Lipid-induced Pore Formation of the Bacillus thuringiensis Cry1Aa Insecticidal Toxin

2001 ◽  
Vol 180 (3) ◽  
pp. 195-203 ◽  
Author(s):  
V. Vié ◽  
N. Van Mau ◽  
P. Pomarède ◽  
C. Dance ◽  
J.L. Schwartz ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Insecticidal Toxin

scholarly journals Cysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

2008 ◽  
Vol 74 (9) ◽  
pp. 2565-2572 ◽  
Author(s):  
Frédéric Girard ◽  
Vincent Vachon ◽  
Gabrielle Préfontaine ◽  
Lucie Marceau ◽  
Yanhui Su ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Membrane Vesicles ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Insect Larvae ◽  
Insecticidal Toxin ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis ◽  
Pore Lining

ABSTRACT Helix α4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the α4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca

2010 ◽  
Vol 104 (3) ◽  
pp. 203-208 ◽  
Author(s):  
Jean-Frédéric Brunet ◽  
Vincent Vachon ◽  
Mireille Marsolais ◽  
Jeroen Van Rie ◽  
Jean-Louis Schwartz ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Insecticidal Toxin

scholarly journals Helix 4 Mutants of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa Display Altered Pore-Forming Abilities

2004 ◽  
Vol 70 (10) ◽  
pp. 6123-6130 ◽  
Author(s):  
Vincent Vachon ◽  
Gabrielle Préfontaine ◽  
Cécile Rang ◽  
Florence Coux ◽  
Marc Juteau ◽  
...  
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Membrane Vesicles ◽  
Amino Acid Position ◽  
Site Directed Mutagenesis ◽  
Insecticidal Toxin ◽  
Mutant Proteins ◽  
Charged Residues ◽  
Α Helix

ABSTRACT The role played by α-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-d-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that α-helix 4 plays a crucial role in the mechanism of pore formation.

scholarly journals No Evidence of an Impact on the Rhizosphere Diazotroph Community by the Expression of Bacillus thuringiensis Cry1Ab Toxin by Bt White Spruce

2007 ◽  
Vol 73 (20) ◽  
pp. 6577-6583 ◽  
Author(s):  
Josyanne Lamarche ◽  
Richard C. Hamelin
Keyword(s):  
Bacillus Thuringiensis ◽  
Bacterial Communities ◽  
White Spruce ◽  
Sequencing Analysis ◽  
Nitrogen Fixing ◽  
Cloning And Sequencing ◽  
Insecticidal Toxin ◽  
Soil Bacterial Communities ◽  
Cry1ab Toxin ◽  
Natural Stands

ABSTRACT Nitrogen fixation is one of the most important roles played by soil bacterial communities, as fixation supplies nitrogen to many ecosystems which are often N limited. As impacts on this functional group of bacteria might harm the ecosystem's health and reduce productivity, monitoring that particular group is important. Recently, a field trial with Bt white spruce, which constitutively expresses the Cry1Ab insecticidal toxin of Bacillus thuringiensis, was established. The Bt white spruce was shown to be resistant to spruce budworm. We investigated the possible impact of these genetically modified trees on soil nitrogen-fixing bacterial communities. The trial consisted of untransformed controls, GUS white spruce (transformed with the β-glucuronidase gene), and Bt/GUS white spruce (which constitutively expresses both the Cry1Ab toxin and β-glucuronidase) in a random design. Four years after planting, soil samples from the control and the two treatments from plantation as well as from two natural stands of white spruce were collected. Diazotroph diversity was assessed by extracting soil genomic DNA and amplifying a region of the nitrogenase reductase (nifH) gene, followed by cloning and sequencing. Analysis revealed that nitrogen-fixing communities did not differ significantly among the untransformed control, GUS white spruce, and Bt/GUS white spruce. Nevertheless, differences in diazotroph diversity were observed between white spruce trees from the plantation site and those from two natural stands, one of which grew only a few meters away from the plantation. We therefore conclude, in the absence of evidence that the presence of the B. thuringiensis cry1Ab gene had an effect on diazotroph communities, that either site and/or field preparation prior to planting seems to be more important in determining diazotroph community structure than the presence of Bt white spruce.

scholarly journals Distribution of Genes Encoding Putative Virulence Factors and Fragment Length Polymorphisms in the vrrA Gene among Brazilian Isolates of Bacillus cereus and Bacillus thuringiensis

2005 ◽  
Vol 71 (12) ◽  
pp. 8107-8114 ◽  
Author(s):  
Viviane Zahner ◽  
Diana Aparecida Cabral ◽  
Adriana Hamond Régua-Mangia ◽  
Leon Rabinovitch ◽  
Gaétan Moreau ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Bacillus Cereus ◽  
Virulence Factors ◽  
Fragment Length ◽  
Tandem Repeats ◽  
Variable Region ◽  
Single Species ◽  
Variable Number ◽  
Insecticidal Toxin ◽  
Reading Frame

ABSTRACT One hundred twenty-one strains of the Bacillus cereus complex, of which 80 were isolated from a variety of sources in Brazil, were screened by PCR for the presence of sequences (bceT, hblA, nheBC, plc, sph, and vip3A) encoding putative virulence factors and for polymorphisms in variable-number tandem repeats (VNTR), using a variable region of the vrrA open reading frame as the target. Amplicons were generated from isolates of B. cereus and Bacillus thuringiensis for each of the sequences encoding factors suggested to play a role in infections of mammals. Intriguingly, the majority of these sequences were detected more frequently in Bacillus thuringiensis than in B. cereus. The vip3A sequence, which encodes an insecticidal toxin, was detected exclusively in B. thuringiensis. VNTR analysis demonstrated the presence of five different fragment length categories in both species, with two of these being widely distributed throughout both taxa. In common with data generated from previous studies examining European, Asian, or North American populations, our investigation of Brazilian isolates supports the notion that B. cereus and B. thuringiensis should be considered to represent a single species.

scholarly journals Monitoring of Bacillus Thuringiensis Cry3Aa Toxin Pore Formation using Artificial Bilayer Array with Fused Brush Border Membrane Vesicles from Colorado Potato Beetle Larvae

2017 ◽  
Vol 112 (3) ◽  
pp. 523a
Author(s):  
Ekaterina Zaitseva ◽  
Gerhard Baaken ◽  
Victor M. Ruiz-Arroyo ◽  
Inmaculada García-Robles ◽  
Camila Ochoa-Campuzano ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Colorado Potato Beetle ◽  
Brush Border ◽  
Pore Formation ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Potato Beetle

scholarly journals Investigation of the pore-forming mechanism of a cytolytic δ-endotoxin from Bacillus thuringiensis

2003 ◽  
Vol 374 (1) ◽  
pp. 255-259 ◽  
Author(s):  
Boonhiang PROMDONKOY ◽  
David J. ELLAR
Keyword(s):  
Bacillus Thuringiensis ◽  
Blood Cells ◽  
Pore Formation ◽  
Response Curve ◽  
Effect Of Temperature ◽  
Membrane Insertion ◽  
Free Solution ◽  
Forming Mechanism ◽  
Close Proximity ◽  
Membrane Bound

Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose–response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 °C were transferred to a toxin-free solution at 37 °C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.

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