nontreated conditions, Bollgard cotton generated statistically similar numbers of large (L4-L5) bollworm larvae compared with the conventional variety; however, Bollgard cotton produced significantly fewer damaged bolls and bollworm adults than the conventional variety. Production of large larvae, damaged bolls, and adults was decreased dramatically by Bollgard II cotton as compared with Bollgard and conventional varieties. When comparing insecticide-treated and nontreated cotton genotypes, both Bt cotton sustained less boll damage than the conventional variety averaged across insecticide regimes; furthermore, Bollgard II cotton had fewer damaged bolls than the Bollgard variety. When averaged across cotton genotypes, pyrethroid oversprays reduced the numbers of damaged bolls compared with the nontreated cotton. Insecticide-treated Bollgard cotton, along with insecticide-treated and nontreated Bollgard II cotton reduced production of bollworm larvae, pupae, and adults. However, the addition of pyrethroid oversprays to Bollgard II cotton seemed to be the best resistance management strategy available for bollworm because no bollworms were capable of completing development under these conditions.

Animals↗
PubMed2006

Analysis of the cellular immune response induced by Bacillus thuringiensis Cry1A toxins in mice: effect of the hydrophobic motif from diphtheria toxin.

Insecticidal Cry1A toxins from Bacillus thuringiensis elicit strong humoral immune response in mice. Previously, we showed that an eight hydrophobic motif amino acid substitution in Domain I did not affect the antibody inducing capacity of the Cry1A toxins, on the contrary, it was enhanced after intranasal immunization. In addition, Cry1A mutants (carrying a substitution of a motif from fragment B of diphtheria toxin into the structurally similar hydrophobic alpha-helix 7 motif of Cry1A toxins) were able to modulate the ratio of IgG subclasses, IgG1/IgG2a. However, the capacity of these toxins to induce cellular immune response has not been studied. Thus, in this work, we investigated the cytokine profile induced after in vitro stimulation with the toxins, in spleen cell cultures from unprimed mice, and intranasally primed mice, with either wild-type Cry1Aa or with mutant toxin Cry1Aa8. Spleen cells from unprimed mice stimulated with Cry1Aa produced very low levels of Th1 (IFN-gamma, IL-12p70) and Th2 type cytokines (IL-10, IL-4), whereas immunization with Cry1Aa8 toxin led to higher production of these cytokines. Restimulation of spleen cells from primed mice with the Cry1Aa induced the production of significant levels of IL-12p70 whereas with Cry1Aa8, IFN-gamma production was stimulated. Interestingly, we found that the capacity of Cry1A toxins to induce cytokine production by lymphocytes was inhibited by N-acetylgalactosamine. Altogether these data demonstrate the immunogenic properties of Cry1A toxins and show that amino acid substitution in Domain I principally affects its ability to induce Th1 cytokines in lymphocytes.

Amino Acid Motifs↗
PubMed2004

The role of Bacillus thuringiensis Cry1C and Cry1E separate structural domains in the interaction with Spodoptera littoralis gut epithelial cells.

The Bacillus thuringiensis delta-endotoxins Cry1C and Cry1E share toxicity against several important lepidopteran species. Their combined use to delay development of resistance in target insects depends on their differential interaction with the gut epithelial cells. The three structural domains and combinations of two consecutive domains of Cry1C and Cry1E were separately expressed in Escherichia coli, and their interactions with the brush border membrane vesicles (BBMV) of Cry1E-tolerant and -susceptible Spodoptera littoralis larvae were studied. About 80% reduction in binding of Cry1E and each of its separate domains to BBMV of Cry1E-tolerant larvae was observed, whereas Cry1C was toxic to all larvae and bound equally to BBMV derived from both Cry1E-tolerant and -susceptible larvae. These results suggest differential interactions of the two toxins with BBMV encompassing all three domains. Comparable binding assays performed with fluorescent Cry1C and Cry1C domain II showed that Cry1C has higher Bmax and lower Kd than Cry1C domain II and further supported the existence of toxin multisite interactions. Competitive binding assays were used to estimate the sequence of interaction events. Cry1C domain II could compete with domain III binding, whereas domain III did not interfere with domain II binding, indicating sequential interactions of domain III and then domain II with the same membrane site. No competition between domain II of Cry1C and Cry1E was observed, confirming the existence of different domain II binding sites for the two toxins. Taken together, all three domains specifically interact with the epithelial cell membrane. The folding of the three-domain toxin probably dictates the sequence of interaction events.

Amino Acid Sequence↗
PubMed2001

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms.

AIMS: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them. METHODS AND RESULTS: Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensis serovars and five more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis. All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia, at the 92.5% DNA relatedness rate. CONCLUSION: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.

Bacillus thuringiensis↗
PubMed1991

Design and use of synthetic oligonucleotide probes in the cloning of delta-endotoxin genes from Bacillus thuringiensis.

A detailed protocol is described for the design and use of synthetic oligonucleotide probes for screening DNA libraries from Bacillus thuringiensis var. kurstaki (strain HD191) for copies of the gene (tox) encoding the insecticidal delta-endotoxin. Two homologous tox genes were identified in this organism; one of these was located on a 75-kb plasmid and the other on a second large plasmid or the bacterial chromosome. A tox gene was isolated as a 6.5-kb HindIII fragment of B. thuringiensis plasmid DNA.

Bacillus thuringiensis↗
PubMed2003

Cloning and expression of the insecticidal crystal protein gene Cry1Ca9 of Bacillus thuringiensis G10-01A from Taiwan granaries.

A new cry gene (cry1Ca9) was cloned and sequenced from a Bacillus thuringiensis isolate native to Taiwan (G10-01A). The cry1C-type gene, designated cry1Ca9, consisted of an open reading frame of 3,567 bp, encoding a protein of 1,189 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 134.7 kDa. The gene sequence was compared against the GenBank nucleotide sequence data base. It was found that the cry1Ca9 gene coded for a 134.7-kDa protoxin which had greater than 99.8% homology with the previously reported cry1Ca1 gene, as only three mismatches were found between the two amino acid sequences. When the Cry1Ca9 toxin was expressed in a crystal-negative strain of B. thuringiensis (cryB-), elliptical crystals were produced. Cell extracts from this recombinant strain appear to have high insecticidal activity against lepidopteran larvae (Plutella xylostella).

Amino Acid Sequence↗
PubMed1990

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.

Bacillus thuringiensis↗
PubMed2000

Development of diagnostic concentrations for monitoring Bacillus thuringiensis resistance in European corn borer (Lepidoptera: Crambidae).

Two candidate diagnostic concentrations of the Cry1Ab and Cry1Ac toxins from Bacillus thuringiensis corresponding to the LC99 and EC99 (effective concentration that causes 99% growth inhibition) for European corn borer, Ostrinia nubilalis (Hübner), were determined based on previously obtained baseline data. Validation experiments using field-collected European corn borer populations from across North America showed that for Cry1Ab, a concentration corresponding to the upper limit of the 95% confidence interval of the LC99, produced mortality > 99% for all populations tested. However, for Cry1Ac, adjustments and further validation are probably necessary. Development of B. thuringiensis resistance monitoring programs that rely on diagnostic techniques are discussed.

Animals↗
PubMed2006

Improvement of Bacillus thuringiensis delta-endotoxins synthesis yields through acquisition of erythromycin resistance.

The acquisition of the erythromycin resistance by Bacillus thuringiensis kurstaki improved yields of delta-endotoxins in sporulating cells ranging from 134 to 215%. Resistance to erythromycin decreased the final spore count by at least 50%. Consequently, erythromycin resistance is an efficient tool for the improvement of bioinsecticides yields with a high ratio of delta-endotoxins to spores.

Bacillus thuringiensis↗
PubMed2000

[Transfer of cry1C gene into Bacillus thuringiensis by electroporation to construct strain with broader insecticidal activity].

Three Transformants were selected by transferring cry1C into Bacillus thuringiensis strain YBT1535. Plasmid profiles, PCR and Southern blot result all proved that cry1C had been transferred into strain YBT1535. Bioassay results showed that the transformants of strain YBT1535 displayed significantly higher toxicity against Spodoptera exigua than strain YBT1535, but the toxicities against Heliothis armigera and Plutella xylostella did not rise except transformant YBT1535-2.

Bacillus thuringiensis↗
PubMed1998

Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence.

The insecticidal activity of Bacillus thuringiensis strains toxic to coleopterous insects is due to Cry3 proteins assembled into small rectangular crystals. Toxin synthesis in these strains is dependent primarily upon a promoter that is active in the stationary phase and a STAB-SD sequence that stabilizes the cry3 transcript-ribosome complex. Here we show that significantly higher yields of Cry3A can be obtained by using dual sporulation-dependent cyt1Aa promoters to drive the expression of cry3Aa when the STAB-SD sequence is included in the construct. The Cry3A yield per unit of culture medium obtained with this expression system was 12.7-fold greater than that produced by DSM 2803, the wild-type strain of B. thuringiensis from which Cry3Aa was originally described, and 1.4-fold greater than that produced by NB176, a mutant of the same strain containing two or three copies of cry3Aa, which is the active ingredient of the commercial product Novodor, used for control of beetle pests. The toxicities of Cry3A produced with this construct or the wild-type strain were similar when assayed against larvae of the cottonwood leaf beetle, Chrysomela scripta. The volume of Cry3A crystals produced with cyt1Aa promoters and the STAB-SD sequence was 1.3-fold that of typical bipyramidal Cry1 crystals toxic to lepidopterous insects. The dual-promoter/STAB-SD system offers an additional method for potentially improving the efficacy of insecticides based on B. thuringiensis.

Bacillus thuringiensis↗
PubMed1991

Deletion by in vivo recombination shows that the 28-kilodalton cytolytic polypeptide from Bacillus thuringiensis subsp. israelensis is not essential for mosquitocidal activity.

The cytA gene encoding the 28-kDa polypeptide of Bacillus thuringiensis subsp. israelensis crystals was disrupted in the 72-MDa resident plasmid by in vivo recombination, thus indicating that homologous recombination occurs in B. thuringiensis. The absence of the 28-kDa protein in B. thuringiensis did not affect the crystallization of the other toxic components of the parasporal body (68-, 125-, and 135-kDa polypeptides). The absence of the 28-kDa protein abolished the hemolytic activity of B. thuringiensis subsp. israelensis crystals. However, the mosquitocidal activity of the 28-kDa protein-free crystals did not differ significantly from that of the wild-type crystals when tested on Aedes aegypti and Culex pipiens larvae. The 28-kDa protein contributed slightly to the toxicity to Anopheles stephensi larvae. This indicates that the 28-kDa protein is not essential for mosquitocidal activity, at least against the three species tested.

Animals↗
PubMedDate not supplied

Transformation studies of Bacillus thuringiensis cryIC gene into a nitrogen-fixing Azospirillum lipoferum.

A lepidopteran toxin gene, cryIC (pSB607) from entomopathogenic Bacillus thuringiensis subsp. aizawai was introduced into nitrogen-fixing Azospirillum lipoferum by transformation. Regeneration of spheroplasts was achieved at 99% with 39% frequency of regeneration. Transformants were screened on NB kanamycin with ampicillin plates and 4 transformants were selected after ten generations. SDS-PAGE and Western blot analysis confirmed the presence of a 68 kDa protein in the transformants. Studies on utilization of carbon sources indicate that glucose and sucrose are the most favorable carbon sources and 2% molasses is the cheap alternate carbon source for the better growth of parent A. lipoferum and transformants.

Azospirillum↗