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PubMedDate not supplied

Characterization of cultured insect cells selected by Bacillus thuringiensis crystal toxin.

Selection for resistance against Bacillus thuringiensis (Bt) Cry1Ac10 in the Trichoplusia ni (Hubner) cell line BTI-TN-5B1-4 (TnH5) was tested, and the development of resistance in the selected cells was like a S-form curve. Monitoring at the Cry1Ac10 50th challenge, the resistance ratio was 1,294-fold as many as that of initial cells. But the resistance to Cry1Ac10 declined gradually when the selection was relaxed. The resistance declined rapidly at the low level of resistance and slowly at the high level of resistance. This resistant cell had high resistance to all the tested solubilized trypsin-treated mixture of crystal multitoxins of B. thuringiensis subsp. aizawai GC-91, an engineering bacterium of Bt, B. thuringiensis subsp. aizawai HD-133 and B. thuringiensis subsp. kurstaki HD-1, and low cross-resistance (19.7-fold) to activated Cry1C. Both N-acetyl-D-galactosamine (GalNAc) and tunicamycin did not inhibit the toxicity of Cry1Ac10 to the susceptible TnH5 cells. Comparison of the total proteins of the selected resistant cells with that of the nonselected susceptible cells by two-dimensional electrophoresis analysis showed that were obvious differences among the 11 protein expressions. These results strongly suggest that there exists an unknown mechanism of resistance in the cell line that was different from the reported mechanisms in insects.

Acetylgalactosamine↗
PubMed2005

New strategy for identification of novel Cry-type genes from Bacillus thuringiensis strains.

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.

Bacillus thuringiensis↗
PubMed2003

Pulsed field gel electrophoresis of chromosomal DNA reveals a clonal population structure to Bacillus thuringiensis that relates in general to crystal protein gene content.

Seventy strains of Bacillus thuringiensis representing 21 serovars were allocated to 38 genomic groups using pulsed field gel electrophoresis (PFGE) of restriction enzyme-digested DNA. There was a broad correlation between PFGE type and serotype for serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, sotto, thuringiensis, and tolworthi, although some serovars included atypical strains. Serovars canadensis and entomocidus were heterogeneous. Detection of crystal protein genes by polymerase chain reaction indicated an approximate correlation between PFGE type and cry gene complement. For example, cry1 products were amplified from DNA from PFGE type 17 strains of serovar aizawai and from PFGE type 23 strains of serovar tolworthi but not from a PFGE 18 strain of aizawai nor from a PFGE type 24 strain of tolworthi. These data suggest a clonal population structure to B. thuringiensis with some consistency of Cry-plasmid composition within PFGE types.

Bacillus thuringiensis↗
PubMed1983

Mechanism of action of Bacillus thuringiensis var israelensis insecticidal delta-endotoxin.

Bacillus thuringiensis var israelensis delta-endotoxin protein active against mosquitoes was inactivated by prior incubation with lipids extracted from Aedes albopictus cells. Experiments with lipid dispersions and multilamellar liposomes showed that the toxin binds to phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine provided these lipids contain unsaturated fatty acids. Phosphatidyl serine binds toxin less efficiently and phosphatidyl inositol, cardiolipin, cerebroside and cholesterol show no affinity for the toxin. The results suggest an insecticidal mechanism in which interaction of toxin with affinity for the toxin. The results suggest an insecticidal mechanism in which interaction of toxin with specific plasma membrane lipids causes a detergent-like rearrangement of the lipids, leading to disruption of membrane integrity and eventual cytolysis.

Aedes↗
PubMed1978

[Comparative characteristics of spore-forming and asporogenic strains of Bacillus thuringiensis].

Comparative characteristics of sporogenous and asporogenous Bacillus thuringiensis strains is carried out. Asporogenous strains are found to differ from wild type strains in a number of criteria, including colony morphology, character of growth on rich and poor media and UV-sensitivity. Sporogenous strains form R colonies, they are more stable and more rare produce variants forming S colonies. S colonies are typical for asporogenous mutants, and under the cultivation in unfavourable conditions (elevated temperature, a shift of pH, a change of an incubation regime) asporogenous strains dissociate with a high frequency into R form. Initial strains, which are multiple auxotrophs, under certain conditions can form "prototrophic" revertants which are unstable when incubated on rich media. Suppressor mutation is supposed to be a possible mechanism of the origination of "prototrophs".

Bacillus thuringiensis↗
PubMedDate not supplied

[Biological characteristics of the variants of Bacillus thuringiensis subsp. galleriae formed during continuous culture].

Bacillus thuringiensis subsp. galleriae S. variants formed during continuous cultivation differ from the parent culture in certain properties. In contrast to the parent R form, their growth in the chemostat does not yield virulent mutants which can cause their lysis on solid media. The chemostat S forms are resistant against virulent phage mutants produced when the R variants are grown under the conditions of continuous cultivation and against a virulent phage obtained from the parent culture 69-6 under the action of vancomycin. The R forms are sensitive to these phages. When the S forms are grown under the chemostat conditions, they do not revert to the R forms. The R and S forms do not differ noticeably in the character of their growth, formation of spores and crystals, and biological activity.

Bacillus thuringiensis↗
PubMedDate not supplied

[Plasmids of Bacillus thuringiensis var. galleriae strain 612].

We studied Bacillus thuringiensis var galleriae, strain 612 plasmids. B. thuringiensis cells contain double-stranded plasmid DNA molecules (ranging of about 12% from total DNA content) with buoyant density 1.59 g/cm3. Plasmid DNA content was constant during the exponential and stationary phases of bacterial growth. The plasmid fractions consist of DNA molecules with molecular weights of 5.9 x 10(6), 10.0 x 10(6), and 110.9 x 10(6) daltons (pVD1, pVD2 pVD3, respectively). Endonuclease EcoRI cuts the plasmids pVD2 and pVD3 into two and four fragments, respectivelyy, but pVDI seemed to be resistent to EcoRI treatment. We found that pVD2 and pVD3 plasmids contain a common DNA fragment with the molecular weight of 6.7 x 10(6) dalton as it was shown by restriction analysis. In contrast, the same plasmids contain the common fragment with molecular weight of 7.5 x 10(6) dalton as shown by heteroduplex analysis. Plasmid pVD3 has a transposon-like structure.

Bacillus thuringiensis↗
PubMed2005

Baseline susceptibility of western corn rootworm (Coleoptera: Crysomelidae) to Cry3Bb1 Bacillus thuringiensis toxin.

Susceptibility to Cry3Bb1 toxin from Bacillus thuringiensis (Bt) was determined for western corn rootworm, Diabrotica virgifera virgifera LeConte, neonates from both laboratory and field populations collected from across the Corn Belt. Rootworm larvae were exposed to artificial diet treated with increasing Cry3Bb1 concentrations, and mortality and growth inhibition were evaluated after 4-7 d. The range of variation in Bt susceptibility indicated by growth inhibition was similar to that indicated by mortality. Although interpopulation variation in susceptibility was observed, the magnitude of the differences was comparable with the variability observed between generations of the same population. In general, the toxin was not highly toxic to larvae and estimated LC50 and EC50 values were several times higher than those reported for lepidopteran-specific Cry toxins by using similar bioassay techniques. These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among rootworm populations and provide a baseline for estimating potential shifts in susceptibility that might result from selection and exposure to Cry3Bb1-expressing corn hybrids.

Animals↗
PubMed1990

Characterization of the Bacillus thuringiensis strains isolated from Taiwan.

Over 100 Bacillus thuringiensis (Bt) isolates which produced phase bright inclusions have been isolated from soil samples from different areas in Taiwan. Three types of crystal proteins were visualized by phase contrast microscopy. Among these isolates, only 14 different types of plasmid profiles have been observed. They all possess a variety of plasmids ranging from a few kb to around 250 kb in size. With respect to the crystal protein profiles, the plasmid profiles, and the shapes of crystal proteins, we found that the majority of our isolates (87%) were different from most of the known Bt strains. Our other two types of isolates (10 and 3%) resembled Bt var. kurstaki HD1 and Bt var. israelensis, respectively. Most of our isolates were active against Bombyx mori (Lepidoptera) and Aedes aegypti (Diptera). Most interestingly, two of our isolates, Nos. 82 and 96, were found highly toxic to Heliothis virescens, even compared with the standard strain, Bt var. kurstaki HD1. Using insecticidal crystal protein (ICP) gene probe from Bt var. aizawai HD-133 to probe the total DNA of our isolates, we observed that at least one plasmid from each of the tested strains reacted with the probe. A 10 kb plasmid from some of our isolates hybridized with the probe. This probably is the first evidence demonstrating that the ICP gene sequence can be found in a low molecular weight plasmid.

Aedes↗
PubMedDate not supplied

Structural and functional studies of a synthetic peptide mimicking a proposed membrane inserting region of a Bacillus thuringiensis delta-endotoxin.

In order to study the mechanism of action of Bacillus thuringiensis delta-endotoxins, a synthetic 31-mer peptide corresponding to the sequence of a putative pore-forming segment of the CrylA(c) toxin was characterized structurally and functionally. The peptide maps onto the central helix (alpha 5) of the six-helix bundle of domain I of the crystal structure of the CryIIIA toxin. CD and NMR spectroscopic studies indicated that the peptide exists as an alpha-helix in methanol and a random coil in water. The peptide associated with liposomes at pH 4.7 and formed discrete, characterizable channels in planar lipid bilayers at low pHs. These channels had a conductance value of 60 picosiemens (pS). It is possible that this helix is a component of the transmembrane pore formed by B. thuringiensis delta-endotoxins in vivo.

Amino Acid Sequence↗
PubMed1981

Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli.

Sau 3A1 partial digestion fragments from Bacillus thuringiensis var. kurstaki HD-1 plasmid DNA were ligated into the BamHI site of the cloning vector pBR322 and transformed into Escherichia coli strain HB101. Colonies presumed to contain recombinant plasmids were screened for production of an antigen that would react with antibody made against B. thuringiensis crystals. One strain, ES12, was isolated by using this procedure. ES12 contains a plasmid of Mr 11 X 10(6) that has DNA sequence homology with pBR322 as well as with Mr 30 X 10(6) and Mr 47 X 10(6) plasmids of B. thuringiensis. It makes a protein antigen, detected by antibodies to crystal, which has the same electrophoretic mobility as the B. thuringiensis crystal protein. Protein extracts of ES12 are toxic to larvae of the tobacco hornworm Manduca sexta.

Bacillus thuringiensis↗
PubMed1991

Sequence of an operon containing a novel delta-endotoxin gene from Bacillus thuringiensis.

An insect larval toxin designated CryII is produced by several subspecies of Bacillus thuringiensis and differs from the other major delta-endotoxins in these bacteria in its size, toxicity profile and presence as part of an operon with three open reading frames (ORF). Such an operon from a novel B. thuringiensis isolate has been cloned and differs from one previously characterized in the following ways: (a) the size and number of amino acid repeats in one of the ORFs; (b) the smaller size of the CryII protoxin and the presence of a unique 110-kDa CryII-related antigen; and (c) high larvicidal activity for a particular Lepidopteran but low activity for a Dipteran. Various subclones of this operon were introduced into a plasmid-free B. thuringiensis strain and only the cryII gene was found to be necessary for protoxin accumulation.

Amino Acid Sequence↗
PubMed2003

The Bacillus thuringiensis linear double-stranded DNA phage Bam35, which is highly similar to the Bacillus cereus linear plasmid pBClin15, has a prophage state.

Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis. Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15. We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage. This state is common, as several B. thuringiensis strains release Bam35-related viruses.

Amino Acid Sequence↗
PubMed1992

The parasporal inclusion of Bacillus thuringiensis subsp. shandongiensis: characterization and screening for insecticidal activity.

The parasporal body of Bacillus thuringiensis subsp. shandongiensis was characterized in terms of its structure, protein composition, and toxicological properties against several types of insects. The crystals of B. thuringiensis shandongiensis appear to consist of a major protein of 144 kDa present in an spherical inclusion, as determined by transmission electron microscopy, titration curve analysis, and SDS-PAGE of the solubilized crystals. A second protein of ca. 60 kDa is present in trace amounts and appears to be associated with a small bar-shaped inclusion. The 144-kDa protein has been characterized by isoelectric point determination, N-terminal amino acid sequence analysis, amino acid analysis, and immunological cross reactivity. Its N-terminal amino acid sequence differed from that of other B. thuringiensis crystal proteins. The 144-kDa protein was not immunologically related to the crystal proteins of two toxic serovars (B. thuringiensis israelensis and B. thuringiensis kurstaki HD-1) and one nontoxic serovar (B. thuringiensis indiana), as shown in immunoblots probed with antiserum raised against the 144-kDa B. thuringiensis shandongiensis protein, the B. thuringiensis israelensis crystal proteins, and the trypsin resistant fragment of B. thuringiensis kurstaki P1 proteins. In contrast to most B. thuringiensis serovars, B. thuringiensis shandongiensis crystals did not dissolve at pH 12. Solubilization was achieved in sodium bicarbonate at pH 8.3 and in the presence of 25 mM dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Diversity of locations for Bacillus thuringiensis crystal protein genes.

The location of crystal protein genes in 22 crystalliferous Bacillus thuringiensis strains representing 14 subspecies was investigated by hybridization of an intragenic restriction fragment from a cloned crystal protein gene to whole plasmid preparations. Hybridization was found to a single plasmid in eight strains, to more than one plasmid in seven strains, and to one or both of two large, unresolved plasmids in two strains. The sizes of the hybridized plasmids ranged from 33 to over 150 megadaltons. In one additional subspecies, hybridization was only to linear DNA fragments, suggesting a chromosomal crystal protein gene, and for four other subspecies, not reported to be toxic to lepidopteran insects, no hybridization was found to either plasmids or to total cell DNA. Hybridization to restriction digests of plasmids and total cell DNA of several strains of subspecies thuringiensis and kurstaki revealed that all homology to the cloned crystal protein gene was plasmid associated and that several of these strains contained multiple regions of homology, implying the presence of multiple crystal protein genes.

Bacillus thuringiensis↗
PubMed1991

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.

Animals↗
PubMed1994

Intracellular proteolysis and limited diversity of the Bacillus thuringiensis CryIA family of the insecticidal crystal proteins.

The current concept of how the highly homologous Bacillus thuringiensis insecticidal crystal protein genes (cry genes) evolved is through recombination among themselves. The cryIA gene family, which is more than 80% identical, consists of only three known genes, even through they are often found together in the same bacterium. To examine the lack of diversity among these genes, recombinatorial chimeric protein toxin genes were constructed and transformed into E. coli, B. subtilis, and B. thuringiensis. Of the nine chimeric proteins examined in this work; three were degraded in E. coli, five in B. subtilis, and seven in B. thuringiensis, suggesting that most Cry proteins resulting from recombination events are degraded by intracellular proteases that are particularly prevalent in B. thuringiensis.

Bacillus subtilis↗
PubMed1990

An in vitro system for testing Bacillus thuringiensis toxins: the lawn assay.

A cell assay system was developed that allows Bacillus thuringiensis delta-endotoxins activated at high pH (10.5) to be tested in vitro without causing alkaline injury to target cells. The assay is carried out on a lawn of gel-suspended cells, requires only 1 microliter of sample per dose, and is quantitative, rapid, and sensitive. The threshold dose for toxicity of B. thuringiensis subsp. kurstaki HD-73 with IPRI-CF-1 cells was 24 pg protein. The assay is also very useful for identifying antibodies which inhibit toxicity and for detecting beta-exotoxin.

Animals↗