RESUMEN
Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.
Asunto(s)
Bacillus thuringiensis/química , Proteínas Bacterianas/farmacología , Toxinas Bacterianas , Endotoxinas/farmacología , Control de Insectos/métodos , Zea mays/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Electroforesis en Gel de Poliacrilamida , Proteínas Hemolisinas , Inmunidad Innata , Immunoblotting , Larva , Modelos Genéticos , Plantas Modificadas Genéticamente , Transformación GenéticaRESUMEN
Nature has provided potent insecticidal toxins as fermentation products of many Bacillus thuringiensis strains. Elucidation of structure-function relationships for this class of natural toxins is in its early stages. Both direct experimentation and application of theoretical structure-function principles emerging from the rapidly growing field of protein structure are accelerating understanding of these toxins. Coupled with the increasing demand for biologically sound pesticides, the benefits from engineering nature's toxins for improved performance set the stage for exciting, fast growth in discovery, characterization and commercialization of new active ingredients for biopesticides and transgenic plants.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Toxinas Bacterianas/química , Evolución Biológica , Endotoxinas/química , Endotoxinas/genética , Animales , Bacillus thuringiensis/fisiología , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Endotoxinas/biosíntesis , Ingeniería Genética/métodos , Variación Genética , Proteínas Hemolisinas , Lepidópteros/efectos de los fármacos , Control Biológico de Vectores , Homología de Secuencia de AminoácidoRESUMEN
The cryIB gene of Bacillus thuringiensis subsp. thuringiensis HD-2 codes for a Mr 139492 protein that is lethal to certain lepidopteran larvae. We used primer extension to map transcriptional initiation sites and found that cryIB was transcribed from two sites that are activated at different times during sporulation. The presumed promoter regions for the two start sites are very similar to the two promoters preceding the cryIA (a) gene, and the in vivo transcriptional start sites were found to be identical. Variable amounts of the full-length cryIB protein were detected by immunoblotting of extracts of recombinant cells of Escherichia coli; larger amounts were found when the TTG translational start codon was changed to ATG and when an htpR- strain of E. coli was used as the recipient for transformation. When expressed in E. coli, the cryIB protein was found to be toxic to the larvae of Artogeia rapae (LC50 of 58 ng/cm2) and exhibited little toxicity to the larvae of Manduca sexta (LC50 greater than 5000 ng/cm2).
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Endotoxinas , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Secuencia de Bases , Codón/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica/genética , Proteínas Hemolisinas , Immunoblotting , Lepidópteros , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Transcripción Genética/genéticaRESUMEN
The lepidopteran-specific, insecticidal crystal proteins of Bacillus thuringiensis vary in toxicity to different species of lepidopteran larvae. We report studies of CryIA(a) and CryIA(c), two related proteins that have different degrees of toxicity to Heliothis virescens yet very similar degrees of toxicity to Manduca sexta. The amino acid differences between these proteins are located primarily between residues 280 and 722. We have constructed a series of chimeric proteins and determined their toxicities to both insects. The most significant findings arise from the replacement of three segments of the cryIA(c) gene with homologous portions of the cryIA(a) gene: codons 332-428, 429-447, and 448-722. Each of these segments contributed substantially and largely additively toward efficacy for H. virescens. However, replacement of the 429-447 segment of cryIA(c) gene with the cryIA(a) sequence resulted in a 27-50-fold reduction in toxicity toward M. sexta whereas the reduction in toxicity to H. virescens was only 3-4-fold. Subdivision of the 429-447 segment and replacements involving residues within this segment reduced toxicity to M. sexta by 5- to more than 2000-fold whereas toxicity to H. virescens was only reduced 3-10-fold. These observations indicate that: 1) different but overlapping regions of the cryIA(c) gene determine specificity to each of the two test insects; 2) some of the examined gene segments interact in determining specificity; and 3) different sequences in the cryIA(a) and cryIA(c) genes are required for maximal toxicity to M. sexta.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Endotoxinas , Genes Bacterianos , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/farmacología , Quimera , Proteínas Hemolisinas , Larva , Lepidópteros/efectos de los fármacos , Datos de Secuencia Molecular , Mariposas Nocturnas/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Plásmidos , Mapeo RestrictivoRESUMEN
The expression in Escherichia coli of a cloned crystal protein gene from Bacillus thuringiensis was investigated through the use of fusions of the crystal protein gene promoter to beta-galactosidase and catechol oxidase genes. Analysis of deletion and insertion derivatives of the crystal protein gene promoter showed that a region of B. thuringiensis DNA located between 87 and 258 base pairs upstream from the transcription initiation site caused reduced transcription from this promoter. Insertion of Tn5 145 base pairs upstream from the transcription initiation site resulted in overproduction of the crystal protein. S1 nuclease mapping experiments failed to detect transcription from an outwardly directed promoter in Tn5, indicating that the overproduction resulted from the disruption or repositioning of the transcription-suppressing region.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas , Endotoxinas , Genes Bacterianos , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , Elementos Transponibles de ADN , ADN Bacteriano/análisis , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Hemolisinas , Mutación , Plásmidos , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Data obtained using several experimental methods (curing, transconjugation, cloning, and hybridization) indicate that crystal protein genes in many subspecies of BT that are toxic to lepidopterans are located on one or more large plasmids; in some subspecies, the gene may be located on the chromosome. Detailed mapping has shown that in three plasmids (each from a different strain) the genes are surrounded by multiple copies of two repeated DNA elements; the arrangement of these elements is the same in the three plasmids. An analysis of the sequence of one of these repeated DNAs strongly suggests that it contains a transposase. Thus, transfer of crystal protein genes between plasmids and/or between plasmids and the chromosome would be possible either by transposition or by recombinational events mediated by the repeated DNAs. Crystal protein genes have been cloned from several plasmids and were expressed in E. coli and B. subtilis, whereas two genes cloned from chromosomal preparations were not expressed. Some of the factors that regulate expression of a plasmid-borne gene in E. coli and B. subtilis have been identified. Very little is known about the role of sporulation genes in regulating expression of the crystal protein gene in B. subtilis or BT. In BT, expression may also be affected by genes on other plasmids. Three homologous crystal protein genes have been identified and cloned from subsp. kurstaki and thuringiensis; different strains of these subspecies may contain one, two, or three of these genes. It seems probable that additional gene families will be found, since the crystals of different subspecies contain immunologically distinguishable proteins. The DNA sequences of the three homologous genes have been published as has the sequence of the crystal protein gene from subsp. sotto. These four genes have regions of identity (the promoter region) and similarity (the N-terminal approximately 280 amino acids, the C-terminal half of the protein, and the terminator). It is interesting that the divergent portions of the molecules are not in precisely the same positions and that all overlap the toxin-encoding portion of the gene. It would be worthwhile to determine if the differences in the amino acid sequence are related to differences in the toxicity and/or the host range of the cloned genes, and to establish how the complement of genes in a given strain contributes to the overall toxicity of that strain.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas , Endotoxinas , Genes Bacterianos , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Dípteros , Proteínas Hemolisinas , Lepidópteros , Control Biológico de VectoresRESUMEN
We have determined the nucleotide sequence of a 4222-base segment of DNA which contains the promoter, the coding region, and the terminator of a crystal protein gene cloned from a Bacillus thuringiensis plasmid. A sequence of 1176 amino acids encoding a Mr 133,500 peptide was deduced from the single open reading frame. This protein-coding region was analyzed for codon usage, predicted hydropathy, and predicted secondary structure. Examination of the base sequence revealed the presence of several inverted and direct repeats located in both the coding and noncoding regions. S1 nuclease mapping was used to locate the transcription termination point at a site following a potentially very stable stem-and-loop structure.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas , ADN Bacteriano/genética , Endotoxinas , Secuencia de Aminoácidos , Toxinas de Bacillus thuringiensis , Secuencia de Bases , Codón/genética , Genes Bacterianos , Proteínas Hemolisinas , Peso Molecular , Conformación Proteica , ARN Bacteriano/genética , ARN Mensajero/genéticaRESUMEN
Crystals of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel contain a Mr 134,000 protoxin which can be cleaved by proteolysis to a peptide of Mr approximately 70,000; this peptide is lethal to lepidopteran larvae. We have analyzed the peptides produced by recombinant Escherichia coli strains bearing deletions and fusions of the protoxin gene in order to delineate the portion of the gene which encodes the toxic peptide. The recombinant strains produced the toxic peptide as well as larger peptides whose size was related to the length of the deleted gene. The results indicate that the amino-terminal 55% of the protoxin protein is sufficient for toxicity. While two different gene fusions to the 10th codon allowed the synthesis of toxic polypeptides, fusions to the 50th codon did not. 3' end deletions up to the 645th codon allowed synthesis of the toxic peptide whereas a deletion to the 603rd codon yielded a non-toxic peptide. Some of the 5' and 3' end alterations to the gene caused changes in the proteolytic cleavage patterns of the polypeptides synthesized by E. coli, suggesting that the alterations led to conformational changes in the proteins. The presence of different 3' end segments affected the levels of synthesis of the altered crystal proteins.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Endotoxinas , Genes Bacterianos , Precursores de Proteínas/genética , Toxinas de Bacillus thuringiensis , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Escherichia coli/genética , Regulación de la Expresión Génica , Proteínas Hemolisinas , Peso Molecular , Conformación ProteicaRESUMEN
The location of crystal protein genes in 22 crystalliferous Bacillus thuringiensis strains representing 14 subspecies was investigated by hybridization of an intragenic restriction fragment from a cloned crystal protein gene to whole plasmid preparations. Hybridization was found to a single plasmid in eight strains, to more than one plasmid in seven strains, and to one or both of two large, unresolved plasmids in two strains. The sizes of the hybridized plasmids ranged from 33 to over 150 megadaltons. In one additional subspecies, hybridization was only to linear DNA fragments, suggesting a chromosomal crystal protein gene, and for four other subspecies, not reported to be toxic to lepidopteran insects, no hybridization was found to either plasmids or to total cell DNA. Hybridization to restriction digests of plasmids and total cell DNA of several strains of subspecies thuringiensis and kurstaki revealed that all homology to the cloned crystal protein gene was plasmid associated and that several of these strains contained multiple regions of homology, implying the presence of multiple crystal protein genes.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas , Endotoxinas , Genes Bacterianos , Toxinas de Bacillus thuringiensis , ADN Bacteriano/análisis , Proteínas Hemolisinas , Hibridación de Ácido Nucleico , PlásmidosRESUMEN
The nucleotide sequence of the promoter region and part of the coding region of the crystal protein gene from Bacillus thuringiensis var. kurstaki HD-1-Dipel has been determined by analysis of a recombinant plasmid from Escherichia coli. The start points for transcription of the gene in B. thuringiensis and in the E. coli strain carrying the recombinant plasmid were located by S1 nuclease mapping. Two adjacent start sites were identified using RNAs synthesized during sporulation of B. thuringiensis: transcription was initiated from one site early in sporulation and from the other site in the middle of sporulation. A good correlation was found between the appearance of the crystal protein gene-specific RNA and the production of the protein, indicating that the gene is primarily under transcriptional control during sporulation. Parallel studies with the recombinant strain of E. coli revealed the presence of only a single species of gene-specific RNA, regardless of the growth phase of the cells; the crystal protein was produced at all stages of growth. The sequence for eight amino acids at the NH2 terminus of the crystal protein was determined and the corresponding coding sequence was located in the DNA sequence. A potential ribosome binding site of 11 nucleotides was found, located three nucleotides upstream from the initiator ATG codon. The deduced sequence for the first 333 amino acids of the crystal protein is presented.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Genes , Biosíntesis de Proteínas , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Colifagos/genética , ADN Recombinante/metabolismo , Escherichia coli/genética , Operón , PlásmidosRESUMEN
Sau 3A1 partial digestion fragments from Bacillus thuringiensis var. kurstaki HD-1 plasmid DNA were ligated into the BamHI site of the cloning vector pBR322 and transformed into Escherichia coli strain HB101. Colonies presumed to contain recombinant plasmids were screened for production of an antigen that would react with antibody made against B. thuringiensis crystals. One strain, ES12, was isolated by using this procedure. ES12 contains a plasmid of Mr 11 X 10(6) that has DNA sequence homology with pBR322 as well as with Mr 30 X 10(6) and Mr 47 X 10(6) plasmids of B. thuringiensis. It makes a protein antigen, detected by antibodies to crystal, which has the same electrophoretic mobility as the B. thuringiensis crystal protein. Protein extracts of ES12 are toxic to larvae of the tobacco hornworm Manduca sexta.