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[Antigenic structure of endotoxins from Bacillus thuringiensis].

The proteins of parasporal crystals of 9 strains of entomopathogenic bacteria of the Bacillus thuringiensis group of different H-serotypes were studied by different methods. Electrophoresis showed that crystals from the bacteria of 1, 3, 4, 5, 8, and 10 serotypes contained 1-2 proteins with a molecular weight of 130-145 kD and an additional fraction with a molecular weight of 55-120 kD. Double-radial immunodiffusion revealed that the crystals of all the serotypes studied are immunologically related. The proteins contained at least 3 types of antigens that can be detected either separately or all together when being tested with different antisera. The ELISA technique showed that the crystal proteins homologous with respect of antigens gave, as a rule, similar titration results.

Antigens, Bacterial↗
PubMed1984

A transposon-like structure related to the delta-endotoxin gene of Bacillus thuringiensis.

A DNA segment (Th-sequence) has been found in several strains of Bacillus thuringiensis. This Th-sequence [3 megadaltons (Md)] induces adjacent deletions when it is located in the pAM beta 1 plasmid derived from Streptococcus faecalis. Electron microscopic examination of reannealed single strands of one plasmid (pMT9) carrying such a deletion revealed that the Th-sequence corresponds to a single-stranded loop (2.8 Md) bounded by a short double-stranded stem (less than 0.2 Md). Southern blotting experiments established that in B. thuringiensis the Th-sequence was generally located on the large plasmid which also harbours the gene coding for the delta-endotoxin (crystal protein). Hybridization and heteroduplex analysis of the extrachromosomal DNA from the berliner 1715 strain demonstrated that the crystal gene and the Th-sequence are located in close vicinity on a 42-Md plasmid and that they are separated by a 1.3-Md DNA segment. This DNA segment is repeated in inverted orientation, once immediately adjacent to the Th-sequence and once 1.8 Md beyond the crystal gene. A model for the organization of these DNA sequences inside a transposon-like structure is proposed.

Bacillus thuringiensis↗
PubMed1991

Nucleotide sequence of a novel delta-endotoxin gene cryIg of Bacillus thuringiensis ssp. galleriae.

A gene cryIg coding for entomocidal protein delta-endotoxin of Bacillus thuringiensis ssp. galleriae str. 11-67 named CryIG has been cloned and sequenced (EMBL accession number X58120). The deduced amino acid sequence that contains 1156 amino acid residues shows only 28% of identical residues, when compared with other delta-endotoxins of the CryI family. The extent of identity is substantially higher for some regions of the sequence ('conserved blocks'), that presumably bear important structural or functional properties. This implies that CryIG delta-endotoxin follows the same type of polypeptide chain folding as other CryI proteins, whereas peculiarities of primary structure help to explain its unique specificity.

Amino Acid Sequence↗
PubMed1999

The oligomeric state of Bacillus thuringiensis Cry toxins in solution.

The molecular mass of different Cry toxins produced by Bacillus thuringiensis bacteria was estimated by size-exclusion chromatography and non-denaturing polyacrylamide gel electrophoresis at neutral and alkaline pH in order to assess the existence of oligomers in solution. We found that Cry1Aa, Cry1Ac, Cry1C, Cry1D and Cry3A toxins exist in solution as a mixture of monomer and high molecular mass aggregates with an apparent molecular mass greater than 600 kDa, that depend on the time elapsed between toxin activation and analysis. Aggregation of toxins by disulfide bonds is unlikely because aggregates are also observed in samples incubated with DTT. These data show that the Cry toxins studied do not form oligomers of less than ten subunits in solution and suggest that oligomer formation may occur after the toxin binds to the receptor and inserts into the membrane.

Bacillus thuringiensis↗
PubMed2001

Genetics of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac.

Laboratory selection increased resistance of pink bollworm (Pectinophora gossypiella) to the Bacillus thuringiensis toxin Cry1Ac. Three selections with Cry1Ac in artificial diet increased resistance from a low level to >100-fold relative to a susceptible strain. We used artificial diet bioassays to test F1 hybrid progeny from reciprocal crosses between resistant and susceptible strains. The similarity between F1 progeny from the two reciprocal crosses indicates autosomal inheritance of resistance. The dominance of resistance to Cry1Ac depended on the concentration. Resistance was codominant at a low concentration of Cry1Ac, partially recessive at an intermediate concentration, and completely recessive at a high concentration. Comparison of the artificial diet results with previously reported results from greenhouse bioassays shows that the high concentration of Cry1Ac in bolls of transgenic cotton is essential for achieving functionally recessive inheritance of resistance.

Animals↗
PubMed2001

Highly toxic and broad-spectrum insecticidal Bacillus thuringiensis engineered by using the transposon Tn917 and protoplast fusion.

The chromosome of the Bacillus thuringiensis strain S184 that was toxic against the third instar larvae of Spodoptera litura with the LC(50) of 9.74 microg/ml was successfully integrated into two genes of cyt1Aa and cry11Aa using the transposon Tn917, yielding the primary engineered strain TnX. The strain TnX was highly toxic to the third instar larvae of Culex pipiens fatigans with the LC(50) of 5.12 ng/ml which was 1.82-fold higher than that of B. thuringiensis subsp. israelensis, but lowly toxic to lepidopterous larvae. By the protoplast fusion of the strain TnX and the strain S184-Tet(r) (resistance to tetracycline), the target engineered strain TnY was obtained. Against the third instar larvae of S. litura, the strain TnY LC(50) was of 4.68 micro g/ml and increased by 2.08-fold in comparison with the parent strain S184. Against the third instar larvae of C. pipiens fatigans, the strain TnY LC(50) was of 103.20 ng/ml. The two target genes of cyt1Aa and cry11Aa integrated into the chromosome were extremely stable and had little possibility of a second transposition. It was unclear whether some factors existing in the parent strain, S184, contributed to the high toxicity of the strains TnX and TnY.

Animals↗
PubMed1998

[Expression characteristic of Bacillus thuringiensis cry1 gene in Pseudomonas fluorescens Pfx-18].

The plasmids of lepidopteran-specific Bacillus thuringiensis strain from our laboratory were hybridized with RNA probe of cry 1Aa EcoR I-F fragment labelled using DIG. Cry1 gene was located in 39.3 MD plasmid. The plasmid was digested with Hind III and analysed by southern blot. It appeared both 7.1 kb and of 6.5 kb positive bands. The 7.1 kb fragment was ligated to broad-host-range vector pSUP106 and transformed into Pseudomonas fluorescens Pfx-18. The cloned strain, LZP-1 was obtained. The plasmids of LZP-1 were analysed by PCR. The results showed that gene-type is cry1Ab. SDS-PAGE analysis demonstrated that LZP-1 could express 66 kD insecticidal crystal protein and some small molecular weight peptides. Bioassay showed that motality of 1000-fold diluted fermentation broth was 33% to 3rd instar plulella xyloslelly larvae.

Animals↗
PubMed1987

[The role of plasmids in the regulation of delta-endotoxin synthesis in Bacillus thuringiensis H14].

The role of plasmids in regulation of delta-endotoxin synthesis by Bacillus thuringiensis H14 was studied. The derivatives of strain Is-1 H14 containing a 4Md plasmid integrated into the chromosome synthesize small crystals and are not toxic for the gnat larvae. The transceptional transfer into this strain of a plasmid coding for crystal synthesis from the strain 69-6 serotype H5 results in restoration of insecticidal activity to the level of the parental strain Is-1. Transcipients activity is increased 10-15 fold in case of 4Md plasmid excision from the chromosome and autonomous functioning. Evidently, 4Md plasmid from the strain Is-1 as well as a plasmid coding for crystal synthesis from the strain 69-6 contains the regulatory elements participating in the expression of crystalline protein genes localized on other plasmids. The existence of two cellular regulatory groups is supposed to result in the significant increase in crystalline protein synthesis.

Bacillus thuringiensis↗
PubMed2004

Cloning, characterization and expression of a new cry 1Ab gene from Bacillus thuringiensis WB9.

A new cry 1Ab-type gene, cry 1Ab17, was cloned from Bacillus thuringiensis WB9 by PCR. Nucleotide sequence indicated that the open reading frames (ORFs) consists of 3471 bases and encodes a protein of 1156 residues with a calculated molecular weight of 130.5 kDa and an pI value of 5.04. Homology comparison revealed that the deduced amino acid sequence of Cry1Ab17 had 95.4% to 99.7% identity with those of the known Cry1Ab proteins. The Cry1Ab17 was one residue longer than the known Cry1Ab (except for Cry1Ab2). Domain I (Tyr(33) to Arg(253)), II (Arg(265) to Phe(462)), III (Asn(464) to Thr(610)) of the Cry1Ab17 were 96.8%, 68.2% and 100% identical to the corresponding domains of Cry1Aa. Additionally, the cry 1Ab17 gene was expressed in Escherichia coli BL21 under the control of T7 promoter and the Cry1Ab17 isolated from the culture medium was toxic to 3rd instar Plutella xylostella larvae.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence and analysis of an insertion sequence from Bacillus thuringiensis related to IS150.

A 5.8-kb DNA fragment encoding the cryIC gene from Bacillus thuringiensis (Bt) subsp. aizawai HD229 was subcloned into the pMex7 vector for expression in Escherichia coli. In addition to the 135-kDa CryIC delta-endotoxin, this DNA fragment also encoded a 30-kDa polypeptide whose open reading frame (orfX) was located less than 200 bp upstream of cryIC. Nucleotide sequencing showed that orfX was truncated at the 5' end, and full sequence was obtained from a second overlapping clone. Sequence analysis showed that orfX could encode a polypeptide closely related to the putative transposase from IS150. OrfX was flanked by a 17-bp imperfect inverted repeat, defining the length of the element as 998 bp. Southern blot analysis revealed that the novel insertion sequence was present in a single copy and located in an identical position immediately upstream of cryIC in plasmid DNA from both Bt subsp. aizawai and entomocidus.

Amino Acid Sequence↗
PubMed1999

Genetic mapping of resistance to Bacillus thuringiensis toxins in diamondback moth using biphasic linkage analysis.

Transgenic plants producing environmentally benign Bacillus thuringiensis (Bt) toxins are deployed increasingly for insect control, but their efficacy will be short-lived if pests adapt quickly. The diamondback moth (Plutella xylostella), a worldwide pest of vegetables, is the first insect to evolve resistance to Bt toxins in open-field populations. A recessive autosomal gene confers resistance to at least four Bt toxins and enables survival without adverse effects on transgenic plants. Allelic variants of this gene confer resistance in strains from Hawaii, Pennsylvania, and the Philippines. Here we exploited the biphasic nature of Lepidopteran genetic linkage to map this gene in diamondback moth with 207 amplified fragment length polymorphisms as DNA markers. We also cloned and sequenced an amplified fragment length polymorphism marker for the chromosome containing the Bt resistance gene. The results provide a powerful tool for facilitating progress in understanding, monitoring, and managing resistance to Bt.

Animals↗
PubMed1989

Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var. tenebrionis.

Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin.

Amino Acid Sequence↗
PubMed1998

CytA protein, a delta-endotoxin of Bacillus thuringiensis subsp. israelensis is associated with DNA.

CytA protein (27 kDa) is produced by Bacillus thuringiensis subsp. israelensis (BTI) and is contained in its inclusion bodies. We previously reported the isolation of 25 kDa portion of CytA protein (p25-CytA protein) and its strong cytotoxic activity to mammalian cells. When p25-CytA protein was applied to an anion-exchange column for further purification, three fractions (M1, M2 and M3) were separated. M1 and M2 fractions were both shown to be 25 kDa protein, while M3 was a high molecular weight complex composed of 25 kDa protein and DNA. Purification and amino acid sequence analysis showed that M1 and M2 fractions were proteins lacking 29 and 31 N-terminal amino acids from CytA protein, respectively, and M3 was M1 protein associated with DNA. DNA was detected in BTI cells co-localizing with inclusion bodies. Both M1 and M2 proteins could bind to double-stranded DNA of BTI genome in vitro; the DNA binding ability of M1 protein was higher than that of M2 protein. These results suggested that CytA protein has DNA binding ability and is associated with DNA in the mother cell.

Amino Acid Sequence↗
PubMed1992

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco.

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 microgram ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.

Bacillus thuringiensis↗
PubMed2004

Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae).

The bacterium Bacillus thuringiensis (Bt) produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584), serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae.

Animals↗
PubMed1993

Site-directed mutations in a highly conserved region of Bacillus thuringiensis delta-endotoxin affect inhibition of short circuit current across Bombyx mori midguts.

Bacillus thuringiensis delta-endotoxins (Cry toxins) are insecticidal proteins of approximately 65 kDa in the proteolytically processed and active form. The structure of one of these toxins, CryIIIA, has been determined by Li et al. [Li, J., Carroll, J. & Ellar, D. J. (1991) Nature (London) 353, 815-821] and contains three domains. It is believed that other delta-endotoxins adopt similar three-dimensional structure. Li et al. proposed that the first domain is the membrane pore-forming domain. Previous work from our laboratory has shown that the second domain is the receptor binding domain, but the function of the third domain is unclear. Site-directed mutagenesis was used to convert the "arginine face" of one of five highly conserved regions, QRYRVRIRYAS of CryIAa (residues 525-535), to selected other residues. This sequence corresponds to the beta-sheet 17 of CryIIIA in the third domain. Mutations in the second and third arginine positions resulted in structural alterations in the protein and were poorly expressed in Escherichia coli. Toxins from genes mutated to replace lysine for the first and fourth arginines were unaltered in expression and structure, as measured by trypsin activation, CD spectra, and receptor binding, but were substantially reduced in their insecticidal properties and inhibition of short circuit current across Bombyx mori midguts. It is proposed that this region plays a role in toxin function as an ion channel.

Amino Acid Sequence↗
PubMed2000

Pesticidal and receptor binding properties of Bacillus thuringiensis Cry1Ab and Cry1Ac delta-endotoxin mutants to Pectinophora gossypiella and Helicoverpa zea.

Bacillus thuringiensis produces several larvicidal crystalline inclusions during sporulation. An understanding of their mechanisms of action is commercially important. In this study, two toxins, Cry1Ab and Cry1Ac, were compared that showed 98% amino acid identity in domain I and II, but differed significantly in domain III. Using site-directed mutagenesis techniques, two conserved loop 2 Arg's ((368)RR(369)) of Cry1Ab and Cry1Ac toxins were replaced with Ala ((368)AR(369), (368)RA(369), (368)AA(369)), Glu ((368)EE(369)), Phe ((368)FF(369)), His ((368)HH(369)), and Lys ((368)KK(369)). The effect of these mutants on structural stability, larvicidal potency, receptor binding, and ionic permeability towards two important cotton pests, pink bollworm (Pectinophora gossypiella) and bollworm (Helicoverpa zea) were analyzed. All seven mutants of Cry1Ab, excluding (368)AR(369), produced a stable protoxin, whereas for Cry1Ac all seven mutants yielded stable protoxin. Results showed that all the stable mutants behaved similarly to the wild type on incubation with trypsin and gut extract of both insect larvae. The Cry1Ab mutants, (368)AR(369), (368)AA(369), (368)FF(369), and (368)HH(369), lost toxicity; (368)EE(369) had reduced toxicity; whereas the more conserved change (368)KK(369) retained the toxicity similar to the wild type towards P. gossypiella. Double mutants of Cry1Ac, (368)AA(369) and (368)FF(369), abolished the toxicity. Double mutant (368)KK(369) of Cry1Ac retained its toxicity against P. gossypiella, whereas single mutants (368)AR(369), (368)RA(369), and (368)HH(369) retained only reduced toxicity. All the mutants of Cry1Ab lost their toxicity against H. zea except (368)KK(369). In Cry1Ac single mutants, (368)AR(369) and (368)RA(369), reduction in the toxicity was observed. A double mutant of Cry1Ac, (368)KK(369), also retained reduced toxicity. All the other double mutants lost their toxicity. Voltage clamping experiments on H. zea midguts provided an additional evidence about the insecticidal property and inhibition of I(sc) across the transepithelial membrane of the insect midgut.

Animals↗
PubMed2000

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

Bacillus↗