Showing papers on "Bacillus thuringiensis published in 1995"

Novel bacillus thuringiensis genes coding toxins active against lepidopteran pests

Abstract: The present invention is drawn to novel toxin genes purified and isolated from Bacillus thuringiensis var kurstaki and given the designations CrylE(c) and CrylE(d). The novel toxin genes encode proteins of approximately 130 kDa in size and are active against Lepidopteran insects. Also included in the invention are the proteins encoded by CrylE(c) and CrylE(d). Further disclosed are recombinant genes and proteins derived from CrylE(c) and CrylE(d). Also provided are biologically pure bacterial strains transformed with at least one of the novel toxin genes which can be used in entomocidal formulations for the control of Lepidopteran insects. Yet another aspect of the invention are plants transformed with at least one of the toxin genes or active fragments thereof, particularly where the transforming sequences have been optimized for expression in maize.

Biochemistry and genetics of insect resistance to Bacillus thuringiensis insecticidal crystal proteins

Abstract: Current knowledge of biochemical mechanisms of insect resistance to Bacillus thuringiensis is reviewed. Available information on resistance inheritance and on patterns of cross-resistance is included. Modification of the binding sites for B. thuringiensis insecticidal crystal proteins has been found in different populations of three insect species. This resistance mechanism seems to be inherited as a single recessive or partially recessive major gene, and the resistance levels reached are high. Altered proteolytic processing of B. thuringiensis crystal proteins has been suggested to be involved in one case of resistance. From the available data it seems that binding site modification is the most significant resistance mechanism under field conditions.

Amplification of a Chimeric Bacillus Gene in Chloroplasts Leads to an Extraordinary Level of an Insecticidal Protein in Tobacco

Abstract: The Bacillus thuringiensis (Bt) crystal toxins are safe biological insecticides, but have short persistance and are poorly effective against pests that feed inside plant tissues. Production of effective levels of these proteins in plants has required resynthesis of the genes encoding them. We report that amplification of an unmodified crylA(c) coding sequence in chloroplasts up to approximately 10,000 copies per cell resulted in the accumulation of an unprecedented 3-5% of the soluble protein in tobacco leaves as protoxin. The plants were extremely toxic to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua. Since the plastid transgenes are not transmitted by pollen, this report has implications for containment of Bt genes in crop plants. Furthermore, accumulation of insecticidal protein at a high level will facilitate improvement in the management of Bt resistant insect populations.

How does Bacillus thuringiensis produce so much insecticidal crystal protein

Abstract: Members of the genus Bacillus are widely used as sources of industrial enzymes, fine biochemicals, antibiotics, and insecticides (for a review, see reference 27). One of these species, Bacillus thuringiensis, accounts for more than 90% of the biopesticides used today (for recent reviews on B. thuringiensis and its toxins, see references 6, 33, and 38). The entomopathogenic properties of this bacterium are due at least in part to the production of d-endotoxins that make up the crystalline inclusions characteristic of B. thuringiensis strains. In 1989, Hofte and Whiteley proposed a classification for d-endotoxins (30). They distinguished four major classes of d-endotoxins (CryI, -II, -III and -IV) and cytolysins (Cyt), found in the crystals of the mosquitocidal strains, on the basis of their insecticidal and molecular properties. The d-endotoxins belonging to each of these classes were grouped in subclasses (A, B, C. . . and a, b, c. . .) according to sequence. Generally, these proteins are toxic for lepidoptera (CryI), both lepidoptera and diptera (CryII), coleoptera (CryIII), and diptera (CryIV). These various insecticidal proteins are synthesized during the stationary phase and accumulate in the mother cell as a crystal inclusion which can account for up to 25% of the dry weight of the sporulated cells (Fig. 1). The amount of crystal protein produced by a B. thuringiensis culture in laboratory conditions (about 0.5 mg of protein per ml) and the size of the crystals (24) indicate that each cell has to synthesize 10 to 2 3 10 d-endotoxin molecules during the stationary phase to form a crystal. This is a massive production of protein and presumably occupies a large proportion of the cell machinery. Nevertheless, sporulation and the associated physiological changes proceed in parallel with d-endotoxin production. The aim of this minireview is to analyze the various mechanisms by which B. thuringiensis accumulates large quantities of toxins as biologically active protein crystals.

Field evaluation of european corn borer control in progeny of 173 transgenic corn events expressing an insecticidal protein from Bacillus thuringiensis

Abstract: The European corn borer [ECB; Ostrinia nubilalis (Hubner)] is an economically significant pest of corn (Zea mays L.). The ability to routinely transform corn has broadened the control options available to include the introduction of resistance genes from sexually incompatible species. In this study, microprojectile bombardment was used to introduce synthetic versions of cryIA insecticidal protein genes from Bacillus thuringiensis subsp. kurstaki (Btk) into embryogenic tissue of the Hi-II (A188/B73 derivative) genotype of corn. Of 715 independent transgenic calli produced, 314 (44%) had insecticidal activity against tobacco hornworm (Manduca sexta L.) larvae. Plants were regenerated, self-pollinated when possible, and crossed to B73. First-generation progeny of 173 independent Btk-protein expressing calli were evaluated under field conditions with artificial ECB infestations in 1992 or 1993. Approximately half (89/173) segregated in a single-gene manner for resistance to first-generation ECB leaf-feeding damage. All of the 89 lines evaluated in 1992 or 1993 for resistance to second-generation ECB exhibited less stalk tunneling damage then the non-transgenic controls. In 1993, 44% (37/77) of the lines tested had ≤ 2.5 cm of tunneling, compared to severe damage (mean = 45.7 cm) in the B73 × Hi-II controls. Experiments are in progress to evaluate the effect of the introduced genes on yield and other agronomic properties

Bacillus cereus and Bacillus thuringiensis isolated in a gastroenteritis outbreak investigation.

Abstract: During investigation of a gastroenteritis outbreak in a chronic care institution, Norwalk virus was found in stool specimens from two individuals and bacterial isolates presumptively identified as Bacillus cereus were isolated from four individuals (including one with Norwalk virus) and spice. Phage typing confirmed all Bacillus clinical isolates were phage type 2. All clinical isolates were subsequently identified as B. thuringiensis when tested as a result of a related study (L. Leroux, personal communication). Eight of 10 spice isolates were phage type 4. All B. cereus and B. thuringiensis isolates showed cytotoxic effects characteristic of enterotoxin-producing B. cereus. An additional 20 isolates each of B. cereus and B. thuringiensis from other sources were tested for cytotoxicity. With the exception of one B. cereus, all showed characteristic cytotoxic patterns.

Contribution of the individual components of the δ-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis

Abstract: The δ-endotoxin crystal of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis contains four major 0-endotoxins. Expression systems were devised to synthesize each of the four toxins at concentrations at which they formed inclusion bodies in an acrystalliferous mutant of Bacillus thuringiensis. The relative activities of these inclusions were then determined against Aedes aegypti larvae. Bioassays of mixtures of the individual toxins revealed a number of synergistic interactions which explained in part why the native crystal is considerably more toxic than any of the individual toxins.

Regulation of insecticidal crystal protein production in Bacillus thuringiensis

Abstract: The production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis normally coincides with sporulation, resulting in the appearance of parasporal crystalline inclusions within the mother cell. In most instances, the temporal and spatial regulation of ICP gene expression is determined at the transcriptional level by mother-cell-specific sigma factors that share homology with sigma E and sigma K from Bacillus subtilis. The cryIII ICP genes are a notable exception; these genes are transcribed from sigma A-like promoters during vegetative growth, are induced or derepressed at the onset of stationary phase, and are overexpressed in sporulation mutants of B. thuringiensis blocked in the phosphorylation of Spo0A, a key regulator of sporulation initiation. Transcription alone, however, cannot account for the impressive ability of this bacterium to accumulate insecticidal proteins. A variety of post-transcriptional and post-translational mechanisms also contribute to the efficient production of ICPs in B. thuringiensis, thus making this bacterium a cost-effective biological control agent.

Insecticidal Activity of the Toxins from Bacillus thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure and Soil Clays.

Abstract: The release of transgenic plants and microorganisms expressing truncated genes from various subspecies of Bacillus thuringiensis that encode active insecticidal toxins rather than inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clays and other soil particles. Toxins from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis, either free or adsorbed at equilibrium or bound on pure clay minerals (montmorillonite or kaolinite) or on the clay size fraction of soil, were toxic to larvae of the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata), respectively. The 50% lethal concentrations (LC(inf50)) of free toxins from B. thuringiensis subsp. kurstaki were higher than those of both bound and adsorbed complexes of these toxins with clays, indicating that adsorption and binding of these toxins on clays increase their toxicity in diet bioassays. The LC(inf50) of the toxin from B. thuringiensis subsp. tenebrionis that was either free or adsorbed on montmorillonite were comparable, whereas the toxin bound on this clay had higher LC(inf50) and the toxin bound on kaolinite had lower LC(inf50) than when adsorbed on this clay. Results obtained with the clay size fraction separated from unamended soil or soil amended with montmorillonite or kaolinite were similar to those obtained with the respective pure clay minerals. Therefore, insecticidal activity of these toxins is retained and sometimes enhanced by adsorption and binding on clays.

Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae).

Abstract: Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp aizawai and B thuringiensis subsp entomocidus was attempted by using laboratory bioassays No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 430 (mu)g/g for the susceptible colony Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B thuringiensis subsp entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects Resistance to CryIC was significantly reduced by the addition of HD-1 spores Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed These results suggest that, in S exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins These results have important implications for devising S exigua resistance management strategies in the field

A comparative review of the mammalian toxicity of Bacillus thuringiensis-based pesticides

Abstract: The extensive mammalian toxicity studies performed to support the safety of Bacillus thuringiensis-containing pesticides clearly demonstrate that the tested isolates are not toxic or pathogenic. Toxicity studies submitted to the US Environmental Protection Agency to support the registration of B. thuringiensis subspecies have failed to show any significant adverse effects in body weight gain, clinical observations, or upon necropsy. Infectivity/pathogenicity studies have shown that the intact rodent immune system responds as expected to eliminate B. thuringiensis gradually from the body after oral, pulmonary or intravenous challenge. Similar clearance patterns were observed with B. subtilis and B. sphaericus, which also were non-toxic to test animals. The results also indicate that the currently used protocols for toxicity/pathogenicity evaluations of micro-organisms in laboratory animals have provided useful and necessary information for risk assessment. The study results raise the issue, however, of whether some reduced set of toxicily/pathogenicity studies could be required for new B. thuringiensis isolates of already registered subspecies, and whether this reduced data set would also apply to subspecies not yet registered as pesticidal active ingredients.

Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spo0A mutant.

Abstract: Overproduction of Encapsulated Insecticidal Crystal Proteins in a Bacillus thuringiensis spoOA Mutant

Resistance to Bacillus thuringiensis CryIA delta-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration.

Abstract: The Bacillus thuringiensis toxin-binding properties of midgut epithelial cells from two strains of Heliothis virescens were compared. One H. virescens strains (YHD2) which was selected against CryIAc toxin had over 10,000-fold resistance to CryIAc toxin relative to the susceptible strain and was cross-resistant to CryIAa and CryIAb. The second H. virescens strain (YDK) was susceptible to these toxins in the order CryIAc > CryIAb > CryIAa. Receptor-binding properties of CryIAa, CryIAb, and CryIAc toxins were compared between the susceptible and resistant strains. Saturation and competition-binding experiments were performed with brush border membrane vesicles prepared from midguts of the susceptible and resistant insects and 125I-labeled toxins. In the susceptible strain, saturable, specific, and high-affinity binding of all three toxins was observed. The relative binding-site concentration was directly correlated with toxicity (CryIAc > CryIAb > CryIAa). In the resistant strains, the binding affinities of CryIAb and CryIAc were similar to that observed with the susceptible strain and ony minor differences in binding-site concentration (Bmax) were observed. The major difference between the two strains was the total lack of binding of CryIAa toxin to the brush border membrane vesicles of the resistant strain. Heterologous competition-binding experiments and ligand blot analysis supported the hypothesis that there were multiple binding sites for the toxins. On the basis of results of the present study, we propose that alterations in binding proteins shared by all three toxins are a major factor in resistance. This suggests that not all receptors of CryIAc might be involved in toxic function.

Resistance: A Threat to the Insecticidal Crystal Proteins of Bacillus thuringiensis

Abstract: Insecticidal crystal proteins (also known as d-endotoxins) synthesized by the bacterium Bacillus thuringiensis Berliner (Bt) are the active ingredient of various environmentally friendly insecticides that are 1) highly compatible with natural enemies and other nontarget organisms due to narrow host specificity, 2) harmless to vertebrates, 3) biodegradable in the environment, and 4) highly amenable to genetic engineering. The use of transgenic plants expressing Bt d-endotoxins has the potential to greatly reduce the environmental and health costs associated with the use of conventional insecticides. The complex mode of action of Bt is the subject of intensive research. When eaten by a susceptible insect d-endotoxin crystals are solubilized in the midgut; proteases then cleave protoxin molecules into activated toxin which binds to receptors on the midgut brush border membrane. Part of the toxin molecule inserts into the membrane causing the midgut cells to leak, swell, and lyse; death results from bacterial septicemia. Insecticides formulated with Bt account for less than 1% of the total insecticides used each year worldwide because of high cost, narrow host range, and comparatively low efficacy. Environmental contamination, food safety concerns, and pest resistance to conventional insecticides have caused a steady increase in demand for Bt-based insecticides. The recent escalation of commercial interest in Bt has resulted in more persistent and efficacious formulations. For example, improved Bt-based insecticides have allowed management of the diamondback moth, Plutella xylostella (L.). Unfortunately this has resulted in the evolution of resistance to d-endotoxins in P. xylostella populations worldwide. The recent appearance of Bt resistance in the field, corroborated by the results of laboratory selection experiments, demonstrates genetically-based resistance in several species of Lepidoptera, Diptera, and Coleoptera. The genetic capacity to evolve resistance to these toxins is probably present in all insects, and the heritability, fitness costs, and stability of the resistance trait are documented in several insect populations. In two strains of Bt-resistant lepidopteran species, mechanisms of resistance involve reductions in the binding of toxin to midgut receptors. Research on other resistant strains suggests that other mechanisms are also involved. Unfortunately, the high stability of the resistance trait, as well as broad spectrum cross-resistance to other d-endotoxins, undermines many potential options for resistance management. Genetically engineered plants, expressing d-endotoxin continuously and at ultrahigh doses, ensure intense and rapid selection of the target insect population. The efficacy of transgenic plants can be preserved only by developing an integrated pest management program that is designed specifically to reduce selection pressure by minimizing exposure to Bt and increasing other mortality factors, thereby slowing the rate of pest adaptation to Bt.

Transgenic broccoli expressing a Bacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

Abstract: We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F1 hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F1 larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.

Natural Variation: A Complicating Factor in Bioassays with Chemical and Microbial Pesticides

Abstract: Our investigation of responses of Colorado potato beetle, Leptinotarsa decemlineata (Say), to Bacillus thuringiensis subsp. tenebrionis over 83 wk; diamondback moth, Plutella xylostello (L.), to B. thuringiensis subsp. kurstaki (cloned into Pseudomonas florescens ) over 37 wk; and western spruce budworm, Choristoneura occidentalis Freeman, to pyrethrins and DDT over 91 generations indicated that ratios at LC50, LC90, and LC99 varied among pesticides tested on the same species and among insect species tested with the same pesticide. Frequencies with which LCs were significantly different (based on bracketing of the value 1.0 by 95% CI of each ratio) compared with the standard (the lowest LC in the data set) were extremely high (>95%) in all tests except those with pyrethrins on western spruce budworm. For example, toxicity ratios for Colorado potato beetle larvae fed B. thuringiensis subsp. tenebrionis were as high as 12.8 at LC50, 37.0 at LC90, and 155 at LC99 over 83 wk; the maximum upper 95% CL for a ratio were 18.6, 168, and 1,000, respectively, at these LCs. We conclude that the extent of natural variation must be investigated before biologically important changes can be identified with any certainty. The conventional practice of using ratios of one LC to another in studies to detect resistance and other biological changes may lead to erroneous conclusions if natural variation in cohorts of a population and subsequent generations of the same genetic strain are unknown.

Cloning and expression of a novel toxin gene from Bacillus thuringiensis subsp. jegathesan encoding a highly mosquitocidal protein.

Abstract: A gene, designated cry11B, encoding a 81,293-Da crystal protein of Bacillus thuringiensis subsp. jegathesan was cloned by using a gene-specific oligonucleotide probe. The sequence of the Cry11B protein, as deduced from the sequence of the cry11B gene, contains large regions of similarity with the Cry11A toxin (previously CryIVD) from B. thuringiensis subsp. israelensis. The Cry11B protein was immunologically related to both Cry11A and Cry4A proteins. The cry11B gene was expressed in a nontoxic strain of B. thuringiensis, in which Cry11B was produced in large amounts during sporulation and accumulated as inclusions. Purified Cry11B inclusions were highly toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi. The activity of Cry11B toxin was higher than that of Cry11A and similar to that of the native crystals from B. thuringiensis subsp. jegathesan, which contain at least seven polypeptides.

Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin gene.

Abstract: Two different 30-nucleotide regions of the cryIAc insecticidal protoxin gene from Bacillus thuringiensis were randomly mutagenized. One region was within one of seven amphipathic helices believed to be important for the formation of ion channels. There was no loss of toxicity for three test insects by any of 27 mutants, a result similar to that obtained previously for mutations within another such helix. Only mutations within a region encoding the central helix have resulted in a substantial number of mutants with low or no toxicity. A second mutagenized region encodes amino acids which are unique to this toxin and are within one of the loops in a portion of the toxin important for specificity. Among 21 different mutations of these 10 residues, only changes of two adjacent serine residues resulted in decreased toxicity which was greater for Manduca sexta than for Heliothis virescens larvae. These mutant toxins bound poorly to the single M. sexta CryIAc vesicle-binding protein and to several of the multiple H. virescens-binding proteins. The loop containing these serines must be involved in the formation of a specific toxin recognition domain.

The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16.

Abstract: The aggregation phenotypes Agr+ and Agr- of Bacillus thuringiensis subsp. israelensis are correlated with a conjugation-like plasmid transfer and characterized by the formation of aggregates when the bacteria are socialized during exponential growth. We present evidence for the association of the Agr+ phenotype with the presence of the large (135-MDa) self-transmissible plasmid pXO16.

Comparative Analysis of the 16S to 23S Ribosomal Intergenic Spacer Sequences of Bacillus thuringiensis Strains and Subspecies and of Closely Related Species.

Abstract: Bacillus thuringiensis spacer regions between the 16S and 23S rRNAs were amplified with conserved primers, designated 19-mer and 23-mer primers. A spacer region of 144 bp was determined for all of 6 B. thuringiensis strains, 7 B. thuringiensis subspecies, and 11 B. thuringiensis field isolates, as well as for the closely related species Bacillus cereus and Bacillus anthracis. Computer analysis and alignment of nucleotide sequences identified three mutations and one deletion in the intergenic spacer region (ISR) of B. thuringiensis subsp. kurstaki HD-1 when compared with ISR sequences from other subspecies. The same differences were identified between the ISR of B. thuringiensis strains and the ISR of B. cereus and B. anthracis. These minor differences do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe.

Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides

Abstract: Strains of Bacillus cereus and B. thuringiensis were tested by the Tecra VIA kit for the ability to produce a diarrhoeal enterotoxin. The strains of B. thuringiensis were isolated from commercial B. thuringiensis-based insecticides (BactimosTM, DiPelTM, FlorbacTM FC, ForayTM 48B, NovodorTM FC, TurexTM, VecTobacTM, XenTariTM). The production of diarrhoeal enterotoxin varied by a factor of more than 100 among the different strains tested. B. cereus (F4433/73) produced the highest amount of enterotoxin and the B. thuringiensis strain isolated from DiPelTM the lowest. The products were tested for their content of diarrhoeal enterotoxin and all products, except MVPTM which does not contain viable B. thuringiensis spores, contained diarrhoeal enterotoxins. The results indicates an potential risk for gastroenteritis outbreak caused by B. thuringiensis.

Inheritance of Resistance to Bacillus thuringiensis subsp. tenebrionis CryIIIA δ-Endotoxin in Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Abstract: We investigated the genetic inheritance of Colorado potato beetle, Leptinotarsa decemlineata (Say), resistance to Bacillus thuringiensis CryIIIA delta-endotoxin. Standard reciprocal crosses and backcrosses between susceptible (S) and resistant (R) strains were used to determine the characteristics of resistance. Analysis of probit lines from the F1 reciprocal crosses indicated that B. thuringiensis delta-endotoxin resistance was inherited autosomaly without maternal effects. We estimated the degree of dominance to be 0.77 and 0.76 for the (R x S) and (S x R) F1 generations, respectively, indicating that B. thuringiensis CryIIIA delta-endotoxin resistance is conferred by incompletely dominant genes. Chi-square analysis of mortality responses of backcrossed offspring suggested that resistance might be caused by more than one locus. The stability of resistance was also studied by testing seventeen generations of resistant beetles after the selection pressure was removed. When the selection pressure was removed, the resistance level of the selected colony decreased after five generations. The resistance level did not decrease further when the selection was removed for > 12 generations.

Transcriptional regulation of the cryIVD gene operon from Bacillus thuringiensis subsp. israelensis.

Abstract: The CryIVD protein is involved in the overall toxicity of the Bacillus thuringiensis subsp. israelensis parasporal inclusions and is one of the four major components of the crystals. Determination of the DNA sequence indicated that the cryIVD gene is the second gene of an operon which includes three genes. The first one encodes a 19-kDa polypeptide and has sequence homology with the orf1 gene of the Bacillus thuringiensis cryIIA and cryIIC operons. The second and third genes have already been identified and encode the CryIVD crystal protein and the P20 polypeptide, respectively. The promoter region was located by deletion analysis, and the 5' end of the mRNA was determined by primer extension mapping. Transcription of the cryIVD gene in B. thuringiensis subsp. israelensis strains is induced 9 h after the beginning of sporulation. Sequence analysis indicated two potential promoters, a strong one and a weak one, recognized respectively by the RNA polymerase associated with the sigma 35 or the sigma 28 factor of B. thuringiensis (sigma E and sigma K of Bacillus subtilis, respectively). Transcriptional lacZ fusion integrated in single copy into the chromosome of various B. subtilis sporulation mutants confirmed the sigma E dependence of cryIVD gene transcription.