The accumulation of Cry toxin protein inside the bacillary body leads to the formation of crystal inclusions in B. thuringiensis. The possible mechanism behind the accumulation of crystal protein by B. thuringiensis is the expression of crystal protein gene via a strong promoter. The promoter is active during both in logarithmic and in stationary phase resulting in the overproduction of the crystal protein. The strains of Bacillus sp. respond to nutritional deficient conditions by either becoming; dormant or by sporulation. On this basis, the cry genes that are expressed in the stationary phase can be divided into two categories; cry genes contingent on spore formation and cry genes that are not related to spore formation (Agaisse and Lereclus, 1995).
The endospore formation in Bacillus sp. takes place in a sporangium which consists of two cellular compartments called as the forespore and the mother cell. The process of endospore development is regulated by a primary sigma factor of vegetative cell and five other sigma factors that appear successively during sporulation. The recognition of gene promoters selectively depends upon the binding of these sigma factors to RNA Polymerase (Helmann et al., 1988, Moran, 1993). The Cry toxin secreted by cryIA gene is an example of cry gene expressed in mother cell of B. thuringiensis and is associated with sporulation. In addition to sigma factors, two overlapping promoters (BtI and BtII) for cryIA were mapped by Wong et al., (1983) which is active during different time points in sporulation phase. The initiation of transcription by the RNA Polymerase from these two promoters carries separate sigma factors (Calogero et al., 1989, Brown and Whiteley, 1990). The different sigma factor mutants of cryIA-lacZ fusion in B. thuringiensis gave either diminished or no β-galactosidase activity. However, a very high β-galactosidase activity was observed in wild cryIA-lacZ fusion of B. thuringiensis, which suggests that strong promoters are involved in the expression of cryIA gene (Bravo et al., 1996). Various studies have shown that some cry genes in different strains of B. thuringiensis carry only BtI or both the promoters and others have regions similar to those in BtI and BtII promoters (Brown and Whiteley, 1988, Brown, 1993, Dervyn et al., 1995, Hajime et al., 1993). Thus, these cry genes can be categorized as sporulation dependent cry genes.
The cryIIIA gene is an example of sporulation independent cry genes as its expression was independent of the sigma factors present during sporulation in both B. thuringiensis and B. subtilis cry gene (Agaisse and Lereclus, 1994a, Salamitou et al., 1996). The expression of cryIIIA was increased in mutant strains of B. thuringiensis which were not able to commence sporulation. (Lereculus et al., 1995). Smith, (1993) identified two different regulators in stationary phase which are required for gene expression in B. subtilis.
Further, the location of a gene on plasmid affects its expression level as the copy number of plasmid has been exploited in recent times for overexpression of a protein. The cry genes are also carried by plasmids which naturally lead to a huge quantity of toxins in various B. thuringiensis strains. The presence of different cry genes varies in different strains of B. thuringiensis which differs in size and shape of the crystal (Lereculus et al., 1993). However, the cloning of cryIAc gene in a strain having other cryI genes lead to lower production of Cry protein as compared to its cloning in cry− strain while there was no decline in its expression when cloned into strains with cryIIIA gene (Baum et al., 1990, Lecadet et al., 1992, Lereculus et al., 1992). Therefore, the expression of cry gene may not be related to the high copy number of plasmid.
mRNA has a specific half life and its degradation affects the expression of a gene. The increase in expression of a protein requires production of a stable mRNA as evident from ompA mRNA that encodes a membrane protein in Escherichia coli having longer half life of 20 minutes as compared to 2-3 minutes for the other mRNAs in E. coli (Nilsson et al., 1984). The mRNAs that encode crystal protein in B. thuringiensis have a half life of ~10 minutes (Glatron et al., 1972). In addition to that, the 3' terminal region of the cryIAa gene acts as a positive retro-regulator in B. thuringiensis as its heterologous expression with a gene increased the mRNAs half life as well as the expression (Wong and Chang, 1986, Wong et al., 1983). The terminal region consists of inverted repeats forming stem loop structure that prevents degradation of mRNA from exonucleases like exoribonuclease PNPase in E. coli (Causton et al., 1994). An ~600bp upstream promoter containing two distinct regions is involved in expression of the cryIIIA gene in B. thuringiensis (De Souza et al., 1993, Agaisse and Lereclus, 1994b). The upstream region was reported to be involved in transcription while the downstream region acts as a 5' mRNA stabilizer which increased the stability of mRNA as well as the expression of protein. (Bechhoffer et al., 1993, Hue et al., 1995, Agaisse and Lereculus, 1996).
Therefore, the present study was undertaken to construct promoter/terminator based expression system harboring promoter and terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1.