Interaction of the Bacillus sphaericus mosquito larvicidal proteins

Abstract

Genes for 51.4- and 41.9-kDa insecticidal proteins of Bacillus sphaericus were separately cloned and expressed in Escherichia coli. Both proteins were required for toxicity. Approximately equal numbers of cells containing the 51.4- and 41.9-kDa proteins produced the greatest toxicity; excess 41.9-kDa protein did not affect toxicity, whereas excess 51.4-kDa protein reduced activity. Larvae were killed when 41.9-kDa protein was fed up to 24 h after the 51.4-kDa protein, but not when the order of feeding was reversed. Radiolabelled toxins bound in approximately equal amounts to the gastric caecum and posterior midgut of Culex quinquefasciatus larvae. Radiolabelled 51.4-kDa protein was rapidly degraded by ca. 12-13 kDa in the larval gut, while 41.9-kDa protein was degraded by 1-2 kDa. Nonreduced toxin extracted from B. sphaericus produced a band on SDS-PAGE of ca. 68-74 kDa that contained both 51.4- and 41.9-kDa proteins based on sequence analysis, and a band of ca. 51 kDa that contained primarily 41.9-kDa protein. Escherichia coli containing 51.4-kDa protein enhanced toxicity of the latter eluted SDS-PAGE band. These proteins may associate very strongly, and trace amounts of 51.4-kDa protein in preparations of 41.9-kDa protein from B. sphaericus may be responsible for the previously reported toxicity of the latter.

Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains

“…Consistent with previous results (Broadwell et al, 1990a, b; Davidson et al, 1990;Oei et al, 1990), when P51 or P42 were fed alone to larvae they were not toxic, even at a concentration of 250 pg ml-l . A combination of the two proteins was toxic to larvae, confirming that the C. Oei, J .…”

“…The surviving larvae were counted after Binding of B. sphaericus toxin to mosquito gut 1519 24 h. Previous results showed that maximum toxicity occurred when the 51 and 42 kDa proteins were present in approximately equal concentrations (Broadwell et al, 1990b;Davidson et al, 1990). Based on this observation, the bioassays were designed such that the 51 kDa protein and its deletion derivatives were fed to the larvae in the presence of an equivalent concentration of 42 kDa protein and vice versa.…”

“…Larvicidal activity in highly toxic strains of B. sphaericus is principally associated with a parasporal crystal which contains several proteins, of which the 51 and 42 kDa proteins (designated P51 and P42 respectively in this study) are the best characterized (Davidson, 1983;Baumann et al, 1985;Berry et al, 1991 ;Baumann et al, 1991). When assayed against mosquito larvae, both proteins were found to be necessary for toxicity, indicating that the B. sphaericus larvicide may act as a binary toxin (Broadwell et al, 1990a, 6;Davidson et al, 1990;Baumann et al, 1991). Following ingestion of the toxin by larvae, the 51 and 42 kDa proteins are processed to lower molecular mass forms of approximately 44 and 39 kDa respectively (Broadwell & Baumann, 1987;Davidson et al, 1987aDavidson et al, , 1990Aly et al, 1989;Broadwell et al, 1990a).…”

“…When assayed against mosquito larvae, both proteins were found to be necessary for toxicity, indicating that the B. sphaericus larvicide may act as a binary toxin (Broadwell et al, 1990a, 6;Davidson et al, 1990;Baumann et al, 1991). Following ingestion of the toxin by larvae, the 51 and 42 kDa proteins are processed to lower molecular mass forms of approximately 44 and 39 kDa respectively (Broadwell & Baumann, 1987;Davidson et al, 1987aDavidson et al, , 1990Aly et al, 1989;Broadwell et al, 1990a). Studies using binary toxin purified from the spores of B. sphaericus, a mixture of the 5 1 and 42 kDa proteins, have shown that toxin binding occurs in the gastric caecum and posterior midgut of Culex larvae (Davidson, 1988).…”

“…Studies using binary toxin purified from the spores of B. sphaericus, a mixture of the 5 1 and 42 kDa proteins, have shown that toxin binding occurs in the gastric caecum and posterior midgut of Culex larvae (Davidson, 1988). However, purification of the individual proteins from B. sphaericus has proved to be problematic (Broadwell et a/., 19906;Davidson et al, 1990), and hence the respective roles of the 51 and 42 kDa proteins in toxin binding have remained unresolved.…”