Insect Control: Biological and Synthetic Agents - Index of

288 8: Mosquitocidal B. sphaericus: Toxins, Genetics, Mode of Action, Use, and Resistance Mechanisms
The replacement of alanine at some sites in the
N- and C-terminal regions of both the BinA and
BinB peptides results in the total loss of mosquitocidal
activity. Surprisingly, toxicity is restored by
mixing two nontoxic derivatives of the same
peptide; i.e., one mutated at the N-terminal end
and the other mutated at the C-terminal end of
either BinA or BinB (Shanmugavelu et al., 1998).
Thus, the altered binary toxins can functionally
complement each other by forming oligomers.
The aggregation of both BinB and BinA has been
analyzed by expressing crystal toxin components
separately or together in homologous or heterologous
Bacillus hosts. The proteins form amorphous
inclusions when expressed independently in
B. subtilis and in B. sphaericus or B. thuringiensis
crystal-negative hosts (Charles et al., 1993; Nicolas
et al., 1993). In contrast, crystals similar to those
produced by natural B. sphaericus strains are found
when the two genes are simultaneously expressed
in either B. sphaericus or B. thuringiensis (Charles
et al., 1993; Nicolas et al., 1993). No crystals can
be detected in B. subtilis when both genes are
present, unless they are fused, eliminating the intergenic
region (Charles et al., 1993). These results
suggest that B. sphaericus and B. thuringiensis, but
not B. subtilis, contain factors that help stabilize
their protein and subsequent crystallization.
In vivo, BinA is slowly converted into a stable
39-kDa protein, whereas BinB is rapidly converted
into a stable 43-kDa fragment (Baumann et al.,
1985; Broadwell and Baumann, 1987). In vitro deletion
analysis was used to delineate the minimal
active fragments of both proteins, which indeed
correspond to the activated fragments (Broadwell
et al., 1990b; Clark and Baumann, 1990; Oei et al.,
1990; Sebo et al., 1990). Thirty-two and 53 amino
acids, at the N- and C-termini of BinB, respectively,
can be eliminated without loss of toxicity (Clark and
Baumann, 1990); deletions of 10 and 17 amino
acids, at the N- and C-terminus of BinA, respectively,
result in a protein similar to the 39-kDa activated
fragment (Figure 2, top; Broadwell et al., 1990b).
Sub-cloning experiments have shown that BinA
alone is toxic for mosquito larvae (C. pipiens), although
the activity is weaker than that of crystals
containing both proteins (Nicolas et al., 1993).
In contrast, BinB alone is not toxic, although its
presence enhances the larvicidal activity of BinA,
suggesting synergy between the two polypeptides
(de la Torre et al., 1989; Broadwell et al., 1990b;
Oei et al., 1990; Nicolas et al., 1993). In vitro assays
confirmed that the activated form of BinA alone
is toxic for C. quinquefasciatus cells, whereas
BinB appears to be inactive; however, no synergy
between the components was observed in vitro
(Baumann and Baumann, 1991). Although the
simultaneous presence of both proteins appears
necessary for full toxicity, the differing activities of
the different B. sphaericus strains towards various
mosquito species depends on the origin of BinA, as
shown by analysis of in vitro mutated toxins (Berry
et al., 1993). Indeed, when amino acids were substituted
in a region centered around position 100 of
BinA, rendering BinA from strains IAB-59 and 2297
similar to that from strain 2362, the activity and
specificity of these mutant toxins towards Culex
and Aedes larvae was comparable, unlike the wildtype,
indicating that this region is involved in specificity.
Taken together, these observations suggest
that BinA is the most important determinant of
specificity and activity.
8.2.2. Mtx Toxins
Three types of Mtx toxin have been described
to date: Mtx1, Mtx2 and Mtx3, with molecular
weights of 97, 31.8 and 35.8 kDa, respectively
(Figure 2, bottom). The genes encoding these proteins
were initially cloned from the SSII-1 strain
(Thanabalu et al., 1991; Thanabalu and Porter,
1995; Liu et al., 1996a), which has a moderate
mosquitocidal activity (Table 1). In contrast to the
crystal toxin genes, mtx genes are expressed during
the vegetative growth phase, and sequences resembling
vegetative promoters from B. subtilis have
been found upstream from each gene (Thanabalu
et al., 1991; Thanabalu and Porter, 1995; Liu et al.,
1996a). lacZ fusion experiments confirmed
these findings (Ahmed et al., 1995). These proteins
possess short N-terminal leader sequences characteristic
of Gram-positive bacterial signal peptides
(Thanabalu et al., 1991; Thanabalu and Porter,
1995; Thanabalu and Porter, 1996). However,
these toxins have been found associated with the
cell membrane of B. sphaericus, indicating little or
no cleavage of the signal sequence. The mature
Mtx1 toxin can be further processed by gut proteases,
leading to two fragments of 27 and 70 kDa,
corresponding to the N- and C-terminal regions,
respectively (Thanabalu et al., 1992). The 70-kDa
fragment possesses three repeated regions of about
90 amino acids (Figure 2, bottom), the function
of which is unknown. The 27-kDa fragment contains
a short putative transmembrane domain.
These toxins do not display any similarity with
each other or with crystal proteins or any other
insecticidal proteins. In contrast, the 27-kDa fragment
shares weak sequence similarities with the
catalytic domains of various bacterial ADPribosyltransferases
(Figure 2, bottom; Thanabalu