Bacillus thuringiensis (Bt) is gram-positive, spore forming, aerobic bacterium present in a wide variety of environments like soil, insect cadavers stored grain products and phylloplane. It produces a kind of proteins during late stage of sporulation, which are accumulated as crystalline inclusions along with spores and are called d-endotoxins. These proteins are known to be toxic to towards larvae of different orders of insect pests (Lepidoptera, Diptera, Coleoptera, Hymenoptera and Homoptera) with different efficacies (Schenpf et al. 1998). The toxicity of Bt is highly specific and it is non-toxic to mammals and beneficial insects. Bt produces other toxins like a-exotoxin b-exotoxin and phospholipases, which are all contributing factors to the virulence of Bt formulations. Bt d-endotoxins or Cry toxins are of particular importance because of their specificity and biodegradability. Bt is known for its insecticidal activity since 1928. Its insecticidal property was first tested on European corn borer. Commercial formulations of Bt have been used to control insect pests in agriculture, horticulture and forestry. Spore-crystal mixtures of Bt strains are used as different formulations to control insect pests.

Hofte and Whiteley (1989) classified the Cry toxins based on their toxicity spectrum (CryI - Lepidoptera; CryII - Diptera + Lepidoptera; CryIII - Coleoptera and CryIV - Diptera). Later, many toxins with other specificities were isolated and it was difficult to accommodate them within these groups. With the advent of molecular biology many genes were sequenced and their toxicity spectrum was studied. To accommodate the growing list of d-endotoxins a new nomenclature has been formulated, wherein each toxin gene/ protein will be having four-letter code, according to their amino acid sequence identity among them (Crickmore et al. 1998). Updated list of the Bt toxin genes can be accessed at http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/. Bt and its d-endotoxins have been extensively studied for their molecular mechanism of action and toxin structure - function relationships.

When nutrients in the medium become depleted Bt undergoes sporulation process and insecticidal proteins are synthesized during late stage of sporulation. These proteins are accumulated inside the cell in the form of crystals of different shapes. These crystals are composed of protoxin molecules, which are bound together by disulphide bonds. When the susceptible larvae consume the crystals, they are solubilized at high pH and protoxin molecules are liberated into the gut environment. The released protoxin molecules are acted upon by midgut proteases like serine proteases at C-terminal and to release toxin molecules of ~60 kDa. The toxin molecule is further trimmed at N- and C- terminal ends to release 578 amino acid molecule The active toxin molecule is composed of three domains each responsible for a particular function in the insect midgut. Sequential action of these domains results in disruption of ionic balance and paralysis of midgut membrane cells. Secondary structures of Bt proteins have been determined for three toxins through X-ray crystallography. Since primary amino acid composition determines the final structure of a protein, closely related proteins, Cry1Aa and Cry3A have super imposable structures with similar mode of action. The tertiary structure of d-endotoxin is comprised of three distinct functional domains connected by a short conserved sequence. Phylogenetic analysis of the domains of d-endotoxins revealed that domain I is the most conserved and domain II is hyper variable among all d-endotoxins (Bravo, 1997).

Traditional bioassays on larvae of insect species show the symptoms and efficacy of Cry toxins on insect larvae. However, an understanding of the molecular mechanisms and site of action of these toxins need clear picture of the structure of Cry toxin. Site directed mutagenesis experiments help in introducing known modifications in genes that encode proteins of interest and producing the proteins in large amount in bacteria (like E. coli) for further analysis. The purified proteins are analyzed for structural modifications and corresponding functional changes using various in vitro techniques like; i) pore forming capacity in artificial membranes and measuring ion channel conductance, ii) binding of toxins to brush border membrane vesicles isolated from susceptible larval midgut using labelled toxins, iii) stability of proteins using proteases like trypsin and larval midgut juice, iv) in vitro toxicity analysis on larvae, v) in vitro toxicity analysis in insect cell lines-lawn assay and vi) prediction of secondary structural changes using CD spectrometry.

Domain I is located in the N-terminal part of the toxin molecule. It comprises of six a-helices with one hydrophobic central helix (a5). It has been known that several other bacterial toxins make pores in the cell membranes (i.e. Colicin A, Diphtheria toxin B subunit and Pseudomonas exotoxin (Parker and Pattus, 1993). pH dependent ion channel formation is a common mode of action of these bacterial toxins. Similar kind of pH dependent conformational changes occurs with Cry toxins also. Model artificial membranes like liposomes, planar lipid bilayers and phosphatidylcholine vesicles are used to assess the pore forming ability of the toxins under in vitro conditions. It was clearly demonstrated that Cry1Ac and Cry3A toxins are involved in ion channel formation in lipid bilayers. Later isolated N-terminal fragment from Cry1Ac toxin was able to form pore on PLB membrane. This was confirmed when domain I of Cry3B2 toxin was analyzed. The pore forming activity of the domain I is also dependent on other domains of the toxin molecule.

Amino acid sequence of Domain II of d-endotoxins is hypervariable and is made of three b-sheets anti-parallel to Domain I. These anti parallel b-sheets form hairpin structures with variable length. These surface exposed hairpin structures are identified as specificity determining regions. It was attributed that the specificity differences among toxins is due to domain II variability. Receptor binding models showed that toxins sharing high homology in the loops of domain II recognize same receptor molecules in larval midgut. BBMV and immunoblotting with labelled toxins showed direct correlation between toxicity and binding of toxins to the midgut receptors. Binding of toxin to the receptor is mediated by a two-step mechanism involving initial reversible biding followed by irreversible binding and membrane insertion. Receptor bound toxin molecule could facilitate additional toxin-toxin interactions. Thus the toxins insert themselves as oligomers. d-endotoxin-binding receptors in the larval midgut are identified as glycoprotein molecules. It has been supported by X-ray crystallographic data that domain II loops showed immunogloblin like structural folds (Li et al. 1991).

Domain III is located in C-terminal part end of the active toxin molecule. It is made of two anti- parallel b-sheets into sandwich structure. Initially it was believed that it is involved in protecting the toxin against insect midgut protease. But from the studies on b-strand structure of other protein molecules it could be assumed that domain III b-sandwich of d-endotoxin can be take part in other functions such as receptor binding and specificity determination and ion channel gating (Schnepf et al. 1998). From X-ray crystallographic studies, salt bridges and hydrogen bonding between domains III and I have been detected. Binding studies using reciprocal hybrids made by exchanging third domain of Cry1Aa with that of Cry1Ac on BBMV showed receptor binding is located in the third domain.

Site directed mutagenesis and domain exchange studies on different d-endotoxins demonstrated the function of each domain. Protein engineering thus not only reveal the mechanism by which d-endotoxins work, but it can also generate toxins with enhanced toxicity. These toxins could be used in resistant management as alternative tools for the toxins already in use to which insect may became resistant by modifying the receptors.

References

BRAVO, A. Phylogenetic relationships of Bacillus thuringiensis delta-endotoxin family proteins and their functional domains. Journal of Bacteriology, May1997, vol. 179, no. 9, p. 2793­2801.

CRICKMORE, N.; ZEIGLER, D.R.; FEITELSON, J.; SCHNEPF, E.; VAN RIE, J.; LERECUS, D.; BAUM, J. and DEAN, D.H. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiology and Molecular Biology Reviews, September 1998, vol. 62, no. 3, p. 807-813.

HÖFTE, H. and WHITELEY, H.R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiological Review, June 1989, vol. 53, no. 2, p. 242­255.

LI, J.; CARROLL, J. and ELLAR, D.J. Crystal Structure of Insecticidal d-Endotoxin from Bacillus thuringiensis at 2.5 Å resolutions. Nature, October 1991, vol. 353, no. 6347, p. 815-821.

PARKER, M.W. and PATTUS, F. Rendering a membrane protein soluble in water: a common packing motif in bacterial protein toxins. Trends Biochemical Sciences, October 1993, vol. 18, no. 10, p. 391-395.

SCHNEPF, H.E.; CRICMORE, N.; VANRIE, J.; LERECLUS, D.; BAUM, J.; FEITELSON, J.; ZFIDER, D.R. and DEAN. D.H. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews, September 1998, vol. 62, no. 3, p. 775­806.