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PubMed1993

Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. galleriae.

A novel cryIC-type gene was isolated from a strain of Bacillus thuringiensis subsp. galleriae. A new polymerase chain reaction (PCR) technique with a set of several oligonucleotide primer pairs specific to the cryIC gene was used to screen a number of B. thuringiensis strains. PCR amplified several DNA fragments ranging from 100 bp to 1 kb for B. thuringiensis strains containing a cryIC gene. PCR fragments amplified from the Bacillus thuringiensis subsp. galleriae HD29 DNA differed from the fragments amplified from other cryIC-containing strains, indicating strain HD29 contained a novel cryIC-type gene. To isolate crystal genes homologous to cryIC, an HD29 gene library was probed with a 984-bp fragment of the amino-terminal coding region of the cryIC gene cloned from Bacillus thuringiensis subsp. aizawai HD229. A putative toxin gene was isolated from a phage that hybridized strongly to the cryIC probe. Translation of the putative toxin DNA sequence revealed an open reading frame of 1,176 amino acids whose predicted molecular mass was 132.8 kDa. Comparisons of the toxin gene sequence with sequences of other cry genes indicated that this gene is a subclass of cryIC. We propose to designate this gene cryIC(b). In Escherichia coli, the cryIC(b) gene produced a protein of approximately 130 kDa toxic to Spodoptera exigua and Trichoplusia ni.

Amino Acid Sequence↗

PubMed1992

[Site-specific restrictases from Bacillus thuringiensis var. Kumantoensis].

The efficiency of bacteriophages CP-54 and CP-55 plating on Bacillus thuringiensis var. kumantoensis H18 (Kum) is decreased about 10-fold as compared with the efficiency of plating on Bacillus thuringiensis var. galleriae H5 (Gal). Bacteriophages having propagated for one cycle in Kum cells might be further grown in this strain without growth restriction. Two site-specific restriction enzymes isolated from Bacillus thuringiensis var. kumantoensis were designated BtkI and BtkII. The endonuclease BtkI recognises the same nucleotide sequence CGCG in DNA as recognised by the restriction endonuclease FnuDII; BtkII recognises the same nucleotide sequence GATC as the endonuclease Sau3A.

Bacillus thuringiensis↗

PubMed2005

Assessment of cry1 gene contents of Bacillus thuringiensis strains by use of DNA microarrays.

A single Bacillus thuringiensis strain can harbor numerous different insecticidal crystal protein (cry) genes from 46 known classes or primary ranks. The cry1 primary rank is the best known and contains the highest number of cry genes which currently totals over 130. We have designed an oligonucleotide-based DNA microarray (cryArray) to test the feasibility of using microarrays to identify the cry gene content of B. thuringiensis strains. Specific 50-mer oligonucleotide probes representing the cry1 primary and tertiary ranks were designed based on multiple cry gene sequence alignments. To minimize false-positive results, a consentaneous approach was adopted in which multiple probes against a specific gene must unanimously produce positive hybridization signals to confirm the presence of a particular gene. In order to validate the cryArray, several well-characterized B. thuringiensis strains including isolates from a Mexican strain collection were tested. With few exceptions, our probes performed in agreement with known or PCR-validated results. In one case, hybridization of primary- but not tertiary-ranked cry1I probes indicated the presence of a novel cry1I gene. Amplification and partial sequencing of the cry1I gene in strains IB360 and IB429 revealed the presence of a cry1Ia gene variant. Since a single microarray hybridization can replace hundreds of individual PCRs, DNA microarrays should become an excellent tool for the fast screening of new B. thuringiensis isolates presenting interesting insecticidal activity.

Bacillus thuringiensis↗

PubMed2005

Distribution of genes encoding putative virulence factors and fragment length polymorphisms in the vrrA gene among Brazilian isolates of Bacillus cereus and Bacillus thuringiensis.

One hundred twenty-one strains of the Bacillus cereus complex, of which 80 were isolated from a variety of sources in Brazil, were screened by PCR for the presence of sequences (bceT, hblA, nheBC, plc, sph, and vip3A) encoding putative virulence factors and for polymorphisms in variable-number tandem repeats (VNTR), using a variable region of the vrrA open reading frame as the target. Amplicons were generated from isolates of B. cereus and Bacillus thuringiensis for each of the sequences encoding factors suggested to play a role in infections of mammals. Intriguingly, the majority of these sequences were detected more frequently in Bacillus thuringiensis than in B. cereus. The vip3A sequence, which encodes an insecticidal toxin, was detected exclusively in B. thuringiensis. VNTR analysis demonstrated the presence of five different fragment length categories in both species, with two of these being widely distributed throughout both taxa. In common with data generated from previous studies examining European, Asian, or North American populations, our investigation of Brazilian isolates supports the notion that B. cereus and B. thuringiensis should be considered to represent a single species.

Animals↗

PubMed1993

Cloning and characterization of a novel Bacillus thuringiensis cytolytic delta-endotoxin.

A gene encoding a major entomocidal polypeptide from Bacillus thuringiensis subspecies kyushuensis delta-endotoxin crystals (CytB) was cloned into Escherichia coli and sequenced. The deduced amino acid sequence gave a predicted molecular mass of 29,236 Da and showed 39% identity and 70% similarity with the 27,371 Da CytA protein from Bacillus thuringiensis subspecies israelensis. The larger size of CytB compared to CytA appears to be due to additional sequence in CytB after the CytA C-terminus. Unlike CytA, CytB was freely expressed in Escherichia coli and formed cytoplasmic inclusions without the need for a "helper" protein. Electron microscopic observation of CytB inclusions revealed them to be generally amorphous, but examples possessing some lattice structure were seen.

Amino Acid Sequence↗

PubMed2000

Molecular and insecticidal characterization of a Bacillus thuringiensis strain isolated during a natural epizootic.

A new Bacillus thuringiensis strain belonging to the serovar aizawai was isolated from a dead larva of the lepidopteran Mythimna loreyi collected in a corn crop in Spain during a natural epizootic. This strain, which was named Leapi01, was compared with the kurstaki and aizawai strains isolated from Dipel(R) and Xentari(R), by electron microscopy, SDS-PAGE, plasmid pattern, PCR and insecticidal activity. This strain showed similar morphological and biochemical characteristics to the standard strains. The content in cry genes of Leapi01 was analysed with a set of general and specific primers recognizing most of the cry genes reported to date. DNA amplification was obtained with primers corresponding to six genes and, to clearly determine the identity of the genes, the amplified fragments were sequenced and corresponded to cry1Aa, cry1Ab, cry1Ca, cry1Da, cry2Ab and cry1Ia. However, the proteins encoded by two of these genes, Cry2 and Cry1I, were not detected in the SDS-PAGE of the purified parasporal bodies. The insecticidal activity of Leapi01 was determined by bioassays against two Lepidoptera species, Helicoverpa armigera and Spodoptera littoralis, that were found to be very susceptible to Leapi01 purified crystals. Since two of the cry genes identified in Leapi01 appear to be silent, other factors may be involved in the toxicity of the strain. As a result of this study, the potential of Leapi01 as biological control agent is discussed, with special emphasis on the high toxicity and relatively broad spectrum activity compared with two B. thuringiensis strains that are the active ingredients of commercial preparations commonly used as bioinsecticides.

Animals↗

PubMed1994

DNA relatedness among Bacillus thuringiensis serovars.

The genetic relationships of Bacillus cereus and of the Bacillus thuringiensis serovars were assessed from measurements of DNA reassociation. A study of 8 to 10 strains each of 13 of the most commonly encountered serovars revealed that the levels of intragroup DNA relatedness for most serovars ranged from 90 to 100%. In contrast, B. thuringiensis serovars canadensis and kenyae consisted of two DNA relatedness groups, each of which exhibited levels of intragroup relatedness of 80% or higher and levels of intergroup relatedness of 60 to 70%. Analyses of DNA relatedness performed with all of the serovars revealed that the taxa were segregated into 11 phena differentiated from each other at about the 65% level; within each phenon the level of relatedness was 80% or higher. Three phena contained strains belonging to more than one serovar; B. thuringiensis serovars alesti and dendrolimus clustered in phenon 1, serovars aizawai, kurstaki, galleriae, and morrisoni clustered in phenon 7, and serovar darmstadiensis and some strains of serovar kenyae clustered in phenon 11. The levels of DNA relatedness between B. cereus and B. thuringiensis strains ranged between 65 and 70%. My results suggest that many of the B. thuringiensis serovars are genetically distinct but closely related.

Bacillus cereus↗

PubMed2006

Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis.

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these "near neighbors" are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.

Bacillus anthracis↗

PubMedDate not supplied

Phenotypic and genetic diversity of Bacillus thuringiensis strains isolated in India active against Spodoptera litura.

Bacillus thuringiensis strains isolated from different agroclimatic regions of India were found to harbor cry1 family genes. Of 831 strains 18 that were found to produce 130- and 68-kDa mol wt proteins in sodium dodecyl sulfate polyacry1amide gel electrophoresis were subjected to bioassay against second instar larvae of Spodoptera litura. According to the time response curve, while the highest toxic activity against S. litura was observed in PBT-782 with an LT50 of 25.46 h, strains PBT-372, PBT-574, PBT-801, and PBT-716 in descending order of merit had LT50 values of 36.81, 48.18, 50.35, and 73.53 h. The results of the field experiment testing the efficacy of different B. thuringiensis strains in controlling S. litura larvae infecting peanut plants showed that the chemical insecticide chlorpyriphos was the most effective in controlling S. litura throughout the study period. However, among B. thuringiensis strains, PBT-372 was superior. All the B. thuringiensis strains except PBT-689 were found to contain cry1Ac1-type gene. However, only nine strains contained cry1Aa1 gene. While cry1Ab1 was present only in PBT-372 and PBT-689, cry1Ca1 was present in PBT-574, PBT-688, PBT-689, and PBT-695. cry1Da1 was detected only in PBT-688 and PBT-692. None of the strains contained cry1Ba1 and cry1Ea1 genes. When polymerase chain reaction analysis using cry1Ca1 primer was performed, PBT-695 produced an unexpected 739-bp product, which showed 33% homology with cry1Ca1 gene between nucleotides 1819 and 2107. Our results indicated that among the field-collected B. thuringiensis strains, PBT-372 harbors multiple cry-type genes and could be employed for biological control of insects.

Animals↗

PubMed2000

Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield.

Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesis were achieved by using cyt1A promoters to drive expression of the 5' half of cry1C that included in the construct the 5' cry3A STAB-SD mRNA stabilizing sequence and the 3' stem-loop transcription terminator. RNA dot blot analysis showed that the STAB-SD and 3' transcriptional termination sequences were important for stabilization of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only the cyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots. The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t protein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC(50)] = 13.2 microg/ml) was lower than that of full-length Cry1C (LC(50) = 1.8 microg/ml). However, transformation of the HD1 isolate of B. thuringiensis subsp. kurstaki with the cry1C-t construct enhanced its toxicity to S. exigua as much as fourfold.

Animals↗

PubMed1994

[Studies on the ecological distribution of Bacillus thuringiensis in forest soils of China].

A total of 384 forest soils samples at 0-5cm depth collected from 8 forest site zones (13 Natural Reserves) in China were examined for the distribution of Bacillus and Bacillus thuringiensis. The 79 cultures (4.21%) in 1873 isolates of Bacillus collected referable to the Bacillus thuringiensis. Soil pH, moisture content and soil nutrient were determined. Rules of the ecological distribution of Bacillus thuringiensis of 8 forest site zones in China were analysed. Toxicities against 6 species of insect were tested. Strain some efficient serains were obtained. It was shown that resource of Bacillus thuringiensis is much fruitful in forest soils of China.

Bacillus thuringiensis↗

PubMed1990

Characterization of the parasporal inclusion of Bacillus thuringiensis subsp. kyushuensis.

Electron microscopy of Bacillus thuringiensis subsp. kyushuensis revealed that the parasporal inclusions are composed of a homogeneous center surrounded by a thick, electron-dense coating. Antibodies directed against the 135- and 65-kilodalton B. thuringiensis subsp. israelensis peptides cross-reacted with the 70- and 26-kilodalton peptides, respectively, of B. thuringiensis subsp. kyushuensis.

Animals↗

PubMed1991

Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins.

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.

Aedes↗

PubMed1991

Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensis delta-endotoxins.

To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand-blot experiments with the three activated CryIA toxins. The CryIA(a) and the CryIA(b) toxins bind the same 170-kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170-kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA(a) or CryIA(b) toxin. This indicates that the sugars are not important for the binding of these toxins. A model for a protein complex binding the B. thuringiensis HD1 ICPs is presented. Our results support the idea that binding proteins on membranes of the gut epithelial cells of H. virescens larvea are important for the specificity of the bacterial toxins.

Animals↗

PubMedDate not supplied

[Extracellular alkaline ribonuclease of Bacillus thuringiensis var. subtoxicus].

Intraspecific selection of Bacillus thuringiensis strains producing extracellular alkaline ribonucleases was carried out. Subtoxicus subspecies with increased expression of the enzyme was detected. A method was developed to isolate preparative amounts of homogeneous extracellular RNase of B. thuringiensis var. subtoxicus. The physico-chemical and catalytic properties of the enzyme was studied and compared with extracellular RNases of others Bacillus species. The conclusion about the structural and evolutional conservation of Bacillus extracellular RNases was drawn.

Amino Acid Sequence↗

PubMed1984

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer.

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.

Bacillus thuringiensis↗

PubMed2000

Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis.

Seventy-four strains of Bacillus thuringiensis thuringiensis representing 24 serovars were examined for the presence of three enterotoxin genes/operons; the non-haemolytic enterotoxin Nhe, the haemolytic enterotoxin hbl and the Bacillus cereus toxin bceT using polymerase chain reaction. The nheBC genes were found in all strains examined, the hblCD genes in 65 of the 74 strains and bceT in 63 strains. There was little consistency of the distribution of enterotoxin loci among strains of the same serovar in serovars that were well represented in our collection. Culture supernatants from all but one strain inhibited protein synthesis in Vero cells, generally with a toxicity equivalent to that seen in strains of B. cereus isolated from incidents of food poisoning. Microbiological Societies.

Animals↗

PubMed1989

Mechanism of action of Bacillus thuringiensis insecticidal delta-endotoxin: interaction with phospholipid vesicles.

Bacillus thuringiensis (Bt) crystal delta-endotoxin from three subspecies and the product of a cloned crystal protein gene were activated in vitro and their interaction with phospholipid liposomes studied. Despite their diverse spectrum of activity, all these toxins were found to cause a rapid increase in the light scattering of liposome suspensions, which reflects a morphological change in the lipid bilayer. When liposomes loaded with radioactive markers were incubated with B. thuringiensis aizawai IC1 toxin, a relatively rapid release of more than 70% of the trapped markers occurred after an initial lag. Activated Bta IC1 and B. thuringiensis israelensis toxins were shown to bind to phospholipid vesicles. Two of the five conserved domains (D1-D5) detectable in the sequence of a range of Bt toxins are predicted to be highly hydrophobic. It is suggested that these, together with an additional conserved hydrophobic region showing structural homology and two predicted amphiphilic helices, play a major part in the interaction of these toxins with target membranes.

Bacillus thuringiensis↗