Mutagenic analysis of a conserved region of domain III in the Cry1ac toxin of Bacillus thuringiensis

L. Masson, Bruce E Tabashnik, A. Mazza, G. Préfontaine, L. Potvin, R. Brousseau, J. L. Schwartz

Research output: Contribution to journalArticle

41 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)194-200
Number of pages7
JournalApplied and Environmental Microbiology
Volume68
Issue number1
DOIs
StatePublished - 2002
Externally publishedYes

Fingerprint

Bacillus thuringiensis
toxin
Arginine
toxins
Lipid Bilayers
Biological Assay
mutants
Glycine
D-Aspartic Acid
toxicity
fold
arginine
aspartic acid
lipid bilayers
Site-Directed Mutagenesis
Ion Channels
Aspartic Acid
Larva
Insects
glycine (amino acid)

ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

Cite this

Mutagenic analysis of a conserved region of domain III in the Cry1ac toxin of Bacillus thuringiensis. / Masson, L.; Tabashnik, Bruce E; Mazza, A.; Préfontaine, G.; Potvin, L.; Brousseau, R.; Schwartz, J. L.

In: Applied and Environmental Microbiology, Vol. 68, No. 1, 2002, p. 194-200.

Research output: Contribution to journalArticle

Masson, L. ; Tabashnik, Bruce E ; Mazza, A. ; Préfontaine, G. ; Potvin, L. ; Brousseau, R. ; Schwartz, J. L. / Mutagenic analysis of a conserved region of domain III in the Cry1ac toxin of Bacillus thuringiensis. In: Applied and Environmental Microbiology. 2002 ; Vol. 68, No. 1. pp. 194-200.
@article{c79a0cf03a5c488db459f9110f744d42,
title = "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.",
author = "L. Masson and Tabashnik, {Bruce E} and A. Mazza and G. Pr{\'e}fontaine and L. Potvin and R. Brousseau and Schwartz, {J. L.}",
year = "2002",
doi = "10.1128/AEM.68.1.194-200.2002",
language = "English (US)",
volume = "68",
pages = "194--200",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "1",

}

TY - JOUR

T1 - Mutagenic analysis of a conserved region of domain III in the Cry1ac toxin of Bacillus thuringiensis

AU - Masson, L.

AU - Tabashnik, Bruce E

AU - Mazza, A.

AU - Préfontaine, G.

AU - Potvin, L.

AU - Brousseau, R.

AU - Schwartz, J. L.

PY - 2002

Y1 - 2002

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0036136953&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036136953&partnerID=8YFLogxK

U2 - 10.1128/AEM.68.1.194-200.2002

DO - 10.1128/AEM.68.1.194-200.2002

M3 - Article

VL - 68

SP - 194

EP - 200

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 1

ER -