Bacillus thuringiensis and its application in agriculture

Presently, a number of approaches to pest control via genetic engineering have been developed and genetically engineered crops expressing insecticidal characteristics are under cultivation for the last 15 years. Use of Bacillus thuringiensis genes encoding o̅ endotoxins with insecticidal characteristics is the major approach and a number of such B. thuringiensis genes have been expressed in crops with variable level of efficiency. It is very crucial to achieve adequate level of B. thuringiensis gene expression to have durable resistance against target insect pests. As with many aspects of genetic engineering, politics can impact on the success of a project involving the development of B. thuringiensis transgenic crops, irrespective of its apparent social, economic or environmental benefits. Public education will be essential to ensure the widespread adoption of genetic adoption technologies in agriculture, and scientists will have to play an active role in this process

Cry3A δ-endotoxin gene mutagenized for enhanced toxicity to spruce bark beetle in a receptor binding loop

Bacillus thuringiensis Cry3A gene was redesigned for high expression in Norwegian spruce and the sequence was slightly modified to allow for simple N- and C- terminal deletions and domain II loop 1 exchange for synthetic oligos. Modified Cry3A toxins from 13 variants of the synthetic gene were expressed in Escherichia coli BL21 and their toxicity on spruce bark beetle larvae was tested using spruce bark sandwiches. Mutant toxins with N-terminal deletion and loop 1 duplication showed increased toxicity.Key words: Bacillus thuringiensis, Ips typographus, Picea abies, resistance

Enhanced sporulation and toxin production by a mutant derivative of Bacillus thuringiensis

Bacillus thuringiensis (Bt) strains along with insecticidal crystal proteins (ICP) also produce proteases, and chitinases during growth and sporulation. These both potentate the activity of ICPs. This work aimed to obtain bioinsecticide over-production and thermotolerant mutant through classical mutagenesis of vegetative cells of Bt by using UV-mutation multiple times. The isolated survivors were screened on the basis of their production of β-glucosidase, delta-endotoxins and biomass in glucose-based medium. Maximum spore and crystal proteins were produced at 40°C with corn steep liquor as nitrogen source and hydrol as a carbon source. The best mutant MUV7 supported significantly (P = 0.0001) higher values of all kinetic parameters than those supported by the wild culture. Lower demand of activation energy (47.4 kJ mol-1) for crystal protein formation revealed that the best mutant was comparable with those of thermophilic group of organisms.Key words: Bioinsecticide, biomass, delta-endotoxin, insecticidal crystal proteins, kinetics, spores

Development Of Bio-Insecticides Based On Local Qatari Bacillus Thuringiensis Strains For The Biological Control Of Harmful Dipteran Disease Vectors

The very friendly bacterium Bacillus thuringiensis (Bt) is the main source of environment-friendly and safe bioinsecticides used in the control of plant pests and animal and human disease vectors. A collection of 441 Qatari Bt strains were characterized and classified based on crystal morphology, plasmid patterns, crystal protein patterns and cry and cyt genes. In summary, the Bti strain QBT220 was recognized as the most efficient against Dipteran insects Aedes Aegypti. Two of its clones obtained by plasmid curing showed an increase of 115% in the -endotoxins. As example of novel Bt strains, QBT674 is a spherical crystal producing strain having cry2 gene but no cuboidal crystals. QBT555 is a Non-Bti strain with molecular profile very different from Bti strains, but expresses proteins like Cry11, Cry10 and Cyt1A. QBT229 showed high cytolytic activity due to five amino acid replacements in its β sheet that enhanced its anti-cancer activity

Isolation and characterization of Bacillus thuringiensis from olive tree-related habitats

Thesis (Master)--Izmir Institute of Technology, Biotechnology, Izmir, 2005Text in English; Abstract: Turkish and Englishxii,115 leavesAbstract:Bacillus thuringiensis (commonlyreferred to as Bt) is a Gram-positive, sporeforming soil bacterium that produces insecticidal crystal proteins during sporulation.These crystals are referred as Bt toxins or -endotoxins. The most important characteristics of the toxins are their insecticidalactivity against many insects. Since their insecticidal potential has been discovered,it has been produced commercially and used as microbial pesticides all over the world.Therefore, the aim of this study was to isolate Btfrom olive tree-related habitats, and to determine the phenotypic andisolate Btfrom olive tree-related habitats, and to determine the phenotypic and genotypic characteristics of the isolates. To accomplish this purpose, 240 samples were collected in the Aegean Region. The phase-contrast microscopy results showed the presence of crystals in 54 environmental samples, corresponding to 100 Bt isolates. The crystal morphologies were spherical, bipyramidal, cuboidal, irregular pointed, and irregular shaped. The greatest proportion of samples yielding this organism was from the soil. The remaining were from olive leaf residue, green olive leaves, animal faeces,dust samples, and olive pomace. The isolates were characterized on the basis of biochemical characters, cry andcyt gene content, plasmid profiling, 16S-ITS rDNA RFLP.Biochemical tests included protease (caseinase and gelatinase), lecithinase, amylase, nuclease, urease, esculinase, arginine dihydrolyse activity; fermentation of sucrose, salicin, mannose, cellobiose, and maltose, production of acetyl-methyl carbinol, methyl red reaction. Polymerase chain reaction (PCR) has been applied for the identification of cry1, cry2, cry4, cry9, cry11, cry13, cyt1, and cyt2 genes. 68% of the isolates amplified cry1 gene; 57% amplified cry4; 20% amplified cry11; 26% amplified cry9; 20% amplified cry2 genes, andnone of the isolates harbored cry13 gene. Cyt1gene was found in 40% of the isolates while cyt2 gene was present in 80% of the isolates. The most abundant genotype of cry genes was cry1 and cry4. Most of the isolates(58%) possessed more than one cry gene. In addition, different combinations of cry andcytgenes were obtained. Plasmid profiling showed the presence of plasmids in all isolatesand the number of plasmids was usually morethan one. Also, the discrimation effect of 16S-ITS rDNA RFLP was tested to differentiate certain isolates and reference strains, which showed similar biochemical characteristics

Kinetics of pore formation by the Bacillus thuringiensis toxin Cry1Ac

AbstractAfter binding to specific receptors, Cry toxins form pores in the midgut apical membrane of susceptible insects. The receptors could form part of the pore structure or simply catalyze pore formation and consequently be recycled. To discriminate between these possibilities, the kinetics of pore formation in brush border membrane vesicles isolated from Manduca sexta was studied with an osmotic swelling assay. Pore formation, as deduced from changes in membrane permeability induced by Cry1Ac during a 60-min incubation period, was strongly dose-dependent, but rapidly reached a maximum as toxin concentration was increased. Following exposure of the vesicles to the toxin, the osmotic swelling rate reached a maximum shortly after a delay period. Under these conditions, at relatively high toxin concentrations, the maximal osmotic swelling rate increased linearly with toxin concentration. When vesicles were incubated for a short time with the toxin and then rapidly cooled to prevent the formation of new pores before and during the osmotic swelling experiment, a plateau in the rate of pore formation was observed as toxin concentration was increased. Taken together, these results suggest that the receptors do not act as simple catalysts of pore formation, but remain associated with the pores once they are formed

Studies on Resistance to Vegetative (Vip3A) and Crystal (Cry1A) Insecticidal Toxins of Bacillus thuringiensis in Heliothis virescens (Fabricius)

Bacillus thuringiensis (Bt) toxins expressed in commercial transgenic crop varieties are all δ-endotoxins (Cry toxins) but the identification of novel vegetative insecticidal proteins (Vip toxins) has extended the range of insecticidal proteins derived from Bt. One such Vip toxin, Vip3A, primarily targets the midgut epithelium cells of susceptible insects as Cry toxins do, although they appear to have different binding sites. The present study investigated the comparative toxicity of Vip3A, Cry1Ab and Cry1Ac against Heliothis virescens (tobacco budworm) and the impact of antibiotics on Bt insecticidal activity. The selection of a resistant Vip3A population led to the determination of cross-resistance, the genetics of resistance and fitness effects. There was very little variability in the natural susceptibility to Vip3A, Cry1Ab and Cry1Ac in the populations tested, although the toxicity of Vip3A was much lower compared to the Cry1A toxins. A Vip3A resistant population was successfully established within 13 selected generations, with little or no cross-resistance to Cry1Ab or Cry1Ac. The inheritance of resistance ranged from almost completely recessive to incompletely dominant with a possible paternal influence, was polygenic and relatively stable. Vip3A resistance showed a fitness benefit, reduced larval development time, and fitness costs, including survival to adult eclosion, reduced egg viability and reduced male mating success. The effects of antibiotics on H. virescens larval susceptibility to Bt toxins varied depending on antibiotic treatment, the Bt toxin used and the larval instar tested. Bt cotton expressing both Vip3A and Cry1Ab to provide activity against a wide range of pest Lepidoptera, including H. virescens, a major cotton pest in the USA is in the process of commercialisation. The present work will help to support a suitable insecticide resistance management strategy for continued use of Bt toxin in transgenic crops

Ser170 of Bacillus thuringiensis Cry1Ab δ-endotoxin becomes anchored in a hydrophobic moiety upon insertion of this protein into Manduca sexta brush border membranes

<p>Abstract</p> <p>Background</p> <p>Three spin-labeled mutant proteins, mutated at the beginning, middle, and end of α-helix 5 of the <it>Bacillus thuringiensis </it>Cry1Ab δ-endotoxin, were used to study the involvement of these specific amino acid residues in ion transport and to determine conformational changes in the vicinity of these residues when the protein was translocated into a biological membrane.</p> <p>Results</p> <p>Amino acid residue leucine 157, located in the N-terminal portion of α-helix 5, showed no involvement in ion transport, and the environment that surrounds the residue did not show any change when transferred into the biological membrane. Serine 170, located in the middle of the α-helix, showed no involvement in ion transport, but our findings indicate that in the membrane-bound state this residue faces an environment that makes the spin less mobile, as opposed to the mobility observed in an aqueous environment. Serine 176, located in the C-terminal end of the α-helix 5 is shown to be involved in ion transport activity.</p> <p>Conclusion</p> <p>Ion transport data for L157, S170, and S176, along with the mobility of the spin-labels, structural characterization of the resulting proteins, and toxicity assays against a target insect, suggest that the toxin undergoes conformational changes upon protein translocation into the midgut membrane. These conformational changes result in the midregion of the α-helix 5 being exposed to a hydrophobic-like environment. The location of these three residues in the toxin suggests that the entire α-helix becomes inserted in the insect midgut membrane.</p

Isolation and molecular characterization of cry gene for Bacillus thuringiensis isolated from soil of gaza strip

Bacillus thuringiensis, insecticide, polymerase chain reactions (PCR), crystalline protein, Cry gene, Gaza strip. Bacillus thuringiensis (Bt) is a rod-shaped, gram-positive, facultative anaerobic, and spore-forming bacterium (Konecka et al., 2007). During sporulation, it produces insecticidal proteins, which are deposited within the sporangium as crystalline aggregates (Crickomre et al., 1998