Contents - Faperta

264 Biotechnological Approaches for Pest Management and Ecological Sustainability
which have been designated as IS231 and IS232 (Lereclus et al., 1992; Lysenko, 1983), and
belong to the IS4 and IS21 family of insertion sequences, respectively (Rezsohazy et al.,
1993; Menou et al., 1990). In B. thuringiensis subsp. israelensis, IS231W is adjacent to the
cry11Aa gene. IS231-related DNA sequences have also been found in strains of B. cereus
Frankland and B. mycoides Flugge (Leonard, Chen, and Mahillon, 1997). IS240 is invariably
present in dipteran-active strains (Rezsohazy et al., 1993). Insertion sequences have also
been found upstream of cry1Ca (Smith et al., 1994) and downstream of cry2Ab (Hodgman
et al., 1993). A transposable element designated as Tn5401 has been isolated from a
coleopteran-active B. thuringiensis (Baum, 1994), which is located downstream of the cry3Aa
gene. Tn4430 mediates the transfer of nonconjugative plasmids by a conduction process
(Green, Battisti, and Thorne, 1989).
Gene Expression and Cry Structure
A common characteristic of all cry genes is their expression during the stationary phase,
and their products accumulate in the cell to form a crystal inclusion, which accounts for 20
to 30% of the dry weight of the sporulated cells. Crystal protein synthesis and accumulation
in B. thuringiensis are controlled by a variety of mechanisms. Sporulation is controlled
by successive activation of sigma factors, which bind the core RNA polymerase to direct
the transcription from sporulation-specifi c promoters. The cry1Aa gene is a sporulationdependent
cry gene expression in the mother cell. Two transcription start sites have been
mapped (BtI and BtII) (Wong, Schnepf, and Whiteley, 1983), of which BtI is active between
T 2 and T 6 stages of sporulation, while BtII is active from T 5 onwards. In vitro transcription
experiments have indicated that at least two other cry genes (cry1Ba and cry2Aa) contain
either BtI alone or BtI with BtII (Brown and Whiteley, 1988).
The expression of cry genes is considered to be sporulation dependent (Poncet et al.,
1997). However, low levels of transcription of cry4Aa, cry4Ba, and cry11Aa genes have been
detected during the transition phase (Yoshisue et al., 1995), and may be controlled by RNA
polymerase (Poncet et al., 1997). The cry3Aa gene from B. thuringiensis var. tenebrionis is
expressed during the vegetative phase, although to a lesser extent than during the stationary
phase (De Souza, Lecadet, and Lereclus, 1993; Malvar, Gawron Burke, and Baum, 1994;
Sekar, 1988). The cry3Aa expression increases in mutant strains incapable of initiating sporulation
(Agaisse and Lereclus, 1994; Malvar and Baum, 1994; Lereclus et al., 1995; Salamitou
et al., 1996). The cry3Aa expression is activated by a non-sporulation-dependent mechanism
during the transition from exponential growth to the stationary phase (Agaisse and
Lereclus, 1994; Salamitou et al., 1996).
The toxin proteins generally form crystalline inclusions in the mother cell, and depending
on protoxin composition, the crystals have various forms: bipyramidal (Cry1), cuboidal
(Cry2), fl at rectangular (Cry3A), irregular (Cry3B), spherical (Cry4A and Cry4B), and rhomboidal
(Cry11A) (Schnepf et al., 1998). The ability of the protoxins to crystallize may
decrease their susceptibility to premature proteolytic degradation. However, the crystals
have to be solubilized rapidly in the insect gut to become biologically active. Structure and
solubility characteristics of the crystal presumably depend on secondary structure of the
protoxin, energy of the disulfi de bonds, and presence of additional B. thuringiensis-specifi c
components. The cysteine-rich C-terminal half of the Cry1 protoxins possibly contributes
to crystal structure through the formation of disulfi de bonds (Bietlot et al., 1990). The
cysteine-rich C-terminal region is absent from the 73 kDa Cry3A protoxins and this protein
forms a fl at, rectangular crystal inclusion in which the polypeptides do not appear to be
linked by disulfi de bridges (Bernhard, 1986). Analysis of the three-dimensional structure