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PubMed2000

[A new resolution vector with cry1Ac10 gene based on Bacillus thuringiensis transposon Tn4430].

A new resolution vector with cry1Ac10 gene based on TnpI-mediated site-specific recombination system of Bacillus thuringiensis(Bt) transposon Tn4430 was developed. The gene cry1Ac10, encoding a protoxin against plutella xylostella larvae, and the gene ori1030, from a plasmid of wide type Bacillus thuringiensis, were inserted into two copy sets of RES sites, named pBMB801. When pBMB801 was introduced into crystal negative Bt host BMB171, antibiotic resistance genes and other non-Bt DNA can be selectively eliminated. This recombinant plasmid was found very stable without antibiotic selection. The resulting strain only contained Bt DNA and is free of antibiotic resistance genes. This strategy should facilitate regulatory approval for its development as a commercial biopesticide.

Bacillus thuringiensis↗
PubMed1993

Transcriptional regulation of the Bacillus thuringiensis subsp. thompsoni crystal protein gene operon.

The two predominant polypeptides of the Bacillus thuringiensis subsp. thompsoni crystal are encoded by the cry40 and cry34 genes. These crystal protein genes are located in an operon. Western analysis (immunoblotting) demonstrated that the operon promoter activity was located in the region upstream of the cry40 gene. The Cry34 protein was expressed only when the upstream promoter region was present. The crystal protein genes are the only cistrons in the operon, and they are expressed during sporulation, with the highest transcript levels detected early in sporulation (1.5 to 3 h after the onset of sporulation). Transcription initiates from two adjacent sites located 84 and 85 bases upstream of the cry40 translational start codon. The B. thuringiensis subsp. thompsoni crystal protein gene operon promoter aligned with other crystal protein gene promoters, which are activated from early to midsporulation and transcribed in vitro by the B. thuringiensis RNA polymerase E sigma 35.

Bacillus thuringiensis↗
PubMed2002

Molecular characterization of local Bacillus thuringiensis strains recovered from Northern Jordan.

A total of 80 isolates of Bacillus thuringiensis were recovered from different habitats of Northern Jordan. These isolates were grouped into three classes based on crystal morphology (spherical, bipyramidal, and both bipyramidal and cuboidal). The isolates that produced spherical crystals were the most common and the most toxic to diptera. SDS-PAGE analysis of the isolates and some reference strains with similar crystal morphology showed similar protein profiles with heterogeneous multiple protein components. The plasmid DNA content of the isolates in comparison with B. thuringiensis serovar israelensis gave a similar single intense DNA band.

Animals↗
PubMed1986

Biochemical and immunological characterization of the cloned crystal toxin of Bacillus thuringiensis var. israelensis.

The protein components of the cloned crystal toxin of Bacillus thuringiensis var. israelensis were separated by polyacrylamide gel electrophoresis under denaturing conditions. Using an antiserum to the solubilized B. thuringiensis var. israelensis crystal protein as a probe, immunological homology between the crystal protein components of B. thuringiensis var. israelensis and those of the recombinant B. megaterium strain VB131 was tested. The results from this study indicate that the crystal inclusion of the recombinant strain contains only the 130 kilodalton protein and not the 68 or the 28 kilodalton proteins of the crystal toxin of B. thuringiensis var. israelensis and that the 130 kilodalton protein is primarily responsible for the mosquitocidal activity of this organism.

Bacillus thuringiensis↗
PubMed1996

Mosquitocidal activity of the CryIC delta-endotoxin from Bacillus thuringiensis subsp. aizawai.

The cloned 135-kDa CryIC delta-endotoxin from Bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against Spodoptera litoralis and Spodoptera frugiperda larvae and in vitro against the S. frugiperda Sf9 cell line. Here, we report that the CryIC delta-endotoxin cloned from B. thuringienesis subsp. aizawai HD-229 and expressed in an acrystalliferous B. thuringiensis strain is also toxic to Aedes aegypti, Anophles gambiae, and Culex quinquefasciatus mosquito larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active delta-endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran delta-endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC delta-endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC delta-endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active delta-endotoxin.

Aedes↗
PubMed1994

Synergism of mosquitocidal toxicity between CytA and CryIVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis.

The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which it is composed (CytA, CryIVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CryIVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bioassays against first instars of Aedes aegypti. The LC50 for the CytA inclusion was 60 ng ml-1, whereas the LC50 for the CryIVD was 85 ng ml-1. In comparison, the LC50s for different combinations of CytA and CryIVD inclusions ranged from 12-15 ng ml-1, 4-5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni is due to synergism among three or four of their major proteins.

Aedes↗
PubMed2000

Bacillus thuringiensis delta-endotoxin proteins show a correlation in toxicity and short circuit current inhibition against Helicoverpa zea.

Pesticidal activity of Bacillus thuringiensis delta-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A, was determined by using the force-feeding bioassay method to 4(th) instar larvae of Helicoverpa zea. H. zea was susceptible to Bt toxins in the order Cry1Ac > Cry1Ab > Cry1Aa > Cry2A with 63.60, 89.04, 159.65, and 375.78 ng/larvae respectively. The abilities of selected Bacillus thuringiensis toxins to inhibit short circuit current (I(SC)) in midgut epithelia of H. zea were also investigated by voltage clamp assay. The voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short circuit current (I(SC)) by activated toxin. A Cry1Aa toxin dilution of 33.3 and 500 ng/ml resulted in inhibition of I(SC) of -2.29 microA/min (lag time 15 min) and -4.48 microA/min (lag time, 2 min) respectively. The Cry1Ab dilution of 25 ng/ml inhibited I(SC) to -1.39 microA/min, a lag time of 14 min, and 333.3 ng/ml dilution resulted in decay of I(SC) -2.49 microA/min, lag time 1 min respectively. The Cry1Ac lower dilution 16.7 ng/ml inhibited I(SC) to -1.39 microA/min, lag time 4 min, and a high dilution 333.3 ng/ml decay I(SC) to -2.44 microA/min, lag time 1 min. The inhibition of I(SC) (-1.10 microA/min, lag time 25) at lower dilution (33.3 ng/ml) and high dilution (500 ng/ml), decay (-2.38 microA/min, lag time 5 min), showed a correlation between toxin concentration and inhibitory response with Cry2A toxin. The lag time decreased with increasing concentration of toxin applied, which is additional evidence of dose response besides direct correlation of toxicity assays and I(SC).

Animals↗
PubMed1992

Isolation and sequence of a 2-kbp miniplasmid from Bacillus thuringiensis var. kurstaki HD-3a3b: relationship with miniplasmids of other B. thuringiensis strains.

The miniplasmid profiles of 18 Bacillus thuringiensis strains belonging to 8 different serotypes were determined using an alkaline hydrolysis method for isolation of low molecular mass plasmids. Nearly all the strains contained covalently closed circular (CCC) DNA species ranging from 2 to 5 species per strain and from 1.5 to 10.5 kbp in size (values corresponding to CCC forms). A 2-kbp plasmid from B. thuringiensis var. kurstaki HD-3a3b futura strain was used in Southern hybridization experiments to analyse relationships among the low molecular mass plasmids of different B. thuringiensis strains. This 2-kbp miniplasmid was present in most strains which show toxicity against lepidoptera. It was not present in those strains toxic against diptera (B. thuringiensis var. israelensis) or coleoptera (B. thuringiensis var. tenebrionis). The 2-kbp miniplasmid from B. thuringiensis var. kurstaki HD-3a3b futura was cloned and fully sequenced. Sequence analysis of the 2058 bp of the miniplasmid revealed the presence of an ORF (630 bp, 210 amino acids in size) that is preceded by a consensus sequence of B. thuringiensis crystal protein gene transcription promoters. No significant homology was observed with known B. thuringiensis toxin nucleic acid sequences or with other known sequences.

Amino Acid Sequence↗
PubMed1982

Formation of Crystalline delta-Endotoxin or Poly-beta-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis.

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of delta-endotoxin on M medium, which contained 330 mug of potassium per ml, but not on ST and ST-a media, each of which contained only 11 mug of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-beta-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH(2)PO(4) (3.7 mM) led to the formation of crystalline delta-endotoxin. The replacement of KH(2)PO(4) with equimolar amounts of KCl, KNO(3), and potassium acetate or an equivalent amount of K(2)SO(4) had a similar effect, whereas the addition of an equimolar amount of NaH(2)PO(4) or NH(4)H(2)PO(4) did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced delta-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-beta-hydroxybutyric acid granules on the media containing </=1 mM potassium. These results clearly indicate that a certain concentration of potassium is essential for the fermentative production of delta-endotoxin by these isolates of B. thuringiensis. Manganese could not be substituted for potassium. Phosphate ions stimulated poly-beta-hydroxybutyric acid formation by strain 290-1. The sporulation of B. thuringiensis and several other Bacillus strains was suppressed on the potassium-deficient ST medium. This suggests that potassium plays an essential role not only in Bacillus cell growth and delta-endotoxin formation but also in sporulation.

Journal Article↗
PubMed2006

Occurrence of parasporin-producing Bacillus thuringiensis in Vietnam.

A total of 63 Bacillus thuringiensis isolates were recovered from urban soils of Hanoi, Vietnam. Of these, 34 were identified to 12 H serogroups. None of the isolates showed larvicidal activities against three lepidopterous insects. Three isolates belonging to the two serovars, colmeri (H21) and konkukian (H34), were highly toxic to larvae of the mosquito Aedes aegypti. Parasporal inclusion proteins of four isolates exhibited cytocidal activities against HeLa cells. Immunologically, proteins of four isolates were closely related to parasporin-1 (Cry31Aa), a parasporal protein that preferentially kills human cancer cells. Haemolytic activities were associated with parasporal proteins of the three mosquitocidal isolates but not with those of the four cancer-cell-killing isolates. PCR experiments and nucleotide sequence analysis revealed that the genes of four anti-cancer isolates are closely related to the gene parasporin-1 (cry31Aa) but are dissimilar to those of the three other existing parasporins. Our results suggest that the soil of northern Vietnam is a good reservoir of parasporin-producing B. thuringiensis.

Animals↗
PubMed1989

Growth & toxicity of Bacillus thuringiensis var israelensis.

Chemically defined media containing glutamic acid, glutamine and aspartic acid at a 20 mM concentration individually supported abundant growth and sporulation of Bacillus thuringiensis var israelensis. The parasporal crystals produced in these media were toxic to fourth instar larvae of Culex pipiens quinquefasciatus Say. The medium containing 20 mM of glutamine induced early sporulation of B. thuringiensis var israelensis. An LC50 value of 3.92 micrograms/l was obtained. Calcium alginate and agar agar immobilized formulations of Bacillus thuringiensis var israelensis exhibited toxicity of 90 per cent and above to C. pipiens quinquefasciatus Say larvae, over a period of 90 days.

Animals↗
PubMedDate not supplied

[Polylysogeny in Bacillus thuringiensis var. Galleriae cultures].

The culture of Bacillus thuringiensis var. galleriae was shown to be polylysogenic. As has been found earlier, the culture has a phage with the original structure of particles whose tail possesses a special substructure called a "collar". The phage was described in detail as a phage of Bac. thuringiensis 1-97 with a "collar". Further studies of the culture of Bac. thuringiensis var. galleriae 1-97 has shown that it also contains another phage which differs sharply from the first one in the morphology of particles, the spectrum of lytic action, and other properties. Electron microscopy of the cultural fluid of the Bac. thuringiensis var. galleriae cultures has revealed in them the presence of different amounts of the particles of both morphological types found in the strain 1-97. These data suggest that the cultures of Bac. thuringiensis var. galleriae are polylysogenic. The methods for the isolation of the new phage are described.

Bacillus thuringiensis↗
PubMed1997

Contribution of the 65-kilodalton protein encoded by the cloned gene cry19A to the mosquitocidal activity of Bacillus thuringiensis subsp. jegathesan.

Two new crystal protein genes, cry19A and orf2, isolated from Bacillus thuringiensis subsp. jegathesan were cloned and characterized. The cry19A gene encodes a 74.7-kDa protein, and the orf2 gene encodes a 60-kDa protein. Cry19A contains the five conserved blocks present in most B. thuringiensis delta-endotoxins. The ORF2 amino acid sequence is similar to that of the carboxy terminus of Cry4 proteins. The cry 19A gene was expressed independently or in combination with orf2 in a crystal-negative B. thuringiensis host. The proteins accumulated as inclusions. Purified inclusions containing either Cry19A alone or Cry19A and ORF2 together were toxic to Anopheles stephensi and Culex pipiens mosquito larvae. They were more toxic to C. pipiens than to A. stephensi. However, inclusions containing Cry19A and ORF2 together were more toxic than inclusions of Cry19A alone but less toxic than the wild-type inclusions of B. thuringiensis subsp. jegathesan.

Amino Acid Sequence↗
PubMed2003

Comparative delta-endotoxins of Bacillus thuringiensis against mosquito vectors (Aedes aegypti and Culex pipiens).

Pure crystals of seven Bacillus thuringiensis field isolates from the Lower Silesia region (Poland) were tested against larvae of Aedes aegypti L. and Culex pipiens L. (Culicidae, Diptera). The crystals of OpQ3 phylloplane isolate (belonging to the first biochemical type of B. thuringiensis subsp. japonensis, yoso, jinghongiensis) killed from 68 +/- 7% to 84 +/- 7% of the fourth instar larvae of A. aegypti. The crystals of two other strains (KpF3 and KpC1) of this group caused mortality between 3 +/- 2% and 70 +/- 7%. The LC50 ranged from 3.2 +/- 0.4 to 34.1 +/- 4.8 microg/ml. The effect of B. thulringiensis wratislaviensis H-47 crystals was the lowest with larval mortality from 0% to 17 +/- 3%. No significant (0%-37 +/- 6%) effect of B. thuringiensis crystals on the larvae of C. pipiens was observed. Our results show that the delta-endotoxins of B. thuringiensis act very specifically.

Aedes↗
PubMed2006

Ubiquity of parasporin-1 producers in Bacillus thuringiensis natural populations of Japan.

Parasporin, a Bacillus thuringiensis parasporal protein, is unique in having a strong cytocidal activity preferential for human cancer cells. In this study, we characterized parasporin activities associated with three novel geographical isolates of B. thuringiensis. Parasporal inclusion proteins of the three isolates were highly toxic to human uterus cervix cancer cells (HeLa), but not to non-cancer uterine smooth muscle cells (UtSMC). Inclusions of the isolates lacked insect toxicity and hemolytic activity against sheep erythrocytes. Ouchterlony immunodiffusion tests revealed that the proteins of the three isolates are immunologically closely related to parasporin-1 (Cry31A), but dissimilar to the three other existing parasporin groups. Our results provide evidence that the parasporin-1-producing organism is a common member in B. thuringiensis populations occurring in natural environments of Japan.

Agglutination Tests↗
PubMed1977

Membrane fractions from the outer layers of spores of Bacillus thuringiensis with toxicity to lepidopterous larvae.

Two membrane fractions, F1 and F2, have been purified from the outer layers of spores of Bacillus thuringiensis. Both fractions contain 6-7% cysteine and appear to be similar in composition. Amino acids account for about 75% of the dry weight, carbohydrate for about 2% and lipids for about 25%. The fractions are both toxic to Pieris brassicae and the toxicity is inactivated by antiserum to the toxic crystal of Bacillus thuringiensis. The fractions can be distinguished by examination under the electron microscope; both fractions show similar hexagonal patterns but with different spacings. The same fractions from an acrystaliferous mutant (cr) were prepared. These were identical in density and in appearance under the electron microscope; the amino acid analysis of fraction F2 from both strains was identical. However, the spores and fractions F1 and F2 from this strain lacked toxicity. Fraction F2 from the cr strain was used to prepare antiserum specific to fraction F2. Using this anti-serum and anticrystal serum, crystal and F2 antigens were shown to appear simultaneously in sporulating cultures. Crystal and F2 antigens appeared some time before the maximum rate of uptake of [35s]cysteine. It is concluded that fraction F2 is derived from the exosporium and that fraction F1 probably originates from the spore coat. The exosporium in Bacillus thuringiensis appears to be synthesised during stages II and III of sporulation although uptake of [35S]cysteine occurs much later.

Amino Acids↗
PubMed1997

Molecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis.

A novel mosquitocidal protein gene, cry20Aa, was cloned from Bacillus thuringiensis subsp. fukuokaensis (H-3a: 3d: 3e). The gene product, Cry20Aa, was naturally truncated and had a molecular mass of 86,138 Da. The Cry20Aa protein possessed five conserved sequence blocks, as do most other insecticidal Cry toxins. However, an amino acid comparison of Cry20Aa with other mosquitocidal toxins, including Cry4A, Cry4B, Cry10A, Cry11A, and Cry11B, demonstrated that Cry20Aa was quite different from other toxins except for the conserved blocks. The N terminus of Cry20Aa was, however, homologous to the N termini of Cry4A and Cry10A. Interestingly, an inverted repeat (IR1) sequence in the open reading frame of the cry20Aa gene caused incomplete expression of Cry20Aa. When this internal IR1 sequence was altered with no change of amino acid sequence, acrystalliferous B. thuringiensis cells transformed with cry20Aa gene dramatically produced crystal inclusions. However, the intact 86-kDa Cry20Aa protein is highly labile, and it is rapidly degraded to polypeptides of 56 and 43 kDa. To increase expression of the cry20Aa gene, the p20 chaperonelike protein and the cyt1Aa promoter were utilized. While p20 did not increase Cry20Aa expression or stability, chimeric constructs in which the cry20Aa gene was under control of the cyt1Aa promoter overexpressed the Cry20Aa protein in acrystalliferous B. thuringiensis. The expressed Cry20Aa protein showed larvicidal activity against Aedes aegypti and Culex quinquefasciatus. However, the mosquitocidal activity was low, probably due to rapid proteolysis to inactive 56- and 43-kDa proteins.

Amino Acid Sequence↗
PubMed1989

[Proteins and proteinase-resistant polypeptides of delta-endotoxins from Bacillus thuringiensis and their insecticidal activity].

Proteins, solubility in alkaline buffer and proteinase-resistant polypeptides (PRP) of parasporal crystals (delta-endotoxin) from Bacillus thuringiensis were investigated. 19 sorts of crystals were divided into 7 types. Difference of crystalline proteins were coincident with their solubility and PRP. The characters of the protein, solubility and PRP were subspecies-specific basically, but in a few of crystals they were strain-specific. There was a closed relation between the biochemistry property of crystal protein and the toxicity in 2 species of insects. Crystal with protein of MW 130-138 kD kD or MW 130-138 kD and 60-65 kD, and PRP of MW 68-75 kD were high toxic in Bombyx mori (Lepidoptera), but most of them only showed weak or no toxicity in Culex fatigans (Diptera). Crystals containing more than 3 sorts of proteins with MW 15-138 kD in major protein and MW 35-75 kD in minor protein and PRP of 35-65 kD had very strong toxicity in Culex fatigans, but no toxicity in Bombyx mori. The construct of the crystal protein and the importance of the character in protein and PRP for the selection of expected strain and for the work of genetic engineering in Bacillus thuringiensis were discussed.

Animals↗