RESUMO
In this study, the endochitinase chiA74 gene lacking its secretion signal peptide sequence (chiA74∆sp) was fused in frame with the sequence coding for the C-terminal crystallization domain and transcription terminator of cry1Ac. The chimeric gene was expressed under the strong pcytA-p/STAB-SD promoter system in an acrystalliferous Cry-B strain of Bacillus thuringiensis and B. thuringiensis subsp. kurstaki HD73. We showed that the chimeric ChiA74∆sp produced amorphous inclusions in both Cry-B and HD73. In addition to the amorphous inclusions putatively composed of the chimera, bipyramidal Cry1Ac crystals, smaller than the wild-type crystal, were observed in recombinant HD73, and chitinase activity was remarkably higher (75-fold) in this strain when compared with parental HD73. Moreover, we observed that lyophilized samples of a mixture containing Cry1Ac, amorphous inclusions, and spores maintained chitinase activity. Amorphous inclusions could not be separated from Cry1Ac crystals by sucrose gradient centrifugation. Interestingly, the chitinase activity of purified Cry1Ac/amorphous inclusions was 51-fold higher compared to purified Cry1Ac inclusions of parental HD73, indicating that the increased enzymatic activity was due primarily to the presence of the atypical amorphous component. The possibility that the chimera is occluded with the Cry1Ac crystal, thereby contributing to the increased endochitinolytic activity, cannot be excluded. Finally, bioassays against larvae of Spodoptera frugiperda with spore/crystals of HD73 or spore-crystal ChiA74∆sp chimeric inclusions of recombinant HD73 strain showed LC50s of 396.86 and 290.25 ng/cm2, respectively. Our study suggests a possible practical application of the chimera in formulations of B. thuringiensis-based lepidopteran larvicides.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Agentes de Controle Biológico , Quitinases/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Corpos de Inclusão/química , Animais , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/biossíntese , Quitinases/biossíntese , Quitinases/metabolismo , Endotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Corpos de Inclusão/ultraestrutura , Larva , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência , Spodoptera/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Animais , Bacillus thuringiensis/fisiologia , Proteínas de Bactérias/genética , Expressão Gênica , Controle de Insetos , Endotoxinas/genética , Proteínas Hemolisinas/genética , Bacillus thuringiensis/ultraestrutura , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Controle de Insetos/métodos , Clonagem Molecular , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidade , Inseticidas , Larva , Mariposas/efeitos dos fármacosRESUMO
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 ß-d-1-thiogalactopyranoside was used to induce the expression of cry1Ab in E. coli BL21(DE3), and consequently, â¼130kDa of Cry1Ab was obtained. Bioassay results indicated that recombinant Cry1Ab at a dose of 1000µgg(-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.
Assuntos
Bacillus thuringiensis/fisiologia , Proteínas de Bactérias/genética , Endotoxinas/genética , Expressão Gênica , Proteínas Hemolisinas/genética , Controle de Insetos , Animais , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Clonagem Molecular , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidade , Controle de Insetos/métodos , Inseticidas , Larva , Mariposas/efeitos dos fármacosRESUMO
Bacillus thuringiensis isolates were obtained from soil samples collected at different sites located in the same region but with different vegetation. The sites showed different frequencies of B. thuringiensis, depending on the type of vegetation. Strains of B. thuringiensis were found to be less common in samples of riparian forest soil than in soil of other types of vegetation. The rate of occurrence of B. thuringiensis in the samples also varied according to the vegetation. These results show that whenever this bacterium was found, it showed a high rate of occurrence, indicating that this species could be better adapted to using soil as a reservoir than other Bacillus species. The presence of cry genes was analyzed by polymerase chain reaction, and genes that exhibited activity against Diptera species were the most commonly found. The isolates obtained were characterized by random amplified polymorphic DNA, and 50% were clustered into clonal groups. These results demonstrated the possible occurrence of a high number of genetically similar strains when samples are collected from the same region, even if they are from locations with different vegetation.
Assuntos
Bacillus thuringiensis/genética , Ecossistema , Variação Genética , Microbiologia do Solo , Animais , Bacillus thuringiensis/classificação , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Filogenia , Plantas/microbiologia , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Bacillus thuringiensis/efeitos da radiação , Bacillus thuringiensis/ultraestrutura , Bacillus thuringiensis/virologia , Toxicidade/análise , Toxicidade/classificação , Toxicidade/métodosRESUMO
Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the Caribbean that produces a spore-crystal complex containing six long appendages, each comprised of numerous thinner filaments approximately 10 nm in diameter and 2.5 µm in length. Each of the multi-filament appendages was attached to a single, small parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages.
Assuntos
Bacillus thuringiensis/classificação , Bacillus thuringiensis/ultraestrutura , Organelas/ultraestrutura , Esporos Bacterianos/ultraestrutura , Bacillus thuringiensis/genética , Bacillus thuringiensis/isolamento & purificação , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Região do Caribe , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Microbiologia do SoloRESUMO
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.
Assuntos
Bacillus thuringiensis/ultraestrutura , Controle Biológico de Vetores , Toxinas de Bacillus thuringiensis/análise , LepidópterosRESUMO
On the basis of the known cry gene sequences of Bacillus thuringiensis, three sets of primers were designed from four conserved blocks found in the delta-endotoxin-coding region. The primer pairs designed amplify the regions between blocks 1 and 5, 2 and 5, and 1 and 4. In silico analyses indicated that 100% of the known three-domain cry gene sequences can be amplified by these sets of primers. To test their ability to amplify known and unknown cry gene sequences, 27 strains from the CINVESTAV (LBIT series) collection showing atypical crystal morphology were selected. Their DNA was used as the template with the new primer system, and after a systematic amplification and sequencing of the amplicons, each strain showed one or more cry-related sequences, totaling 54 different sequences harbored by the 27 strains. Seven sequences were selected on the basis of their low level of identity to the known cry sequences, and once cloning and sequencing of the complete open reading frames were done, three new cry-type genes (primary ranks) were identified and the toxins that they encode were designated Cry57Aa1, Cry58Aa1, and Cry59Aa1 by the B. thuringiensis Toxin Nomenclature Committee. The rest of the seven sequences were classified Cry8Ka2, Cry8-like, Cry20Ba1, and Cry1Ma1 by the committee. The crystal morphology of the selected strains and analysis of the new Cry protein sequences showed interesting peculiarities.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Insetos/genética , Receptores de Superfície Celular/genética , Bacillus thuringiensis/ultraestrutura , Proteínas de Bactérias , Sequência de Bases , Clonagem Molecular , Biologia Computacional , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Twelve Bacillus thuringiensis (Bt) strains, isolated from larvae and soil samples in Argentina, were molecularly and phenotypically characterized and their insecticidal activities against Spodoptera frugiperda and Peridroma saucia were determined. One isolate--Bt RT--produced more than 93% mortality on first instar larvae of both species, which was higher than that produced by the reference strain Bt 4D1. Bt RT carried a different cry gene profile than Bt 4D1. Scanning electron microscopy showed the presence of bipyramidal and cuboidal crystals. Phenotypic characterization revealed lytic enzymes that could contribute to Bt pathogenicity.
Assuntos
Bacillus thuringiensis/isolamento & purificação , Bacillus thuringiensis/patogenicidade , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Infecções por Bactérias Gram-Positivas/veterinária , Proteínas Hemolisinas/toxicidade , Lepidópteros/efeitos dos fármacos , Lepidópteros/microbiologia , Animais , Argentina , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Endotoxinas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/ultraestrutura , Larva/microbiologia , Microscopia Eletrônica de Varredura , Microbiologia do Solo , Spodoptera/efeitos dos fármacos , Spodoptera/microbiologia , Análise de SobrevidaRESUMO
Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-microm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.