Insecticidal toxins of Bacillus thuringiensis and their genes
Authors: Jyoti Raina and Ajaykumara K.M
Ph.D Research Scholar, GBPUAT, Pantnagar, Uttarakhand
Bacillus thuringiensis (Bt) is a motile, gram positive, spore forming, crystalliferous and facultative bacterium that in addition to endospores, produces a proteinaceous parasporal crystal in the sporangium at the time of sporulation. These proteinaceous crystal contains 1or 2 proteins in crystalline form, which possess insecticidal properties and those proteins are well known as Bt toxins. Depending upon the strain and sub-species, the crystal inclusion may be composed of one or more δ-endotoxins (Cry proteins), ß-endotoxin or α-exotoxin, which are variously toxic to the larvae of Lepidoptera or Coleopteran or Diptera. There are more recent reports of B. thuringiensis isolates active against nematodes and livestock ectoparasites. In addition to the Cry genes, Cyt genes, genes encoding chitinase, vegetative insecticidal proteins have also been cloned from B. thuringiensis and studied for insecticidal properties.
Classification of B. thuringiensis crystal proteins
The insecticidal activity of Bt serotypes is dependent on insecticidal proteins or toxins produced by them. Hofte and Whiteley (1989) claasified Cry gene on the basis of host specificity of these toxins and on their structural similarities. They proposed a nomenclature keeping the Cry epithet (for designating crystal), followed by a Roman numeral indicating the insect class against which the encoded toxin is active and by two alphabetic designations to indicate relative amino acid homology. They proposed 5 major classes of insecticidal crystal proteins, i.e Cry I (Lepidoptera specific toxin), Cry II (Lepidoptera and Diptera specific toxin), Cry III (Coleoptera specific toxin), Cry IV (Diptera specific toxin), Cry V (Coleptera and Lepidoptera specific toxin), Cry VI (Nematode specific toxin), Cyt A (cytolytic and Haemolytic toxin). Whereas, Crickmore et al.(1996) introduced a new system of classification which is solely based on amino acid homology, where each protoxin acquired a name consisting of the mnemonic Cry (or Cyt) and four hierarchical ranks consisting of numbers, capital letters, lower case letters and numbers (e.g. Cry25Aa1) and parentheses are removed.
These genes encodes for insecticidal crystal proteins. These insecticidal proteins (δ-endotoxins) are stomach poisons and toxic only after ingestion by the susceptible insects. The crystals of delta endotoxins are stable proteins, which easily breaks up by the alkaline pH> 9.5 and highly reductive environment of mid gut of lepidopteran larva and so obtained protein is known as protoxin of 130 kDa. This protoxin further cleaved by gut protease enymes (mainly serine protease), which cleave 29 amino acids from N-terminals and 500 amino acids from C-terminals and further leads to the formation of 60-70 kDa toxin(trypsin resistant fragment). This toxin can easily pass through peritrophic membrane of mid gut and irreversibly binds to the specific receptor in the target tissue, the apical brush border membrane of the columnar cells followed by insertion of the toxin and formation of pores which allows rapid fluxes of ions specially the K+. Ion movements through this pore disrupt potassium and pH gradients and lead to lyses of the epithelium, gut paralysis, insect will stop feeding and die due to starvation.
These genes encode for Cytolytic and Haemolytic toxin (Insecticidal crystal protein). These proteins are smaller than that of Cry protein i.e 20-27 kDa in size. Mode of action of these proteins is similar to that of Cry proteins. These are also activated in alkaline gut pH. But these proteins donot require receptors for pore formation, get automatically inserted into phospholipid bilayer and forms pores of 1.0nm Diameter. Pore formation leads to ionic imbalance, gut paralysis in insects and insect died due to starvation.
Few, B. thuringiensis strains produce and excrete chitinase into the culture media. Synergistic actions between Cry protein and chitinase have been demonstrated. Co-expression of heterologous chitinase gene in B. thuringiensis also has been demonstrated to increase the insecticidal activity of B. thuringiensis. Three chitinase gene from three different strains of B. thuringiensis viz. pakistani, kenyae and HD1 have been cloned and properties of the encoded protein have been discussed in detail (Arora et al., 2003).
This gene encodes for novel class of protein called as vegetative insecticidal protein produced by
Bacillus thuringiensis during mid log phase to sporulation phase. Size of
Vip is about 88.6 kDa in size. Though symptoms produced by
Vips are similar to those caused by
Cry proteins. But,
Vip develop symptoms over a 48-72 hrs after ingestion, whereas, in case of
Cry protein, it takes only 16-24 hrs for the symptoms to appear. This is found to be toxic to tobacco budworm, corn earworm and black cutworm.
Arora, N., Ahmad, T., Rajagopal, R. and Bhatnagar, R.K. 2003. A constitutively expressed 36 kDa exochitnase from B. thuringiensis HD-1. Biochemical and Biophysical Research Communication. 307: 620-625.
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