Bacillus thuringiensis

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Bacillus thuringiensis:

Bacillus thuringiensis Presented by: Sunil Subramanya A.E .

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Bacillus thuringiensis (or Bt ) is a facultative anaerobic, gram-positive, soil-dwelling bacterium , commonly used as a biological alternative to a pesticide ; alternatively, the Cry toxin may be extracted and used as a pesticide.

Characteristics of Bt ::

Characteristics of Bt : Bt subspecies can synthesize more than one parasporal inclusion. The parasporal inclusions are formed by different insecticidal crystal proteins (ICP). The crystals have various shapes ( bipyramidal , cuboidal , flat rhomboid, spherical or composite with two crystal types), depending on their ICP composition.

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During sporulation many Bt strains produce crystal proteins ( proteinaceous inclusions), called δ-endotoxins (Cry proteins), which are encoded by cry genes, and have insecticidal action. This has led to their use as insecticides, and more recently to genetically modified crops using Bt genes. In most strains of B. thuringiensis the cry genes are located on the plasmid.

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Cry toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles), hymenoptera ( wasps , bees , ants and sawflies ) and nematodes . Different domains of the ICP are responsible for host susceptibility (receptor recognition) and toxicity (pore formation).

Scientific Classification::

Scientific Classification: Kingdom: Eubacteria Phylum: Firmicutes Class : Bacilli Order : Bacillales Family : Bacillaceae Genus : Bacillus Species: thuringiensis (Berliner 1915)

Genome structure:

Genome structure B. thuringiensis has a circular chromosome and a GC-content of approximately 32%~35% . It has a genome size of between 5.2–5.8 Megabases . It is a facultative anaerobic organism. It has many plasmids and Bt's strains harbors a diverse range of plasmids that vary in number and in size ( 2–200kb).

Cell structure and metabolism :

Cell structure and metabolism B. thuringiensis is gram-positive. it has a thick cell wall that is comprised of peptidoglycan (amino acid polypeptide and a sugar). Between the cell wall and the plasma membrane is a small section called the periplasmic space which is essential for biosynthesis and protection.

History ::

History : B. thuringiensis was first discovered in 1901 by Japanese biologist Shigetane Ishiwata , most abundantly found in grain dust from silos and other grain storage facilities. In 1911, B. thuringiensis was rediscovered in Germany by Ernst Berliner, who isolated it as the cause of a disease called Schlaffsucht (excessive sleeping) in flour moth caterpillars, collected in the German province of Thuringia.

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Bt first became available as a commercial insecticide in France in 1938, and in the 1950s it entered commercial use in the USA. Whalon and McGaughey in 1998 showed that each strain of Bt produces a unique crystal protein which is encoded by a single gene located in the plasmid.

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Gene Crystal shape Protein size(kDa) Insect activity cry I ………….. [ subgroups: A(a), A(b), A(c), B, C, D, E, F, G] Bipyramidal 130-138 lepidoptera larvae cry II …………. [subgroups A, B, C] Cuboidal 69-71 lepidoptera and diptera cry III….……… [subgroups A, B, C] flat/irregular 73-74 coleoptera cry IV………… [subgroups A, B, C, D] Bipyramidal 73-134 diptera cry V-IX Various 35-129 various

Habitats ::

Habitats : Many different Bt subspecies have been isolated from dead or dying insects mostly from the orders Coleoptera , Diptera and Lepidoptera, but many subspecies have also been isolated from soil, leaf surfaces and other habitats. The carcasses of dead insects often contain large quantities of spores and ICPs that may enter the environment.

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The coleopteran-active and lepidopteran -active Bt subspecies are primarily associated with the soil and phylloplane (leaf surfaces), whereas the dipteran-active Bt subspecies are commonly found in aquatic environments. In the environment, the spores persist and vegetative growth may occur when conditions are favourable and nutrients are available.

Isolation and Culture ::

Isolation and Culture : For Soil Sample : One gram of each sample is suspended in 10 ml sterile distilled water and pasteurized at 80 0 C for 30 min. For the selection of B. thuringiensis , 1 ml of each suspension is added to 10 ml of Luria- Bertani broth buffered with 0.25 M sodium acetate pH 6.8. The suspensions are incubated at 30 o C for 4 h and then heated at 80 o C for 3 min.

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Suspensions were diluted and plated on T3 medium (per liter: 3 g tryptone , 2 g tryptose , 1.5 g yeast extract, 0.05 M sodium phosphate pH 6.8, and 0.005 g of MnCl2). After incubation at 30 o C for 24 h, the colonies showing similar morphology were selected and examined under phase-contrast microscope to determine the presence of parasporal inclusions and spores.

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For Leaf Sample : The leaves from different horticultural crops are washed gently in sterile distil water to remove dust and superficially adhering microflora . Then, leaves are put inside a 250mL conical flask containing 100mL sterile distil water and rotated at 250rpm, at 30 0 C for 5hrs. The suspension was poured into sterile centrifugation tubes and centrifuged at 10,000rpm, at 4 0 C for 15mins.

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The pellets are washed with 5mL of LB broth buffered with 0.25M Na-acetate and the entire contents poured into a 100mL conical flask containing 5mL of LB broth buffered with 0.25M Na-acetate and rotated in a rotary shaker at 250rpm, at 30 0 C for 4hrs. NOTE : The procedure given for soil sample can be followed for leaf, seed dust and other samples also.

Media compositions ::

Media compositions : LB Broth and Agar (Morris et.al., 1998) Tryptone - 10 g Yeast Extract - 5 g NaCl - 5 g DW - 1000 mL Before sterilization, pH is adjusted to 7.0 and 15g agar is added to prepare LB agar.

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Buffered LB-broth with 0.25M sodium acetate buffer : 2.5M Sodium acetate (34.02g/100mL water) 2.5M acetic acid (0.8mL/99.2mL water) 2.5M Na-acetate is added to 2.5M acetic acid until the pH reaches 6.8. This buffer should be freshly prepared and added to the broth before use.

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Buffered T3 medium with 0.05M sodium phosphate buffer : NaH2PO4 – 6.9g in 100mL water NaH2PO4 _ 7.1g in 100mL water Add solution A to B until Ph 6.8 is reached. NA and NB may also be needed.

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T3 medium : (Morris et.al., 1998) Tryptose - 3 g Tryptone - 2 g Yeast Extract - 15 g MgCl - 0.005 g

Mode of Action of Bt::

Mode of Action of Bt: The sporulated Bt with ICP or spore-ICP complexes must be ingested by a susceptible insect larva followed by solubilisation, and processing from a protoxin to an activated toxin core in the insect digestive fluid. The toxin core travels across the peritrophic matrix and the C-terminal region binds to specific receptors called cadherins on the brush border membrane of the gut cells, resulting in pore formation by the N-terminal domain. Accumulation of toxin oligomers results in toxin insertion in the membrane, pore formation, osmotic cell shock, septicaemia and ultimately insect death .

Commercial Products, Production and Application:

Commercial Products, Production and Application Conventional Bt products, which utilize naturally-occurring Bt strains, account for approximately 90% of the world . Each year some 13 000 tonnes are produced using aerobic fermentation technology . Conventional Bt products have been targeted primarily against lepidopteran pests of agricultural and forestry crops; however in recent years, Bt strains active against coleopteran pests have also been marketed. Strains of Bt active against dipteran vectors of parasitic and viral diseases are being used in public health programmes .

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Commercial Bt formulations may be applied as an insecticide to foliage, soil, water environments or food storage facilities. After the application of a Bt subspecies to an ecosystem, the vegetative cells and spores may persist at gradually decreasing concentrations for weeks, months or years as a component of the natural microflora . The ICPs, however, are rendered biologically inactive within hours or days.

Is it Dangerous?:

Is it Dangerous? No Because stability of the bt protien is upto 60 0 c only means boiling of Bt product denatures the Bt protien . Bt- protiens are specific to insect species particularly lepidoptereans and those receptors are not presnt in humans. Alkaline gut p H is required for conversion of protien to toxin but it is acidic in human

Advantages : :

Advantages : There are several advantages in expressing Bt toxins in transgenic Bt crops: The level of toxin expression can be very high thus delivering sufficient dosage to the pest. The toxin expression is contained within the plant system and hence only those insects that feed on the crop perish. The toxin expression can be modulated by using tissue-specific promoters , and replaces the use of synthetic pesticides in the environment.

Disadvantages ::

Disadvantages : Bt is susceptible to degradation by sunlight. Specific activity of Bt insecticides may limit their use on Crops where problems with several pests occur, including non-susceptible insects (aphids, grasshoppers) Since Bt does not kill rapidly , users may incorrectly assume that it is ineffective a day or two days after treatment. This, however, is merely a perceptual problem. Bt-based products tend to have a shorter shelf life than other insecticides.

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