RESUMO
Bacillus thuringiensis (Bt) is one of the most promising biological control agents used commercially. Its products can contribute to reducing ecological and environmental problems associated with the use of chemical pesticides. Among the limiting factors of using Bt as bioinsecticide are the costs and ensuring its biological activity, which may vary according to the strain and culture conditions. This systematic review aimed to collect state-of-the-art information on the production of Bt endotoxins and to score the methodological feasibility of the data obtained, thus highlighting possible incoherencies. In order to consolidate recent findings and guide future studies, a total of 47 original articles from the last 10 years was analysed, with special attention being given to corroborating data, identifying inconsistencies and suggesting future adjustments so as to increase data reliability. With a maximum score of 8 points, three production parameters were classified on the following scale: preferable (score: 2), adequate (score: 1) and inadequate (score: 0), and another two parameter were classified as adequate (score: 1) or inadequate (score: 0). No article scored more than 6 out of the maximum of 8, thus reflecting the need for more detailed studies regarding Bt endotoxin production. The lack of standardization of methods and units of measurement also have made a comparison of results and an overall analysis difficult. Standards are suggested in the present study. The inclusion of bioassays and quantifying toxin via alkaline dilution are strongly recommended for studies of this nature, along with LC50 expressed in mg/L. Sixteen articles (34%) did not use either of these suggested methods, which indicates the need for further supporting studies. These findings reinforce the need for robust studies in this area, which could include the development of more affordable and effective bioinsecticides, thus increasing their competitiveness against insecticides derived from unsustainable sources.
Assuntos
Toxinas de Bacillus thuringiensis/biossíntese , Bacillus thuringiensis/metabolismo , Endotoxinas/biossíntese , Animais , Toxinas de Bacillus thuringiensis/análise , Bioensaio , Agentes de Controle Biológico , Bases de Dados Factuais , Endotoxinas/análise , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Controle Biológico de VetoresRESUMO
Our objective was to determine whether a recombinant chitinase ChiA74∆sp of Bacillus thuringiensis and its truncated versions (ChiA74∆sp-60, ChiA74∆sp-50) could be produced in B. thuringiensis HD1 with no detrimental effect on the size and insecticidal activity of the native bipyramidal Cry crystal. chiA-p, the promoter used to drive chitinase gene expression, was active during vegetative growth of Cry-B. HD1 recombinants showed increases from ~7- to 12-fold in chitinase activity when compared with parental HD1 and negligible or no effect on the volume of bipyramidal crystals was observed. HD1/ChiA74∆sp-60 showed increases from 20% to 40% in the yield of Cry1A per unit of culture medium when compared with parental HD1 and HD1/ChiA74∆sp-50, HD1/ChiA74∆sp. Inclusion bodies presumably composed of the enzyme attached to native Cry1A crystals of recombinant strains were observed; these inclusions were likely responsible for the enhancements in chitinase activity. Western blot analysis using polyclonal anti-ChiA74∆sp showed a weak signal with proteins of ~50â¯kDa in sporulated and lysed cells of recombinant strains. Bioassays against Spodoptera frugiperda using sporulated/lysed samples of the recombinant strains did not show statistically significant differences in LC50s when compared with HD1.
Assuntos
Proteínas de Bactérias/genética , Quitinases/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Recombinantes/genética , Spodoptera/efeitos dos fármacos , Animais , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Endotoxinas/biossíntese , Endotoxinas/química , Endotoxinas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas Hemolisinas/biossíntese , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacologia , Corpos de Inclusão/genética , Inseticidas/química , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Spodoptera/patogenicidadeRESUMO
In this study, the endochitinase chiA74 gene lacking its secretion signal peptide sequence (chiA74∆sp) was fused in frame with the sequence coding for the C-terminal crystallization domain and transcription terminator of cry1Ac. The chimeric gene was expressed under the strong pcytA-p/STAB-SD promoter system in an acrystalliferous Cry-B strain of Bacillus thuringiensis and B. thuringiensis subsp. kurstaki HD73. We showed that the chimeric ChiA74∆sp produced amorphous inclusions in both Cry-B and HD73. In addition to the amorphous inclusions putatively composed of the chimera, bipyramidal Cry1Ac crystals, smaller than the wild-type crystal, were observed in recombinant HD73, and chitinase activity was remarkably higher (75-fold) in this strain when compared with parental HD73. Moreover, we observed that lyophilized samples of a mixture containing Cry1Ac, amorphous inclusions, and spores maintained chitinase activity. Amorphous inclusions could not be separated from Cry1Ac crystals by sucrose gradient centrifugation. Interestingly, the chitinase activity of purified Cry1Ac/amorphous inclusions was 51-fold higher compared to purified Cry1Ac inclusions of parental HD73, indicating that the increased enzymatic activity was due primarily to the presence of the atypical amorphous component. The possibility that the chimera is occluded with the Cry1Ac crystal, thereby contributing to the increased endochitinolytic activity, cannot be excluded. Finally, bioassays against larvae of Spodoptera frugiperda with spore/crystals of HD73 or spore-crystal ChiA74∆sp chimeric inclusions of recombinant HD73 strain showed LC50s of 396.86 and 290.25 ng/cm2, respectively. Our study suggests a possible practical application of the chimera in formulations of B. thuringiensis-based lepidopteran larvicides.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Agentes de Controle Biológico , Quitinases/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Corpos de Inclusão/química , Animais , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/biossíntese , Quitinases/biossíntese , Quitinases/metabolismo , Endotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Corpos de Inclusão/ultraestrutura , Larva , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência , Spodoptera/crescimento & desenvolvimentoRESUMO
This study aimed to determine the influence of vector structure on dual Bt gene expression and establish an efficient expression vector using Cry1Ac and Cry3A genes. Four vectors (N4, N5, N10, and S23) were developed and used for genetic transformation of tobacco to obtain insect-resistant transgenic lines. The vectors were constructed using the MAR structure, applying different promoter and enhancer sequences, and changing the transgene open-reading frame sequence. The average Cry1Ac toxalbumin expression quantity was 67 times higher in N5 than in N4 transgenic lines (8.77 and 0.13 µg/g, respectively). In contrast, the average Cry3A toxalbumin expression quantity was 1.5 times higher in N4 than in N5 lines (12.70 and 8.21 µg/g, respectively). The sequences of both Bt genes significantly influenced toxalbumin expression, although upstream Bt genes presented lower expression levels. The average Cry1Ac toxalbumin content was 13 times higher in the transgenic lines of AtADH 5'-non-translated sequence N5 (8.77 mg/g) than in the omega N10 lines (0.67 mg/g). Furthermore, the average Cry1Ac toxalbumin content was 5 times higher in MAR N5 than in non-MAR S23 lines (8.77 and 1.63 mg/g, respectively). The average Cry3A toxalbumin content was 1.3 times higher in N5 than in S23 lines (8.21 and 6.48 mg/g, respectively). Moreover, toxalbumin expression levels differed significantly among the S23-transformed lines. The MAR structure applied on both ends of the genes increased both the level and stability of exogenous gene expression. In conclusion, N5 was the most optimal of the four tested vectors.
Assuntos
Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Lepidópteros/fisiologia , Nicotiana/genética , Folhas de Planta/genética , Agrobacterium tumefaciens/genética , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/biossíntese , Endotoxinas/biossíntese , Expressão Gênica , Vetores Genéticos , Proteínas Hemolisinas/biossíntese , Herbivoria , Larva/fisiologia , Controle Biológico de Vetores , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Nicotiana/metabolismo , Transformação Genética , TransgenesRESUMO
Bt cotton plants expressing Cry1Ac protein have high specificity for the control of lepidopteran larvae. However, studies conducted in several countries have shown these plants have a differential impact on nontarget herbivores. The aim of this study was to compare the colonization rates and population abundance of the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae) and the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), in plots of Bt (Nuopal) and non-Bt cotton (Delta Opal) in an experimental field in Brasilia, DF, Brazil. No difference was observed in the preference and colonization by winged aphids to plants from the two treatments. There was no significant difference in abundance of wingless aphids or in the production of winged aphids between treatments. Apparently, the parameters that control factors such as fecundity, survival, and dispersal were similar on both Bt and non-Bt plants. Monitoring of plants for coccinellids, a specialist predator of aphids, and ants that act on the dispersal of aphids among plants showed no significant difference between Bt and non-Bt plants, supporting the inference above. Regarding the effect on boll weevil, there was also no significant difference between treatments in the total number of fruiting structures attacked in each plot, the percentage of fruiting structures attacked per plant or on the number of weevils emerging from fruits with boll weevil damage from egg-laying, when damaged fruit samples were held in the laboratory. Based on these results, we conclude that there is no impact of Bt cotton crop expressing Cry1Ac on the nontarget herbivores tested under field conditions.
Assuntos
Distribuição Animal , Afídeos/fisiologia , Gossypium/parasitologia , Gorgulhos/fisiologia , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/biossíntese , Brasil , Endotoxinas/biossíntese , Gossypium/metabolismo , Proteínas Hemolisinas/biossíntese , Plantas Geneticamente Modificadas/metabolismoRESUMO
The growth kinetics, sporulation, and toxicity of Bacillus thuringiensis var. israelensis were evaluated through the analysis of batch cultures with different dissolved oxygen (DO) profiles. Firstly, DO was maintained constant at 5%, 20%, or 50% throughout fermentation in order to identify the most suitable one to improve the main process parameters. Higher biomass concentration, cell productivity, and cell yield based on glucose were obtained with 50% DO. The higher aeration level also resulted in higher spore counts and markedly improved the toxic activity of the fermentation broth, which was 9-fold greater than that obtained with 5% DO (LC(50) of 39 and 329 mg/L, respectively). Subsequently, using a two-stage oxygen supply strategy, DO was kept at 50% during the vegetative and transition phases until the maximum cell concentration was achieved. Then, DO was changed to 0%, 5%, 20%, or 100% throughout sporulation and cell lysis phases. The interruption of oxygen supply strongly reduced the spore production and thoroughly repressed the toxin synthesis. On the contrary, when DO was raised to 100% of saturation, toxic activity increased approximately four times (LC(50) of 8.2 mg/L) in comparison with the mean values reached with lower DO levels, even though spore counts were lower than that from the 50% DO assay. When pure oxygen was used instead of normal air, it was possible to obtain 70% of the total biomass concentration achieved in the air assays; however, cultures did not sporulate and the toxin synthesis was consequently suppressed.
Assuntos
Bacillus thuringiensis/crescimento & desenvolvimento , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/biossíntese , Endotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Oxigênio/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Toxinas de Bacillus thuringiensis , Biomassa , Meios de Cultura/química , FermentaçãoRESUMO
Bacillus thuringiensis (Bt) has been used as a biopesticide in agriculture, forestry and mosquito control because of its advantages of specific toxicity against target insects, lack of polluting residues and safety to non-target organisms. The insecticidal properties of this bacterium are due to insecticidal proteins produced during sporulation. Despite these ecological benefits, the use of Bt biopesticides has lagged behind the synthetic chemicals. Genetic improvement of Bt natural strains, in particular Bt recombination, offers a promising means of improving efficacy and cost-effectiveness of Bt-based bioinsecticide products to develop new biotechnological applications.
Assuntos
Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/biossíntese , Vias Biossintéticas/genética , Endotoxinas/biossíntese , Engenharia Genética , Proteínas Hemolisinas/biossíntese , Insetos/microbiologia , Animais , Bacillus thuringiensis/patogenicidade , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Controle de Insetos/métodos , Controle Biológico de Vetores/métodos , Recombinação GenéticaRESUMO
Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5' mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (+/-0.214) and 1.356 (+/-0.247) mum in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (+/-0.222) and 1.139 (+/-0.202) mum in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 x 10(9), 1.3 x 10(9)), compared to HD-73-pEHchiA74 (4.9 x 10(9), 5.3 x 10(9)) and HD-73 (6.8 x 10(9), 8.8 x 10(9)) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.
Assuntos
Bacillus thuringiensis/enzimologia , Bacillus thuringiensis/fisiologia , Proteínas de Bactérias/biossíntese , Quitinases/biossíntese , Endotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Esporos Bacterianos/crescimento & desenvolvimento , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/ultraestrutura , Quitina/metabolismo , Quitinases/genética , Contagem de Colônia Microbiana , Expressão Gênica , Proteínas Hemolisinas/ultraestrutura , Microscopia Eletrônica de Transmissão , Transformação BacterianaRESUMO
A linear relationship between total solid concentration (TSC), delta-endotoxin production [Cry = 0.2795(TSC)-0.2472, R2 = 0.8644] and poly-beta-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R2 = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between delta-endotoxin and PHB accumulation [Cry = 2.1573(PHB)-1.1248, R2 = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l(-1) was required before the onset of delta-endotoxin production.