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1.
Talanta ; 236: 122813, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635209

RESUMO

Bacillus thuringiensis (Bt) is used as a bioinsecticide since it effectively kills insect larvae. Bt is also genetically similar to Bacillus cereus (Bc), a well recognized foodborne human pathogen; they are both members of the Bacillus cereus group (BC group). Although approved Bt bioinsecticide products have been confirmed to be non-pathogenic to humans, close monitoring of Bt during dissemination is important for cost considerations and to limit impact on biodiversity towards nontarget organisms. As such, developing rapid, sensitive, and specific tools for quantitative detection of Bt spores during and following spray operations is highly desirable. The goals of this study were to investigate commercially available detection reagents for sensitivity and selectivity in detecting Bt spores, and then functionalize a surface of (001) GaAs used in photonic biosensing. To achieve these goals, we (1) screened commercial antibodies for their capacity to bind recombinant proteins from Bt spores, (2) screened antibodies and aptamers for their sensitivity and selectivity against Bt spores, and (3) tested the efficiency of selected antibodies and aptamers in capturing Bt spores on the surface of functionalized GaAs biochips. Seven genes encoding Bt spore proteins were cloned and expressed in Escherichia coli. The binding of each purified spore antigen was tested by commercially available polyclonal and monoclonal antibodies claimed to exclusively target spores. Of the seven targets, Bacillus collagen-like protein A, was the most abundant protein on Bt spores and demonstrated the strongest binding affinity to all test antibodies. The commercial antibodies (Abs) were also tested for specificity to BC Group versus non-BC Group spores. Three of six commercial antibodies showed selectivity to Bt spores, with recombinant Abs providing the most robust lower range of detection (102 to 6 × 103 spores/mL). The sensitivity and selectivity of three published DNA aptamer sequences demonstrated a wide range of detection sensitivity for Bt spores. Two of the three test aptamers also showed reasonable selectivity towards Bt spores while the third demonstrated reactivity to non-BC Group B. megaterium and B. subtilis. Of the reagents tested, a thiolated aptamer and llama recombinant Ab showed highest Bt spore capture efficiency as measured by spore coverage of the GaAs surface. These results confirm that the selected aptamer and llama rAb can be considered strong candidates for the development of GaAs-based biosensing devices.


Assuntos
Bacillus anthracis , Bacillus thuringiensis , Técnicas Biossensoriais , Arsenicais , Bacillus thuringiensis/genética , Gálio , Humanos , Esporos Bacterianos
2.
J Agric Food Chem ; 69(40): 11816-11824, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34596393

RESUMO

The continued success of pest control using insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) in transgenic plants was threatened by the evolution of resistance. Previous studies suggested that polycalin from Plutella xylostella could bind to Cry1Ac toxin as a potential receptor. In this study, a fragment of P. xylostella polycalin (Pxpolycalinf, G2209-A2942) containing a carboxyl-terminal GPI-anchored signal peptide was cloned and expressed. Purified Pxpolycalinf retained the binding ability to Cry1Ac and synergized Cry1Ac toxicity to the third larvae of P. xylostella in bioassays. Moreover, the polyclonal antibody of Pxpolycalinf decreased the Cry1Ac activity after being fed together with normal food. Further, the ELISA results showed the concentration-dependent binding of Pxpolycalinf to P. xylostella brush border membrane vesicles (BBMV). Spodoptera frugiperda 9 (Sf9) cells expressing Pxpolycalinf were not susceptive to Cry1Ac, whereas Pxpolycalinf increased Cry1Ac cytotoxicity to Sf9 cells expressing P. xylostella ATP-dependent binding cassette transporter C2 (PxABCC2). Immunolocalization presented the binding of Pxpolycalinf to the Sf9 cell membrane, and ELISA showed the concentration-dependent binding of Pxpolycalinf to Sf9 cell extraction. These results here provide the first evidence that a fragment of P. xylostella polycalin, a potential receptor of Cry1Ac, synergizes Cry1Ac toxicity to P. xylostella larvae and Sf9 cells expressing PxABCC2.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas de Insetos/genética , Resistência a Inseticidas , Larva/genética , Mariposas/genética
3.
Ecotoxicol Environ Saf ; 225: 112721, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34478987

RESUMO

Previous studies reported adverse effects of genetically engineered maize that produces insecticidal Cry proteins from Bacillus thuringiensis (Bt) on the water flea Daphnia magna. In the current study, effects of flour, leaves, or pollen from stacked Bt maize that contains six Bt proteins (SmartStax) in two plant backgrounds on life table parameters of D. magna were investigated. Adverse effects were observed for Bt maize flour, originating from different production fields and years, but not for leaves or pollen, produced from plants grown concurrently in a glasshouse. Because leaves contained eight to ten times more Cry protein than flour, the effects of the flour were probably not caused by the Cry proteins, but by compositional differences between the plant backgrounds. Furthermore, considering the natural range of variation in the response of D. magna to conventional maize lines, the observed effects of Bt maize flour were unlikely to be of biological relevance. Our study demonstrates how Cry protein effects can be separated from plant background effects in non-target studies using Bt plant material as the test substance and how detected effects can be judged for their biological relevance.


Assuntos
Bacillus thuringiensis , Animais , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Daphnia/genética , Endotoxinas/genética , Endotoxinas/toxicidade , Farinha , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Plantas Geneticamente Modificadas , Zea mays/genética
4.
Ecotoxicol Environ Saf ; 226: 112805, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34592526

RESUMO

During the production and application of Bacillus thuringiensis (Bt) transgenic crops, large doses of insecticidal Bt toxic proteins are expressed continuously. The multi-interfacial behaviors of Bt proteins entering the environment in multi-media affects their states of existence transformation, transport and fate as well as biological and ecological impacts. Because both soil matrix and organisms will be exposed to Bt proteins to a certain extent, knowledge of the multi-interfacial behaviors and affecting factors of Bt proteins are vital not only for understanding the source-sink distribution mechanisms, predicting their bio-availability, but also for exploring the soil safety and environmental problems caused by the interaction between Bt proteins and soil matrix. This review summarized and analyzed various internal and external factors that affect the adsorption/ desorption and degradation of Bt proteins in the environment, so as to understand the multi-interfacial behaviors of Bt proteins. In addition, the reasons of concentration changes of Bt proteins in soil are discussed. This review will also discuss the existing knowledge of the combined effects of Bt proteins and other pollutants in environment. Finally, discussing the factors that should be considered when assessing the environmental risk of Bt proteins, thus to further improve the understanding of the environmental fate of Bt proteins.


Assuntos
Bacillus thuringiensis , Adsorção , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas , Solo
5.
Antonie Van Leeuwenhoek ; 114(11): 1759-1770, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34491485

RESUMO

Bacillus thuringiensis is the most successful microbial insecticide against different pests in agriculture and vectors of diseases. Its activity is mostly attributed to the Cry proteins expressed during its sporulation phase. However, these proteins are not exclusive to B. thuringiensis. Some cry genes have been found in other Bacillus species, or even in other genera. In this work, cry genes were searched in 223 acrystalliferous bacillaceous strains. From these strains 13 amplicons were obtained, cloned, and sequenced; however, only 6 amplicons tested positive for cry-like genes, and the 6 isolates showed to be the same strain. We report the characterization of an unusual strain of B. cereus (LBIC-004) which is unable to form protein inclusions during the sporulation phase. LBIC-004 showed a high identity to B. cereus using the sequences of 16S rRNA, gyrB and hag genes; in addition, a unique plasmid pattern of the strain was obtained. A 1953-bp cry gene was identified, coding for a 651 amino acid protein with a molecular weight of 74.9 kDa. This protein showed a predicted three-domain structure, similar to all Cry proteins. However, the amino acid sequence of the protein showed only 41% identity its highest hit: the Cry8Ca1 protein, indicating the uniqueness of this cry-like gene. It was cloned and transferred into a mutant acrystalliferous B. thuringiensis strain which was used in bioassays against Caenorhabditis elegans, Aedes aegypti, Manduca sexta and Phyllophaga sp. The recombinant strain showed no crystal formation and no toxicity to the tested species.


Assuntos
Bacillus cereus , Bacillus thuringiensis , Bacillus cereus/genética , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Plasmídeos , RNA Ribossômico 16S/genética
6.
BMC Genomics ; 22(1): 639, 2021 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-34479486

RESUMO

BACKGROUND: Resistance of pest insect species to insecticides, including B. thuringiensis (Bt) pesticidal proteins expressed by transgenic plants, is a threat to global food security. Despite the western corn rootworm, Diabrotica virgifera virgifera, being a major pest of maize and having populations showing increasing levels of resistance to hybrids expressing Bt pesticidal proteins, the cell mechanisms leading to mortality are not fully understood. RESULTS: Twenty unique RNA-seq libraries from the Bt susceptible D. v. virgifera inbred line Ped12, representing all growth stages and a range of different adult and larval exposures, were assembled into a reference transcriptome. Ten-day exposures of Ped12 larvae to transgenic Bt Cry3Bb1 and Gpp34/Tpp35Ab1 maize roots showed significant differential expression of 1055 and 1374 transcripts, respectively, compared to cohorts on non-Bt maize. Among these, 696 were differentially expressed in both Cry3Bb1 and Gpp34/Tpp35Ab1 maize exposures. Differentially-expressed transcripts encoded protein domains putatively involved in detoxification, metabolism, binding, and transport, were, in part, shared among transcripts that changed significantly following exposures to the entomopathogens Heterorhabditis bacteriophora and Metarhizium anisopliae. Differentially expressed transcripts in common between Bt and entomopathogen treatments encode proteins in general stress response pathways, including putative Bt binding receptors from the ATP binding cassette transporter superfamily. Putative caspases, pro- and anti-apoptotic factors, as well as endoplasmic reticulum (ER) stress-response factors were identified among transcripts uniquely up-regulated following exposure to either Bt protein. CONCLUSIONS: Our study suggests that the up-regulation of genes involved in ER stress management and apoptotic progression may be important in determining cell fate following exposure of susceptible D. v. virgifera larvae to Bt maize roots. This study provides novel insights into insect response to Bt intoxication, and a possible framework for future investigations of resistance mechanisms.


Assuntos
Bacillus thuringiensis , Besouros , Praguicidas , Animais , Bacillus thuringiensis/genética , Sobrevivência Celular , Besouros/genética , Endotoxinas/toxicidade , Resistência a Inseticidas , Larva/genética , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Regulação para Cima , Zea mays/genética
7.
Neotrop Entomol ; 50(5): 804-811, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34398398

RESUMO

Bacillus thuringiensis (Bt)-based bioinsecticides and transgenic plants expressing proteins with insecticidal activity (Cry and Vip) have been successfully used in several integrated pest management programs worldwide. Lepidoptera comprise some of the most economically important insect pests of the major agricultural crops. In this study, the toxicity of 150 Bt strains was evaluated against Helicoverpa armigera (Hübner) larvae. Eight strains (426, 520B, 1636, 1641, 1644, 1648, 1657 and 1658) showed high insecticide activity against H. armigera and were therefore tested against Anticarsia gemmatalis (Hübner), Spodoptera cosmioides (Walker), Chrysodeixis includens (Walker), and Diatraea saccharalis (Fabricius) larvae. Our results showed that most of the Bt strains were also toxic to these lepidopteran species. The biochemical and molecular analyses of these strains revealed that they had a similar protein profile; however, their cry and vip gene contents were variable. In addition, the median lethal concentration (LC50) of the selected strains indicated that the strains 1636, 1641, and 1658 were the most effective against H. armigera, showing LC50 values of 185.02, 159.44, and 192.98 ng/cm2, respectively. Our results suggest that the selected Bt strains have great potential to control the lepidopteran pests H. armigera, A. gemmatalis, D. saccharalis, S. cosmioides, and C. includes.


Assuntos
Bacillus thuringiensis , Agentes de Controle Biológico , Mariposas , Controle Biológico de Vetores , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas/toxicidade , Proteínas Hemolisinas , Larva/microbiologia , Mariposas/microbiologia
8.
Acta Trop ; 223: 106088, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34389325

RESUMO

Some Bacillus thuringiensis (Bt) strains produce dipteran-active toxins and can control larval mosquitoes. We identified a novel mosquitocidal toxin named Xpp81Aa1 with the thioredoxin domain from Bt strain HSY204. This toxin has very little sequence similarity to the three-domain Cry toxin and Cyt toxins and has significant toxicity to Aedes aegypti larvae. A safety assessment indicated that the Xpp81Aa1 toxin has no cytocidal activity against red blood cells and did not induce allergic reactions. The Xpp81Aa1 toxin exhibited a synergistic effect in combination with Cry2Aa and Cry4Aa protein toxins. Thus, the Xpp81Aa1 toxin could be a good candidate for mosquito control applications to reduce the mosquito-borne disease.


Assuntos
Aedes , Toxinas de Bacillus thuringiensis , Controle de Mosquitos , Animais , Bacillus thuringiensis , Toxinas de Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Larva
9.
Arch Microbiol ; 203(9): 5387-5396, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34390357

RESUMO

Keratinases are a group of proteases of great industrial significance. To take full advantage of Bacillus species as an inherent superior microbial producer of proteases, we performed the ribosome engineering to improve the keratinase synthesis capacity of the wild-type Bacillus thuringiensis by inducing streptomycin resistance. Mutant Bt(Str-O) was identified as a stable keratinase overproducer. Comparative characterization of the two strains revealed that, although the resistance to Streptomycin increased by eight-fold in MIC, the mutant's resistance to other commonly used antibiotics was not affected. Furthermore, the mutant exhibited an enhanced keratinase synthesis (1.5-fold) when cultured in a liquid LB medium. In the whole feather degradation experiment, the mutant could secret twofold keratinase into the medium, reaching 640 U/mL per 107 CFU. By contrast, no significant differences were found in the scanning electron microscopic analysis and spore formation experiment. To understand the genetic factors causing these phenotypic changes, we cloned and analyzed the rpsL gene. No mutation was observed. We subsequently determined the genome sequences of the two strains. Comparing the rpsL gene revealed that the emergence of streptomycin resistance was not necessarily dependent on the mutation(s) in the generally recognized "hotspot." Genome-wide analysis showed that the phenotypic changes of the mutant were the collective consequence of the genetic variations occurring in the regulatory regions and the non-coding RNA genes. This study demonstrated the importance of genetic changes in regulatory regions and the effectiveness of irrational ribosome engineering in creating prokaryotic microbial mutants without sufficient genetic information.


Assuntos
Bacillus thuringiensis , Estreptomicina , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Mutação , Peptídeo Hidrolases/genética , Sequências Reguladoras de Ácido Nucleico , Estreptomicina/farmacologia
10.
Environ Sci Technol ; 55(18): 12504-12516, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34460233

RESUMO

It is generally believed that Bacillus thuringiensis var. israelensis (Bti) biopesticides are harmless to non-target organisms; however, new research shows controversial results. We exposed acutely and chronicallyLithobates sylvaticusandAnaxyrus americanus tadpoles until metamorphic climax to VectoBac 200G (granules) and VectoBac 1200L (aqueous suspension) at 300-20,000 ITU/L covering field-relevant concentrations and higher. The data show that the exposure parameters tested did not affect significantly the survival, total length, total weight, hepatosomatic index, gonadosomatic index, the expression of genes of interest (i.e., related to xenobiotic exposure, oxidative stress, and metamorphosis), and the intestine tissue layer detachment ofL. sylvaticusandA. americanus in a concentration-response pattern. In contrast, VectoBac 200G significantly increased the median time to metamorphosis ofL. sylvaticus tadpoles by up to 3.5 days and decreased the median by up to 1 day inA. americanus. VectoBac 1200L significantly increased the median time to metamorphosis ofL. sylvaticusandA. americanustadpoles by up to 4.5 days. Also, the exposure to VectoBac 200G and 1200L altered the intestine bacterial community composition inA. americanus at application rates recommended by the manufacturer, which led to an increase in the relative abundance of Verrucomicrobia, Firmicutes, Bacteroidetes, and Actinobacteria. Changes in the intestine microbiota might impact the fitness of individuals, including the susceptibility to parasitic infections. Our results indicate that the effect of Bti commercial products is limited; however, we recommend that Bti-spraying activities in amphibian-rich ecosystems should be kept minimal until there is more conclusive research to assess if the changes in the time to metamorphosis and microbiota can lead to negative outcomes in amphibian populations and, eventually, the functioning of ecosystems.


Assuntos
Bacillus thuringiensis , Microbioma Gastrointestinal , Animais , Agentes de Controle Biológico , Ecossistema , Humanos , Larva , Controle Biológico de Vetores
11.
Ecotoxicol Environ Saf ; 223: 112569, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34352582

RESUMO

Transgenic-Bacillus thuringiensis (Bt) crops express insecticidal proteins, which can accumulate in plants and soil where they may influence microbial populations. The impact of Bt crops on bacterial communities has only been assessed under short-term, and results have been contradictory. Here, we analyzed the bacterial communities in three niches, rhizosphere soil (RS), root endosphere (RE) and leaf endosphere (LE), of three Bt rice and their non-Bt parental lines for three consecutive years by high-throughput sequencing. In principal coordinate analysis (PCoA) and PERMANOVA (Adonis) analysis, operational taxonomic units (OTUs) were clustered primarily by niche type and differed significantly in the RE and LE but not in the RS between each of three Bt lines compared with the non-Bt rice line, and not in each respective niche among the three Bt rice lines. The bacterial communities in the RS of different rice lines over the 3 years were clustered mainly by year rather than by lines. The differential bacterial taxa among the lines did not overlap between years, presumably because Cry proteins are rapidly degraded in the soil. A network analysis of RS bacterial communities showed that the network complexity and density for the three Bt rice lines did not decrease compared with those for the non-Bt line. In conclusion, our results demonstrated that bacterial communities differed significantly in RE and LE between Bt and non-Bt rice lines, but the differences were mild and transient, and had no adverse impact on RS over the 3 years. This study provides favorable evidence in support of the commercialization of Bt rice.


Assuntos
Bacillus thuringiensis , Oryza , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Oryza/genética , Plantas Geneticamente Modificadas , Rizosfera
12.
Arch Insect Biochem Physiol ; 108(2): e21834, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34288075

RESUMO

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), is a major pest of potato plants worldwide and is notorious for its ability to develop resistance to insecticides. Cry3 toxins synthesized by Bacillus thuringiensis ssp. tenebrionis have been used successfully to manage this pest. Resistance to Cry toxins is a concerning problem for many insect pests; therefore, it is important to determine the mechanisms by which insects acquire resistance to these toxins. Cadherin-like and ABC transporter proteins have been implicated in the mode of action of Cry toxins as mutations in these genes render lepidopterans resistant to them; however, clear consensus does not exist on whether these proteins also play a role in Cry3 toxin activity and/or development of resistance in coleopterans. In the current study, we identified the L. decemlineata orthologues of the cadherin (LdCAD) and the ABCB transporter (LdABCB1) that have been implicated in the mode of action of Cry toxins in other coleopterans. Suppression of LdABCB1 via RNA interference reduced toxin-related larval mortality, whereas partial silencing of LdCAD did not. Our results suggest that the ABCB is involved in the mode of action of Cry3Aa toxins; however, no evidence was found to support the role of cadherin as a receptor of Cry3Aa in L. decemlineata.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/farmacologia , Besouros , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Caderinas/genética , Caderinas/metabolismo , Besouros/efeitos dos fármacos , Besouros/metabolismo , Besouros/microbiologia , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/metabolismo , Larva/microbiologia , Controle Biológico de Vetores , Interferência de RNA
13.
Pest Manag Sci ; 77(11): 5236-5245, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34310008

RESUMO

BACKGROUND: Resistance evolution of lepidopteran pests to Bacillus thuringiensis (Bt) toxins produced in maize and cotton is a significant issue worldwide. Effective toxin stewardship requires reliable detection of field-evolved resistance to enable the implementation of mitigation strategies. Currently, visual estimates of maize injury are used to document changing susceptibility. In this study, we evaluated an existing maize injury monitoring protocol used to estimate Bt resistance levels in Helicoverpa zea (Lepidoptera: Noctuidae). RESULTS: We detected high interobserver variability across multiple injury metrics, suggesting that the precision and accuracy of maize injury detection could be improved. To do this, we developed a computer vision-based algorithm to measure H. zea injury. Algorithm estimates were more accurate and precise than a sample of human observers. Moreover, observer estimates tended to overpredict H. zea injury, which may increase the false-positive rate, leading to prophylactic insecticide application and unnecessary regulatory action. CONCLUSIONS: Automated detection and tracking of lepidopteran resistance evolution to Bt toxins are critical for genetically engineered crop stewardship to prevent the use of additional insecticides to combat resistant pests. Advantages of this computerized screening are: (i) standardized Bt injury metrics in space and time, (ii) preservation of digital data for cross-referencing when thresholds are reached, and (iii) the ability to increase sample sizes significantly. This technological solution represents a significant step toward improving confidence in resistance monitoring efforts among researchers, regulators and the agricultural biotechnology industry.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Computadores , Endotoxinas/genética , Proteínas Hemolisinas/genética , Humanos , Resistência a Inseticidas/genética , Mariposas/genética , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Zea mays/genética
14.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198929

RESUMO

The molecular mechanisms of insect resistance to Cry toxins generated from the bacterium Bacillus thuringiensis (Bt) urgently need to be elucidated to enable the improvement and sustainability of Bt-based products. Although downregulation of the expression of midgut receptor genes is a pivotal mechanism of insect resistance to Bt Cry toxins, the underlying transcriptional regulation of these genes remains elusive. Herein, we unraveled the regulatory mechanism of the downregulation of the ABC transporter gene PxABCG1 (also called Pxwhite), a functional midgut receptor of the Bt Cry1Ac toxin in Plutella xylostella. The PxABCG1 promoters of Cry1Ac-susceptible and Cry1Ac-resistant strains were cloned and analyzed, and they showed clear differences in activity. Subsequently, a dual-luciferase reporter assay, a yeast one-hybrid (Y1H) assay, and RNA interference (RNAi) experiments demonstrated that a cis-mutation in a binding site of the Hox transcription factor Antennapedia (Antp) decreased the promoter activity of the resistant strain and eliminated the binding and regulation of Antp, thereby enhancing the resistance of P. xylostella to the Cry1Ac toxin. These results advance our knowledge of the roles of cis- and trans-regulatory variations in the regulation of midgut Cry receptor genes and the evolution of Bt resistance, contributing to a more complete understanding of the Bt resistance mechanism.


Assuntos
Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/genética , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Receptores de Superfície Celular/genética , Animais , Bacillus thuringiensis/genética , Endotoxinas/genética , Lepidópteros/efeitos dos fármacos , Lepidópteros/genética , Mutação/genética , Regiões Promotoras Genéticas/genética
15.
J Agric Food Chem ; 69(36): 10407-10418, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34319733

RESUMO

Insect-resistant genetically modified organisms have been globally commercialized for the last 2 decades. Among them, transgenic crops based on Bacillus thuringiensis crystalline (Cry) toxins are extensively used for commercial agricultural applications. However, less emphasis is laid on quantifying Cry toxins because there might be unforeseen health and environmental concerns. Immunoassays, being the preferred method for detection of Cry toxins, are reviewed in this study. Owing to limitations of traditional colorimetric enzyme-linked immunosorbent assay, the trend of detection strategies shifts to modified immunoassays based on nanomaterials, which provide ultrasensitive detection capacity. This review assessed and compared the properties of the recent advances in immunoassays, including colorimetric, fluorescence, chemiluminescence, surface-enhanced Raman scattering, surface plasmon resonance, and electrochemical approaches. Thus, the ultimate aim of this study is to identify research gaps and infer future prospects of current approaches for the development of novel immunosensors to monitor Cry toxins in food and the environment.


Assuntos
Bacillus thuringiensis , Técnicas Biossensoriais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Imunoensaio
16.
Microb Pathog ; 158: 105093, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34271121

RESUMO

Bacillus thuringiensis is the most popular mosquitocidal bacteria, strains of which are effective against almost all mosquito larvae. It has host specificity and thus, has no adverse effect on non-target species of the ecosystem. Culex tritaeniorhynchus, a vector of Japanese encephalitis (JE), breeds in vast area of rice fields in Burdwan district of West Bengal, India, which has already confronted JE epidemic. Entomological investigation and ecological studies on this vector mosquito showed that JE epidemic may reoccur anytime in the area. A strain of Bt (BU55) was isolated from rice field soil, efficacy was tested against Cx. tritaeniorhynchus and mosquitocidal role was confirmed against Cx. quinquefascistus also. The LC50 of Bacillus thuringiensis BU55 against Cx. tritaeniorhynchus and Cx. quinquefascistus after 72 h was 8.59 ml (final dose 2.49 x107 CFU/ml) and 7.52 ml (final dose 2.20 x 107 CFU/ml), respectively. Insecticidal crystal protein profile of BU55 produced 136.89, 64.80, 43.45, 33.65 and 26.98 kDa bands. Among them 136.89, 64.29, 26.98 kDa proteins are comparable to actual toxins viz. Cry1Ac (138.3 kDa, Lepidoptera specific), Cry4D (68.0 kDa, Diptera specific) and Cyt (27.4 kDa, Diptera specific). The results clearly showed that the Bt strain is a potent dipteran larvicide and can be used against the JE vectors to control the disease.


Assuntos
Bacillus thuringiensis , Culex , Oryza , Animais , Ecossistema , Índia , Mosquitos Vetores , Solo
17.
Curr Microbiol ; 78(9): 3483-3493, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34272975

RESUMO

Lon protease, an intracellular protease, plays a key role in cell homeostasis in bacteria and is involved in numerous physiological processes. In this work, we aimed to study the impact of Lon on the production of endotoxins and stress response in Bacillus thuringiensis, which is an important bioinsecticide alternative for toxic chemicals. For this purpose, lon gene was cloned into a multi-copy vector with its original promoter and transcriptional terminator and expressed in B. thuringiensis serovar israelensis ATCC 35,646. Our results showed that the recombinant lon gene transcribed and translated efficiently and the resulting protein was active. Although the sporulation efficiency of the recombinant strain was found to be reduced and its mobility impaired, overexpression of the lon gene triggered the production of endotoxin. Together with increased biofilm formation, recombinant strain exhibited significantly better adaptation to osmotic and heat shock stresses and UV exposure compared to wild type and the control strain with empty plasmid. This study suggested a possible link between Lon protease and the production of insecticide and stress response in B. thuringiensis and provides a platform for future studies focusing on enhancing bio-insecticidal production using this bacterium.


Assuntos
Bacillus thuringiensis , Protease La , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Plasmídeos , Protease La/genética
18.
GM Crops Food ; 12(1): 382-395, 2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-34193022

RESUMO

The idea of enhanced methanol production from cell wall by pectin methyl esterase enzymes (PME) combined with expression of cry genes from Bacillus thuringiensis as a strategy to improve insect pest control in cotton is presented. We constructed a cassette containing two cry genes (cry1Fa and Cry32Aa) and two pme genes, one from Arabidopsis thaliana (AtPME), and other from Aspergillus. niger (AnPME) in pCAMBIA1301 plant expression vector using CAMV-35S promoter. This construction was transformed in Eagle-2 cotton variety by using shoot apex-cut Agrobacterium-mediated transformation. Expression of cry genes and pme genes was confirmed by qPCR. Methanol production was measured in control and in the cry and pme transformed plants showing methanol production only in transformed plants, in contrast to the non-transgenic cotton plants. Finally, insect bioassays performed with transgenic plants expressing cry and pme genes showed 100% mortality for Helicoverpa armigera (cotton bollworm) larvae, 70% mortality for Pectinophora gossypiella (pink bollworm) larvae and 95% mortality of Earias fabia, (spotted bollworm) larvae, that was higher than the transgenic plants expressing only cry genes that showed 84%, 49% and 79% mortality, respectively. These results demonstrate that Bt. cry-genes coupled with pme genes are an effective strategy to improve the control of different insect pests.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Gossypium/genética , Proteínas Hemolisinas/genética , Resistência a Inseticidas , Larva , Metanol , Plantas Geneticamente Modificadas
19.
Int J Mol Sci ; 22(14)2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-34299280

RESUMO

Invertebrate pests, such as insects and nematodes, not only cause or transmit human and livestock diseases but also impose serious crop losses by direct injury as well as vectoring pathogenic microbes. The damage is global but greater in developing countries, where human health and food security are more at risk. Although synthetic pesticides have been in use, biological control measures offer advantages via their biodegradability, environmental safety and precise targeting. This is amply demonstrated by the successful and widespread use of Bacillusthuringiensis to control mosquitos and many plant pests, the latter by the transgenic expression of insecticidal proteins from B. thuringiensis in crop plants. Here, I discuss the prospects of using bacterial and fungal toxins for pest control, including the molecular basis of their biocidal activity.


Assuntos
Bacillus thuringiensis/química , Insetos/efeitos dos fármacos , Nematoides/efeitos dos fármacos , Controle Biológico de Vetores/métodos , Doenças das Plantas/prevenção & controle , Toxinas Biológicas/farmacologia , Animais , Humanos , Insetos/microbiologia , Nematoides/microbiologia , Doenças das Plantas/parasitologia
20.
FEMS Microbiol Ecol ; 97(7)2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34117749

RESUMO

Recent discovery of endophytic strains of Bacillus thuringiensis significantly improves the knowledge on its ecology. It also may be a new source for the isolation of insecticidal strains. This report shows the characterization of two endophytic, highly insecticidal strains of B. thuringiensis. Strains LBIT-1250L and LBIT-1251P were isolated from lavender and Poinsettia sap, respectively. Their parasporal crystals were very similar in morphology to those shown by serotypes israelensis and kurstaki, respectively. Bioassays on Aedes aegypti fourth instar larvae and on Manduca sexta first instar larvae, respectively, showed significantly higher levels of toxicity than those of their standard counterparts, IPS-82 (israelensis) and HD-1 (kurstaki) strains, respectively. Characterization of both strains included the sequencing of flagellin (hag) gene, plasmid and Bc Rep-PCR patterns and crystal protein content. All four characterization features indicated that LBIT1250L is highly related to the IPS-82 standard (serotype H-14: israelensis); while the LBIT-1251P was highly related to the HD-1 standard (serotype H-3a3b3c kurstaki). These results indicate that endophytic strains of B. thuringiensis may be a new source of potential insecticidal strains and opens more in-depth studies about the role of this bacterium in such a specialized habitat.


Assuntos
Aedes , Bacillus thuringiensis , Inseticidas , Animais , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas , Larva
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