Mutagenic Analysis of a Conserved Region of Domain III in the Cry1Ac Toxin of Bacillus thuringiensis

ABSTRACT We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.

[1]  J. Schwartz,et al.  Helix 4 of the Bacillus thuringiensis Cry1Aa Toxin Lines the Lumen of the Ion Channel* , 1999, The Journal of Biological Chemistry.

[2]  A. Aronson,et al.  Aggregation of Bacillus thuringiensisCry1A Toxins upon Binding to Target Insect Larval Midgut Vesicles , 1999, Applied and Environmental Microbiology.

[3]  L. Masson,et al.  One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[4]  X. J. Chen,et al.  Site-directed mutations in a highly conserved region of Bacillus thuringiensis delta-endotoxin affect inhibition of short circuit current across Bombyx mori midguts. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[5]  S. Sohi,et al.  An in vitro system for testing Bacillus thuringiensis toxins: the lawn assay. , 1990, Journal of invertebrate pathology.

[6]  H. R. Whiteley,et al.  Insecticidal crystal proteins of Bacillus thuringiensis , 1989 .

[7]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[8]  B. H. Knowles,et al.  Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ-endotoxins with different insect specificity , 1987 .

[9]  J. Schwartz,et al.  Restriction of intramolecular movements within the Cry1Aa toxin molecule of Bacillus thuringiensis through disulfide bond engineering , 1997, FEBS letters.

[10]  Y. Shai,et al.  The Assembly and Organization of the α5 and α7 Helices from the Pore-forming Domain of Bacillusthuringiensis δ-Endotoxin , 1995, The Journal of Biological Chemistry.

[11]  H. R. Whiteley,et al.  Insecticidal crystal proteins of Bacillus thuringiensis. , 1989, Microbiological reviews.

[12]  B. Tabashnik,et al.  Resistance to Toxins from Bacillus thuringiensis subsp. kurstaki Causes Minimal Cross-Resistance to B. thuringiensis subsp. aizawai in the Diamondback Moth (Lepidoptera: Plutellidae) , 1993, Applied and environmental microbiology.

[13]  W. Stiekema,et al.  Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots , 1996, Applied and environmental microbiology.

[14]  F. S. Walters,et al.  Ion channel activity of N-terminal fragments from CryIA(c) delta-endotoxin. , 1993, Biochemical and biophysical research communications.

[15]  J. Schwartz,et al.  Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation. , 1995, Journal of molecular biology.

[16]  H. Höfte,et al.  Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins , 1990, Applied and environmental microbiology.

[17]  C. Hofmann,et al.  Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin , 1997, Applied and environmental microbiology.

[19]  L. Masson,et al.  Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1. , 1990, The Biochemical journal.

[20]  H. R. Whiteley,et al.  Specificity-determining regions of a lepidopteran-specific insecticidal protein produced by Bacillus thuringiensis. , 1990, The Journal of biological chemistry.

[21]  J. Schwartz,et al.  Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers , 1993, The Journal of Membrane Biology.

[22]  N. Crickmore,et al.  Bacillus thuringiensis and Its Pesticidal Crystal Proteins , 1998, Microbiology and Molecular Biology Reviews.

[23]  D. Ellar,et al.  Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution. , 1991, Nature.

[24]  Y. Shai,et al.  Bacillus thuringiensis cytolytic toxin associates specifically with its synthetic helices A and C in the membrane bound state. Implications for the assembly of oligomeric transmembrane pores. , 1997, Biochemistry.

[25]  Y. Shai,et al.  The assembly and organization of the alpha 5 and alpha 7 helices from the pore-forming domain of Bacillus thuringiensis delta-endotoxin. Relevance to a functional model. , 1995, The Journal of biological chemistry.

[26]  D. Dean,et al.  Domain III exchanges of Bacillus thuringiensis CryIA toxins affect binding to different gypsy moth midgut receptors. , 1995, Biochemical and biophysical research communications.

[27]  M. G. Wolfersberger,et al.  Site-directed mutations in the third domain of Bacillus thuringiensis delta-endotoxin CryIAa affect its ability to increase the permeability of Bombyx mori midgut brush border membrane vesicles , 1996, Applied and environmental microbiology.

[28]  L. Masson,et al.  Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay system , 1994, Molecular microbiology.

[29]  D. Dean,et al.  Location of the Bombyx mori specificity domain on a Bacillus thuringiensis delta-endotoxin protein. , 1989, Proceedings of the National Academy of Sciences of the United States of America.