title: A novel Bacillus thuringiensis (PS149B1) containing a Cry34Ab1/Cry35Ab1 binary toxin specific for the western corn rootworm Diabrotica virgifera virgifera LeConte forms ion channels in lipid membranes.
abstract: The binary Bacillus thuringiensis PS149B1 insecticidal crystal (Cry) protein is comprised of two components, Cry34Ab1, a 14-kDa protein, and Cry35Ab1, a 44-kDa protein, the combination of which forms a novel binary toxin active on western corn rootworm larvae. The permeabilizing behavior of the native binary toxin and its two individual components expressed as recombinant proteins was studied using calcein efflux determination in liposomes and by ion channel activity measurements in planar lipid bilayers (PLBs). Data obtained with solubilized native PS149B1 binary protein revealed it to be a pore-forming toxin that can permeabilize liposomes and form ion channels ( approximately 300-900 pS) in PLBs at pH 5.5 but not pH 9.0. The 14-kDa component of the toxin also formed ion channels ( approximately 15-300 pS) at pH 5.5 but did not insert easily in PLBs. While the 44-kDa moiety did seldomly form resolvable ion channels ( approximately 15-750 pS) in PLBs, it did destabilize the membranes. It showed pH-dependent truncation to a stable 40-kDa protein. The purified 40-kDa truncated product formed channels ( approximately 10-450 pS) in PLBs at pH 5.5. At that same pH, while a 3:1 molar mixture (14:44 kDa) of the individual components of the toxin induced channel activity that resembled that of the 14-kDa component alone, the 3:1 molar mixture of the 14-kDa component and 40-kDa truncated product induced channel activity ( approximately 20-800 pS) similar to that of PS149B1 in planar lipid bilayers. We conclude that the overall membrane permeabilization process of Cry34Ab1/Cry35Ab1 is a result of ion channel formation.
authors.affiliation: Biotechnology Research Institute, National Research Council, Montreal, Quebec, Canada H4P 2R2.
authors.forename: Luke
authors.initials: L
authors.lastname: Masson
authors.forename: George
authors.initials: G
authors.lastname: Schwab
authors.forename: Alberto
authors.initials: A
authors.lastname: Mazza
authors.forename: Roland
authors.initials: R
authors.lastname: Brousseau
authors.forename: Lena
authors.initials: L
authors.lastname: Potvin
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D001517:Coleoptera / Q000378:metabolism* / Q000382:microbiology*; D004553:Electric Conductivity; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D006460:Hemolysin Proteins; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011489:Protein Denaturation
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D005452:Fluoresceins; D006460:Hemolysin Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D010446:Peptide Fragments; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C007740:fluorexon