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PubMed1998

Molecular and phenotypic characterization of bacillus thuringiensis isolated from leaves and insects

Bacillus thuringiensis isolates from the phylloplane of organically cultivated cabbage were characterized using molecular and phenotypic methods. Of the 58 isolates under study, 31 belonged to serovar kurstaki, 16 did not react with any of the currently recognized antisera, 7 reacted with known antisera, and 4 could not be serotyped as they were nonmotile. Round crystals were found in 26 isolates, while bipyramidal crystals were found in the remaining 32 isolates, all of which had activity to lepidopteran larvae. Further, one isolate with unknown serotype and round crystals had lepidopteran activity. Colony hybridization was found to be a useful tool for screening the isolates for specific gene homologies and showed good correlation with the phenotypic observations. Polymerase chain reaction (PCR) was used for confirmation of the colony hybridization data, in most cases with concordant results. However, in one case some of the colony hybridization data could not be confirmed by PCR, due to DNA sequence variations in the binding area of one of the primers. The random amplified polymorphic DNA (RAPD) analysis showed that isolates otherwise indistinguishable could be distinguished by this method. However, the method was not able to distinguish the 31 kurstaki isolates. Further, the kurstaki isolates could not be distinguished from the B. thuringiensis serovar kurstaki HD-1 strain used in commercial products for lepidopteran control. One of the isolates was a serovar israelensis, but no genes encoding dipteran activity could be detected, and the RAPD analysis revealed that the DNA fingerprint of this israelensis isolate deviated from the israelensis ONR60A isolate used in commercial products. In conclusion we find that a molecular method like colony hybridization is suitable for screening large collections of bacteria. When colony hybridization data are combined with RAPD analyses isolates can be grouped based on genetic potential and DNA fingerprint, whereby further characterizations by PCR and the more labourious phenotypic methods can be performed more effectively. Copyright 1998 Academic Press.

Journal Article↗
PubMed2001

Regeneration of cultured midgut cells after exposure to sublethal doses of toxin from two strains of Bacillus thuringiensis.

Toxin from two strains of Bacillus thuringiensis (Bt), AA 1-9 and HD-73, caused dose-dependent destruction of cultured midgut cells from Heliothis virescens larvae. HD-73 toxin was more effective although, at the doses used, not all cells were killed. After 2 days of exposure to 0.8 pg/µl AA 1-9 or 0.06 pg/µl HD-73, columnar and goblet cell numbers declined to ca 20% of controls. In contrast, stem and differentiating cells increased to 140-200% of controls. The dynamic of depletion and replacement depended on toxin type and concentration. Two days after toxin was washed out, ratios of cell types returned to approximate control levels, suggesting rapid population corrections in vitro. Regulation of the ratio of cell types in each population, and the rate of proliferation and differentiation of stem cells was induced by the cultured midgut cells themselves. Controls and cells treated with toxin from Bt strain AA 1-9 were stained using a polyclonal antibody to Lepidopteran midgut differentiation factor 1 (MDF1). With Bt toxin, 1.5 times more cells stained for MDF1, suggesting increased synthesis of this differentiation factor during increased stem cell differentiation. The response of cultured midgut cells to Bt toxin injury is similar to injured vertebrate tissues dependent on stem cells for replacement and healing.

Journal Article↗
PubMed1993

Biotinylation of Bacillus thuringiensis Insecticidal Crystal Proteins.

Biotinylation of Bacillus thuringiensis insecticidal crystal proteins (ICPs) was evaluated for its potential use in an alternative ICP screening method and in the characterization of ICP receptors. In vivo biological activity of CryIA(b), as inferred from bioassays with Manduca sexta and Ostrinia nubilalis and from histopathological effects on O. nubilalis midgut cells induced by force feeding, was not affected by biotinylation at moderate biotinylation ratios. A competitive radioreceptor assay showed that there was only a minor reduction in binding affinity of biotin-labeled CryIA(b) for M. sexta brush border membrane vesicles. On midgut tissue sections, the binding pattern along the midgut epithelium and the staining intensity of biotinylated ICPs detected with streptavidin-enzyme conjugate were virtually identical to the binding pattern and staining intensity of native CryIA(b) detected with antibodies. The specificity of biotinylated ICP binding to larval midgut tissue was demonstrated by performing homologous competition experiments. The relationship between different ICP receptor types in Plutella xylostella, as inferred from radioligand binding studies, was confirmed by the results of heterologous competition experiments performed with biotinylated and native ICPs.

Journal Article↗
PubMed1995

The assembly and organization of the alpha 5 and alpha 7 helices from the pore-forming domain of Bacillus thuringiensis delta-endotoxin. Relevance to a functional model.

The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA delta-endotoxin contains two helices, alpha 5 and alpha 7, that are highly conserved within all different Cry delta-endotoxins. To gain information on the mode of action of delta-endotoxins, we have used a spectrofluorimetric approach and characterized the structure, the organization state, and the ability to self-assemble and to co-assemble within lipid membranes of alpha 5 and alpha 7. Circular dichroism (CD) spectroscopy revealed that alpha 7 adopts a predominantly alpha-helical structure in methanol, similar to what has been found for alpha 5, and consistent with its structure in the intact molecule. The hydrophobic moment of alpha 7 is higher than that calculated for alpha 5; however, alpha 7 has a lesser ability to permeate phospholipids as compared to alpha 5. Binding experiments with 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labeled peptide demonstrated that alpha 7 binds to phospholipid vesicles with a partition coefficient in the order of 10(4) M-1 similar to alpha 5, but with reduced kinetics and in a noncooperative manner, as opposed to the fast kinetics and cooperativity found with alpha 5. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that, in their membrane-bound state, alpha 5 self-associates but alpha 7 does not, and that alpha 5 coassembles with alpha 7 but not with an unrelated membrane bound alpha-helical peptide. Furthermore, resonance energy transfer experiments, using alpha 5 segments, specifically labeled in either the N- or C-terminal sides, suggest a parallel organization of alpha 5 monomers within the membranes. Taken together the results are consistent with an umbrella model suggested for the pore forming activity of delta-endotoxin (Li, J., Caroll, J., and Ellar, D. J. (1991) Nature 353, 815-821), where alpha 5 has transmembrane localization and may be part of the pore lining segment(s) while alpha 7 may serve as a binding sensor that initiates the binding of the pore domain to the membrane.

4-Chloro-7-nitrobenzofurazan↗
PubMed2005

Identification of cry-type genes on 20-kb DNA associated with Cry1 crystal proteins from Bacillus thuringiensis.

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.

Bacillus thuringiensis↗
PubMed1985

Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae.

The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.

Aedes↗
PubMed1986

Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific delta-endotoxin.

The lepidopteran-specific P1 delta-endotoxin of Bacillus thuringiensis var. kurstaki HD-1 was activated in vitro using insect gut proteases and found to be highly specific for the lepidopteran cell line Choristoneura fumiferana CF1 among a wide range of lepidopteran and dipteran cell lines tested. The toxicity of P1 against CF1 cells is inhibited by N-acetylgalactosamine (GalNAc), and the lectins soybean agglutinin (SBA) and wheat-germ agglutinin. Protein blotting was used to identify a glycoprotein of 146 X 10(3) Mr in the plasma membrane of CF1 cells, capable of binding both the toxin and SBA, which is specific for GalNAc. This glycoprotein was labelled using galactose oxidase and sodium boro-[3H]hydride and solubilized in Triton X-100 before partial purification by affinity chromatography on SBA-agarose. We propose that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific P1 delta-endotoxin of B. thuringiensis var. kurstaki HD-1.

Bacillus thuringiensis↗
PubMed2005

Occurrence and diversity of mosquitocidal strains of Bacillus thuringiensis.

Ever since the discovery of the first Bacillus thuringiensis strain capable of killing mosquito larvae, namely, B. thuringiensis var israelensis, there are several reports from different parts of the world about the occurrence of mosquitocidal strains belonging to different subspecies/serotypes numbering thirty-six. The main sources of these wild type strains are soils/sediments, plants, animal feces, sick/moribund insects and waters. The toxicity of the strains within a subspecies/serotype varied widely. Some of the strains exhibited toxicity to mosquitoes as well as lepidopterans and dipterans (including mosquitoes) as well as plant parasitic nematodes.

Animals↗
PubMed1996

Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis.

The sigE and sigK genes, encoding the sporulation-specific sigma factors sigma 35 and sigma 28 of Bacillus thuringiensis, were each disrupted by inserting a gene conferring resistance to kanamycin into their coding sequences. The B. thuringiensis SigE- and sigK- mutant strains were blocked at different sporulation stages and were unable to sporulate. The SigE-strain was blocked at stage II of sporulation, whereas the SigK- strain was blocked at stage IV. The expression of a cryIAa'-'lacZ transcriptional fusion was analysed in these genetic backgrounds and it was found that both sigma factors are involved in the in vivo transcription of this gene. However, the SigK- strain harbouring the cryIAa gene produced amounts of toxin similar to those produced by the B. thuringiensis Spo+ strain. The toxins accumulated in the mother cell compartment to form a crystal inclusion which remained encapsulated within the cell wall. Thus, transcription from the sigma E-dependent promoter alone (Bt I promoter) is sufficient to support high levels of toxin production in B. thuringiensis.

Bacillus thuringiensis↗
PubMed2000

Screening and characterization of Bacillus thuringiensis isolates from Brazil for the presence of coleoptera-specific cry genes.

Forty-three Bacillus thuringiensis isolates from Brazil and 3 from Argentina were screened, using the polymerase chain reaction (PCR), for various coleoptera-specific cry genes. Seven isolates produced specific and/or nonspecific DNA fragments in a PCR reaction with primers specific for two coleopteran cry genes and 4 of these produced DNA fragments with primers specific for 7 known coleopteran cry genes. These isolates showed, by electron microscopy, the presence of spherical crystals. They also showed proteins of around 70 kDa which were immunologically similar to the Cry3Aa protein from B. thuringiensis subsp. tenebrionis. The 3 isolates from Argentina were toxic to T. molitor, and although no isolate from Brazil showed toxicity, they might show toxicity to another insect species.

Animals↗
PubMedDate not supplied

Toxicity studies of microbial insecticide Bacillus thuringiensis var. kenyae in rats, rabbits, and fish.

Bacillus thuringiensis var. kenyae (B.t.k.) is a microbial insecticide effective against lepidopteran pest species. Acute oral toxicity in rats and acute dermal toxicity, ocular irritation, skin irritation in rabbits were studied for the wettable powder formulation of B.t.k. In addition, toxicity of the wettable powder formulation was also studied in fresh water fish (Gambussia affinis). The results of these studies indicate that this wettable powder formulation of B.t.k. is nontoxic and nonirritant to rats, rabbits, and fish.

Administration, Cutaneous↗
PubMed1991

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var Sotto in Escherichia coli.

During sporulation, Bacillus thuringiensis var. Sotto produces a parasporal crystalline protein which is toxic for the silk-worm, Bombyx mori and the cotton leaf worm, Spodoptera littoralis. The gene coding this crystal protein is present in a single plasmid. The plasmid DNA was isolated, purified and physically mapped using restriction endonuclease enzymes (R.E.). The gene coding the delta-endotoxin was inserted into Escherichia coli-Jm103, using cloning vector pUC8. Transformed E. coli cells were found to synthesize the delta-endotoxin as demonstrated by the pathogenicity of the transformed cells against 4th instar larvae of S. littoralis.

Animals↗
PubMedDate not supplied

[Bacillus thuringiensis plasmid DNA].

Plasmid DNAs were isolated from Bacillus thuringiensis and their physico-chemical properties were studied. The DNAs were purified using ultracentrifugation in a CsCl2 density gradient. The molecular weight of these extrachromosomal DNAs determined by electrophoresis in 0.6% agarose gel and by electron microscopy was 6 . 10(6)--10 . 10(6) daltons; a DNA with a molecular weight of 90 . 10(6) daltons was also found. Digestion with restriction endonuclease EcoRI yielded fragments of the plasmid DNAs with cohesive ends; these were further identified by means of electrophoresis in 1% agarose gel.

Bacillus thuringiensis↗
PubMed2004

[Control of chironomids in water by immobilized Bacillus thuringiensis var. israelensis de Barjac].

A strain from Bacillus thuringiensis var. israelensis de Barjac (B. t. i), having a fatal toxicity to chironomids, was immobilized in the PVA-H3BO3 gel beads confected with a bit of sodium alginate and activated carbon. The result showed that optimal weight ratios were 10% PVA, 1% sodium alginate and 20% activated carbon, respectively. When 4% calcium chloride saturated boric acid solution (pH=6.7) was used as gelatin agent for gelating 24 h, the produced gel beads had good performance in mechanical strength. This method is proved to be simple and cheap. The toxicity of the beads to chironomids is high and the release is slow.

Animals↗
PubMed2003

[Cloning and expression of ICP cry1AB16 gene of Bacillus thuringiensis].

With two pairs of primers designed on the basis of the sequence of cry1 and cry1Ab gene from Bacillus thuringiensis (Bt), a novel insecticidal crystal protein crylAb gene of Bt was amplified from bacillus strain AC-11 by using Long Template PCR System. Southernblot further confirmed that the novel gene existed in plasmid of the strain. The amplified fragment was sequenced and compared with cry1Ab1 gene in EMBL/GenBank. The results showed that eight nucleotides and seven amino acid residues were different from that of cry1Ab1, suggesting that it was a new crylAb gene, which was designated cry1Ab16 in GenBank (Accession No. AF375608). The cry1Ab16 gene was cloned into the E. coli expression vector pQE30, creating the recombinant plasmid pQCT, which was then transformed into E. coli M15. Westernblot analysis showed that Cry1Ab16 protein, induced by IPTG was expressed in the strain M15 (pQCT) with the molecular mass of approximate 130 kD, but Cry1Ab16 was unstable and was mostly degraded into about 65 kD protein. Bioassay showed that the LC50 of Cry1Ab16 against the third instar lavae of Plutella xylostella with a spreaded method was 258.3 mg/L, and it could also inhibit the growth of Spodoptera larvae.

Amino Acid Sequence↗
PubMed1993

Investigations on possible resistance in Aedes vexans field populations after a 10-year application of Bacillus thuringiensis israelensis.

In the Upper Rhine Valley (Germany), Bacillus thuringiensis var. israelensis has been widely used against floodwater mosquitoes over an area of approximately 500 km2 for more than 10 years. The susceptibility of larvae of Aedes vexans field populations in 3 untreated (Lake Constance) and 3 treated areas (Upper Rhine Valley) was assessed by means of bioassays with B.t.i. (Bactimos WP, 6,000 AAU/mg), following WHO guidelines. Log-probit analyses and statistical evaluations of the data showed that the LC50 values as well as slopes of bioassays of the larvae deriving from the different areas showed no significant differences. Two populations in the treated area were even more susceptible than populations from the untreated areas. These results have been confirmed by resistance ratios, which were less than one in all tests carried out.

Aedes↗
PubMed1995

Adhesion and cytotoxicity of Bacillus thuringiensis to cultured Spodoptera and Drosophila cells.

Two Bacillus thuringiensis strains were tested for the ability to adhere to cultured Spodoptera and Drosophila insect cells. The wild-type strain is virulent and motile and readily adheres to and kills both types of insect cells. The avirulent mutant strain, which lacks flagella and several other of the proposed virulence factors, does not adhere to the insect cells and does not kill these cells efficiently. Purified flagella bind to insect cells; addition of anti-flagella serum abolishes cell binding and reduces the killing efficiency. Both bacteria and culture supernatant from the wild-type strain, but not from the mutant, are cytotoxic. Of the factors known to be reduced in the avirulent mutant, such as flagellin and phosphatidylcholine (PC) degrading or phosphatidylinositol-specific phospholipase C (PLC), only PC-PLC is shown to be cytotoxic.

Animals↗