Mapping of the entomocidal fragment of Spodoptera-specific Bacillus thuringiensis toxin CryIC

Abstract

Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI proteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins.

Molecular Genetic Manipulation of Truncated Cry1C Protein Synthesis in Bacillus thuringiensis To Improve Stability and Yield

“…However, this report contained no data on the relative toxicity of Cry1C-t or full-length Cry1C molecules to S. littoralis. The sequence of the Cry1C-t used in the present study was identical to the sequence of the Cry1C-t described previously (19). Here, it is shown that Cry1C-t produced in B. thuringiensis is also toxic to larvae of the beet armyworm, S. exigua, although the toxicity of the truncated protein was 7-to 12.6-fold less than that of the full-length Cry1C (Table 3).…”

“…5), which were degraded to 31-kDa peptides (Fig. 3B), were possibly the translation products resulting from a second in-frame ATG codon, as noted previously (19).…”

“…It has been reported previously that Cry1C-t inclusions produced in E. coli were toxic to Spodoptera littoralis (19). However, this report contained no data on the relative toxicity of Cry1C-t or full-length Cry1C molecules to S. littoralis.…”

“…In previous studies it has been shown that truncated versions of Cry1 proteins are unstable and have little or no toxicity compared to the toxicity of full-length toxins (1,15,19,21). The instability is probably due to the inability of these proteins to fold and crystallize properly.…”

Susceptibility of a Field-Derived, Bacillus thuringiensis -Resistant Strain of Diamondback Moth to In Vitro-Activated Cry1Ac Toxin

“…Proteolytic activation of Cry1 protoxins involves the removal of peptide sequences from both N and C termini. The observations that a mutant toxin in which Nterminal cleavage is prevented has reduced toxicity (4) and that N-terminally truncated toxins are toxic to Escherichia coli expression hosts (4,9) suggest that N-terminal cleavage may be an important part of the toxic mechanism.…”