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Bacillus thuringiensis: Potential Microbial Biopesticide
- 1. BACILLUS THURINGIENSIS - BIOLOGY, CLASSIFICATION, MODE OF ACTION Presented By Dr. Rajib Kumar De Principal Scientist Crop Protection Division I.C.A.R.-C.R.I.J.A.F. , Barrackpore.
- 2. Bacillus thuringiensis: Potential Microbial Biopesticide INTRODUCTION Bacillus thuringiensis is Gram + ve bacteria. Belongs to Genus 2, Bacillus in Group 18 in Bergy’s Manual of Determinative Bacteriology (9th edition.) Special feature of Bt is it produces proteinaceous crystal in cell during sporulation, which in turn produces a toxin known as delta endotoxin. According to variation in flagellum antigen and other characteristics 70 serotypes and 83 sub spp are identified (Lacedet et al 1991.)
- 3. History of Bacillus thuringiensis. Japanese biologist, Shigetane Ishiwatari while investigating the cause of the sotto disease (sudden-collapse disease) in silkworms was first to isolate the bacterium Bacillus thuringiensis (Bt) as the cause of the disease in 1901. Ernst Berliner isolated a bacteria that had killed a Mediterranean flour moth in 1911, and rediscovered Bt. He named it Bacillus thuringiensis, after the German town Thuringia where the moth was found. Ishiwatari had named the bacterium Bacillus sotto in 1901 but the name was later ruled invalid. In 1915, Berliner reported the existance of a crystal within Bt, but the activity of this crystal was not discovered until much later. Farmers started to use Bt as a pesticide in 1920. France soon started to make commericialized spore based formulations called Sporine in 1938. Sporine, at the time was used primarly to kill flour moths. In 1956, researchers, Hannay, Fitz-James and Angus found that the main insecticidal activity against lepidoteran (moth) insects was due to the parasporal crystal. In the US, Bt was used commercially starting in 1958. By 1961, Bt was registerd as a pesticide to the EPA.
- 4. Biology of Bacillus thuringiensis Upon sporulation B. thuringiensis, forms crystals of proteinaceous insecticidal δ-endotoxins (called crystal proteins or Cry proteins), which are encoded by cry genes. In most strains of B. thuringiensis, the cry genes are located on a plasmid (in other words, cry is not a chromosomal gene in most strains). These plasmids are usually 1-12 in number and are of molecular weight 2-200kb. The crystal genes also produce VIP’s, Thuringiensin (beta-exotoxin).
- 5. CLASSIFICATION OF BACILLUS THURINGIENSIS Cry toxin Insect Order Cry I Lepidoptera Cry II Lepidoptera & Diptera Cry III Coleoptera Cry IV Diptera In 1989, Hofte and Whiteley based on diversity of amino acid sequence and toxicity divided 42 crystal proteins into 5 groups that were further divided into 14 sub groups (13 Cry sub groups and 1Cyt.)
- 6. Number of genes 32 Cry genes 113 Cry genes 2 Cyt genes Till September 28, 2011, 280 ICP genes belonging to 34 classes have been reported which include Gene Crystal shape Protein size(kDa) Insect activity Cry I [several 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
- 7. NEW CLASSIFICATION OF BACILLUS THURINGIENSIS Cry I of Hofte and Whiteley was renamed from Cry1A to Cry1K Cry II of Hofte and Whiteley was renamed from Cry2A(sub groups a, b, c) Cry III of Hofte and Whiteley was renamed from Cry3A(except for CryIII C renamed as Cry7A) Cry IV of Hofte and Whiteley was renamed from Cry4A(except for Cry IV C renamed as Cry10A and Cry IV D renamed as Cry11A) For further information regarding Bt classification please visit http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/
- 8. STRUCTURAL MORPHOLOGY OF CRY PROTEINS Cry proteins are composed of 1,100-1,200 amino-acids of 130- 140kDa mol.wt. Full length of the cry protein is not required for exerting toxicity. The toxin has two terminals: N-terminal and C-terminal. N-terminal C-terminal Hydrophobic Forms alpha –helix Contributes toxicity High diversity in amino acid sequence and sequence homology is about 40-90% Hydrophilic Forms beta –sheets Mainly supports formation of parasporal crystals Highly conserved amino acid sequence and sequence homology is 90%
- 9. Relative length of Cry protoxins and position of protease digestion. White boxes represent the protoxin and striped boxes represent the activated toxin. Solid arrows show the amino- and carboxy- terminal cleavage sites of the activated toxins. Doted arrows show the intramolecular cleavages. Cleavage of Cry1A at residue 51 resulted in loss of helix α-1 and pre-pore formation. Cleavage of Cry4B resulted in two fragments of 18 and 46 kDa, while Cry11A resulted in two fragments of 34 and 32 kDa.
- 10. TOXIN ACTIVATION 1-28th /29th amino acid residues from N-terminal side and 600th- 1150th amino acid residues from C –terminal is cleaved by trypsin enzymes of the insect mid gut to form a toxin core of 60kDa.
- 11. Recent studies on the delta-endotoxin structure show that it has three domains. Domain I is a bundle of 7 alpha-helices, some or all of which can insert into the gut cell membrane, creating a pore through which ions can pass freely. Domain II consists of three antiparallel beta-sheets, similar to the antigen-binding regions of immunoglobulins, suggesting that this domain binds to receptors in the gut. Domain III is a tightly packed beta-sandwich which is thought to protect the exposed end (C-terminus) of the active toxin, preventing further cleavage by gut proteases.
- 12. Three dimensional structures of insecticidal toxins produced by Bacillus thuringiensis Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa, Cry4Bb and Cyt2A.
- 13. MODE OFACTION OF Bt. 1. pH requirement of alkaline condition. 2. Release of toxin 1. Solubilisation of toxins by breaking the disulphide linkages 2. Activation of protoxin to active toxin 3. Binding of active toxin to BBMV’s receptors mainly cadherins, APN, ALP, GPI (glycosylated phosphotidyl inositol ) of midgut epithelial cells. 4. Formation of pores in cell membrane (two models are proposed) 1. Penknife model. 2. Umbrella model. 5. Cell lysis (two theories are proposed) 1. Proton Perill theory 2. Osmotic cell lysis theory.
- 14. Receptor molecules of Cry1A proteins. CADR, cadherin receptor; APN, aminopeptidase-N, ALP, alkaline phosphatase, GCR, 270 kDa glyco-conjugate receptor.
- 15. Schematic representation of Bt mode of action
- 16. Mode of action of Bt
- 17. Model of the mode of action of Cry and Cyt toxins. Panel A, sequential interaction of Cry toxins with different receptor molecules in lepidopteran larvae. (1) Solubilization and activation of the toxin; (2). Binding of monomeric Cry toxin to the first receptor (CADR or GCR), conformational change is induced in the toxin and α-helix 1 is cleaved; (3) Oligomer formation; (4) Binding of oligomeric toxin to second receptor (GPI-APN or GPI-ALP), a conformational change occurs and a molten globule state of the toxin is induced; (5) insertion of the oligomeric toxin into lipid rafts and pore formation. Panel B, role of Cyt and Cry toxins in the intoxication of dipteran larvae. (1) Cry and Cyt toxins are solubilized and activated; (2) Cyt toxin inserts into the membrane and Cry toxin binds to receptors located in the membrane (ALP or Cyt toxin); (3) oligomerization of the Cry toxin is induced; (4) oligomer is inserted into the membrane resulting in pore formation.
- 18. Theories for mode of action of Bt
- 19. Bt: Mode of action in transgenic crops
- 20. Mode of action of Bt The mode of action of the Cry1A toxin and a comparison with the Cry1AMod toxin. The main difference is the formation of the oligomeric structure that in one case requires the interaction with the cadherin receptor and in the other is independent of this receptor.
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