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Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.

Aedes↗

Photoprotection of Bacillus thuringiensis kurstaki from ultraviolet irradiation.

Irradiation of Bacillus thuringiensis var. kurstaki HD1 at 300-350 nm for up to 12 hr using a photochemical reactor results in a rapid loss of its toxicity to larvae of Heliothis armigera. Photoprotection of the toxic component was obtained by adsorption of cationic chromophores such as acriflavin (AF), methyl green, and rhodamine B to B. thuringiensis. AF gave the best photoprotection and a level of 0.42 mmol/g dye absorbed per gram of B. thuringiensis was highly toxic even after 12 hr of ultraviolet (uv) irradiation as compared to the control (77.5 and 5% of insect mortality, respectively). Ultraviolet and Fourier-transform infrared spectroscopic studies indicate molecular interactions between B. thuringiensis and AF. The nature of these interactions and energy or charge transfer as possible mechanisms of photoprotection are discussed. It is speculated that tryptophan residues are essential for the toxic effect of B. thuringiensis. It is suggested that photoprotection is attained as energy is transferred from the excited tryptophan moieties to the chromophore molecules.

Bacillus thuringiensis↗

Cloning and sequence analysis of Bacillus thuringiensis gene fragments isolated by restriction digest PCR.

OBJECTIVE: To clone and analyze Bacillus thuringiensis gene fragments isolated by restriction digest PCR (RD-PCR). METHOD: Specific primers were designed to amplify the genes of Bacillus thuringiensis israelensis (Bti), and the PCR products were classified and re-amplified by RD-PCR to obtain the fragments for subsequent purification and cloning into the pMD18-T vectors, followed by rapid identification. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. RESULTS: Sequence analysis showed that all the fragments amplified were Bti genes. CONCLUSION: RD-PCR is reliable in breaking down large gene fragments into confined and shorter gene fragments for preparing microarray probes.

Bacillus thuringiensis↗

[Distribution, homology and cloning of cryptic Bacillus thuringiensis plasmids].

The 69-6 strain of Bacillus thuringiensis subsp. galleriae harbours at least 7 cryptic plasmids (pBTG1 - pBTG7) with molecular lengths 8,4 to 15,7 kb. According to hybridization analysis, the plasmid pBTG2 (8,7 kb) and other plasmids of the same host strain as well as cryptic plasmids of the strains belonging to 10 other serotypes of Bac. thuringiensis share detectable homology. As shown by the data of heteroduplex analysis, about 60% of pBTG1 and pBTG2 genomes have homologous DNA sequences. These data point out tht some plasmid genes are conserved in Bac. thuringiensis. BasmHI-, EcoRI- and HindIII-generated fragments of Bac. thuringiensis subsp. galleriae strain 69-6 are cloned on the pBR325 vehicle in the cells of Escherichia coli.

Bacillus thuringiensis↗

Two delta-endotoxin genes, cry9Da and a novel related gene, commonly occurring in Lepidoptera-specific Bacillus thuringiensis Japanese isolates that produce spherical parasporal inclusions.

Six Lepidoptera-specific Bacillus thuringiensis isolates, which belong to the four H serovars (sotto, fukuokaensis, canadensis, and galleriae) and produce spherical parasporal inclusions, were examined for assignment of the classes of the delta-endotoxin genes. Gene analysis was conducted by PCR technique with primers designed to probe the genes cry9Ca and cry9Da. The data revealed that the delta-endotoxin of a serovar canadensis isolate is encoded by the gene cry9Da, while those of the five other strains are encoded by an undescribed delta-endotoxin gene. DNA fragments from five strains had an identical 1917-bp nucleotide sequence, covering the four conserved regions and a partial sequence of the block 5 region. The deduced amino acid sequence exhibited a 70.6% homology to that of the corresponding region of the Cry9Ea delta-endotoxin protein which is active on the order Lepidoptera, and a 63.1% homology to the Cry9Ca protein highly toxic to the noctuid lepidopterans. The results showed that Japanese isolates of B. thuringiensis producing spherical parasporal inclusions with Lepidoptera-specific activity are categorized into two groups: one produces the class Cry9Da protein and the other a novel delta-endotoxin allied to the class Cry9. It also appeared that heterogeneous multiple H serovars are involved in each group.

Animals↗

Use of cellular fatty acid analysis to characterize commercial brands of Bacillus thuringiensis var. israelensis.

The cellular fatty acid composition of Bacillus thuringiensis var. israelensis (B.t.i.) from 5 commercial brands (Vectobac, Acrobe, Skeetal, Bactimos and Teknar), as well as of the current International Standard for B.t.i. (IPS 82), was determined using a Hewlett-Packard Microbial Identification System. The original strain of B.t.i., B.t. var. kurstaki, B.t. var. thuringiensis, B.t. var. morrisoni and Bacillus sphaericus strain 2362 were used as outgroups. Acrobe, Bactimos, Teknar, Vectobac and IPS 82 consisted of the same strain. Skeetal represented a different strain than the other commercially produced B.t.i. Our results indicate that cellular fatty acid analysis can be used to distinguish among the forms of B.t.i. produced by various manufacturers.

Bacillus thuringiensis↗

Cry29A and Cry30A: two novel delta-endotoxins isolated from Bacillus thuringiensis serovar medellin.

Two novel crystal protein genes from a highly mosquitocidal Bacillus thuringiensis serovar medellin strain were cloned and sequenced. The corresponding proteins, Cry29A and Cry30A, were nontoxic when tested individually against the mosquito species bioassayed (Aedes aegypti, Culex pipiens and Anopheles stephensi). However, Cry29A synergized the toxicity of Cry11Bb against Aedes aegypti by a four-fold factor.

Amino Acid Sequence↗

The occurrence and properties of Bacillus thuringiensis isolated from free-living animals.

AIMS: To assess the prevalence and properties of Bacillus thuringiensis isolated from the intestines of small mammals. METHODS AND RESULTS: Bacillus thuringiensis was found in 11% of rodents and 17% of insectivores. Using PFGE of chromosomal DNA, SDS-PAGE of whole-cell proteins and biochemical tests (API system), 12 isolates and three reference strains were classified. Numerical analysis revealed 61% and 89% similarity of protein profiles and biochemical properties of the bacilli, respectively. The results of PFGE were consistent with the outcomes of the analysis of protein profiles. CONCLUSIONS: Although B. thuringiensis is not common in the intestines of small mammals, it is heterogeneous at the genotypic and phenotypic level. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here help to explain the diversity and the ecological significance of B. thuringiensis. Future study should focus on the toxic activity of the isolated strains.

Animals↗

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.

Bacillus anthracis↗

Transformation of Bacillus thuringiensis by electroporation.

Plasmids were transformed by electroporation into various strains of Bacillus thuringiensis with frequencies of up to 10(5) transformants/micrograms. pC 194 transformed all strains tested at a high frequency and cells could be stably transformed with pC194 and pUB110 simultaneously by electroporation with a frequency of 10(2) pC194+ pUB110 transformants/micrograms DNA. Low transformation frequencies observed with some plasmids, especially those grown initially in Escherichia coli, could be increased by passage through B. thuringiensis, B. thuringiensis var. israelensis and in acrystalliferous mutant of the same strain transformed at frequencies of 10(4)-10(5)/micrograms DNA with most of the plasmids tested. A cloned israelensis 27-kDa delta-endotoxin gene was introduced into the israelensis acrystalliferous mutant and a kurstaki acrystalliferous mutant by electroporation. Both transformants were shown to express the endotoxin gene and to be toxic to Aedes aegypti larvae.

Bacillus thuringiensis↗

Reversal of resistance to Bacillus thuringiensis in Plutella xylostella.

Continued success of the most widely used biopesticide, Bacillus thuringiensis, is threatened by development of resistance in pests. Experiments with Plutella xylostella (diamondback moth), the first insect with field populations resistant to B. thuringiensis, revealed factors that promote reversal of resistance. In strains of P. xylostella with 25- to 2800-fold resistance to B. thuringiensis compared with unselected strains, reversal of resistance occurred when exposure to B. thuringiensis was stopped for many generations. Reversal of resistance was associated with restoration of binding of B. thuringiensis toxin CryIA(c) to brush-border membrane vesicles and with increased biotic fitness. Compared with susceptible colonies, revertant colonies had a higher proportion of extremely resistant individuals. Revertant colonies responded rapidly to reselection for resistance. Understanding reversal of resistance will help to design strategies for extending the usefulness of this environmentally benign insecticide.

Animals↗

Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuringiensis subsp. morrisoni PG-14 expressed in B. thuringiensis subsp. kurstaki by using the shuttle vector pHT3101.

The mosquitocidal properties of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni PG-14 are attributable to protein inclusions grouped together within a parasporal body. In both of these strains, the mosquitocidal activity resides in proteins with molecular masses of 27, 72, 128, and 135 kDa. In an attempt to determine the toxicity of each protein, the shuttle vector pHT3101 was used to express the cryIVD gene (encoding the 72-kDa CryIVD protein) from B. thuringiensis subsp. morrisoni in an acrystalliferous mutant of B. thuringiensis subsp. kurstaki. With this system, parasporal inclusions of the 72-kDa protein were obtained that were comparable in size, shape, and toxicity to those produced by parental B. thuringiensis subsp. morrisoni. The inclusions were bar shaped, measured 500 by 300 by 150 nm, and were easily visible with phase-contrast microscopy by 16 h of cell growth. A 50% lethal concentration of 64 ng/ml for these inclusions was determined in bioassays against fourth instars of Culex quinquefasciatus, which was similar to the 50% lethal concentration of 55 ng/ml obtained for the 72-kDa inclusion from B. thuringiensis subsp. israelensis. In contrast, expression of the cryIVD gene in Escherichia coli was very low and only detectable by immunoblot analysis. These results demonstrate that the pHT3101-B. thuringiensis expression system can be used to express the CryIVD protein in quantities and with properties comparable to that obtained with the natural host. This system may prove useful for the expression of other B. thuringiensis proteins and, in particular, for reconstitution experiments with inclusions produced by the mosquitocidal subspecies of B. thuringiensis.

Animals↗

Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles.

A rapid analysis of Bacillus thuringiensis strains predictive of insecticidal activity was established by using polymerase chain reaction (PCR) technology. Primers specific to regions of high homology within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each insecticidal class. Predictions of insecticidal activity were made on the basis of the electrophoretic patterns of the PCR products. Included in the screen were PCR primers specific for cryI, cryIII, and cryIV genes, which are insecticidal for lepidopterans, coleopterans, and dipterans, respectively. Known B. thuringiensis strains as well as unidentified strains isolated from soil and insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a single PCR reaction containing 12 to 20 primers capable of distinguishing between the different insecticidal genes. Insecticidal activity predicted by the PCR screen was found to correspond with the insecticidal activity of insect bioassays. In addition to identifying strains with known insecticidal genes, the PCR screen can identify strains with altered electrophoretic patterns containing potentially novel genes.

Animals↗

Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer: Holotrichia parallela.

A new Bacillus thuringiensis strain, Bt185, was isolated from HeBei soil samples in China. Observations after transmission electron microscopy found that the strain produced spherical parasporal inclusions similar to that of the B. thuringiensis subsp. japonensis Buibui strain, which showed toxicity to both Anomala corpulenta and Popillia japonica. The plasmid profile seen on an agarose gel revealed that Bt185 contained six large bands of 191 kb, 161 kb, 104 kb, 84 kb, 56 kb, and 37 kb. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed one major band with an estimated molecular mass of 130 kDa. Polymerase chain reaction-restriction fragment length polymorphism results showed that a novel cry8-type gene sequence was found in the Bt185 strain. When we screened for this novel gene sequence, an additional novel cry8-type gene was isolated, having a partial sequence of 2340 bp and encoding a protein of 780 amino acids. Bioassay results showed that Bt185 had no toxicity against several Coleopteran and Lepidopteran pests. However, Bt185 exhibited toxicity against larvae of the Asian cockchafer, Holotrichia parallela. This is the first report of the occurrence of a Bacillus strain that has insecticidal activity against Holotrichia parallela larvae.

Animals↗

The mosquito larvicidal activity of 130 kDa delta-endotoxin of Bacillus thuringiensis var. israelensis resides in the 72 kDa amino-terminal fragment.

Bacillus thuringiensis var. israelensis produces 130 kDa delta-endotoxin which is highly toxic to mosquito-larvae. The mosquito-larvicidal activity was delineated by sequential deletions from ends of the 1136 amino acids delta-endotoxin. A maximum of 459 amino acids could be removed from the carboxy-terminal of the toxin without a significant loss of the larvicidal activity. However, no more than 38 amino acids could be deleted from the amino-terminal without losing the toxicity. The truncated peptide of 72 kDa exhibited similar toxicity to the 130 kDa toxin and was between 39th and 677th amino acids.

Amino Acid Sequence↗

Restriction of intramolecular movements within the Cry1Aa toxin molecule of Bacillus thuringiensis through disulfide bond engineering.

Disulfide bridges were introduced into CrylAa, a Bacillus thuringiensis lepidopteran toxin, to stabilize different protein domains including domain I alpha-helical regions thought to be involved in membrane integration and permeation. Bridged mutants could not form functional ion channels in lipid bilayers in the oxidized state, but upon reduction with beta-mercaptoethanol, regained parental toxin channel activity. Our results show that unfolding of the protein around a hinge region linking domain I and II is a necessary step for pore formation. They also suggest that membrane insertion of the hydrophobic hairpin made of alpha-helices 4 and 5 in domain I plays a critical role in the formation of a functional pore.

Bacillus thuringiensis↗

Functional analysis of block 5, one of the highly conserved amino acid sequences in the 130-kDa CryIVA protein produced by Bacillus thuringiensis subsp. israelensis.

There are five amino acid sequences highly conserved among Bacillus thuringiensis delta-endotoxins. We have changed the amino acid residues in block 5, one of the conserved sequences, of CryIVA. When the amino acid residues with charged side chains were replaced by others, the amount of production of the altered CryIVA protein was markedly decreased. It is suggested that the decrease is caused by the unstable conformation of the altered CryIVA protein molecule, as judged by digestion with trypsin and thermolysin. On the other hand, the substitution of amino acid residues in block 5 did not affect the insecticidal activity of CryIVA. These results strongly suggest that block 5 of CryIVA is one of the stability-determining elements of the protoxin molecule.

Amino Acid Sequence↗

Expression of delta-endotoxin cryIA(c) gene of Bacillus thuringiensis in Escherichia coli and Streptomyces lividans.

The cryIA(c) gene of Bacillus thuringiensis was isolated from plasmid pOS1000. In order to obtain a proper cloning site and open reading frame, some DNA sequences preceding the initiating codon of the gene were replaced by synthetic oligonucleotide sequences. The isolated cryIA(c) was cloned into E. coli expression vector pKK223-3, and production of CryIA(c) protein was detected after induction by IPTG. A recombinant plasmid, pHZ1256, was constructed by insertion of the cryIA(c) gene into Streptomyces vector pHZ1272. pHZ1256 was introduced into Streptomyces lividans, and the production of CryIA(c) protein was confirmed by Western blotting after thiostrepton induction. A bioassy experiment showed that the CryIA(c) protein produced by E. coli and S. lividans caused 93% and 57% mortality to Plutella xylostella, respectively.

Animals↗