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Samir Jaoua - One of the best experts on this subject based on the ideXlab platform.
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Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium
Brazilian Journal of Microbiology, 2013Co-Authors: Saoussen Ben Khedher, Samir Jaoua, Nabil ZouariAbstract:In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on Delta-Endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for Delta-Endotoxin production consists of 30 g L -1 starch, 30 g L -1 soya bean an d9gL -1 sodium chloride. When compared to the basal production medium, an improvement in Delta-Endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg Delta-Endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg Delta-Endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.
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Correlation between Delta-Endotoxin and proteolytic activities produced by Bacillus thuringiensis var. kurstaki growing in an economic production medium
Biocontrol Science and Technology, 2013Co-Authors: Karim Ennouri, Saoussen Ben Khedher, Samir Jaoua, Nabil ZouariAbstract:Abstract Bacillus thuringiensis is a Gram positive bacterium that produces an insecticidal crystalline protein making it one of the most important biocontrol agents for pest management. Bioinsecticides based on B. thuringiensis were produced by fermentation processes in liquid media. Cultural conditions controlling proteolytic activities in different culture media were investigated to study the possible correlations between B. thuringiensis production of proteases and Delta-Endotoxins in a low-cost complex medium. Aeration appeared to play an important role in Delta-Endotoxin production. The correlation between proteolytic activity and aeration does not seem to be reliable. A negative correlation (correlation coefficient =− 0.774) was established between protease activity and Delta-Endotoxin production. In order to prove this correlation, protease hypo-producing and overproducing mutants were isolated through random mutagenesis of two wild strains, BUPM13 and BUPM5, by using nitrous acid. Interestingly, d...
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Construction of a Bacillus thuringiensis genetically-engineered strain harbouring the secreted Cry1Ia Delta-Endotoxin in its crystal
Biotechnology Letters, 2011Co-Authors: Mariam Dammak, Samir Jaoua, Slim TounsiAbstract:Unlike other Bacillus thuringiensis Cry proteins, Cry1Ia does not form a crystal since it is a secreted Delta-Endotoxin. We have engineered a Cry1Iac chimeric protein by substituting the C -terminal part of Cry1Ia by the corresponding Cry1Ac part. When expressed in an acrystalliferous B. thuringiensis strain, Cry1Iac did not crystallize, but when expressed in the crystalliferous strain BNS3, the chimeric protein co-crystallized with the endogenous Cry1A Delta-Endotoxins forming a typical bipyramidal crystal. The integration of Cry1Ia in the composition of the crystal of BNS3 led to an increase of its Delta-Endotoxin production (13%) and to an improvement (60%) of its toxicity against Agrotis ipsilon .
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improvement of bacillus thuringiensis bioinsecticide production by sporeless and sporulating strains using response surface methodology
New Biotechnology, 2011Co-Authors: Saoussen Ben Khedher, Samir Jaoua, Amel Kamoun, Nabil ZouariAbstract:Statistical experimental designs, involving a Plackett–Burman design followed by a rotatable central composite design were used to optimize the culture medium constituents for Bacillus thuringiensis bioinsecticide production. This was carried out by using firstly an asporogenic strain and extrapolated to some sporeless and sporulating strains. Initial screening of production parameters was performed and the variables with statistically significant effects on Delta-Endotoxin production were identified: glucose, glycerol, yeast extract and MnSO4. These variables were selected for further optimization by response surface methodology. The obtained results revealed that the optimum culture medium for Delta-Endotoxin production consists of 22.5 g/l of glucose, 4.8 g/l of glycerol, 5.8 g/l of yeast extract and 0.008 g/l of MnSO4. Under these conditions, Delta-Endotoxin production was 2130 and 2260 mg/l into 250 and 1000 ml flask respectively, which represent more than 38% improvement in toxin production over the basal medium (1636 mg/l). Such medium composition was shown to be suitable for overproducing Delta-Endotoxins by sporeless and sporulating strains.
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Overproduction of Delta-Endotoxins by Sporeless Bacillus thuringiensis Mutants Obtained by Nitrous Acid Mutagenesis
Current microbiology, 2010Co-Authors: Saoussen Ben Khedher, Nabil Zouari, Nadia Messaddeq, Patrick Schultz, Samir JaouaAbstract:Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-Endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and Delta-Endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.
Nabil Zouari - One of the best experts on this subject based on the ideXlab platform.
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Multiple linear regression and artificial neural networks for Delta-Endotoxin and protease yields modelling of Bacillus thuringiensis
3 Biotech, 2017Co-Authors: Karim Ennouri, Rayda Ben Ayed, Mohamed Ali Triki, Ennio Ottaviani, Maura Mazzarello, Fathi Hertelli, Nabil ZouariAbstract:The aim of the present work was to develop a model that supplies accurate predictions of the yields of Delta-Endotoxins and proteases produced by B. thuringiensis var . kurstaki HD-1. Using available medium ingredients as variables, a mathematical method, based on Plackett–Burman design (PB), was employed to analyze and compare data generated by the Bootstrap method and processed by multiple linear regressions (MLR) and artificial neural networks (ANN) including multilayer perceptron (MLP) and radial basis function (RBF) models. The predictive ability of these models was evaluated by comparison of output data through the determination of coefficient ( R ^2) and mean square error (MSE) values. The results demonstrate that the prediction of the yields of Delta-Endotoxin and protease was more accurate by ANN technique (87 and 89% for Delta-Endotoxin and protease determination coefficients, respectively) when compared with MLR method (73.1 and 77.2% for Delta-Endotoxin and protease determination coefficients, respectively), suggesting that the proposed ANNs, especially MLP, is a suitable new approach for determining yields of bacterial products that allow us to make more appropriate predictions in a shorter time and with less engineering effort.
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Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium
Brazilian Journal of Microbiology, 2013Co-Authors: Saoussen Ben Khedher, Samir Jaoua, Nabil ZouariAbstract:In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on Delta-Endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for Delta-Endotoxin production consists of 30 g L -1 starch, 30 g L -1 soya bean an d9gL -1 sodium chloride. When compared to the basal production medium, an improvement in Delta-Endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg Delta-Endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg Delta-Endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.
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Correlation between Delta-Endotoxin and proteolytic activities produced by Bacillus thuringiensis var. kurstaki growing in an economic production medium
Biocontrol Science and Technology, 2013Co-Authors: Karim Ennouri, Saoussen Ben Khedher, Samir Jaoua, Nabil ZouariAbstract:Abstract Bacillus thuringiensis is a Gram positive bacterium that produces an insecticidal crystalline protein making it one of the most important biocontrol agents for pest management. Bioinsecticides based on B. thuringiensis were produced by fermentation processes in liquid media. Cultural conditions controlling proteolytic activities in different culture media were investigated to study the possible correlations between B. thuringiensis production of proteases and Delta-Endotoxins in a low-cost complex medium. Aeration appeared to play an important role in Delta-Endotoxin production. The correlation between proteolytic activity and aeration does not seem to be reliable. A negative correlation (correlation coefficient =− 0.774) was established between protease activity and Delta-Endotoxin production. In order to prove this correlation, protease hypo-producing and overproducing mutants were isolated through random mutagenesis of two wild strains, BUPM13 and BUPM5, by using nitrous acid. Interestingly, d...
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improvement of bacillus thuringiensis bioinsecticide production by sporeless and sporulating strains using response surface methodology
New Biotechnology, 2011Co-Authors: Saoussen Ben Khedher, Samir Jaoua, Amel Kamoun, Nabil ZouariAbstract:Statistical experimental designs, involving a Plackett–Burman design followed by a rotatable central composite design were used to optimize the culture medium constituents for Bacillus thuringiensis bioinsecticide production. This was carried out by using firstly an asporogenic strain and extrapolated to some sporeless and sporulating strains. Initial screening of production parameters was performed and the variables with statistically significant effects on Delta-Endotoxin production were identified: glucose, glycerol, yeast extract and MnSO4. These variables were selected for further optimization by response surface methodology. The obtained results revealed that the optimum culture medium for Delta-Endotoxin production consists of 22.5 g/l of glucose, 4.8 g/l of glycerol, 5.8 g/l of yeast extract and 0.008 g/l of MnSO4. Under these conditions, Delta-Endotoxin production was 2130 and 2260 mg/l into 250 and 1000 ml flask respectively, which represent more than 38% improvement in toxin production over the basal medium (1636 mg/l). Such medium composition was shown to be suitable for overproducing Delta-Endotoxins by sporeless and sporulating strains.
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Overproduction of Delta-Endotoxins by Sporeless Bacillus thuringiensis Mutants Obtained by Nitrous Acid Mutagenesis
Current microbiology, 2010Co-Authors: Saoussen Ben Khedher, Nabil Zouari, Nadia Messaddeq, Patrick Schultz, Samir JaouaAbstract:Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-Endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and Delta-Endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.
Dhouha Ghribi - One of the best experts on this subject based on the ideXlab platform.
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improvement of bacillus thuringiensis Delta Endotoxin production by overcome of carbon catabolite repression through adequate control of aeration
Enzyme and Microbial Technology, 2007Co-Authors: Dhouha Ghribi, Nabil Zouari, Hassen Trabelsi, Samir JaouaAbstract:Abstract Over-production of Delta-Endotoxin by Bacillus thuringiensis , strain BNS3, entomopathogenic towards lepidoptera through overcome of catabolic repression of its synthesis, was investigated into full-controlled 3 l fermenter using simple and complex media. Equilibrium between density of the vegetative cells and their ability to synthesize toxins during sporulation was shown to be necessary to take into account. By application of various dissolved oxygen profiles during fermentation, it was clear that the level of dissolved oxygen saturation in the fermentation broth affected cell density as well as Delta-Endotoxin synthesis. Indeed, the adequate dissolved oxygen profiles to be followed throughout the fermentation for high production of bioinsecticides were determined using the two different media. It was found out that aeration rates corresponding to 60% and 70% oxygen saturation during the first 6 h of fermentation should be applied into 15 g l −1 glucose-based medium and 42 g l −1 gruel-based one, respectively. Then, 40% oxygen saturation should be ensured up to the end of fermentation, independently of the carbon source origin. With higher oxygen saturation values, cell densities were increased, but Delta-Endotoxin synthesis yields were strongly reduced. Cells produced with low aeration into the medium acquired higher capacities to synthesize toxins during sporulation. Interestingly, by following adequate oxygen profiles, it was possible to use increased initial glucose or gruel concentrations, without significant decrease of Delta-Endotoxin production yields. Consequently, the adequate control of dissolved oxygen in the culture media of B. thuringiensis allowed at least partial overcome of the carbon catabolite repression which seemed to be an apparent regulation, mostly due to the rate of use of energy generated during a first step of high growth of B. thuringiensis cells. These results are of great importance from practical point of view, since it could be possible to produce large quantities of insecticidal crystal proteins during large-scale fermentation, contributing to the reduction of B. thuringiensis insecticides production cost. Interestingly, in bioinsecticides produced with adequate aeration profiles, spores have lower yield, which is in favor of the dissemination of less spores into the environment.
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Use of sea water as salts source in starch- and soya bean-based media, for the production of Bacillus thuringiensis bioinsecticides
Process Biochemistry, 2007Co-Authors: Dhouha Ghribi, W. Trigui, Nabil Zouari, Samir JaouaAbstract:Abstract In order to reduce the cost of Bacillus thuringiensis bioinsecticide-process production , a new medium only composed of starch, soya bean and diluted sea water, providing particularly 7.5 g/l NaCl, was optimized. The results obtained with cultures carried out into 1000 ml shake flasks showed 7% improvement of Delta-Endotoxin production in such cheap medium compared to that containing a mixture of pure minerals instead of sea water. Interestingly, this new formulated medium was shown to be efficient for Delta-Endotoxin production by several B. thuringiensis strains exhibiting various larvicidal activities towards either Diptera or Lepidoptera. Moreover, the use of such medium for large scale production of bioinsecticides was also evidenced in an automated full controlled 3 l fermenter. So that it was shown that sea water did not affect such requirements and have positive effect on growth, sporulation and Delta-Endotoxin synthesis. This should contribute to a significant reduction of the cost of B. thuringiensis bioinsecticide production and utilisation.
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Improvement of bioinsecticides production through adaptation of Bacillus thuringiensis cells to heat treatment and NaCl addition
Journal of Applied Microbiology, 2005Co-Authors: Dhouha Ghribi, Nabil Zouari, Samir JaouaAbstract:ABSTRACT D. GHRIBI, N. ZOUARI AND S. JAOUA. 2005. Aims: The present work aimed to increase yields of Delta-Endotoxin production through adaptation of Bacillusthuringiensis cells to heat shock and sodium chloride and to investigate their involvements in bioinsecticidesproduction improvement.Methods and Results: Growing B. thuringiensis cells were heat treated after different incubation times to studythe response of the adaptative surviving cells in terms of Delta-Endotoxin synthesis. Similarly, adaptation of B.thuringiensis cells to sodium chloride was investigated. Adaptation to combined stressors was also evaluated. Whenapplied separately in the glucose-based medium, 20-min heat treatment of 6-h-old cultures and addition of 7 g l )1 NaCl at the beginning of the incubation gave respectively 38 and 27% Delta-Endotoxin production improvements.Heat shock improved toxin synthesis yields, while NaCl addition improved Delta-Endotoxin production byincreasing the spore titres without significant effect on toxin synthesis yields. Cumulative improvements (66%) wereobtained by combination of the two stressors at the conditions previously established for each one. Interestingly,when the similar approach was conducted by using the large scale production medium based on gruel and fishmeal, 17, 8 and 29% Delta-Endotoxin production improvements were respectively, obtained with heat shock,NaCl and combined stressors.Conclusions: Heat treatment of vegetative B. thuringiensis cells and NaCl addition to the culture media improvedbioinsecticides production. Heat treatment increased toxin synthesis yields, while addition of NaCl increasedbiomass production yields. Cumulative improvements of 66 and 29% were obtained in glucose and economicproduction media, respectively.Significance and Impact of the Study: Overproduction of bioinsecticides by B. thuringiensis could beobtained by the combination of heat treatment of vegetative cells and addition of NaCl to the culture medium. Thisshould contribute to a significant reduction of the cost of B. thuringiensis bioinsecticides production andutilization, and also manage for higher toxin content in the bioinsecticides, which is very interesting from a practicalpoint of view because fewer spores would be disseminated into the ecosystem.Keywords: Bacillus thuringiensis, bioinsecticides, Delta-Endotoxin, heat shock, improvement, NaCl, stress.
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Improvement of bioinsecticides production through mutagenesis of Bacillus thuringiensis by u.v. and nitrous acid affecting metabolic pathways and/or Delta‐Endotoxin synthesis
Journal of Applied Microbiology, 2004Co-Authors: Dhouha Ghribi, Nabil Zouari, Samir JaouaAbstract:AIMS: The present work aimed to obtain bioinsecticide over-producing mutants through classical mutagenesis of vegetative cells of Bacillus thuringiensis (Bt) by using u.v. and nitrous acid, and to evidence the involvement of cell metabolism in Delta-Endotoxin synthesis. METHODS AND RESULTS: Vegetative cells of Bt were treated by nitrous acid (0.17 mg ml(-1)) or exposed to u.v. rays (emitted at a wave length of 240 nm). The isolated survivors were screened on the basis of the production of Delta-Endotoxins and biomass in glucose and/or in gruel-based media at two aeration conditions. Bioinsecticide over-producing mutants were obtained with high frequencies because random mutations were shown to affect cell metabolism at different pathways related to the regulation of Delta-Endotoxin synthesis. CONCLUSIONS: Classical mutagenesis of Bt cells lead to the isolation of a large variety of Delta-Endotoxin over-producing mutants that could be classified into six groups based on the location of the mutations, particularly in metabolism pathways and Delta-Endotoxin synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: High frequencies of Delta-Endotoxin over-producing mutants of Bt could be obtained through classical mutagenesis of vegetative cells. This should contribute to a significant reduction of production and utilization costs of Bt bioinsecticides.
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improvement of bioinsecticides production through mutagenesis of bacillus thuringiensis by u v and nitrous acid affecting metabolic pathways and or Delta Endotoxin synthesis
Journal of Applied Microbiology, 2004Co-Authors: Dhouha Ghribi, Nabil Zouari, Samir JaouaAbstract:D. GHRIBI, N. ZOUARI AND S. JAOUA. 2004. Aims: The present work aimed to obtain bioinsecticide over-producing mutants through classical mutagenesis of vegetative cells of Bacillus thuringiensis (Bt) by using u.v. and nitrous acid, and to evidence the involvement of cell metabolism in Delta-Endotoxin synthesis. Methods and Results: Vegetative cells of Bt were treated by nitrous acid (0AE17 mg ml )1 ) or exposed to u.v. rays (emitted at a wave length of 240 nm). The isolated survivors were screened on the basis of the production of DeltaEndotoxins and biomass in glucose and/or in gruel-based media at two aeration conditions. Bioinsecticide overproducing mutants were obtained with high frequencies because random mutations were shown to affect cell metabolism at different pathways related to the regulation of Delta-Endotoxin synthesis. Conclusions: Classical mutagenesis of Bt cells lead to the isolation of a large variety of Delta-Endotoxin overproducing mutants that could be classified into six groups based on the location of the mutations, particularly in metabolism pathways and Delta-Endotoxin synthesis. Significance and Impact of the Study: High frequencies of Delta-Endotoxin over-producing mutants of Bt could be obtained through classical mutagenesis of vegetative cells. This should contribute to a significant reduction of production and utilization costs of Bt bioinsecticides.
David J Ellar - One of the best experts on this subject based on the ideXlab platform.
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Role of proteolysis in determining potency of Bacillus thuringiensis Cry1Ac Delta-Endotoxin.
Applied and environmental microbiology, 2000Co-Authors: Daniel J. Lightwood, David J Ellar, Paul JarrettAbstract:Bacillus thuringiensis protein Delta-Endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac Delta-Endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of approximately 56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of approximately 58, approximately 40, and approximately 20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.
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Mosquitocidal activity of the CryIC Delta-Endotoxin from Bacillus thuringiensis subsp. aizawai.
Applied and environmental microbiology, 1996Co-Authors: G P Smith, J D Merrick, E J Bone, David J EllarAbstract:The cloned 135-kDa CryIC Delta-Endotoxin from Bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against Spodoptera litoralis and Spodoptera frugiperda larvae and in vitro against the S. frugiperda Sf9 cell line. Here, we report that the CryIC Delta-Endotoxin cloned from B. thuringienesis subsp. aizawai HD-229 and expressed in an acrystalliferous B. thuringiensis strain is also toxic to Aedes aegypti, Anophles gambiae, and Culex quinquefasciatus mosquito larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active Delta-Endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran Delta-Endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC Delta-Endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC Delta-Endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active Delta-Endotoxin.
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the receptor for bacillus thuringiensis cryla c Delta Endotoxin in the brush border membrane of the lepidopteran manduca sexta is aminopeptidase n
Molecular Microbiology, 1994Co-Authors: Peter J K Knight, Neil Crickmore, David J EllarAbstract:A 120 kDa glycoprotein in the larval midgut membrane of the lepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) Delta-Endotoxin, has been purified by a combination of protoxin affinity chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin in the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.
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An analysis of Bacillus thuringiensis Delta-Endotoxin action on insect-midgut-membrane permeability using a light-scattering assay
European journal of biochemistry, 1993Co-Authors: Joe Carroll, David J EllarAbstract:Changes in the membrane permeability of Manduca sexta midgut brush-border-membrane vesicles (BBMV) after addition of Bacillus thuringiensis Delta-Endotoxins were studied using osmotic swelling experiments, volume changes being monitored as the change in 90 degrees light scattering. Typically, control BBMV exhibited limited permeability for sucrose and salts (KCl), while being permeable for urea and glucose. The action of Delta-Endotoxin was examined using proteolytically activated Cry-IA(c) and CryIB toxins. CryIA(c) produced a marked change in the solute permeability of M. sexta BBMV, significant effects being observed at 3.75 pmol/mg BBMV. The permeability change was relatively non-selective with cations, anions and neutral solutes all traversing the membrane to an increased extent in the presence of CryIA(c). In contrast, the CryIB toxin had no effect on BBMV permeability.
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crystal structure of insecticidal Delta Endotoxin from bacillus thuringiensis at 2 5 a resolution
Nature, 1991Co-Authors: Joe Carroll, David J EllarAbstract:The structure of the Delta-Endotoxin from Bacillus thuringiensis subsp. tenebrionis that is specifically toxic to Coleoptera insects (beetle toxin) has been determined at 2.5 A resolution. It comprises three domains which are, from the N- to C-termini, a seven-helix bundle, a three-sheet domain, and a beta sandwich. The core of the molecule encompassing all the domain interfaces is built from conserved sequence segments of the active Delta-Endotoxins. Therefore the structure represents the general fold of this family of insecticidal proteins. The bundle of long, hydrophobic and amphipathic helices is equipped for pore formation in the insect membrane, and regions of the three-sheet domain are probably responsible for receptor binding.
M Gomez - One of the best experts on this subject based on the ideXlab platform.
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transgenic sweet potato plants carrying the Delta Endotoxin gene from bacillus thuringiensis var tenebrionis
Plant Science, 1998Co-Authors: R Moran, R Garcia, A Lopez, Z Zaldua, J Mena, M Garcia, R Armas, D Somonte, J Rodriguez, M GomezAbstract:Abstract Some clones of sweet potato ( Ipomoea batatas L. ), transgenic plants of the cultivar ‘Jewel’, carrying the cry IIIA gene from Bacillus thuringiensis subsp. tenebrionis (B.t.t), were obtained. Molecular tests and biological activity experiments against sweet potato weevil (Cylas formicarius), were performed . The cry IIIA Delta-Endotoxin gene was cloned under the regulation of the 35S CaMV promoter and the TMV Omega fragment. The neomycin phosphotransferase ( npt II) gene for kanamycin resistance was used as selection marker. An efficient protocol for in vitro plant regeneration, which combines naphtalenacetic acid (NAA) and 6-benzilaminopurin (BAP), was established. An Agrobacterium tumefaciens -mediated transformation protocol for sweet potato was implemented. An increase of the transformation frequency of sweet potato, compared to that previously reported, was obtained. Biological activity experiments to evaluate the insecticidal capacity of transgenic plants were performed against sweet potato weevil under both controlled, and field conditions. Plants from the two best resulting clones were tested by molecular methods such as RNA. dot blot and Western blot.