Open Access
The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles on toxin oligomerization and pore formation
(American Chemical Society, 2011) Rodríguez Almazán, Claudia; Ruiz de Escudero Fuentemilla, Íñigo; Cantón, Pablo Emiliano; Muñoz Garay, Carlos; Pérez, Claudia; Gill, Sarjeet S.; Soberón, Mario; Bravo, Alejandra; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a β-sheet. Regarding to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the β-sheet allowing insertion of β-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects in Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, N-terminal region is able to induce aggregation of Cyt1Aa toxin in solution. Finally, Cterminal domain composed mainly of β-strands, is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation while the C-terminal domain in the interaction of the toxin with the lipid membrane.
Open Access
Potential of the Bacillus thuringiensis toxin reservoir for the control of Lobesia botrana (Lepidoptera: tortricidae), a major pest of grape plants
(American Society for Microbiology, 2006) Ruiz de Escudero Fuentemilla, Íñigo; Estela, Anna; Escriche, Baltasar; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua, FP2000-5497
The potential of Bacillus thuringiensis Cry proteins to control the grape pest Lobesia botrana was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from B. thuringiensis, was the protein most toxic to L. botrana larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 μg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 μg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of B. thuringiensis sprays or expressed together in transgenic plants.
Open Access
Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism
(Elsevier, 2011) Cantón, Pablo Emiliano; Zanicthe Reyes, Esmeralda; Ruiz de Escudero Fuentemilla, Íñigo; Bravo, Alejandra; Soberón, Mario; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop 6–E K198A, E204A and 7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop -8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.
Open Access
UV protection and insecticidal activity of microencapsulated Vip3Ag4 protein in Bacillus megaterium
(Elsevier, 2024-06-17) Palma Dovis, Leopoldo; Ruiz de Escudero Fuentemilla, Íñigo; Mañeru Oria, Francisco Javier; Berry, Colin; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
In this study, secretable Vip3Ag4 protein was encapsulated in Bacillus megaterium and used for quantitative bioassays, in order to determine the UV photoprotective capacity of the cell, for preventing inactivation of the insecticidal activity of the protein. The non-encapsulated and purified protein was exposed to the UV light showing a LC50 of 518 ng/cm2 against Spodoptera littoralis larvae, whereas the exposed encapsulated protein exhibited 479 ng/cm2. In addition to the capability to accumulate Vip3 proteins for the development of novel insecticidal formulates, the B. megaterium cell has demonstrated to provide moderate protection against the deleterious action of UV light.
Open Access
Vip3C, a novel class of vegetative insecticidal proteins from Bacillus thuringiensis
(American Society for Microbiology, 2012) Palma Dovis, Leopoldo; Hernández Rodríguez, C.; Maeztu Martínez, Mireya; Hernández Martínez, Patricia; Ruiz de Escudero Fuentemilla, Íñigo; Escriche, Baltasar; Muñoz Labiano, Delia; Rie, Jeroen van; Ferré, Juan; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Three vip3 genes were identified in two Bacillus thuringiensis Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 μg/cm2.