RESUMEN
As an alternative to chemical insecticides, gut bacteria of insects could be used to control insect pests. In this study, bacteria associated with Tuta absoluta, an invasive species that has developed resistance to chemical insecticides, were isolated, and their potential for pest control was investigated. We isolated 13 bacteria from larvae of the pest and identified the isolates on the basis of their morphological, physiological, biochemical, and molecular characteristics as Bacillus thuringiensis (Ta1-8), Staphylococcus petrasii (Ta9), Citrobacter freundii (Ta10), Chishuiella changwenlii (Ta11), Enterococcus casseliflavus (Ta12), and Pseudomonas tremae (Ta13). A laboratory screening test at 109 cfu/ml showed that B. thuringiensis (Bt) isolates caused more than 90% mortality after 3 days. Among the isolates, Bt-Ta1 showed the highest mortality in a short time. The LC50 and LC90 values for Bt-Ta1 were estimated to be 1.2 × 106 and 2 × 109 cfu/ml, respectively. Detailed characterization of Bt-Ta1 revealed that it is one of the serotypes effective on lepidopterans and contains the genes cry1Aa, cry2Aa, and vip3Aa, which encode lepidopteran toxic proteins. Bt-Ta1 isolate has been shown to have the potential to be used in the integrated management of Tuta absoluta.
Asunto(s)
Bacillus thuringiensis , Insecticidas , Lepidópteros , Animales , Insecticidas/farmacología , Especies Introducidas , LarvaRESUMEN
Insecticidal transgenes, when incorporated and expressed in plants, confer resistance against insects by producing several products having insecticidal properties. Protease inhibitors, lectins, amylase inhibitors, and chitinase genes are associated with the natural defenses developed by plants to counter insect attacks. Several toxin genes are also derived from spiders and scorpions for protection against insects. Bacillus thuringiensis Berliner is a microbial source of insecticidal toxins. Several methods have facilitated the large-scale production of transgenic plants. Bt-derived cry, cyt, vip, and sip genes, plant-derived genes such as lectins, protease inhibitors, and alpha-amylase inhibitors, insect cell wall-degrading enzymes like chitinase and some proteins like arcelins, plant defensins, and ribosome-inactivating proteins have been successfully utilized to impart resistance to insects. Besides, transgenic plants expressing double-stranded RNA have been developed with enhanced resistance. However, the long-term effects of transgenes on insect resistance, the environment, and human health must be thoroughly investigated before they are made available for commercial planting. In this chapter, the present status, prospects, and future scope of transgenes for insect pest management have been summarized and discussed.
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Bacillus thuringiensis , Quitinasas , Insecticidas , Animales , Humanos , Insectos/genética , Insecticidas/metabolismo , Transgenes , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Lectinas/genética , Quitinasas/genética , Proteínas Bacterianas/metabolismo , Endotoxinas/farmacología , Proteínas Hemolisinas/genética , Control Biológico de VectoresRESUMEN
Effective control of diseases transmitted by Aedes aegypti is primarily achieved through vector control by chemical insecticides. However, the emergence of insecticide resistance in A. aegypti undermines current control efforts. Arachnid venoms are rich in toxins with activity against dipteran insects and we therefore employed a panel of 41 spider and 9 scorpion venoms to screen for mosquitocidal toxins. Using an assay-guided fractionation approach, we isolated two peptides from the venom of the tarantula Lasiodora klugi with activity against adult A. aegypti. The isolated peptides were named U-TRTX-Lk1a and U-TRTX-Lk2a and comprised 41 and 49 residues with monoisotopic masses of 4687.02 Da and 5718.88 Da, respectively. U-TRTX-Lk1a exhibited an LD50 of 38.3 pmol/g when injected into A. aegypti and its modeled structure conformed to the inhibitor cystine knot motif. U-TRTX-Lk2a has an LD50 of 45.4 pmol/g against adult A. aegypti and its predicted structure conforms to the disulfide-directed ß-hairpin motif. These spider-venom peptides represent potential leads for the development of novel control agents for A. aegypti.
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Venenos de Araña , Ponzoñas , Animales , Ponzoñas/farmacología , Brasil , Mosquitos Vectores , Péptidos/farmacología , Insectos , Venenos de Araña/toxicidad , Venenos de Araña/químicaRESUMEN
Cry11Aa and Cyt1Aa are two pesticidal toxins produced by Bacillus thuringiensis subsp. israelensis. To improve our understanding of the nature of their oligomers in the toxic actions and synergistic effects, we performed the atomic force microscopy to probe the surfaces of their natively grown crystals, and used the L-weight filter to enhance the structural features. By L-weight filtering, molecular sizes of the Cry11Aa and Cyt1Aa monomers obtained are in excellent agreement with the three-dimensional structures determined by x-ray crystallography. Moreover, our results show that the layered feature of a structural element distinguishes the topographic characteristics of Cry11Aa and Cyt1Aa crystals, suggesting that the Cry11Aa toxin has a better chance than Cyt1Aa for multimerization and therefore cooperativeness of the toxic actions.
Asunto(s)
Bacillus thuringiensis , Endotoxinas , Endotoxinas/química , Endotoxinas/toxicidad , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidad , Proteínas Bacterianas/química , Bacillus thuringiensis/químicaRESUMEN
The insecticidal toxin complex (Tc) proteins are produced by several insect-associated bacteria, including Yersinia enterocolitica strain W22703, which oscillates between two distinct pathogenicity phases in invertebrates and humans. The mechanism by which this high-molecular-weight toxin is released into the extracellular surrounding, however, has not been deciphered. In this study, we investigated the regulation and functionality of a phage-related holin/endolysin (HE) cassette located within the insecticidal pathogenicity island Tc-PAIYe of W22703. Using the Galleria mellonella infection model and luciferase reporter fusions, we revealed that quorum sensing contributes to the insecticidal activity of W22703 upon influencing the transcription of tcaR2, which encodes an activator of the tc and HE genes. In contrast, a lack of the Yersinia modulator, YmoA, stimulated HE gene transcription, and mutant W22703 ΔymoA exhibited a stronger toxicity toward insect larvae than did W22703. A luciferase reporter fusion demonstrated transcriptional activation of the HE cassette in vivo, and a significantly larger extracellular amount of subunit TcaA was found in W22703 ΔymoA relative to its ΔHE mutant. Using competitive growth assays, we demonstrated that at least in vitro, the TcaA release upon HE activity is not mediated by cell lysis of a significant part of the population. Oral infection of Caenorhabditis elegans with a HE deletion mutant attenuated the nematocidal activity of the wild type, similar to the case with a mutant lacking a Tc subunit. We conclude that the dual holin/endolysin cassette of yersiniae is a novel example of a phage-related function adapted for the release of a bacterial toxin. IMPORTANCE Members of the genus Yersinia cause gastroenteritis in humans but also exhibit toxicity toward invertebrates. A virulence factor required for this environmental life cycle stage is the multisubunit toxin complex (Tc), which is distinct from the insecticidal toxin of Bacillus thuringiensis and has the potential to be used in pest control. The mechanism by which this high-molecular-weight Tc is secreted from bacterial cells has not been uncovered. Here, we show that a highly conserved phage-related holin/endolysin pair, which is encoded by the genes holY and elyY located between the Tc subunit genes, is essential for the insecticidal activity of Y. enterocolitica and that its activation increases the amount of Tc subunits in the supernatant. Thus, the dual holY-elyY cassette of Y. enterocolitica constitutes a new example for a type 10 secretion system to release bacterial toxins.
Asunto(s)
Toxinas Bacterianas , Insecticidas , Mariposas Nocturnas , Yersinia enterocolitica , Animales , Humanos , Yersinia enterocolitica/genética , Caenorhabditis elegans/metabolismo , Mariposas Nocturnas/microbiología , Toxinas Bacterianas/metabolismo , Insectos , Insecticidas/metabolismo , Luciferasas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
A toxin complex consists of a high-molecular-weight group of toxins that exhibits insecticidal activity against insect pests. These toxins are a promising alternative to Bacillus thuringiensis (Bt) toxins that have been extensively utilized in insect pest control. Herein, a codon-optimized insecticidal gene (tccZ) (381 bp) identified in Pantoea ananatis strain MHSD5 (a bacterial endophyte previously isolated from Pellaea calomelanos) was ligated into the pET SUMO expression vector and expressed in Escherichia coli BL21 (DE3). We report the success of cloning the tccZ gene into the pET SUMO vector and ultimately the transformation into E. coli BL21 (DE3) competent cells. However, despite conducting a time course of expression as well as isopropyl ß-d-1-thiogalactopyranoside (IPTG) dosage optimization to determine optimal conditions for expression, TccZ protein expression could not be detected on Stain-Free and Coomassie-stained SDS-PAGE gels.
RESUMEN
Afidopyropen, a newly identified chemical, is a derivative of pyripyropene A, which is produced by the filamentous fungus Penicillium coprobium. It is a promising novel pesticide applied against whiteflies in agriculture. In this study, the reversion and selection, cross-resistance patterns, synergistic effects, and fitness costs of afidopyropen resistance were studied in a field-developed resistant population of B. tabaci. Compared to a reference MED-S strain, the field-developed resistant Haidian (HD) population showed 36.5-fold resistance to afidopyropen. Significant reversion of resistance to afidopyropen was found in the HD population when it was kept with no selective pressure of the insecticide. The HD-Afi strain, developed from the HD population with afidopyropen pressure, developed 104.3-fold resistance to afidopyropen and significant cross-resistance to sulfoxaflor. Piperonyl butoxide (PBO) largely inhibited afidopyropen resistance in the HD-Afi strain, which indicates that P450 monooxygenase could be involved in the resistance. Significant fitness costs associated with afidopyropen resistance were observed in HD-Afi. This study indicates that a rotation of afidopyropen with other chemical control agents could be useful for impeding afidopyropen resistance in B. tabaci. In addition, we expanded upon the understanding of resistance to afidopyropen, offering evidence suggesting the importance of devising better strategies for the management of whiteflies.
Asunto(s)
Hemípteros , Insecticidas , Animales , Compuestos Heterocíclicos de 4 o más Anillos , Resistencia a los Insecticidas , Insecticidas/metabolismo , Insecticidas/farmacología , Lactonas/metabolismoRESUMEN
The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD50 determined for Ct-IT1 is 3.81 µg/100 mg of cricket weight, but low amounts of peptides (0.8 µg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant ß-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method.
Asunto(s)
Insecticidas , Venenos de Escorpión , Secuencia de Aminoácidos , Animales , Péptidos , EscorpionesRESUMEN
The nemertide toxins from the phylum Nemertea are a little researched family of neurotoxins with potential for development as biopesticides. Here we report the recombinant production of nemertide α-1 (α-1), a 65-residue inhibitor cystine knot (ICK) peptide from Lineus longissimus, known to target insect voltage-gated sodium channels. The insecticidal activity of α-1 was assessed and compared with the well characterised ICK venom peptide, ω-atracotoxin/hexatoxin-Hv1a (Hv1a). α-1 elicited potent spastic paralysis when injected into cabbage moth (Mamestra brassicae) larvae; conferring an ED50 3.90 µg/larva (10.30 nmol/g larva), followed by mortality (60% within 48 h after 10 µg injection). By comparison, injection of M. brassicae larvae with recombinant Hv1a produced short-lived flaccid paralysis with an ED50 over 6 times greater than that of α-1 at 26.20 µg/larva (64.70 nmol/g larva). Oral toxicity of α-1 was demonstrated against two aphid species (Myzus persicae and Acyrthosiphon pisum), with respective LC50 values of 0.35 and 0.14 mg/mL, some 6-fold lower than those derived for recombinant Hv1a. When delivered orally to M. brassicae larvae, α-1 caused both paralysis (ED50 11.93 µg/larva, 31.5 nmol/g larva) and mortality. This contrasts with the lack of oral activity of Hv1a, which when fed to M. brassicae larvae had no effect on feeding or survival. Hv1a has previously been shown to be non-toxic by injection to the beneficial honeybee (Apis mellifera). By contrast, rapid paralysis and 100% mortality was observed following injection of α-1 (31.6 nmol/g insect). These results demonstrate the great potential of naturally occurring non-venomous peptides, such as α-1, for development as novel effective biopesticides, but equally highlights the importance of understanding the phyletic specificity of a given toxin at an early stage in the quest to discover and develop safe and sustainable pesticides.
Asunto(s)
Insecticidas , Mariposas Nocturnas , Venenos de Araña , Canales de Sodio Activados por Voltaje , Animales , Abejas , Insecticidas/toxicidad , LarvaRESUMEN
The bacterial strain M7D1T was isolated from samples of the rhizosphere of desert bloom plants on the Atacama region located in northern Chile as part of a study intended to isolate nitrifying bacteria in this adverse environment. It was previously identified as belonging to the Pseudomonas fluorescens group. In this study, the phylogenetic analysis of the 16s RNA, gyrA, rpoB and rpoD genes confirmed that this strain belongs to this group, especially Sub Group (SG) Koreensis, but it represents a potential new species. Additionally, the average nucleotide identity confirmed this as the highest identity value (0.92) with Pseudomonas moraviensis LMG 24280, which is lower than the 0.94 threshold established to classify two strains within the same species. The strain M7D1T shared a similar fatty acids methyl ester profile than the type strains of other Pseudomonas spp. previously described. Furthermore, it can be differentiated phenotypically from other related species of SG P. koreensis. Based on these results, the existence of a new species of Pseudomonas is demonstrated, for which the name Pseudomonas atacamensis is proposed. This strain presented a set of genes associated with plant growth-promoting rhizobacteria and it is a good candidate to be used for recovery of contaminated soils. However, more studies are required to demonstrate whether this bacterium is non-pathogenic, can survive in the presence of toxic compounds and promote growth or help to the stress management of plants.
Asunto(s)
Filogenia , Pseudomonas/clasificación , Pseudomonas/aislamiento & purificación , Rizosfera , Microbiología del Suelo , Técnicas de Tipificación Bacteriana , Composición de Base , Chile , ADN Bacteriano/genética , Ácidos Grasos/análisis , Genes Bacterianos/genética , Genoma Bacteriano , Hibridación de Ácido Nucleico , Pseudomonas/citología , Pseudomonas/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Vegetatively expressed insecticidal proteins (VIPs) produced by Bacillus thuringiensis fall into several classes of which the third, VIP3, is known for their activity against several key Lepidopteran pests of commercial broad acre crops and because their mode of action does not overlap with that of crystalline insecticidal proteins. The details of the VIP3 structure and mode of action have remained obscure for the quarter century that has passed since their discovery. In the present article, we report the first crystal structure of a full-length VIP3 protein. Crystallization of this target required multiple rounds of construct optimization and screening-over 200 individual sequences were expressed and tested. This protein adopts a novel global fold that combines domains with hitherto unreported topology and containing elements seemingly borrowed from carbohydrate-binding domains, lectins, or from other insecticidal proteins.
Asunto(s)
Bacillus thuringiensis/química , Proteínas Bacterianas/química , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Pliegue de ProteínaRESUMEN
Cry3Bb toxin from Bacillus thuringiensis is an important insecticidal protein due to its potency against coleopteran pests, especially rootworms. Cadherin, a protein in the insect midgut epithelium, is a receptor of Cry toxins; in some insect species toxin-binding domains of cadherins-synergized Cry toxicity. Previously, we reported that the DvCad1-CR8-10 fragment of Diabrotica virgifera virgifera cadherin-like protein (GenBank Accession #EF531715) enhanced Cry3Bb toxicity to the Colorado Potato Beetle (CPB), Leptinotarsadecimlineata (L. decimlineata). We report that individual CR domains of the DvCad1-CR8-10 fragment were found to have strong binding affinities to α-chymotrypsin-treated Cry3Bb. The dissociation constant (Kd) of Cry3Bb binding to the CR8, CR9, and CR10 domain was 4.9 nM, 28.2 nM, and 4.6 nM, respectively. CR8 and CR10, but not CR9, enhanced Cry3Bb toxicity against L. decimlineata and the lesser mealworm Alphitobius diaperinus neonates. In-frame deletions of the DvCad1-CR10 open reading frame defined a high-affinity binding and synergistic site to a motif in residues I1226-D1278. A 26 amino acid peptide from the high affinity Cry3Bb-binding region of CR10 functioned as a Cry3Bb synergist against coleopteran larvae.
Asunto(s)
Cadherinas/metabolismo , Escarabajos/efectos de los fármacos , Endotoxinas/toxicidad , Proteínas de Insectos/metabolismo , Péptidos/toxicidad , Tenebrio/efectos de los fármacos , Animales , Sitios de Unión , Larva/efectos de los fármacos , Control Biológico de VectoresRESUMEN
A PCR-RFLP method was used to identify cry2A toxin genes in a collection of 300 strains of Bacillus thuringiensis. From 81 genes identified, the vast majority appeared to be cry2Aa or cry2Ab, however three showed a different pattern and were subsequently cloned and sequenced. The gene cloned from strain HD395 was named cry2Ba2. Since the proteins encoded by the genes cloned from LS5115-3 and DS415 shared >95% sequence identity with existing toxins their genes were named cry2Aa17 and cry2Ab29 respectively by the toxin nomenclature committee. Despite this overall similarity these two toxins resembled natural hybrids, with Cry2Ab29 resembling Cry2Ab for the majority of the protein but then showing identity to Cry2Aa for the last 66 amino acids. For Cry2Aa17, Domains II and III most closely resembled Cry2Aa (99% identity) whilst Domain I was identical to that of Cry2Ab. The toxicity of the recombinant toxins was tested against Aedes aegypti and Spodoptera exigua, and it was found that the toxicity profile of Cry2Aa17 more closely matched the profile of Cry2Ab than that of Cry2Aa, thus implicating Domain I in specificity determination. This association of Domain I with toxicity was confirmed when hybrids were made between Cry2Aa and Cry2Ab.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Aedes , Animales , Toxinas de Bacillus thuringiensis , Bioensayo , Clonación MolecularRESUMEN
Sodalis praecaptivus is a close relative and putative environmental progenitor of the widely distributed, insect-associated, Sodalis-allied symbionts. Here we show that mutant strains of S. praecaptivus that lack genetic components of a quorum-sensing (QS) apparatus have a rapid and potent killing phenotype following microinjection into an insect host. Transcriptomic and genetic analyses indicate that insect killing occurs as a consequence of virulence factors, including insecticidal toxins and enzymes that degrade the insect integument, which are normally repressed by QS at high infection densities. This method of regulation suggests that virulence factors are only utilized in early infection to initiate the insect-bacterial association. Once bacteria reach sufficient density in host tissues, the QS circuit represses expression of these harmful genes, facilitating a long-lasting and benign association. We discuss the implications of the functionality of this QS system in the context of establishment and evolution of mutualistic relationships involving these bacteria.
Asunto(s)
Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/veterinaria , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Percepción de Quorum/genética , Percepción de Quorum/fisiología , Virulencia/genética , Gorgojos/microbiología , 4-Butirolactona/análogos & derivados , 4-Butirolactona/genética , 4-Butirolactona/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Quitinasas/metabolismo , ADN Bacteriano/genética , Modelos Animales de Enfermedad , Enterobacteriaceae/crecimiento & desarrollo , Enterobacteriaceae/patogenicidad , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Insecticidas/metabolismo , Insecticidas/farmacología , Fenotipo , Análisis de Secuencia de ARN , Sobrevida , Simbiosis , Transcriptoma , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Gorgojos/efectos de los fármacosRESUMEN
BACKGROUND: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. RESULTS: The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. CONCLUSIONS: Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.
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Proteínas Bacterianas/química , Proteínas Bacterianas/toxicidad , Endotoxinas/química , Endotoxinas/toxicidad , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidad , Plaguicidas/toxicidad , Proteínas Citotóxicas Formadoras de Poros/química , Homología Estructural de Proteína , Animales , Toxinas de Bacillus thuringiensis , Bioensayo , Caenorhabditis elegans/efectos de los fármacos , Cristalografía por Rayos X , Disulfuros/metabolismo , Modelos Moleculares , Plaguicidas/química , Conformación Proteica , Tripsina/metabolismoRESUMEN
The inhibitor cystine knot (ICK) is an unusual three-disulfide architecture in which one of the disulfide bonds bisects a loop formed by the two other disulfide bridges and the intervening sections of the protein backbone. Peptides containing an ICK motif are frequently considered to have high levels of thermal, chemical and enzymatic stability due to cross-bracing provided by the disulfide bonds. Experimental studies supporting this contention are rare, in particular for spider-venom toxins, which represent the largest diversity of ICK peptides. We used ω-hexatoxin-Hv1a (Hv1a), an insecticidal toxin from the deadly Australian funnel-web spider, as a model system to examine the contribution of the cystine knot to the stability of ICK peptides. We show that Hv1a is highly stable when subjected to temperatures up to 75 °C, pH values as low as 1, and various organic solvents. Moreover, Hv1a was highly resistant to digestion by proteinase K and when incubated in insect hemolymph and human plasma. We demonstrate that the ICK motif is essential for the remarkable stability of Hv1a, with the peptide's stability being dramatically reduced when the disulfide bonds are eliminated. Thus, this study demonstrates that the ICK motif significantly enhances the chemical and thermal stability of spider-venom peptides and provides them with a high level of protease resistance. This study also provides guidance to the conditions under which Hv1a could be stored and deployed as a bioinsecticide.
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Ciclotidas/química , Cisteína/química , Motivos Nodales de Cisteina , Venenos de Araña/química , Animales , Agentes de Control Biológico , Conformación Proteica , Estabilidad Proteica , Arañas/químicaRESUMEN
Pseudomonas chlororaphis HT66, a plant growth-promoting rhizobacterium that produces phenazine-1-carboxamide with high yield, was compared with three genomic sequenced P. chlororaphis strains, GP72, 30-84 and O6. The genome sizes of four strains vary from 6.66 to 7.30 Mb. Comparisons of predicted coding sequences indicated 4833 conserved genes in 5869-6455 protein-encoding genes. Phylogenetic analysis showed that the four strains are closely related to each other. Its competitive colonization indicates that P. chlororaphis can adapt well to its environment. No virulence or virulence-related factor was found in P. chlororaphis. All of the four strains could synthesize antimicrobial metabolites including different phenazines and insecticidal protein FitD. Some genes related to the regulation of phenazine biosynthesis were detected among the four strains. It was shown that P. chlororaphis is a safe PGPR in agricultural application and could also be used to produce some phenazine antibiotics with high-yield.
RESUMEN
Photorhabdus temperata subsp. thracensis 39-8(T), a symbiotic bacterium from an entomopathogenic nematode Heterorhabditis bacteriophora, is a novel bacterium harboring insect pathogenicity. Herein, we present the complete genome sequence of strain 39-8(T), which consists of one circular chromosome of 5,147,098 bp with a GC content of 44.10%. This genetic information will provide insights into biotechnological applications of the genus Photorhabdus producing insecticidal toxins, leading to the enhanced commercial bioinsecticide in agricultural pest control.
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Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Genoma Bacteriano/genética , Insecticidas , Photorhabdus/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , Control Biológico de VectoresRESUMEN
Three insecticidal toxin complex (tc)-like genes were identified in Vibrio parahaemolyticus 13-028/A3, which can cause acute hepatopancreatic necrosis disease in penaeid shrimp. The three genes are a tcdA-like gene (7710 bp), predicted to code for a 284-kDa protein; a tcdB-like gene (4272 bp), predicted to code for a 158-kDa protein; and a tccC3-like gene (2916 bp), predicted to encode a 107-kDa protein. All three predicted proteins contain conserved domains that are characteristic of their respective Tc proteins. By RT-PCR, all three tc-like genes were found to be expressed in this bacterium. Through genome walking and the use of PCR to join contigs surrounding these three genes, a genomic island (87 712 bp, named tc-GIvp) was found on chromosome II localized next to the tRNA Gly. The GC content of this island, which is not found in other Vibrio species, is 40%. The tc-GIvp is characterized to have 60 ORFs encoding regulatory or virulence factors. These include a type 6 secretion protein VgrG, EAL domain-containing proteins, fimbriae subunits and assembly proteins, invasin-like proteins, peptidoglycan-binding proteins, and Tc proteins. The tc-GIvp also contains 21 transposase genes, suggesting that it was acquired through horizontal transfer from other organisms.
Asunto(s)
Toxinas Bacterianas/genética , Islas Genómicas/genética , Penaeidae/microbiología , Vibrio parahaemolyticus/genética , Factores de Virulencia/genética , Animales , Mapeo Cromosómico , Insecticidas , Sistemas de Lectura Abierta/genética , Vibrio parahaemolyticus/patogenicidadRESUMEN
Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.