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... Colonies with morphological features that are peculiar to the Bacillus spp. group (whitish, not bright, dry, rough, surface, and irregular border) were preselected and examined by means of a phase-contrast microscope (Leitz, Diaplan, Germany). Staining of the preselected colonies was performed using Coomassie Brilliant Blue G-250 solution [17,30]. Colonies containing bacillary forms producing spores were selected as Bacillus spp. ...
... Protein extraction was based on separation in biphasic system [30]. Crystal purity was highlighted after staining with Coomassie Brilliant Blue solution under photonique microscope. ...
Article
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Parasporins (PS), a class of non-insecticidal and non-hemolytic crystal proteins of Bacillus thuringiensis (Bt), are being explored as promising anti-cancer agents due to their specific toxicity to cancer cells. This work is considered as a first initiative aiming at investigating Algerian soil Bt isolates’ activity and cytotoxic potential against cancer cells. A total of 48 Bacillus spp. were isolated from different sites in Algeria. Phenotypic and biochemical tests, 16S rDNA molecular identification, and microscopic observation of crystal have confirmed the identification of Bt for ten strains. A screening for non-hemolytic crystalline proteins was performed. Extraction, purification, and activation of non-hemolytic proteins by chromatographic analysis yielded several polypeptides of different molecular weights. A purified PS1, with pro-protein of 81 kDa and several peptides with different molecular weights (18–58 kDa) after activation by trypsin, has been identified from the strain BDzG. The NH2-terminal sequence deciphered in BLAST analysis showed homology to a Bt PS1 protein. Moreover, the screening of parasporin-1 (PS1) gene has also been performed. Cytocidal activity against human epithelial type 2 (HEp2) cells, considered to originate from a human laryngeal carcinoma, was observed with an IC50 equal to 2.33 μg/ml, while moderate cytotoxicity against adenocarcinomic human alveolar basal epithelial (A549) cells has been shown with IC50 equal to 18.54 μg/ml. No cytotoxicity against normal cells was noted. Fluorescence microscopy revealed a condensed or fragmented chromatin indicating the apoptotic death of HEp2 cells. Thus, Bt PS-producer isolated from Algerian soil might have a potential to join the arsenal of natural anti-cancer drugs with high therapeutic potential.
... Raised bilirubin is destructive to the body, and it is anything but a sign of a few genuine ailments. The bilirubin content have been measured by Diazoreagent (5,6,7,8,9). As a result, it was observed that bilirubin levels significantly decreased in the treated rats. ...
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ARTICLE INFO ABSTRACT The Cry proteins produced by Bacillus thuringiens is are viewed as profoundly explicit insecticidal proteins. Decided to be of no risk for mankind and livestock because of their particular poisonousness, the proteins have been used as an organic pesticide and brought into hereditarily adjusted plants. The immediate impacts of enacted Cry proteins on mammalian cells have not yet been completely affirmed. Therefore, in this study we employed primary tissue homogenates for biochemical analysis preferences in several spheres like enzyme activities for oxidative stress, total protein concentration, alkaline phosphatase activity, serum glutamic oxaloacetate and pyruvate concentrations etc. There were no such significant differences observed between the weights of the organs of the treated and control rats, though the increase in total protein concentration and catalase activity indicates onset of oxidative stress and increase in Acid phosphatase levels in spleen and stomach comprises ceased enzyme activities for prostate.
... Total proteins were isolated using detergent-free methods as described elsewhere [53]. Spore samples were pelleted and washed with 1 × PBS. ...
Article
Bacillus thuringiensis (Bt) is used as a bioinsecticide since it effectively kills insect larvae. Bt is also genetically similar to Bacillus cereus (Bc), a well recognized foodborne human pathogen; they are both members of the Bacillus cereus group (BC group). Although approved Bt bioinsecticide products have been confirmed to be non-pathogenic to humans, close monitoring of Bt during dissemination is important for cost considerations and to limit impact on biodiversity towards nontarget organisms. As such, developing rapid, sensitive, and specific tools for quantitative detection of Bt spores during and following spray operations is highly desirable. The goals of this study were to investigate commercially available detection reagents for sensitivity and selectivity in detecting Bt spores, and then functionalize a surface of (001) GaAs used in photonic biosensing. To achieve these goals, we (1) screened commercial antibodies for their capacity to bind recombinant proteins from Bt spores, (2) screened antibodies and aptamers for their sensitivity and selectivity against Bt spores, and (3) tested the efficiency of selected antibodies and aptamers in capturing Bt spores on the surface of functionalized GaAs biochips. Seven genes encoding Bt spore proteins were cloned and expressed in Escherichia coli. The binding of each purified spore antigen was tested by commercially available polyclonal and monoclonal antibodies claimed to exclusively target spores. Of the seven targets, Bacillus collagen-like protein A, was the most abundant protein on Bt spores and demonstrated the strongest binding affinity to all test antibodies. The commercial antibodies (Abs) were also tested for specificity to BC Group versus non-BC Group spores. Three of six commercial antibodies showed selectivity to Bt spores, with recombinant Abs providing the most robust lower range of detection (10² to 6×10³ spores/mL). The sensitivity and selectivity of three published DNA aptamer sequences demonstrated a wide range of detection sensitivity for Bt spores. Two of the three test aptamers also showed reasonable selectivity towards Bt spores while the third demonstrated reactivity to non-BC Group B. megaterium and B. subtilis. Of the reagents tested, a thiolated aptamer and llama recombinant Ab showed highest Bt spore capture efficiency as measured by spore coverage of the GaAs surface. These results confirm that the selected aptamer and llama rAb can be considered strong candidates for the development of GaAs-based biosensing devices.
... This procedure was repeated four times. Afterward, the pellet was washed by adding 5 mL of sterile water in order to remove cellular debris (Mounsef, J. R., et al., 2014). Finally, the pellet was re-suspended in 1 mL of sterile water and the concentrations of the purified delta-endotoxins were determined according to the method of Bradford ). ...
Thesis
Les études de la diversité et de la richesse bactérienne des sols constituent un enjeu primordial pour développer des bio pesticides efficaces en lutte biologique contre les diptères phytoravageurs et vecteurs de maladie (malaria, dengue…). Dans ce manuscrit, la caractérisation de la diversité bactérienne des sols de chênes et de pins Libanais a été étudiée par comparaison aux mêmes sols d’Autriche, une zone géographique différente et de climat différent. La composition en classes et genres du phylum des Firmicutes et son abondance dans les différents sols étudiés ont été particulièrement caractérisées par des indices de diversité (alpha, bêta, OTUs…) et des méthodes moléculaires (SSCP et qRT-PCR). A partir des sols de pins et de chênes Libanais et précisément à partir de ce phylum, des souches de Bacillusthuringiensis ont été criblées pour leur activité insecticides contre les diptères,Culex pipiens, Aedes albopictus et Anopheles gambiae. Deux souches de Bt ont été sélectionnées. La souche A23 possède toxicité in vivo plus élevée que la souche de référence Bacillus thuringiensis isarelensis (Bti). Cette souche n’a cependant pas révélé de différences génétiques et moléculaires par rapport àBti. La souche H3, quant à elle, a été étudiée étant non cytolytique et présentant une activité insecticide in vivo contre les diptères mais à fortes concentrations par rapport à Bti. Cette souche a révélé un contenu génique et protéique différent de Bti. H3 a probablement un nouveau variant du gène cry4B et du gène cry40connu pour avoir une activité diptéricide. Des investigations supplémentaires sont nécessaires afin de confirmer ces résultats et de comprendre les mécanismes de toxicité de ces deux souches.
... After 48 h, the crystals and spores of the different strains of B. thuringiensis were separated according to a protocol developed by Monsef et al. [34]. ...
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In this paper, we report a morphological study of the crystals and spores of different shapes synthesized by seven different strains of Bacillus thuringiensis. Crystals and spores were separated after 48 h of culture on T3 agar medium and imaged under a scanning electron microscope (SEM). Sizes of the crystals and spores were determined using Image J software. The results showed that crystal and spore sizes were normally distributed. In addition, the volumes and aspect ratios of the crystals and spores were calculated. The statistical analysis of the data showed the variability of the size distribution and morphological data of the crystals produced by the analyzed strains. Furthermore, variations in spore size and shape within the same serovar were observed, indicating that, perhaps, there are still some unexplored differences between strains of this serovar, making them less identical than what was believed so far.
... Whole-crystal protein identification. In order to identify the proteins composing the H3 crystal, H3 spores and crystals were first separated using a hexane-based method as described by Rahbani Mounsef et al. (79). Hexane treatment was repeated until a 1:10 spore/crystal ratio was obtained. ...
Article
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Bacillus thuringiensis has emerged as a major bioinsecticide on the global market. It offers a valuable alternative to chemical products classically utilized to control pest insects. Despite the efficiency of several strains and products available on the market, the scientific community is always on the lookout for novel toxins that can replace or supplement the existing products. In this study, H3, a novel B. thuringiensis strain showing mosquitocidal activity, was isolated from Lebanese soil and characterized in vivo at genomic and proteomic levels. H3 parasporal crystal is toxic on its own but displays an unusual killing profile with a higher 50% lethal concentration (LC50) than the reference B. thuringiensis serovar israelensis crystal proteins. In addition, H3 has a different toxicity order: it is more toxic to Aedes albopictus and Anopheles gambiae than to Culex pipiens. Whole-genome sequencing and crystal analysis revealed that H3 can produce 11 novel Cry proteins, 8 of which are assembled in genes with an orf1-gap-orf2 organization, where orf2 is a potential Cry4-type crystallization domain. Moreover, pH3-180, the toxin-carrying plasmid, holds a wide repertoire of mobile genetic elements that amount to ca. 22% of its size., including novel insertion sequences and class II transposable elements. Two other large plasmids present in H3 carry geneticdeterminants for the production of many interesting molecules—such as chitinase, cellulase, and bacitracin—that may add up to H3 bioactive properties. This paper therefore reports a novel mosquitocidal Bacillus thuringiensis strain with unusual Cry toxin genes in a rich mobile DNA environment © 2021 Fayad et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license.
... Spore-crystal mixtures were obtained according to the protocol described previously 41 . For SDS-PAGE analysis, the crystals were purified using hexane and low speed centrifugation according to the previously described method 42 www.nature.com/scientificreports www.nature.com/scientificreports/ ...
Article
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Bacillus thuringiensis serovar israelensis (Bti) is used to control insect vectors of human and animal diseases. in the present study, the toxicity of four strains of Bti, named T0124, T0131, T0137, and T0139, toward Aedes aegypti and Culex quinquefasciatus larvae was analyzed. The T0131 strain showed the highest larvicidal activity against A. aegypti (Lc 50 = 0.015 µg/ml) and C. quinquefasciatus larvae (Lc 50 = 0.035 µg/ml) when compared to the other strains. Furthermore, the genomic sequences of the four strains were obtained and compared. These Bti strains had chromosomes sizes of approximately 5.4 Mb with GC contents of ~35% and 5472-5477 putative coding regions. Three small plasmids (5.4, 6.8, and 7.6 kb) and three large plasmids (127, 235, and 359 kb) were found in the extrachromosomal content of all four strains. the Snp-based phylogeny revealed close relationship among isolates from this study and other Bti isolates, and SNPs analysis of the plasmids 127 kb did not reveal any mutations in δ-endotoxins genes. This newly acquired sequence data for these Bti strains may be useful in the search for novel insecticidal toxins to improve existing ones or develop new strategies for the biological control of important insect vectors of human and animal diseases.
... The obtained pellet was resuspended in a saline solution, the organic solvent was added again, and the same procedure was repeated 3 times. Finally, the pellet was washed twice with cold distilled water by centrifuge at 6000 RPM for 10 min [23]. ...
Article
Objective: The objective of the study was to isolate and identify Bacillus thuringiensis (Bt) from Bt cotton rhizosphere soil and analyze its larvicidal activity against Anopheles mosquito larvae.Methods: Soil samples were collected from Bt cotton field, and B. thuringiensis was isolated and characterized by biochemical and microscopical characterization. Crystal (Cry) proteins were extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and its larvicidal activity was checked by mortality analysis. Effective Bt isolates were identified by 16S rRNA sequencing.Results: A total of 24 isolates were characterized as Bacillus sp. by biochemical characterization. Further, parasporal inclusion and Cry proteins were extracted, and it was quantified by Bradford assay. The precipitated Cry proteins were analyzed by SDS-PAGE and the results have indicates Cry I protein at 100kDa, Cry3 protein at 44kDa, and cytolytic protein (Cyt) at 29kDa in most of the isolates. Larvicidal activity was checked by the 4th instar Anopheles mosquito larvae. Among the tested isolates, RBL10 and RBL20 have shown the highest percentage of larvicidal activity at 96 and 83%, respectively. Further, the two isolates RBL10 and RBL20 were identified as Bt by 16S rRNA sequencing.Conclusion: B. thuringiensis producing Cry and Cyt proteins posses potential larvicidal activity against Anopheles mosquito larvae which has biological and economic importance in mosquito control.
... For proteolytic activation , the solubilized protoxins were digested with 1:50 trypsin (Sigma) (trypsin/protoxin) at 37 @BULLET C for 2 h. In the case of Cry2Ab, crystals inclusions were purified using a hexane and centrifugation method as described [13]. Briefly, the spore/crystal pellet was suspended in 300 mM NaCl, 10 mM EDTA, 1 mM PMSF, 10% hexane solution and sonicated 1 min three times with 5 min repose in ice between each pulse of sonication. ...
Article
Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC-MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.
... Three strains identified as Bt, registered as D10 MKA , H3 MKA , and G6 MKA [23] and isolated from the Lebanese soil, are selected . One of them produces large spherical parasporal crystals with a diameter on the order of 1 μm while the others produce smaller spherical crystals. ...
Article
In this paper, we report experimental results where laser speckle grain size and degree of light polarization are used to distinguish different sizes of Bacillus thuringiensis spherical crystals and different concentration within fermentation products. Three strains of Bacillus thuringiensis, isolated from the Lebanese soil, are selected. After fermentation, crystals and spores are separated using a technique based on flotation in order to obtain large quantities of relatively pure crystals. Crystals are then embedded in an agarose matrix gel and illuminated by a laser. Speckle patterns are recorded and analyzed. Results show that when crystals concentrations increase in the sample, the values of light linear polarization degree and speckle grain size decrease, and the negative values of light circular polarization degree increase. A transition from a Rayleigh scattering regime to a Mie regime is also observed. Furthermore, when the diameter of spherical crystal increases, the normalized value of light linear polarization degree and the grain size diminish drastically for a given concentration of crystals in the matrix, whereas the normalized value of light circular polarization degree increases. This optical characterization constitutes an unambiguous signature of spherical crystals produced by fermentations of different Bacillus thuringiensis strains and shows that speckle may potentially be used at an industrial scale as a low-cost and non-invasive technique in order to characterize crystals geometry and to evaluate the yield of crystal production within fermentation.
Article
Mosquitoes need to be eradicated as they can spread deadly diseases. Cry toxic proteins from Bacillus and zinc oxide nanoparticles also can tremendously control pest and bacterial pathogens. With this reference, the Ac-ZnO NPs was effectively synthesized using Acorus calamus rhizomes extract where after incorporated with bacterial cry toxic protein (Btp) to produce Btp-Ac-ZnO nanocomposites. The XRD and FTIR, disclose the crystalline form with an average size of 17.47 nm and the possible biomolecules of Btp-Ac-ZnO NCs. SEM and TEM make known the well agglomerated and cone shape of Btp-Ac-ZnO NCs. The NCs show concentration-dependent antioxidant activity. Btp-Ac-ZnO NCs drastically arrest the formation of biofilm by the pathogenic bacteria such E. faecalis, S. aureus, P. aeruginosa, and P. vulgaris at 100 μg/mL. All the above, the Btp-Ac-ZnO NCs exhibits superior larvicidal activity against three mosquito vectors namely Ae. aegypti, An. stephensi and Cx. quinquefasciatus with LC50 values of 43.76, 39.60 and 37.13 μg/mL respectively. Besides, the biological enzymes are significantly reduced in the treated larvae than that of untreated one, which indicates the effect of Btp-Ac-ZnO NCs. Since, the Btp-Ac-ZnO NCs could be utilized against the pathogenic bacteria, and its biofilm structure, and also in the vector control sectors.
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Vip3Aa was first identified as a protein secreted during the vegetative growth phase of Bacillus thuringiensis (Bt) bacteria and which shows high insecticidal toxicity against lepidopteran insect pests (Estruch et al., 1996). Bt strains formulated as bio-insecticides only had low amounts of Vip3Aa secreted to the medium. Here, we report that Vip3Aa proteins produced by three different Bt strains, including an industrial strain, were indeed not secreted to the culture solution when grown in sporulation medium, but were retained in the mother cell compartment. In order to further investigate the Vip3Aa secretion and location, we grew the strains in rich medium. We found that in rich medium, a fraction of Vip3Aa was secreted, suggesting that Vip3Aa secretion is nutrient-dependent. Regardless of the growth conditions, we found that Vip3Aa retained in cell pellets exhibited high toxicity against Spodoptera frugiperda larvae. Hence, we speculate that the accumulation of Vip3Aa protein in the mother cell compartment under sporulation conditions could still be used as an efficient strategy for industrial production in commercial Bt strains.
Chapter
To purify the crystal protein, the protein is required in solution and it is possible by breaking the cells or tissues of the protein. There are various methods available for the purpose of purification and organic solvent extraction is one of them. The choice of the method employed depends on the sturdy of the cells and on its protein fragility. Through this method of organic solvent extraction, protein found in the solvent is the soluble protein which can be then separated from its DNA and cell membrane through the process of centrifugation.
Book
This manual details the techniques involved in the study of plant microbe interactions (PMI). Covering a wide range of basic and advanced techniques associated with research on biological nitrogen fixation, microbe-mediated plant nutrient use efficiency, the biological control of plant diseases and pests such as nematodes, it will appeal to postgraduate students, research scholars and postdoctoral fellows, as well as teachers from various fields, including pathology, entomology and agronomy. It consists of five broad sections featuring different units. Information panels at the beginning of each unit present essential knowledge as well as advances in a particular topic. The manual can also serve as a textbook for undergraduate courses like Techniques for Plant-Microbe Interactions; Biological Control of Plant Diseases; and Nutrient Use Efficiency. Providing basic insights and working protocols from all related disciplines, this unique laboratory manual is a valuable resource for researchers interested in investigating PMI.
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The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC50 of Lip and HD1 were 33.27 and 128.61 μg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.
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A simple and a rapid technique to separate intact crystals from spores and cell debris of Bacillus thuringiensis using a carboxymethyl cellulose (CM-cellulose) column is described. B. thuringiensis subsp. kurstaki, a toxic strain to lepidopteran insects that produces small bipyramidal crystals, and B. thuringiensis subsp. yunnanensis, a nontoxic strain producing unusually large bipyramidal and spindle-shaped crystals were used as test organisms. Spores and cell debris were washed in sodium acetate and sodium phosphate buffers and the crystals bound to the CM-cellulose were eluted with Tris-EDTA buffer, regardless of their size and shape. Light microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purity of the crystal was > 98%. The percent recoveries of crystals from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. yunnanensis were approximately 14 and 25, respectively.
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A method is described for the large-scale purification of the Bacillus thuringiensis protein crystal by zonal gradient centrifugation. NaBr gradients are employed in a Beckman J21-B centrifuge equipped with a JCF-Z rotor.
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Spores and parasporal crystals of Bacillus thuringiensis can be separated at moderate centrifugation speeds (10,000 to 12,000 rpm) in gradients of Renografin or sodium diatrizoate.
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Bacillus thuringiensis var israelensis parasporal crystal delta-endotoxin was purified by ultracentrifugation on a discontinuous sucrose gradient. Native delta-endotoxin crystals showed no detectable toxicity in the vitro and in vivo systems that are described. By contrast alkali-solubilized crystal delta-endotoxin caused rapid cytological and cytopathological changes in Aedes albopictus, Choristoneura fumiferana 63 CF1, Spodoptera frugiperda and Trichoplusia ni cell lines as observed by phase-contrast microscopy and vital staining. Mouse fibroblasts, primary pig lymphocytes and three mouse epithelial carcinoma cell types showed a similar response to the alkali-soluble crystal delta-endotoxin. In addition the soluble crystal delta-endotoxin protein caused haemolysis of rat, mouse, sheep, horse and human erythrocytes. Intravenous administration of the alkali-soluble crystal delta-endotoxin to Balb. c mice at a dose rate of 15-30 micrograms of protein per gram body weight resulted in rapid paralysis followed by death within 12h. Subcutaneous inoculation of 15-30 micrograms of protein per gram body weight resulted in death of suckling mice in 2-3 h. The alkali-solubilized crystal delta-endotoxin was not toxic however, when administered per os. A comparison is made with a similar alkali-soluble fraction from the parasporal crystal delta-endotoxin of B. thuringiensis var kurstaki. With the exception of the Lepidopteran cell line, Choristoneura fumiferana 63 CF1, this soluble crystal delta-endotoxin protein showed no in vitro or in vivo toxicity, and no haemolytic activity.
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During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.
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Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins in agriculture and forest management. We here identified a crystal protein that is non-insecticidal and non-hemolytic but has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g. highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa. In slices of liver and colon cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected. The cytocidal effect of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does occur when the cell damage proceeded slowly. The amino acid sequence deduced from the nucleotide sequence of the cloned gene of the protein has little sequence homology with the insecticidal crystal proteins of B. thuringiensis. These observations raise the presence of a new group of the B. thuringiensis toxin and the possibility of new applications for the protein in the medical field.
Article
Bacillus thuringiensis (Bt) is a delta-endotoxins producing bacterium that inhibits the digestive process of many insect orders such as; lepidopteran, dipteran and coleopteran. A comparative study between sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and DNA hybridization was performed on 25 chosen different strains of B. thuringiensis. Based on patterns from these molecular tools, the 25 isolates where grouped based on insect killing capability. Soil samples from Tennessee were collected and analyzed for the presence of B. thuringiensis. Fifty six (56) of more than one hundred isolates collected were initially processed using the sodium acetate and a crystal protein staining procedure. Proteinaceous and genomic profiles were gathered for 25 isolates which were named Bt1 thru Bt25. DNA was extracted, quantified, amplified, cloned and sequenced. Alignment studies were performed on the sequenced products. This sequencing data helped decipher which of the Bt isolates belonged to the resembling parent category. The spore/crystal mixtures produced during sporulation were harvested by centrifugation at 10,000 g at 4°C. The spores and crystals were then separated using a discontinuous sucrose gradient with ultracentrifugation. The separation was confirmed by polarized microscopy. The crystal proteins were quantified and then separated using sodium dodecyl sulphate-polyacrylamide gel electrophresis (SDS-PAGE). A correlation was achieved between genomic and proteomic profiles which directly helped in grouping the 25 isolates. The compilation of data suggest that 14 of the 25 isolates resembled Bacillus thuringiensis subspecies kurstaki, four of the 25 isolates resembled B. thuringiensis subspecies israelensis while just one of the 25 isolates resembled B. thuringiensis subspecies tenebrionis. The overwhelming majority of the Bt isolates collected in our study resembled B. thuringiensis subspecies kurstaki. This can be due to the over use of genetically modified Bt corn in the late 1980’s which was subsequently dispersed into the environment.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
Spores from severalBacillus species displayed a strong affinity for hexadecane and other hydrophobic solvents. The binding ofBacillus subtilis spore suspensions to octyl-Sepharose was enhanced by ammonium sulfate and other salts, but was inhibited by detergents. Treatment of spore suspensions with strong denaturants promoted their adherence to hexadecane, presumably by exposing hydrophobic residues in coat proteins. The hydrophobic characteristics of spores may be important in the ecological adaptation of certain bacteria.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
A method is described for the purification of Bacillus thuringiensis protein crystals by Ludox gradient centrifugation. This method is simple, inexpensive, fast, and efficient compared with other techniques. It has been successfully used to purify and characterize the protein crystals from several B. thuringiensis strains.
  • J R Mounsef
J.R. Mounsef et al. / Journal of Microbiological Methods 107 (2014) 147–149
Effects of Carbon:Nitrogen Ratio and Oxygen on the Growth Kinetics of Bacillus thuringiensis and Yield of Bioinsecticidal Crystal Protein
  • T Anderson
Anderson, T., 1990. Effects of Carbon:Nitrogen Ratio and Oxygen on the Growth Kinetics of Bacillus thuringiensis and Yield of Bioinsecticidal Crystal Protein(M.Sc Thesis) The University of Western Ontario, London, Canada.
Growth Kinetics of Bacillus thuringiensis Batch, Fed Batch and Continuous Bioreactor Cultures(M.Sc Thesis) The University of Western Ontario
  • D Rivera
Rivera, D., 1998. Growth Kinetics of Bacillus thuringiensis Batch, Fed Batch and Continuous Bioreactor Cultures(M.Sc Thesis) The University of Western Ontario, London, Canada.