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PubMed2000

Control of aedes aegypti breeding in desert coolers and tires by use of Bacillus thuringiensis var. Israelensis formulation.

Three different formulations of Bacillus thuringiensis var. israelensis (Bti) were evaluated for their efficacy against immature Aedes aegypti in desert coolers and tires. Three formulations, viz., VectoBac tablets, VectoBac granules, and Bacticide powder, at the application rate of 0.75, 2, and 1 g per cooler, respectively, and VectoBac tablets at 0.75 and 0.375 g per tire, were evaluated. In coolers and tires, 100% reduction in the abundance of late larval instars of Ae. aegypti was observed for a period of 2 and 3 wk, respectively. The possibility of using tablets and capsules filled with Bti granules and powder formulation by individuals or communities for control of Ae. aegypti breeding has been discussed in view of the increasing outbreaks of dengue and dengue hemorrhagic fever in India. Use of these formulations over conventional methods is better and more user-friendly.

Aedes↗
PubMed2003

Characterization of flagellar antigens and insecticidal activities of Bacillus thuringiensis populations in animal feces.

In total, 287 Bacillus thuringiensis isolates, recovered from feces of 28 zoo-maintained animal species, were examined for flagellar (H) antigenicity and insecticidal activity. Serologically, 209 isolates (72.8%) were allocated to the 8 H serogroups, 4 were untypable, and 74 were untestable. Among the 8 H serotypes detected, H3abc (serovar kurstaki) predominated at a high frequency of 88.0%, followed by H6 (serovar entomocidus) with a frequency of 7.7%. Insecticidal activity was associated with 67.2% of the fecal populations: 188 isolates were toxic to both Bombyx mori (Lepidoptera: Bombycidae) and Aedes aegypti (Diptera: Culicidae), 2 isolates were specific for B. mori, and 3 isolates were toxic to A. aegypti only. Of the isolates with dual toxicity, 97.9% belonged to the serovar kurstaki, producing bipyramidal parasporal inclusions. All of the H7 (serovar aizawai) isolates were toxic to both insects.

Animals↗
PubMed2006

Discovery of a significant optical chromatographic difference between spores of Bacillus anthracis and its close relative, Bacillus thuringiensis.

A significant difference between two closely related Bacillus spores has been discovered using optical chromatography. This difference can be harnessed for the separation of microscopic particles using opposing laser and fluid flow forces. Particles of different size, composition, and shape experience different optical and fluid forces and come to rest at unique equilibrium positions where the two forces balance. Separations in excess of 600 mum have been observed between Bacillus anthracis Sterne strain and its genetic relative, Bacillus thuringiensis. These findings open new possibilities for detection and characterization of the biological warfare agent, B. anthracis, the causative agent of anthrax, the deadly mammalian disease. The large optical separation between these species is surprising given their close genetic relationship but may be explained by differences in their shape and exosporium morphology, which may result in differences in fluid drag force. The observation of large differences due to less common variables indicates the complex nature of the force balance in optical chromatography, which may in the future be used to separate and characterize microbiological samples. In general, the discovery of such large differences between such closely related biological species suggests new possibilities for the separation and characterization of microorganisms using the full range of emerging techniques that employ radiation pressure (optical filtering, laser tweezers, optical chromatography, etc.).

Bacillus anthracis↗
PubMed2006

Identification and characterization of a polysaccharide deacetylase gene from Bacillus thuringiensis.

One polysaccharide deacetylase gene was cloned from Bacillus thuringiensis and designated pdaA. Disruption of pdaA did not affect vegetative growth and sporulation but obviously affected spore germination. When L-alanine was added into the spore suspension, the spores of the pdaA disruption mutant showed a slow and partial reduction in absorbance at OD600 and became phase pale gray compared with phase dark of the wild-type strain. In contrast with the outgrowing of wild-type spores after germination, the pdaA mutant spores were blocked at the stage of spore germination. Transmission electron micrographs revealed a significant difference between the pdaA mutant and the wild-type strain in the spore cortex. Introduction of the pdaA gene into the pdaA disruption mutant complemented the germination-negative phenotype. Reverse transcription--polymerase chain reaction showed that pdaA was transcribed after incubation for 10 h in CCY medium.

Amidohydrolases↗
PubMed2005

Cost-effective production of Bacillus thuringiensis by solid-state fermentation.

Production of Bacillus thuringiensis (Bt) was standardized on wheat bran based media in 250 ml Erlenmeyer flasks. Scale-up of Bt production on the best medium in plastic tubs with aeration at 8 h intervals starting 16 h after incubation yielded a significant increase in spore count and toxin content of the product. Maximum lysis of Bt cells was obtained by 60 h of incubation at 30 degrees C. This protocol was suitable for production of Bt strains and local isolates. The Bt produced proved highly effective at 0.1% concentration against larvae of castor semilooper, Achaea janata L, resulting in complete mortality by three days in laboratory bioassays. In field trials, the population of castor semilooper larvae on the castor bean crop was reduced significantly by three days after application. The cost for material production of 1 kg of Bt was approximately US dollars 0.70.

Animals↗
PubMedDate not supplied

Exploration of receptor binding of Bacillus thuringiensis toxins.

Wild type and mutant toxins of Bacillus thuringiensis delta-endotoxins were examined for their binding to midgut brush border membrane vesicles (BBMV). CryIAa, CryIAb, and CryIAc were examined for their binding to Gypsy moth (Lymantria dispar) BBMV. The binding of CryIAa and CryIAc was directly correlated with their toxicity, while CryIAb was observed to have lower binding than expected from its toxicity. The latter observation confirms the observation of Wolfersberger (1990). The "rule" of reciprocity of binding and toxicity is apparently obeyed by CryIAa and CryIAc, but broken by CryIAb on L. dispar. Alanine substitutions were made in several positions of the putative loops of CryIAa to test the hypothesis that the loops are intimately involved in binding to the receptor. The mutant toxins showed minor shifts in heterologous binding to Bombyx mori BBMV, but not enough to conclude that the residues chosen play critical roles in receptor binding.

Amino Acid Sequence↗
PubMed2005

Starch industry wastewater-based stable Bacillus thuringiensis liquid formulations.

Liquid formulations were developed from Bacillus thuringiensis (Bt)-fermented broths of starch industry wastewater (SIW) and of soya medium. Stability studies were carried out for 1 yr. Storage stability was tested by studying various physical and chemical (e.g., viscosity, particle size, corrosion, and suspendibility) and biological (e.g., microbial contamination, viable spores, and entomotoxicity) parameters at different pH levels and temperatures. Three suspending agents, sorbitol, sodium monophosphate, and sodium metabisulfite, were added to fermented broth in different concentrations. Sorbitol and sodium monophosphate in the ratio 3:1 was the best suspending agent combination for both formulations. Starch industry wastewater fermentation yielded cell and viable spore counts 10- and 4-fold greater than those from soya medium, respectively, and a 1.7-fold increase in entomotoxicity. However, both formulations started deteriorating at pH 6 and 6.5 and 40 and 50 degrees C. There were no signs of corrosion and microbial contamination in both types of formulations.

Animals↗
PubMed1990

Directed mutagenesis of selected regions of a Bacillus thuringiensis entomocidal protein.

Comparison of the sequences of Bacillus thuringiensis entomocidal toxins of widely differing specificity reveals six conserved domains. The role of one of the most highly conserved domains (D1) located near the N-terminus has been investigated by site directed mutagenesis at two positions. Although preliminary results indicate that the capacity of the mutants to bind to putative receptors on the plasma membrane of susceptible cells was unaffected, toxicity in vivo was reduced by 70-80%. The role of the highly hydrophobic segment exposed at the N-terminus of the toxin after proteolytic activation was investigated by substituting two aspartate residues for phenylalanine and valine located adjacent to each other in the centre of this segment. The toxicity of the resulting mutant protein was only 40% of the unmutated toxin but again preliminary results suggest that binding to putative receptors was unaffected. These results suggest that regions close to the N-terminus of this and similar toxins may play an important role in the membrane insertion event which is believed to follow receptor recognition.

Animals↗
PubMed2002

Cloning and expression of the vegetative insecticidal protein (vip3V) gene of Bacillus thuringiensis in Escherichia coli.

A genomic library of Bacillus thuringiensis var. kurstaki (B.t.k.) was constructed and a positive clone harboring the full-length gene encoding a novel vegetative insecticidal protein (Vip3V) was characterized. The vip3V gene was subcloned into pET-22b(+) vector and overexpressed in Escherichia coli to an extent of about 30% of the total protein. While transcription was influenced by the T7 promoter of the vector, synthesis of Vip3V in E. coli host occurred from the B.t.k. ribosomal binding site (rbs) found 917bp downstream of the insert and not from the E. coli rbs of the vector. The expressed Vip3V protein was found in the soluble and periplasmic fractions as well as in the inclusion bodies. A simplified anion-exchange chromatographic method for the purification of Vip3V using step gradient or one-step elution was developed. The purified protein showed broad-spectrum activity against some of the lepidopteran larvae tested and did not show any activity against the larvae of silkworm (Bombyx mori) and mosquito (Culex quinquefasciatus).

Amino Acid Sequence↗
PubMed1990

Bacillus thuringiensis insecticidal delta-endotoxin: diversity of crystal proteins and its relatedness to the toxicity spectrum.

Bacillus thuringiensis strains produce crystal delta-endotoxins which exhibit a diverse toxicity spectrum. In order to explore the basis of toxin specificity, a comparison of the activity of 13 strains belonging to seven serotypes was made against three insect species. The delta-endotoxin crystals were purified and their polypeptide composition analyzed by SDS-PAGE. Among the strains studied, the delta-endotoxins consist of a variety of crystal proteins in the 60-144 KDa size range. On the basis of molecular mass, endotoxins maybe grouped into two classes; one contained both high (125-144 KDa, P1) and medium sized (60-66 KDa, P2) proteins and a second class consisting of only the high Mr polypeptides. Immunoblotting with B. aizawai P1 antiserum revealed antigenic cross-reaction with one or more of the polypeptides in 125-144 KDa range in all the strains studied. When the crystal proteins from different strains were immunoblotted with kurstaki P2 antiserum, none of the P1 protein crossreacted suggesting that the P1 and P2 proteins are not structurally related. However, the B. kurstaki P2 antiserum crossreacted with 66 KDa proteins in some other strains which underlines a structural homology in this class of the toxic polypeptides. Toxicity studies revealed that the high Mr P1 proteins of all the strains in this study were active against lepidopteran (Pieris brassicae and Diacrisia obliqua) larvae. B. thuringiensis aizawai strains exhibit a dual toxicity associated with the high Mr (130-135 KDa; P1) proteins. The P2 crystal proteins (60-66 KDa) also showed dual toxicity against the lepidopteran and dipteran larvae but were found to be structurally and immunologically distinct.

Animals↗
PubMed1995

Promotion of cytotoxic T-cell generation in mixed leukocyte culture by phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis.

Phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis, which cleaves phosphatidylinositol or glycosylphosphatidylinositol on the external cell surface to generate a second messenger for intracellular signal transduction (S. Rahman et al., FEBS Lett. 303:193-196, 1992), was found to preferentially promote the generation of alloantigen-specific cytotoxic T lymphocytes in mixed leukocyte culture. PIPLC affected an early stage of cytotoxic T-lymphocyte activation in culture, and there was no evidence of any soluble cellular mediators of this PIPLC action. PIPLC neither enhanced overall cell proliferation nor noticeably promoted interleukin-2 and -4 production in mixed leukocyte culture. The relative population size of Ly-2+ T cells was increased, however, in a late mixed leukocyte culture with PIPLC. In addition, PIPLC enhanced an anti-CD3 monoclonal antibody-induced early increase in [Ca2+]i. These results suggest a new parasite (bacterium)-oriented mechanism for enhancing antigen-driven host cytotoxic T-lymphocyte immunity which does not include promotion of interleukin-2 production.

Animals↗
PubMed2001

Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil.

AIMS: Detection and identification of new antagonistic activities towards Bacillus cereus and relatives. METHODS AND RESULTS: Twenty Bacillus thuringiensis strains were screened for their capacity to express bacteriocin-like agents. Strain BMG1.7, isolated from soil, showed an antagonistic activity called thuricin 7. Thuricin 7 was active against several species of the genus Bacillus, including three of the four known B. thuringiensis/B. cereus bacteriocin producers, as well as against Streptococcus pyogenes and Listeria monocytogenes strains. Antimicrobial activity was lost after treatment with proteinase K. The active protein had an apparent molecular weight of 11.6 kDa, and was secreted at the end of the exponential growth phase. Thuricin 7 retained 55% of the activity after incubation at 98 degrees C for 30 min. The mode of action of thuricin 7 was shown to be bactericidal and bacteriolytic. CONCLUSION: Thuricin 7 is a novel bacteriocin produced by a newly isolated Bacillus thuringiensis strain BMG1.7. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of thuricin 7 indicate that it is a new bacteriocin which may have interesting biotechnological applications due to its relatively large activity spectrum.

Bacillus cereus↗
PubMed2005

Isolation and toxicity of Bacillus thuringiensis from potato-growing areas in Bolivia.

Bacillus thuringiensis was isolated from 116 samples collected in high altitude potato-growing areas in Bolivia. In these regions, main potato pests are the potato tuberworm Phthorimaea operculella, and the Andean weevils Premnotrypes latithorax and Rhigopsidius tucumanus. B. thuringiensis was found in 60% of the samples. The main percentage of samples with B. thuringiensis was found in larvae of R. tucumanus (78%). Bioassays were performed with 112 isolates. None resulted toxic to either larvae or adults of the two Andean weevils. However, 18 isolates from this study showed more toxicity against the beet armyworm Spodoptera exigua than the standard strain var. kurstaki isolated from DELFIN. Among these isolates, three were also effective against P. operculella, conferring better or equal protection to the tubers than the reference strain HD-1 isolated from DIPEL. The most toxic strains against S. exigua and P. operculella were characterized in terms of serotyping, crystal morphology, protein profile, and cry gene content. PCR was performed with primers amplifying genes from the cry1, cry2, cry3, cry4, cry7, 8, and cry9Aa families. The toxic strains presented bipyramidal crystals, at least a band of 130kDa in SDS-PAGE, and showed an amplification product with cry1 family primers. One of the isolates did not amplify with any specific primer belonging to known cry1 genes. Restriction Fragment Length Polymorphism (RFLP) confirmed the presence of a novel gene and sequence comparison showed that this gene had homology to cry1G.

Animals↗
PubMed2005

Cloning and characterization of two novel genes, cry24B and s1orf2, from a mosquitocidal strain of Bacillus thuringiensis serovar sotto.

Two new crystal protein genes, cry24B and s1orf2, were cloned from a mosquitocidal Bacillus thuringiensis serovar sotto strain. The cry24B and s1orf2 genes encoded a 76-kDa and 62-kDa protein, respectively. The Cry24B protein retained five conserved regions commonly found in the existing Cry proteins. The amino acid sequence of the S1ORF2 had a high homology to that of the ORF2 protein of B. thuringiensis serovar jegathesan. Southern hybridization experiments with a cry24B gene-specific probe revealed that these genes are located on two large plasmids of > 100 kb. When the two genes, cry24B and s1orf2, were expressed in an acrystalliferous B. thuringiensis host, the proteins were synthesized and accumulated as inclusions. These inclusions exhibited no larvicidal activities against three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens molestus. Likewise, the inclusions contained no cytocidal activity against HeLa cells.

Animals↗
PubMed1983

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis.

Three crystalliferous (Cry+) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry-) mutants, either induced or spontaneously derived from a single Cry+ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry+ and Cry- variants were analyzed by both a cleared lysate- and a modified Eckhardt lysate-electrophoresis technique. All of the Cry- mutants derived from the Cry+ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry- variants. All three Cry+ strains, including the parent of the Cry- strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry+ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry- derivatives differed in other respects from the pattern for their parent strain. The various Cry+ and Cry- strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.

Bacillus thuringiensis↗
PubMed1987

Alteration of Trichostrongylus colubriformis egg permeability by Bacillus thuringiensis israelensis toxin.

A toxin from the bacterium Bacillus thuringiensis israelensis is lethal to nematode eggs. Exposure of eggs of the ruminant nematode Trichostrongylus colubriformis to the toxin significantly increased the eggs' permeability to radiolabeled phenylalanine within 2 hr. Calcium chloride inhibited the toxin-induced change in egg permeability. Iodine staining of eggs that were exposed to the microbial toxin revealed that egg permeability was altered within 5 min and was dependent on the dose of toxin. Addition of 34 mM sucrose, 17 mM sodium chloride, or 17 mM potassium chloride to the eggs' medium increased the toxin's lethality. Exopeptidase activity in eggs of T. colubriformis was reduced significantly after exposure to the B. t. israelensis toxin. Tetrodotoxin, tetraethylammonium chloride, ouabain, 4-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid (SITS), 4,4'-diisothiocyano-2,2'disulfonic acid stilbene (DIDS), valinomycin, and sodium vanadate, which affect membrane transport, had no significant effect on the activity of B. t. israelensis toxin for eggs. Likewise, a series of nucleotides and their derivatives had no effect on the toxin's activity. Ovicidal activity of the microbial toxin was increased by 4-aminopyridine (4.4 X), but was decreased by furosemide (97 X), nigericin (263 X), or monensin (125 X). Microscopic measurement of T. colubriformis eggs after treatment with the microbial toxin revealed no significant size change.

Animals↗
PubMed1989

Characterization of the mammalian toxicity of the crystal polypeptides of Bacillus thuringiensis subsp. israelensis.

Solubilized crystal polypeptide preparations of Bacillus thuringiensis subsp. israelensis (BTI) were fractionated by immunoaffinity chromatography using a bound monoclonal antibody formed against the 28K crystal polypeptide. The 28K polypeptide was confirmed to be hemolytic and to possess low mosquitocidal activity against Aedes aegypti larvae. By comparison, the 28K polypeptide was more potent than the solubilized BTI crystals in male Swiss Webster mice, as the LD50 values were (p less than 0.05) 0.77 and 2.33 mg protein/kg body wt, respectively. Acute administration of the 28K polypeptide (mg/kg, ip) produced severe hypothermia and bradycardia in the mouse. No evidence for cooperativity between the 28K and other crystal polypeptides was observed. Preliminary histological examination of the mouse hearts exposed to the 28K polypeptide did not reveal any specific lesion, suggesting that the deficient cardiac performance might be a secondary physiological response. Gross pathological examination of mice as well as Sprague-Dawley rats acutely treated with equivalent doses of solubilized BTI crystal preparations revealed focal to segmental reddened and edematous areas within the small intestine. Histopathology indicated that the major lesion was in the jejunum. Contrary to expectations from in vitro hemolysis assays, cytolysis of mouse red and white blood cells was not detectable after in vivo exposure to the BTI solubilized proteins. The present results indicate that the 28K polypeptide is the mammalian toxic component of BTI crystals.

Aedes↗
PubMed1985

[The activity of Bacillus thuringiensis var. israelensis against black fly larvae under field conditions in South Bohemia].

The effectiveness of Bacillus thuringiensis var. israelensis spore suspension against black fly larvae was tested in two regulated brooks in the Ceské Budĕjovice district, South Bohemia. The sites under study were small, regulated, paved brooks of trapezoidal profile, with vegetation of different density, and with stream flow rates ranging from 40 to 90 cm/sec. Lyophilized spores (produced by Roger Belon) at a final concentration of 1 X 10(5) spores per ml were used. The spore efficacy was determined in both natural and artificial substrates in the field and under laboratory conditions, on the larvae collected at the control sites. A 90-100% mortality of the larvae was recorded in all parts of the brooks examined both in the natural samples studied in laboratory and in those kept on artificial substrates. In the area of fading spore suspension action, a higher percentage of the last-instar larvae was found in larval population which may be ascribed, in agreement with other authors, to an increased susceptibility of younger larvae to the preparation. The results of the tests demonstrated that Bacillus thuringiensis var. israelensis is an effective agent to control the black fly species occurring in Central Europe and may be of value mainly in the control of some species of veterinary importance overpopulated in regulated brooks.

Animals↗