Showing papers on "Bacillus thuringiensis published in 2003"
TL;DR: The sequencing and analysis of the type strain B. cereus ATCC 14579 together with the gapped genome of B. anthracis A2012 enables the comparative analysis to clarify the phylogeny of the cereus group, and the latter to determine plasmid-independent species-specific markers.
Abstract: Bacillus cereus is an opportunistic pathogen causing food poisoning manifested by diarrhoeal or emetic syndromes1. It is closely related to the animal and human pathogen Bacillus anthracis and the insect pathogen Bacillus thuringiensis, the former being used as a biological weapon and the latter as a pesticide. B. anthracis and B. thuringiensis are readily distinguished from B. cereus by the presence of plasmid-borne specific toxins (B. anthracis and B. thuringiensis) and capsule (B. anthracis). But phylogenetic studies based on the analysis of chromosomal genes bring controversial results, and it is unclear whether B. cereus, B. anthracis and B. thuringiensis are varieties of the same species2 or different species3,4. Here we report the sequencing and analysis of the type strain B. cereus ATCC 14579. The complete genome sequence of B. cereus ATCC 14579 together with the gapped genome of B. anthracis A20125 enables us to perform comparative analysis, and hence to identify the genes that are conserved between B. cereus and B. anthracis, and the genes that are unique for each species. We use the former to clarify the phylogeny of the cereus group, and the latter to determine plasmid-independent species-specific markers.
841 citations
TL;DR: Onfarm field trials carried out with Bacillus thuringiensis (Bt) cotton in different states of India show that the technology substantially reduces pest damage and increases yields.
Abstract: Onfarm field trials carried out with Bacillus thuringiensis (Bt) cotton in different states of India show that the technology substantially reduces pest damage and increases yields. The yield gains are much higher than what has been reported for other countries where genetically modified crops were used mostly to replace and enhance chemical pest control. In many developing countries, small-scale farmers especially suffer big pest-related yield losses because of technical and economic constraints. Pest-resistant genetically modified crops can contribute to increased yields and agricultural growth in those situations, as the case of Bt cotton in India demonstrates.
762 citations
TL;DR: The success of Bt crops exceeds expectations of many, but does not preclude resistance problems in the future, and violations of key assumptions of the refuge strategy (low resistance allele frequency and recessive inheritance) may occur in some cases.
Abstract: Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) grew on >62 million ha worldwide from 1996 to 2002. Despite expectations that pests would rapidly evolve resistance to such Bt crops, increases in the frequency of resistance caused by exposure to Bt crops in the field have not yet been documented. In laboratory and greenhouse tests, however, at least seven resistant laboratory strains of three pests (Plutella xylostella [L.], Pectinophora gossypiella [Saunders], and Helicoverpa armigera [Hubner]) have completed development on Bt crops. In contrast, several other laboratory strains with 70- to 10,100-fold resistance to Bt toxins in diet did not survive on Bt crops. Monitoring of field populations in regions with high adoption of Bt crops has not yet detected increases in resistance frequency. Resistance monitoring examples include Ostrinia nubilalis (Hubner) in the United States (6 yr), P. gossypiella in Arizona (5 yr), H. armigera in northern China (...
533 citations
TL;DR: Toxicity in nematodes correlates with damage to the intestine, consistent with the mechanism of crystal toxin action in insects, and structure–function analyses indicate that one novel nematicidal crystal protein can be engineered to a small 43-kDa active core.
Abstract: Bacillus thuringiensis (Bt) crystal proteins are pore-forming toxins used as insecticides around the world. Previously, the extent to which these proteins might also target the invertebrate phylum Nematoda has been mostly ignored. We have expressed seven different crystal toxin proteins from two largely unstudied Bt crystal protein subfamilies. By assaying their toxicity on diverse free-living nematode species, we demonstrate that four of these crystal proteins are active against multiple nematode species and that each nematode species tested is susceptible to at least one toxin. We also demonstrate that a rat intestinal nematode is susceptible to some of the nematicidal crystal proteins, indicating these may hold promise in controlling vertebrate-parasitic nematodes. Toxicity in nematodes correlates with damage to the intestine, consistent with the mechanism of crystal toxin action in insects. Structure–function analyses indicate that one novel nematicidal crystal protein can be engineered to a small 43-kDa active core. These data demonstrate that at least two Bt crystal protein subfamilies contain nematicidal toxins.
440 citations
TL;DR: The results reported here identify the cadherin gene as a leading target for DNA-based screening of resistance to Bt crops in lepidopteran pests.
Abstract: Evolution of resistance by pests is the main threat to long-term insect control by transgenic crops that produce Bacillus thuringiensis (Bt) toxins. Because inheritance of resistance to the Bt toxins in transgenic crops is typically recessive, DNA-based screening for resistance alleles in heterozygotes is potentially much more efficient than detection of resistant homozygotes with bioassays. Such screening, however, requires knowledge of the resistance alleles in field populations of pests that are associated with survival on Bt crops. Here we report that field populations of pink bollworm (Pectinophora gossypiella), a major cotton pest, harbored three mutant alleles of a cadherin-encoding gene linked with resistance to Bt toxin Cry1Ac and survival on transgenic Bt cotton. Each of the three resistance alleles has a deletion expected to eliminate at least eight amino acids upstream of the putative toxin-binding region of the cadherin protein. Larvae with two resistance alleles in any combination were resistant, whereas those with one or none were susceptible to Cry1Ac. Together with previous evidence, the results reported here identify the cadherin gene as a leading target for DNA-based screening of resistance to Bt crops in lepidopteran pests.
427 citations
TL;DR: Testing a unique model system consisting of Bt transgenic broccoli plants and the diamondback moth, Plutella xylostella, shows that resistance to pyramided two-gene plants was significantly delayed as compared with resistance to single-Gene plants deployed in mosaics, and to Cry1Ac toxin when it was the first used in a sequence.
Abstract: Preventing insect pests from developing resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops is a major challenge for agriculture. Theoretical models suggest that plants containing two dissimilar Bt toxin genes ('pyramided' plants) have the potential to delay resistance more effectively than single-toxin plants used sequentially or in mosaics. To test these predictions, we developed a unique model system consisting of Bt transgenic broccoli plants and the diamondback moth, Plutella xylostella. We conducted a greenhouse study using an artificial population of diamondback moths carrying genes for resistance to the Bt toxins Cry1Ac and Cry1C at frequencies of about 0.10 and 0.20, respectively. After 24 generations of selection, resistance to pyramided two-gene plants was significantly delayed as compared with resistance to single-gene plants deployed in mosaics, and to Cry1Ac toxin when it was the first used in a sequence. These results have important implications for the development and regulation of transgenic insecticidal plants.
413 citations
TL;DR: It is suggested that long-term regional pest suppression after deployment of Bt crops may also contribute to reducing the need for insecticide sprays.
Abstract: Despite the potentially profound impact of genetically modified crops on agriculture and the environment, we know little about their long-term effects. Transgenic crops that produce toxins from Bacillus thuringiensis (Bt) to control insects are grown widely, but rapid evolution of resistance by pests could nullify their benefits. Here, we present theoretical analyses showing that long-term suppression of pest populations is governed by interactions among reproductive rate, dispersal propensity, and regional abundance of a Bt crop. Supporting this theory, a 10-year study in 15 regions across Arizona shows that Bt cotton suppressed a major pest, pink bollworm (Pectinophora gossypiella), independent of demographic effects of weather and variation among regions. Pink bollworm population density declined only in regions where Bt cotton was abundant. Such long-term suppression has not been observed with insecticide sprays, showing that transgenic crops open new avenues for pest control. The debate about putative benefits of Bt crops has focused primarily on short-term decreases in insecticide use. The present findings suggest that long-term regional pest suppression after deployment of Bt crops may also contribute to reducing the need for insecticide sprays.
317 citations
TL;DR: Resistance to Bt appears to be costly and there is a rapid decline of resistance in populations collected from greenhouses and maintained in the laboratory without selection, which will require sporadic use of Bt–based sprays or alternatively use of sprays containing other Bt toxins.
Abstract: The microbial insecticide Bacillus thuringiensis ( Bt ) has become the mainstay of non–chemical control of Lepidopteran pests, either as sprays or through the incorporation of Bt toxins into transgenic crops. Given the wide use of Bt , it is striking that currently only one pest species, Plutella xylostella , has been reported to have developed significant resistance to Bt outside the laboratory. By contrast, we report here the frequent and rapid development of resistance to B. thuringiensis kurstaki (Dipel, Abbott) in populations of cabbage loopers, Trichoplusia ni , in commercial greenhouses. Resistance to Bt appears to be costly and there is a rapid decline of resistance in populations collected from greenhouses and maintained in the laboratory without selection. Management of pests resistant to Bt in vegetable greenhouses will require sporadic use of Bt –based sprays or alternatively use of sprays containing other Bt toxins.
302 citations
TL;DR: The results suggest that extended pre‐ and post‐commercial monitoring are necessary to assess the long‐term impact of Bt toxin in transgenic plant residues on soil organisms.
Abstract: Large quantities of Bacillus thuringiensis (Bt) corn plant residue are left in the field after harvest, which may have implications for the soil ecosystem Potential impacts on soil organisms will also depend on the persistence of the Bt toxin in plant residues Therefore, it is important to know how long the toxin persists in plant residues In two field studies in the temperate corn-growing region of Switzerland we investigated degradation of the Cry1Ab toxin in transgenic Bt corn leaves during autumn, winter and spring using an enzyme-linked immunosorbent assay (ELISA) In the first field trial, representing a tillage system, no degradation of the Cry1Ab toxin was observed during the first month During the second month, Cry1Ab toxin concentrations decreased to ∪ 20% of their initial values During winter, there was no further degradation When temperatures again increased in spring, the toxin continued to degrade slowly, but could still be detected in June In the second field trial, representing a no-tillage system, Cry1Ab toxin concentrations decreased without initial delay as for soil-incorporated Bt plants, to 38% of the initial concentration during the first 40 days They then continued to decrease until the end of the trial after 200 days in June, when 03% of the initial amount of Cry1Ab toxin was detected Our results suggest that extended pre- and post-commercial monitoring are necessary to assess the long-term impact of Bt toxin in transgenic plant residues on soil organisms
266 citations
TL;DR: Three laboratory strains of Helicoverpa armigera (Hübner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain to preserve genetic diversity, and genetic analysis confirmed that resistance in the BX strain of H. armigersa is incompletely recessive.
Abstract: Three laboratory strains of Helicoverpa armigera (Hubner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain. These strains were cultured on artificial diet containing the Cry1Ac protoxin of Bacillus thuringiensis using three different protocols. When no response to selection was detected after 7–11 generations of selection, the three strains were combined by controlled mating to preserve genetic diversity. The composite strain (BX) was selected on the basis of growth rate on artificial diet containing Cry1Ac crystals. Resistance to Cry1Ac was first detected after 16 generations of continuous selection. The resistance ratio (RR) peaked approximately 300-fold at generation 21, after which it declined to oscillate between 57- and 111-fold. First-instar H. armigera from generation 25 (RR = 63) were able to complete their larval development on transgenic cotton expressing Cry1Ac and produce fertile adults. There appeared to be a fitness cost associated with resistance on cotton and on artificial diet. The BX strain was not resistant to the commercial Bt spray formulations DiPel and XenTari, which contain multiple insecticidal crystal proteins, but was resistant to the MVP formulation, which only contains Cry1Ac. The strain was also resistant to Cry1Ab but not to Cry2Aa or Cry2Ab. Toxin binding assays showed that the resistant insects lacked the high affinity binding site that was detected in early generations of the strain. Genetic analysis confirmed that resistance in the BX strain of H. armigera is incompletely recessive.
231 citations
TL;DR: An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis.
Abstract: The three species of the group 1 bacilli, Bacillus anthracis, B. cereus, and B. thuringiensis, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis. Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B. anthracis Ames. Ninety-three DNA sequences that were present in B. anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B. anthracis strains. These sequences map to distinct loci on the B. anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B. anthracis.
TL;DR: Actin, aminopeptidase N, and membrane alkaline phosphatase were confirmed as accurate protein identifications through western blots and Peptide mass fingerprints were generated for several spots identified as Cry1Ac binding proteins and GPI-anchored proteins and these fingerprints were used for database searches.
TL;DR: This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions and the PCR screening strategy presented here led to identify a putative novel cry11B gene.
Abstract: The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented. Characterization was based on their insecticidal activity against Aedes aegypti, Culex quinquefasciatus, and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for cry and cyt genes encoding proteins active against mosquitoes (cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40). Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A. aegypti and C. quinquefasciatus larvae than B. thuringiensis subsp. israelensis and displayed high similarities with the B. thuringiensis subsp. israelensis used in this study with regard to protein and plasmid profiles and the presence of cry genes. Strain 147-8906 has activity against A. aegypti similar to that of B. thuringiensis subsp. israelensis but has different protein and plasmid profiles. This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions. Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene.
TL;DR: This work compares the largest B. thuringiensis PCR-based screenings, and the natural occurrence of cry genes among native strains, and discusses the use of PCR for the identification of novel cry genes, as well as the potential of novel technologies for the characterization of B.ThuringiensIS strains.
Abstract: The polymerase chain reaction (PCR) is a molecular tool widely used to characterize the insecticidal bacterium Bacillus thuringiensis. This technique can be used to amplify specific DNA fragments and thus to determine the presence or absence of a target gene. The identification of B. thuringiensis toxin genes by PCR can partially predict the insecticidal activity of a given strain. PCR has proven to be a rapid and reliable method and it has largely substituted bioassays in preliminary classification of B. thuringiensis collections. In this work, we compare the largest B. thuringiensis PCR-based screenings, and we review the natural occurrence of cry genes among native strains. We also discuss the use of PCR for the identification of novel cry genes, as well as the potential of novel technologies for the characterization of B. thuringiensis strains.
TL;DR: The results support the hypothesis that physiological adaptation of insects and resistance to Bt is multifaceted, including protein modification and changes in the synthesis of specific larval gut proteins.
TL;DR: A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1i-type gene fragments.
Abstract: A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis δ-endotoxin nomenclature committee.
TL;DR: The δ-endotoxins are a superfamily of proteins that occur as crystalline inclusions in the spore-forming bacterium Bacillus thuringiensis that are of considerable interest as environmentally safe insecticides.
Abstract: The δ-endotoxins are a superfamily of proteins that occur as crystalline inclusions in the spore-forming bacterium Bacillus thuringiensis ([35][1]). These toxins are of considerable interest as environmentally safe insecticides. Historically, B. thuringiensis toxins have been divided into two
TL;DR: It is suggested that resistance is probably rare enough in France and the northern US corn belt for the high-dose plus refuge strategy to delay resistance to Bt maize.
Abstract: Farmers, industry, governments and environmental groups agree that it would be useful to manage transgenic crops producing insecticidal proteins to delay the evolution of resistance in target pests. The main strategy proposed for delaying resistance to Bacillus thuringiensis (Bt) toxins in transgenic crops is the high-dose/refuge strategy. This strategy is based on the unverified assumption that resistance alleles are initially rare ( 1,200 isofemale lines of Ostrinia nubilalis Hubner (Lepidoptera: Crambidae) collected in France and the US corn belt during 1999–2001. In none of the isofemale lines did we detect alleles conferring resistance to Bt maize producing the Cry1Ab toxin. A Bayesian analysis of the data indicates that the frequency of resistance alleles in France was 80%. In the northern US corn belt, the frequency of resistance to Bt maize was 90%. Only 95 lines have been screened from the southern US corn belt, so these data are still inconclusive. These results suggest that resistance is probably rare enough in France and the northern US corn belt for the high-dose plus refuge strategy to delay resistance to Bt maize.
TL;DR: The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B. sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria.
Abstract: Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A. Moreover, although intensive use of B. sphaericus against Culex populations in the field can result in high levels of resistance, most of this can be suppressed by combining this bacterial species with Cyt1A; the latter enables the binary toxin of this species to enter midgut epithelial cells via the microvillar membrane in the absence of a midgut receptor. The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B. sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria.
TL;DR: Considering the additive interaction of toxins, a relatively simple insect resistance‐monitoring procedure was proposed for the monitoring of commercial cotton varieties expressing both toxins.
Abstract: Laboratory studies were performed to characterize the lepidopteran toxicity of cotton plants expressing two different toxin proteins from Bacillus thuringiensis (Bt), in order to assess insect resistance management implications of a commercial, two-toxin transgenic cotton An independent and additive interactive effect of two Bt δ-endotoxins expressed by the transgenic cotton variety 15985 was demonstrated by examining the responses of Heliothis virescens (F), Helicoverpa zea (Boddie), and Spodoptera frugiperda (JE Smith) larvae to field- or greenhouse-grown tissue from genetic near-isolines, which expressed Cry1A only, Cry2Ab only, or both toxins In all cases, the Cry2Ab component was the larger contributor to total toxicity in the two-toxin isoline Toxin-specific, quantitative enzyme-linked immunosorbent assay (ELISA) tests confirmed that the levels of each toxin in tissues of the two-toxin isoline were not statistically different (P > 005) from the levels found in the corresponding tissues of the respective single-toxin isoline Resistance management considerations were discussed Considering the additive interaction of toxins, a relatively simple insect resistance-monitoring procedure was proposed for the monitoring of commercial cotton varieties expressing both toxins
TL;DR: It is suggested that ingested corn DNA and Cry1Ab protein were not totally degraded in the gastrointestinal tract, as shown by their presence in a form detectable by PCR or immunological tests.
Abstract: Genetically modified corn has been approved as an animal feed in several countries, but information about the fate of genetically modified DNA and protein in vivo is insufficient. Genetically modified corn Bt11 is developed by inserting a recombinant DNA sequence encoding insecticidal Cry1Ab protein from Bacillus thuringiensis subsp. kurstaki. We examined the presence of corn intrinsic and recombinant cry1Ab gene by PCR, and the Cry1Ab protein by immunological tests in the gastrointestinal contents of five genetically modified corn Bt11-fed and five nongenetically modified corn-fed pigs. Fragments of corn zein (242 bp), invertase (226 bp) and of ribulose-1,5-bisphosphate carboxylase/ oxygenase genes (1,028 bp) were detected in the gastrointestinal contents of both Bt11 and nongenetically modified corn-fed pigs. Fragments of recombinant cry1Ab gene (110 bp and 437 bp) were detected in the gastrointestinal contents of the Bt11-fed pigs but not in the control pigs. Neither corn intrinsic nor cry1Ab gene fragments were detected in the peripheral blood by PCR. The gastrointestinal contents were positive for Cry1Ab protein by ELISA, immunochromatography, and immunoblot; however, these methods did not work for blood and precluded conclusions about any potential absorption of the protein. These results suggest that ingested corn DNA and Cry1Ab protein were not totally degraded in the gastrointestinal tract, as shown by their presence in a form detectable by PCR or immunological tests.
TL;DR: Some strains with moderate to high biopesticide activity against Spodoptera frugiperda and Premnotrypes vorax insects were identified, this being important to explore future microbial strategies for the control of these crop pests in the region.
TL;DR: Two transgenic rice lines, KMD1 and KMD2, containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner, exhibited high and stable resistance against natural infestations by the RLF, and showed no symptoms of damaged leaves throughout the rice-growing season.
TL;DR: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers.
Abstract: A . C H E R I F , S . C H E H I M I , F . L I M E M , B . M . H A N S E N , N . B . H E N D R I K S E N , D . D A F F O N C H I O A N D A . B O U D A B O U S . 2003. Aims: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers. Methods and Results: Bacillus thuringiensis ssp. entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi. Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase. Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121� C. Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization. After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12AE4 kDa was isolated. The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells. Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B. thuringiensis HD9 was not toxic against Vero cells. Conclusions: Entomocin 9 is a novel heat-stable, bacteriocin produced by B. thuringiensis HD9. The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments. Significance and Impact of the Study: New finding on entomocin 9 would make B. thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry.
TL;DR: Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes.
TL;DR: The results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding, which may question the effectiveness of gene stacking in delaying evolution of resistance.
Abstract: One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.
TL;DR: The results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.
Abstract: Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC(50)], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC(50) congruent with 114 ng/ml) and C. pipiens (LC(50) 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or AEDES: These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.
TL;DR: The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus and ChiA71 from Bacillin thuringiensis serovar pakistani, and showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chit in-binding domain.
Abstract: The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5αF′. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2°C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.
TL;DR: Compared toxin binding in resistant and susceptible strains of Pectinophora gossypiella, a major pest of cotton worldwide, shows that the resistance fits the "mode 1" pattern of resistance described previously in Plutella xylostella, Plodia interpunctella, and Heliothis virescens.
TL;DR: This antimicrobial peptide, referred to as thuricin 439, acts as a bacteriocidal peptide and exhibits an apparent narrow range of inhibitory activity, essentially only affecting growth of Bacillus cereus and B. thuringiensis strains.
Abstract: Bacillus thuringiensis strain B439 produces a bacteriocin-like inhibitory substance in its growth medium. This antimicrobial peptide, referred to as thuricin 439, acts as a bacteriocidal peptide and exhibits an apparent narrow range of inhibitory activity, essentially only affecting growth of Bacillus cereus and B. thuringiensis strains. It remains active over a relatively wide pH and temperature range, showing no loss of activity following heat treatments up to 80°C. Purification of thuricin 439 was achieved using several chromatographic steps, which resulted in the identification of two peptides with inhibitory activity. These two peptides were shown to possess identical N-terminal sequences, but different molecular masses.