RESUMEN
The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringiensis and their interactions with cell membrane has not been defined. To elucidate the involvement of toxin-membrane and toxin-receptor interactions in cytotoxicity, we established a cell-based system utilizing High Five insect cells stably expressing BT-R1, the cadherin receptor for Cry1Ab toxin. Cry1Ab toxin is incorporated into cell membrane in both oligomeric and monomeric form. Monomeric toxin binds specifically to BT-R1 whereas incorporation of oligomeric toxin is nonspecific and lipid dependent. Toxin oligomers in the cell membrane do not produce lytic pores and do not kill insect cells. Rather, cell death correlates with binding of the Cry1Ab toxin monomer to BT-R1, which apparently activates a Mg2+-dependent cellular signaling pathway.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Cadherinas/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidad , Manduca/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Muerte Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quelantes/farmacología , Ácido Edético/farmacología , Ácido Egtácico/farmacología , Endotoxinas/antagonistas & inhibidores , Proteínas Hemolisinas , Proteínas de Insectos/metabolismo , Magnesio/metabolismo , Manduca/efectos de los fármacos , Unión Proteica , Receptores de Superficie Celular/antagonistas & inhibidores , Transducción de Señal , Especificidad por SustratoRESUMEN
Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R(1) (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K(d) approximately 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R(1) revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R(1) cDNA, subsequently killing the cells. Recruitment of BT-R(1) by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Cadherinas/metabolismo , Endotoxinas/metabolismo , Proteínas de Insectos , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Sitios de Unión , Células COS , Chlorocebus aethiops , Proteínas Hemolisinas , Larva , Manduca , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/aislamiento & purificación , TransfecciónRESUMEN
The cadherin-related receptor of Manduca sexta, BT-R(1), for the Cry1A family of Bacillus thuringiensis insecticidal toxins, was expressed in cultured Spodoptera frugiperda (Sf21) insect cells utilizing the expression vector deltaOp-gp64. Recombinant BT-R(1) was released by the Sf21 cells in soluble form into the culture medium and represents approximately 58% of total BT-R(1) produced by the cells. The soluble protein was purified by affinity chromatography using Cry1Ab toxin coupled to Sepharose 4B. The apparent molecular mass of purified soluble recombinant BT-R(1) is 195 kDa. Radiolabeled toxin bound to purified soluble BT-R(1) with a K(d) value of 1.1 nM, which is similar to that of both membrane-bound BT-R(1) in Sf21 cells and natural BT-R(1) from M. sexta larval midgut tissue. Binding of radiolabeled toxin to soluble BT-R(1) was competitively inhibited by unlabeled Cry1Ab toxin but not by other Cry toxins as was observed also for membrane-bound BT-R(1). The recombinant soluble protein was stable in culture medium for at least 3 days at 27 degrees C and for 7 days at 4 degrees C and exhibited toxin-binding properties similar to the natural protein. Apparently, neither membrane association nor the extent of glycosylation influences the binding affinity and specificity of BT-R(1). Approximately 1 mg of purified BT-R(1) was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) Sf21 cells.
Asunto(s)
Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Cadherinas/química , Endotoxinas/metabolismo , Proteínas de Insectos , Manduca , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Spodoptera , Animales , Toxinas de Bacillus thuringiensis , Unión Competitiva , Línea Celular , Proteínas Hemolisinas , Ligandos , Receptores de Superficie Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Especificidad por Sustrato , TermodinámicaRESUMEN
The identity of the physiologically important Cry1A receptor protein(s) in the lepidopteran Manduca sexta has been a matter of dispute due to the multiple proteins which bind the Cry1Ac toxin. Cry1Aa, Cry1Ab, and Cry1Ac exhibit essentially identical toxicities toward M. sexta larvae and show a high degree of sequence and presumed structural identities. These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity. Investigators in our laboratory previously demonstrated that the cloned 210-kDa glycoprotein BT-R1 binds all three Cry1A toxins (T. P. Keeton and L. A. Bulla, Jr., Appl. Environ. Microbiol. 63:3419-3425, 1997). This protein remains a common binding protein even after being subjected to various midgut membrane preparation and processing protocols. The method used to isolate proteins from the M. sexta larval midgut in no significant way affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1A toxin to Cry1Ac binding proteins other than BT-R1. Alterations in blot substrate and blocking, hybridization, and washing buffers support these conclusions. Collectively, these results indicate that in M. sexta the cadherin-like BT-R1 protein is a common high-affinity receptor protein for the Cry1A family of toxins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Endotoxinas/metabolismo , Proteínas de Insectos , Manduca/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Tampones (Química) , Sistema Digestivo/metabolismo , Proteínas Hemolisinas , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Peso Molecular , Control Biológico de Vectores , Receptores de Superficie Celular/química , Receptores de Superficie Celular/aislamiento & purificaciónRESUMEN
Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210 kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp, berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210 kDa protein, termed BT-R1 (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R1, the gene encoding the 210 kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R1 as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R1. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R1, whereas the other is a closely related homologue.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Southern Blotting , Endotoxinas/metabolismo , Genes de Insecto , Proteínas de Insectos , Manduca/genética , Receptores de Superficie Celular/genética , Animales , Toxinas de Bacillus thuringiensis , Mapeo Cromosómico , Sondas de ADN , Marcadores Genéticos , Proteínas Hemolisinas , Resistencia a los Insecticidas , Modelos Genéticos , Polimorfismo Genético , Receptores de Superficie Celular/metabolismoRESUMEN
The Manduca sexta receptor for the Bacillus thuringiensis Cry1Aa, Cry1Ab, and Cry1Ac toxins, BT-R1, has been expressed in heterologous cell culture, and its ligand binding characteristics have been determined. When transfected with the BT-R1 cDNA, insect and mammalian cell cultures produce a binding protein of approximately 195 kDa, in contrast to natural BT-R1 from M. sexia, which has an apparent molecular weight of 210 kDa. Transfection of cultured Spodoptera frugiperda cells with the BT-R1 cDNA imparts Cry1A-specific high-affinity binding activity typical of membranes prepared from larval M. sexta midguts. Competition assays with BT-R1 prepared from larval M. sexta midguts and transiently expressed in cell culture reveal virtually identical affinities for the Cry1Aa, Cry1Ab, and Cry1Ac toxins, clearly demonstrating the absolute specificity of the receptor for toxins of the lepidopteran-specific Cry1A family. BT-R1 therefore remains the only M. sexta Cry1A binding protein to be purified, cloned, and functionally expressed in heterologous cell culture, and for the first time, we are able to correlate the Cry1Aa, Cry1Ab, and Cry1Ac toxin sensitivities of M. sexta to the identity and ligand binding characteristics of a single midgut receptor molecule.
Asunto(s)
Bacillus thuringiensis/metabolismo , Toxinas Bacterianas , Proteínas de Insectos , Manduca/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Unión Competitiva , Células COS , Línea Celular , Endotoxinas/genética , Endotoxinas/metabolismo , Expresión Génica , Proteínas Hemolisinas , Humanos , Ligandos , Manduca/genética , Receptores de Superficie Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecciónRESUMEN
BT-R1, the Manduca sexta midgut receptor for the crystal toxin Cry1Ab produced by Bacillus thuringiensis ssp. berliner, was partly purified by gel filtration from M. sexta brush border membrane vesicles in the presence of the detergent CHAPS. Fractions containing BT-R1 were tested for their stability against degradation as indicated by retention of Cry1Ab binding on ligand blots. At 4 degrees C and pH 7.4 in the presence of Ca2+, BT-R1 was stable for up to 48 h but a 65% loss of binding was observed after 100 h. Under the same conditions, no loss of binding was observed in the presence of EGTA after 100 h. Cry1Ab binding decreased markedly as pH increased from 6 to 10 for incubations of 24 h at 4 degrees C. Increasing the temperature of incubation from 4 to 37 degrees C also decreased Cry1Ab binding. Neither metal ions nor free sulfhydryl groups are involved in Cry1Ab binding to BT-R1. A trypsin-like, metal-ion-dependent proteolytic activity co-eluted with BT-R1 during gel filtration. This endoproteolytic activity was unaltered by the addition of Cry1Ab. BT-R1 did not co-elute with peaks of aminopeptidase, alkaline phosphatase, alpha-glucosidase, beta-glucosidase and beta-galactosidase activities. When BT-R1 in the gel filtration fraction was further purified on a Mono Q anion exchange column, partial separation of the trypsin-like activity from BT-R1 was observed. BT-R1 could be removed from the appropriate Mono Q fraction by immunoprecipitation with only a slight decrease in this activity. These results demonstrate that there is no copurification of BT-R1 and these enzymes and that BT-R1 is unlikely to form complexes with them. Binding of Cry1Aa and Cry1Ac to BT-R1 in gel filtration fractions is similar to that of Cry1Ab, indicating that BT-R1 may be the high-affinity receptor for the Cry1A toxins. Binding of Cry1Ab to a 120 kDa protein has not been observed in this study.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Cadherinas/metabolismo , Endotoxinas/metabolismo , Proteínas de Insectos , Receptores de Superficie Celular/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Sistema Digestivo , Proteínas Hemolisinas , Concentración de Iones de Hidrógeno , Manduca/metabolismo , Receptores de Superficie Celular/aislamiento & purificación , Tripsina/metabolismoRESUMEN
Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the hornworm. BT-R1 shares sequence similarity with the cadherin superfamily of proteins.
Asunto(s)
Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Cadherinas/química , Cadherinas/genética , Endotoxinas/metabolismo , Proteínas de Insectos , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Unión Competitiva , Northern Blotting , Línea Celular , Clonación Molecular , Secuencia de Consenso , Sistema Digestivo/metabolismo , Drosophila , Expresión Génica , Proteínas Hemolisinas , Humanos , Cinética , Manduca , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Control Biológico de Vectores , Biosíntesis de Proteínas , Receptores de Superficie Celular/química , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Biopesticides based on the bacterium Bacillus thuringiensis have attracted wide attention as safe alternatives to chemical insecticides. In this paper, we report, for the first time, the identification of a single binding protein from a coleopteran insect, Tenebrio molitor, that is specific for the cryIII toxin of B. thuringiensis. The protein appeared as a single band of 144 kDa on radioligand and immunoblots of total proteins extracted from brush border membrane vesicles of the midgut of T. molitor. Radiolabelled cryIIIA toxin bound to the protein with a Kd value of 17.5 nM and could be specifically blocked by unlabelled toxin but not by toxins from other subspecies of B. thuringiensis. This study lays the groundwork to clone the cryIIIA toxin binding protein and to determine the molecular mechanism(s) of toxin action.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Endotoxinas/metabolismo , Hormonas de Insectos/metabolismo , Tenebrio/metabolismo , Animales , Bacillus thuringiensis , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/toxicidad , Unión Competitiva , Proteínas Portadoras/aislamiento & purificación , Sistema Digestivo/metabolismo , Endotoxinas/toxicidad , Proteínas Hemolisinas , Hormonas de Insectos/aislamiento & purificación , Radioisótopos de Yodo , Cinética , Larva , Peso Molecular , Ensayo de Unión Radioligante , Especificidad de la EspecieRESUMEN
Two variants of insertion sequence IS231, named IS231G and H, were isolated from Bacillus thuringiensis subsp. darmstadiensis 73-E-10-2 (BTD2), an isolate toxic to dipteran insects, and characterized by DNA sequence analysis. They are encoded consecutively as direct repeats on an EcoRI fragment of 5.6 kilo base pairs. Direct tandem repeats of IS231 elements have not been previously reported. Both elements are closely related to other members of the IS231 family that have been isolated from B. thuringiensis strains toxic to lepidopteran as well as to dipteran insects. A close correlation exists between the evolutionary relationships of the IS231 sequences determined to data and the toxicity spectrum of the host cell. Probing of BTD2 DNA with a radiolabeled IS231G fragment demonstrated that IS231 elements are located on 55- and 34-MDa plasmids as well as on chromosomal DNA. Chromosomal DNA, but not plasmids, from BTD2 also hybridizes to another, unrelated insertion sequence, IS240, from B. thuringiensis subsp. israelensis, an isolate toxic to dipteran insects. BTD2, therefore, contains IS elements once thought to reside exclusively in either dipteran- or lepidopteran-specific subspecies of B. thuringiensis.
Asunto(s)
Bacillus thuringiensis/genética , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Bacillus thuringiensis/clasificación , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Secuencia de Bases , Sondas de ADN , ADN Bacteriano/clasificación , Endotoxinas/genética , Proteínas Hemolisinas , Control de Insectos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Plásmidos/genética , Precursores de Proteínas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ADNRESUMEN
Biopesticides based on the bacterium Bacillus thuringiensis have attracted wide attention as safe alternatives to chemical pesticides. In this paper, we report, for the first time, the identification and purification of a single binding protein from a lepidopteran insect, Manduca sexta, that is specific for a cryIA toxin of B. thuringiensis. The purified protein appeared as a single band of 210 kDa on a two-dimensional gel, had a pI of approximately 5.5, and stained with Schiff's reagent. The band material was sensitive to proteolytic digestion and was rich with acidic amino acids, indicating its protein nature. Radiolabeled toxin bound to the protein with a Kd value of 708 pM and could be specifically blocked by unlabeled toxin but not by toxins from other subspecies of B. thuringiensis. This study lays the groundwork to clone the toxin binding protein and to determine the molecular mechanism(s) of toxin action.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Endotoxinas , Hormonas de Insectos , Hormonas de Insectos/metabolismo , Mariposas Nocturnas/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/toxicidad , Unión Competitiva , Proteínas Portadoras/aislamiento & purificación , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Proteínas Hemolisinas , Hormonas de Insectos/aislamiento & purificación , Insectos/efectos de los fármacos , Cinética , Peso Molecular , Mariposas Nocturnas/efectos de los fármacosRESUMEN
Bacillus thuringiensis subspecies kurstaki (Btk) and subspecies berliner (Btb) both produce lepidopteran-specific larvicidal protoxins with different activities against the same insect species. Toxic activity resides in the amino-terminal half of both protoxins, whereas the carboxy-terminal half of the molecules is not required for toxicity. The protoxins are 90% homologous, with a major cluster of differences in the amino-terminal half, and a 26 consecutive amino-acid insertion within the carboxy-terminal half of the Btk protoxin. Protoxin chimeras composed of the amino-terminal half of one subspecies and the carboxy-terminal half of the other were generated. Wild-type and chimeric protoxins were compared in bioassays against tobacco hornworm larvae. The amino-terminal half, the toxin itself, dictates specific larvicidal activity.
Asunto(s)
Bacillus thuringiensis/genética , Toxinas Bacterianas/genética , Precursores de Proteínas/genética , Animales , Toxinas Bacterianas/toxicidad , Quimera , Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Larva , Mariposas Nocturnas/efectos de los fármacos , Plásmidos , Precursores de Proteínas/toxicidad , Mapeo RestrictivoRESUMEN
Two proteins from parasporal crystals of Bacillus thuringiensis subsp. israelensis were purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography. The larger of the two proteins (molecular weight, 68,000) was not cytolytic, whereas the smaller protein (molecular weight, 28,000) was highly cytolytic when assayed against rat erythrocytes. When these proteins were assayed against larvae of the yellow fever mosquito, Aedes aegypti, the larger protein was at least 100-fold more toxic than the smaller protein. Although proteolytic activity was not detected in solubilized crystals nor in purified protein preparations, the toxin (molecular weight, 68,000) was readily degraded to smaller, nontoxic molecules, even when maintained at 4 degrees C. Mixtures of the two purified proteins were significantly more toxic to mosquito larvae than was either protein alone. Thus, it is likely that both the mosquitocidal and the cytolytic protein play roles in the overall insecticidal action of the parasporal crystal produced by this bacterium.
Asunto(s)
Aedes , Bacillus thuringiensis/análisis , Proteínas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/aislamiento & purificación , Control Biológico de Vectores , Animales , Proteínas Bacterianas/análisis , Toxinas Bacterianas/análisis , Cromatografía en Gel , Electroforesis en Gel de PoliacrilamidaRESUMEN
One of the challenges in the application of biotechnology to pest control is the identification of agents found in nature which can be used effectively. Biotechnology offers the potential of developing pesticides based on such agents which will provide environmentally sound and economically feasible insect control alternatives. Such an agent, the insect pathogen Bacillus thuringiensis, is the subject of intense investigations in several laboratories. Insecticides which use the entomocidal properties of B. thuringiensis are currently produced and sold worldwide; new products are currently in the development stage. Herein, the biology and genetics of B. thuringiensis and the problems associated with current products are critically reviewed with respect to biotechnology. Moreover, the economic and regulatory implications of technologically advanced products are evaluated.
Asunto(s)
Bacillus thuringiensis , Control Biológico de Vectores/métodos , Secuencia de Aminoácidos , Bacillus thuringiensis/genética , Bacillus thuringiensis/fisiología , Toxinas Bacterianas/genética , Toxinas Bacterianas/aislamiento & purificación , Secuencia de Bases , Biotecnología , ADN Bacteriano/genética , Datos de Secuencia Molecular , Control Biológico de Vectores/legislación & jurisprudenciaRESUMEN
A 3778-bp DNA sequence of the insecticidal protoxin gene coding sequence and flanking regions from Bacillus thuringiensis subspecies berliner 1715 has been determined. The protoxin is composed of 1155 amino acids, deduced from the nucleotide sequence, and has a calculated molecular mass of 130,615 daltons. To determine the DNA portion that encodes toxicity, sequential deletions were constructed from the 3' end of the coding region using nuclease Bal-31. Using these mutants in an insect bioassay, we found that an amino-terminal 612-amino-acid peptide is toxic, whereas, a 603-amino-acid peptide is not toxic to insects. Ninety percent of the amino acid residues were homologous to the protoxins from closely related subspecies kurstaki HD-1-Dipel and sotto. The differences occurred both in the amino-terminal half, or toxic portion, and in the carboxy-terminal half. These differences were clustered in several regions. From comparative analysis of subspecies berliner and kurstaki, we propose a model whereby the protoxin molecule is divided into distinct structural and functional domains. These domains may be responsible for the differences in specific toxicities and spectra of insect host range among these subspecies.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Endotoxinas , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Toxinas de Bacillus thuringiensis , Secuencia de Bases , Mapeo Cromosómico , Genes Bacterianos , Proteínas Hemolisinas , Peso Molecular , Conformación Proteica , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.
Asunto(s)
Bacillus thuringiensis/crecimiento & desarrollo , Toxinas Bacterianas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Precursores de Proteínas/aislamiento & purificación , Bacillus thuringiensis/enzimología , Cristalización , Cinética , Peso Molecular , Inhibidores de Proteasas/farmacología , Esporas Bacterianas/fisiologíaRESUMEN
The mobility of 13C specifically labeled branched chain end groups of iso-even fatty acids in intact, live Bacillus thuringiensis cells was studied by 13C nuclear magnetic resonance spectroscopy. This study apparently represents the first direct observation of branched chain carbon atoms in living cells. End groups were labeled using DL-[beta, delta, delta'-13C]valine as a precursor chain initiator for iso-even fatty acid synthesis after using L-[delta, delta'-14C]L-valine to determine optimal conditions for labeling of the membrane fatty acid end groups. Cell survival in the NMR was determined for various lengths of time at 28 and 39 degrees C. Subsequently, 13C-labeled vegetative cells, sporulating cells (three stages of development), and purified mature spores were analyzed by 13C NMR using corresponding unlabeled cells as controls. Spin lattice relaxation times (T1) were obtained for the enriched iso-branched region at 23.3 ppm and for the natural abundance peak for the glycerol backbone (carbons 1 and 3) of the membrane lipids at 61.7 ppm. The T1 of the glycerol carbons (0.08 s) did not change significantly with stage of development or temperature. The T1 of the iso-even enriched end group changed dramatically from vegetative cells (0.70s) to sporulating cells (0.28 s) at 28 degrees C. A decrease in the T1 was also observed at 39 degrees C from 0.91 s for vegetative cells to 0.54 s for sporulating cells. Accompanying the reduced mobility indicated by the T1 values, there was a general decline in the signal-to-noise ratios of identically acquired spectra as sporulation continued which culminated in the lack of discernible plasma membrane lipid resonances in purified mature spores. The progressive loss of signal appeared to have resulted from a continuous decline in the fraction of plasma membrane fatty acids with sufficient mobility to give signals above background.
Asunto(s)
Bacillus thuringiensis/fisiología , Ácidos Grasos/análisis , Lípidos de la Membrana/análisis , Bacillus thuringiensis/análisis , Espectroscopía de Resonancia Magnética , Esporas BacterianasRESUMEN
Escherichia coli strains harboring deletion mutations of the insecticidal protoxin gene of Bacillus thuringiensis subsp. berliner 1715 were constructed. Although these strains did not produce intact protoxin, cell extracts from one of the mutants were extremely toxic to tobacco hornworm (Manduca sexta) larvae, indicating that only a part of the protoxin gene is required for insecticidal activity.
Asunto(s)
Bacillus thuringiensis/genética , Toxinas Bacterianas/genética , Control Biológico de Vectores , Precursores de Proteínas/genética , Escherichia coli/genéticaRESUMEN
Bacillus thuringiensis subsp. israelensis produces, during sporulation, protein inclusion bodies of wide ranging sizes, all of which are toxic to mosquitoes. Two proteins are present in the smallest protein bodies (less than 0.2 micron dia.), but the number of proteins increases with increasing size of protein bodies. The largest bodies (greater than 1.5 micron dia.) contain seven proteins. All of the proteins are synthesized at different times during sporulation and are added to developing protein bodies in a stepwise manner. The protein component responsible for mosquitocidal activity is a 65,000-dalton protein, that is present in all of the protein bodies.