Insecticidal Activity and Processing in Larval Gut Juices of Genetically Engineered 130-kDa Proteins of<i>Bacillus thuringiensis</i>subsp.<i>aizawai</i>

Nakamura, Keiko; Murai-Nishioka, Rika; Shimizu, Masatoshi; Oshie, Kazuyuki; Mikitani, Kenichi; Oeda, Kenji; Ohkawa, Hideo

doi:10.1271/bbb.56.1

Cited by 16 publications

(3 citation statements)

References 9 publications

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“…Similarly, replacements of charged D670 and E673 residues of block 5 (corresponding to D608 and E611 in CryIC) result in CryIVA toxins with reduced conformational stability (Nishimoto et al 1994). In common with the I610T exchange in the B4 mutant, a R610Q replacement in CryIA(b) (corresponding to K609 of CryIC; Nakamura et al 1992) affects the common DR/KIE sequence of block 5 (Fig. 1) and abolishes protease resistance.…”

Section: Discussionmentioning

confidence: 99%

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

Strizhov¹,

Keller

Konez-Kálmán³

et al. 1996

Mol Gen Genet

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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.

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Section: Discussionmentioning

confidence: 99%

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

Strizhov¹,

Keller

Konez-Kálmán³

et al. 1996

Mol Gen Genet

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“…In the case of @IA(b), upon proteolytic digestion, the protease-resistant 60 kDa fragment cannot be produced from the altered protoxin carrying the mutation at Arg601, which correspond to Lys671 of block 5 of CryIVA [23]. However, upon treatment with trypsin or gut extracts, the 45 kDa fragment can be produced from the 130 kDa protein of the mutant CryIVA-Lys671Ala (data not shown).…”

Section: Discussionmentioning

confidence: 92%

Functional analysis of block 5, one of the highly conserved amino acid sequences in the 130‐kDa CryIVA protein produced by Bacillus thuringiensis subsp. israelensis

et al. 1994

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There are five amino acid sequences highly conserved among Bacillus thuringiensis δ‐endotoxins. We have changed the amino acid residues in block 5, one of the conserved sequences, of CryIVA. When the amino acid residues with charged side chains were replaced by others, the amount of production of the altered CryIVA protein was markedly decreased. It is suggested that the decrease is caused by the unstable conformation of the altered CryIVA protein molecule, as judged by digestion with trypsin and thermolysin. On the other hand, the substitution of amino acid residues in block 5 did not affect the insecticidal activity of CryIVA. These results strongly suggest that block 5 of CryIVA is one of the stability‐determining elements of the protoxin molecule.

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“…The CrylC e~stals produced from the E. coli recombinant at 37 ° C were predonfinantly amorphous shaped (not shown), possibly due to temperature effects on protein assembly (29). Other groups also have documented the presence of small peptides associated with various toxins expressed in recombinant E. coil (10,27,29). Eater fractions contained CryIC purified to a single protein band as determined by SDS-PAGE.…”

Section: Discussionmentioning

confidence: 94%

Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp. midgut epithelial cells in vitro

Wang

McCarthy

1997

In Vitro Cell.Dev.Biol.-Animal

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A sensitive lactate dehydrogenase (LDH) assay was modified to determine the cytolytic activity of Bacillus thuringiensis CryIC and CryIAc delta endotoxins to viable collagenase-dissociated midgut epithelial cells (MEC) from larvae of Spodoptera frugiperda and Spodoptera exigua. The MEC preparations from these Spodoptera sp. consisted predominantly of columnar cells (65-75%) and goblet cells (25-35%). Time course microscopy experiments indicated that only the columnar cells became swollen during CryIC toxin incubation. Also, comparative cytotoxicity studies were run with cell lines of nonmidgut origin established from S. frugiperda (SF21AE) and S. exigua (SEUCR1A). Optimum conditions for the cytotoxicity assay were similar for MEC and cell lines of both species, and were met in an assay in which 0.1-ml cell concentrations (8.5 +/- 0.5 x 10(4) cells) were incubated with toxin dilutions (0.01-20 micrograms) for 1 h at 24 degrees C at a final pH of 7.8. The Spodoptera sp. MEC were twofold more sensitive to CryIC (68% lysis) than CryIAc (32% lysis) at optimum toxin levels (2.5-5 micrograms). Also, the SEUCR1A cells were more sensitive (2.3-fold) to CryIC (70% lysis) than CryIAc (30% lysis) at optimum toxin levels of 5-10 micrograms. The SF21AE cells, however, were twofold less sensitive to CryIC (30% lysis) than SEUCR1A cells and response to CryIAc and CryIC was similar. Immunoblot analysis of either Spodoptera sp. MEC or brush border membrane vesicles (BBMV) identified seven CryIC binding proteins with molecular mass of 137, 120, 115, 68, 65, 63, and 45 kDa. Occasionally, a 148-kDa protein band was observed. The CryIAc toxin bound to two proteins on MEC and BBMV with molecular mass of 137 and 120 kDa.

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Insecticidal Activity and Processing in Larval Gut Juices of Genetically Engineered 130-kDa Proteins ofBacillus thuringiensissubsp.aizawai

Cited by 16 publications

References 9 publications

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

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

Functional analysis of block 5, one of the highly conserved amino acid sequences in the 130‐kDa CryIVA protein produced by Bacillus thuringiensis subsp. israelensis

Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp. midgut epithelial cells in vitro

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