Expression of cry1Ab gene from a novel Bacillus thuringiensis strain SY49-1 active on pest insects

ABSTRACT In this study, the cry1Ab gene of previously characterized and Lepidoptera-, Diptera-, and Coleoptera-active Bacillus thuringiensis SY49-1 strain was cloned, expressed and individually tested on Ephestia kuehniella (Lepidoptera: Pyralidae) and Plodia interpunctella (Lepidoptera: Pyralidae) larvae. pET-cry1Ab plasmids were constructed by ligating the cry1Ab into pET28a (+) expression vector. Constructed plasmids were transferred to an Escherichia coli BL21 (DE3) strain rendered competent with CaCl2. Isopropyl β-D-1-thiogalactopyranoside was used to induce the expression of cry1Ab in E. coli BL21(DE3), and consequently, ∼130 kDa of Cry1Ab was obtained. Bioassay results indicated that recombinant Cry1Ab at a dose of 1000 µg g-1 caused 40% and 64% mortality on P. interpunctella and E. kuehniella larvae, respectively. However, the mortality rates of Bt SY49-1 strains' spore-crystal mixture at the same dose were observed to be 70% on P. interpunctella and 90% on E. kuehniella larvae. The results indicated that cry1Ab may be considered as a good candidate in transgenic crop production and as an alternative biocontrol agent in controlling stored product moths.

Characterization of a Mutant Bacillus thuringiensis d-endotoxin with enhanced stability and toxicity / Caracterización de una delta endotoxina mutante de Bacillus thuringiensis con estabilidad y toxicidad aumentadas

The centrally located a-helix 5 of Bacillus thuringiensis d-endotoxins is critical for insect toxicity through ion-channel formation. We analyzed the role of the highly conserved residue Histidine 168 (H168) using molecular biology, electrophysiology and biophysical techniques. Toxin H168R was ~3-fold more toxic than the wild type (wt) protein whereas H168Q was 3 times less toxic against Manduca sexta. Spectroscopic analysis revealed that the H168Q and H168R mutations did not produce gross structural alterations, and that H168R (Tm= 59 °C) was more stable than H168Q (Tm= 57.5 °C) or than the wt (Tm= 56 °C) toxins. These three toxins had similar binding affinities for larval midgut vesicles (Kcom) suggesting that the differences in toxicity did not result from changes in initial receptor binding. Dissociation binding assays and voltage clamping analysis suggest that the reduced toxicity of the H168Q toxin may result from reduced insertion and/or ion channel formation. In contrast, the H168R toxin had a greater inhibition of the short circuit current than the wt toxin and an increased rate of irreversible binding (kobs), consistent with its lower LC50 value. Molecular modeling analysis suggested that both the H168Q and H168R toxins could form additional hydrogen bonds that could account for their greater thermal stability. In addition to this, it is likely that H168R has an extra positive charge exposed to the surface which could increase its rate of insertion into susceptible membranes.

La a-Hélice 5 del domino I de las d-endotoxinas de Bacillus thuringiensis, es crítica para la toxicidad de las toxinas contra insectos al participar en la formación de canales iónicos. La participación en la función tóxica del residuo Histidina 168 (H168) –el cual es altamente conservado– fue estudiada mediante técnicas de biología molecular, electrofisiología y biofísica. La toxina mutante H168R fue ~ 3 veces más tóxica que la toxina silvestre (ts) en Manduca sexta, mientras que H168Q fue 3 veces menos tóxica. Los análisis espectroscópicos indicaron que las mutaciones no producen alteraciones estructurales significativas y que la toxina H168R (Tm= 59 °C) es más estable que las toxinas H168Q (Tm= 57.5 °C) y wt (Tm= 56 °C). Las tres toxinas exhibieron uniones de afinidad similares (Kcom) en vesículas de intestino de larvas de insecto, indicando que las diferencias en la toxicidad no se deben a cambios en la unión inicial al receptor. Los ensayos de unión/disociación y fijación de voltaje mostraron que la reducción de la toxicidad de la toxina H168Q se puede atribuir a una disminución en la inserción y/o en la formación de canales iónicos. De otro lado, H168R mostró una inhibición a la corriente de corto circuito mayor que la ts y un aumento en unión irreversible (kobs), lo cual es consistente con un menor valor de CL50. La modelación molecular sugiere que H168Q y H168R forman puentes de hidrógeno adicionales, lo que les confiere mayor estabilidad térmica. Adicionalmente, es probable que H168R tenga una carga positiva extra expuesta en la superficie, lo cual aumentaría su tasa de inserción en membranas susceptibles.

Seleção e caracterização molecular de Bacillus thuringiensis Berliner com atividade tóxica para Trichoplusia ni Huebner (Lepidoptera: Noctuidae) / Selection and molecular characterization of Bacillus thuringiensis Berliner with toxic activity against Trichoplusia ni Huebner (Lepidoptera: Noctuidae)

Trichoplusia ni é uma praga polífaga importante em plantios de crucíferas, soja e algodão. O presente estudo objetivou selecionar e caracterizar por método molecular isolados de Bacillus thuringiensis (Bt) com potencial para atuar com agentes de controle biológico de T. ni. Para os bioensaios de patogenicidade, uma alíquota com 3 x 108 esporos/mL de suspensão de Bt de cada isolado foi aplicada na superfície do disco de dieta artificial, previamente distribuída em placas de acrílico com 50 lagartas, distribuídas em 5 repetições. Nos bioensaios para a obtenção da CL50, apenas os isolados com 100% de mortalidade foram pré-selecionados, sendo testadas as seguintes concentrações: 102, 5 x 102, 103, 2 x 103, 4 x 103, 6 x 103, 8 x 103 esporos/mL, sendo os tratamentos compostos por 120 lagartas, distribuídas em 3 repetições. Foi feita caracterização molecular para detectar os genes cry1, cry2 e Vip para os isolados que obtiveram mortalidade acima de 95%. Os isolados HD-1 (Padrão), Bt-1043N-V, Bt-1034F, Bt-1009K, Bt-1000, Bt-969A causaram 100% de mortalidade nos testes de patogenicidade e CL50 de 1,17 x 103, 1,45 x 103, 1,46 x 103, 1,01 x 103, 9,43 x 102, 1,22 x 103, respectivamente. Não foram encontrados genes cry1, cry2 e Vip nos isolados testados, podendo outras toxinas Cry estar causando a mortalidade de T. ni, visto que os isolados testados são específicos para a ordem Lepidoptera. Estes isolados mostraram potencial para o controle de T. ni, sendo virulentos a este inseto, com potencial para serem utilizados em programa de manejo desta praga.

Trichoplusia ni is a polyphagous pest that is becoming a major pest in plantations of cruciferous crops, soybeans and cotton. This study was aimed to select and molecularly characterize efficient isolates of Bacillus thuringiensis (Bt) for the control of T. ni. For the bioassays of pathogenicity, an aliquot with a 3 x 108 spores/mL suspension of Bt of each isolate was applied on the surface of the artificial diet disk, previously distributed on acrylic plates with 50 larvae, distributed in 5 repetitions. In bioassays to obtain the LC50, only isolates with 100% mortality were preselected, and tests were carried out at the concentrations 102, 5 x 102, 103, 2 x 103, 4 x 103, 6 x 103, and 8 x 103 spores/mL, and the treatments consisting of 120 larvae, distributed in 3 repetitions. A molecular characterization was performed to detect the genes cry1, cry2 and Vip for the isolates which obtained mortality over 95%. Isolates HD-1 (Standard), Bt-1043N-V, Bt-1034F, Bt-1009K, Bt-1000 and Bt-969A caused 100% mortality in the test for pathogenicity and presented an LC50 of 1.17 x 103, 1.45 x 103, 1, 46 x 103, 1.01 x 103, 9.43 x 102, 1.22 x 103, respectively. Genes cry1, cry2 and Vip were not found in the isolates tested, and other Cry toxins may have been causing the mortality of T. ni, since the isolates tested are specific for the Lepidoptera order. These isolates showed potential for the control of T. ni, being aggressive to this insect, with a potential to be used in a pest management program for this species.

Diversity of Bacillus thuringiensis strains isolated from coffee plantations infested with the coffee berry borer Hypothenemus hampei

The coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) was first reported infecting Costa Rican coffee plantations in the year 2000. Due to the impact that this plague has in the economy of the country, we were interested in seeking new alternatives for the biological control of H. hampei, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 202 B. thuringiensis isolates obtained from Costa Rican coffee plantations infested with H. hampei were analyzed through crystal morphology of the crystal inclusions and SDS-PAGE of 6-endotoxins, while 105 strains were further evaluated by PCR for the presence cry, cyt and vip genes. Most of the Bt strains showed diverse crystal morphologies: pleomorphic (35%), oval (37%), bipyramidal (3%), bipyramidal and oval (12%), bipyramidal, oval and pleomorphic (10%) and bipyramidal, oval and cubic (3%). The SDS-PAGE analyses of the crystal preparations showed five strains with delta-endotoxin from 20 to 40 kDa, six from 40 to 50 kDa, seven from 50 to 60 kDa, 19 from 60 to 70 kDa, 29 from 70 to 100 kDa and 39 from 100-145 kDa. PCR analyses demonstrated that the collection showed diverse cry genes profiles having several genes per strain: 78 strains contained the vip3 gene, 82 the cry2 gene, 45 the cry1 and 29 strains harbored cry3-cry7 genes. A total of 13 strains did not amplified with any of the cry primers used: cry1, cry2, cry3-7, cry5, cry11, cry12 and cry14. Forty-three different genetic profiles were found, mainly due to the combination of cry1A genes with other cry and vip genes. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against H. hampei and other insect pests of agricultural importance.

Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin

Characterization of the inseticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt) subsp. medellin (Btmed) was perfomed and compared to solubilized crystal proteins of isolates 1884 of B. thuringiensis subsp. israelensis (Bti) and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm). In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI) was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3. Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3. An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200). Three main protein peaks were observed on the chromatogram. The first peak had two main proteins that migrate between 90 to 100kDa. These proteins are apparently not common to other Bt strains isolated to date. The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak independently, showed toxicity against 1 st instar Culex quinquefasciatus larvae. Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity. These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae. When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed cross reaction was the 28 kDa protein. These data suggest that Btmed could be an alternative bacterium for mosquito control programs in case mosquito larval resistance emerges to Bti toxic proteins.