RESUMO
Mosquitoes are the most notorious arthropod vectors of viral and parasitic diseases for which approximately half the world's population, ~4,000,000,000, is at risk. Integrated pest management programs (IPMPs) have achieved some success in mitigating the regional transmission and persistence of these diseases. However, as many vector-borne diseases remain pervasive, it is obvious that IPMP successes have not been absolute in eradicating the threat imposed by mosquitoes. Moreover, the expanding mosquito geographic ranges caused by factors related to climate change and globalization (travel, trade, and migration), and the evolution of resistance to synthetic pesticides, present ongoing challenges to reducing or eliminating the local and global burden of these diseases, especially in economically and medically disadvantaged societies. Abatement strategies include the control of vector populations with synthetic pesticides and eco-friendly technologies. These "green" technologies include SIT, IIT, RIDL, CRISPR/Cas9 gene drive, and biological control that specifically targets the aquatic larval stages of mosquitoes. Regarding the latter, the most effective continues to be the widespread use of Lysinibacillus sphaericus (Ls) and Bacillus thuringiensis subsp. israelensis (Bti). Here, we present a review of the health issues elicited by vector mosquitoes, control strategies, and lastly, focus on the biology of Ls and Bti, with an emphasis on the latter, to which no resistance has been observed in the field.
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The chitinase ChiA74 is synthesized by Bacillus thuringiensis and possesses a modular organization composed of four domains. In the C-terminal of the enzyme is located the chitin-binding domain (CBD), which has not been isolated as a single unit or characterized. Here, we aimed to isolate the ChiA74's CBD as a single unit, determine the binding properties, and evaluate its antimicrobial and hemolytic activities. We cloned the ChiA74's CBD and expressed it in Escherichia coli BL21. The single domain was purified, analyzed by SDS-PAGE, and characterized. The recombinant CBD (rCBD) showed a molecular mass of â¼14 kDa and binds strongly to α-chitin, with Kd and Bmax of â¼4.7 ± 0.9 µM and 1.5 ± 0.1 µmoles/g chitin, respectively. Besides, the binding potential (Bmax/Kd) was stronger for α-chitin (â¼0.31) than microcrystalline cellulose (â¼0.19). It was also shown that the purified rCBD inhibited the growth of the clinically relevant Gram-negative bacteria (GNB) Vibrio cholerae, and V. parahemolyticus CVP2 with minimum inhibitory concentrations (MICs) of 121 ± 9.9 and 138 ± 3.2 µg/mL, respectively, and of one of the most common GNB plant pathogens, Pseudomonas syringae with a MIC of 230 ± 13.8 µg/mL. In addition, the rCBD possessed antifungal activity inhibiting the conidia germination of Fusarium oxysporum (MIC = 192 ± 37.5 µg/mL) and lacked hemolytic and agglutination activities against human erythrocytes. The significance of this work lies in the fact that data provided here show for the first time that ChiA74's CBD from B. thuringiensis has antimicrobial activity, suggesting its potential use against significant pathogenic microorganisms. Future works will be focused on testing the inhibitory effect against other pathogenic microorganisms and elucidating the mechanism of action.
Assuntos
Bacillus thuringiensis , Quitinases , Humanos , Bacillus thuringiensis/química , Bactérias Gram-Negativas/metabolismo , Antifúngicos/química , Quitina/química , Quitinases/genética , Quitinases/farmacologia , Quitinases/químicaRESUMO
The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.
Assuntos
Bacillaceae , Bacillus , Culex , Praguicidas , Animais , Bacillaceae/química , Bacillaceae/metabolismo , Controle de Mosquitos , Larva/metabolismoRESUMO
The three most important commercial bacterial insecticides are all derived from subspecies of Bacillus thuringiensis (Bt). Specifically, Bt subsp. kurstaki (Btk) and Bt subsp. aizawai (Bta) are used to control larval lepidopteran pests. The third, Bt subsp. israelensis (Bti), is primarily used to control mosquito and blackfly larvae. All three subspecies produce a parasporal body (PB) during sporulation. The PB is composed of insecticidal proteins that damage the midgut epithelium, initiating a complex process that results in the death of the insect. Among these three subspecies of Bt, Bti is unique as it produces the most complex PB consisting of three compartments. Each compartment is bound by a multilaminar fibrous matrix (MFM). Two compartments contain one protein each, Cry11Aa1 and Cyt1Aa1, while the third contains two, Cry4Aa1/Cry4Ba1. Each compartment is packaged independently before coalescing into the mature spherical PB held together by additional layers of the MFM. This distinctive packaging process is unparalleled among known bacterial organelles, although the underlying molecular biology is yet to be determined. Here, we present structural and molecular evidence that the MFM has a hexagonal pattern to which Bti proteins Bt152 and Bt075 bind. Bt152 binds to a defined spot on the MFM during the development of each compartment, yet its function remains unknown. Bt075 appears to be derived from a bacteriophage major capsid protein (MCP), and though its sequence has markedly diverged, it shares striking 3-D structural similarity to the Escherichia coli phage HK97 Head 1 capsid protein. Both proteins are encoded on Bti's pBtoxis plasmid. Additionally, we have also identified a six-amino acid motif that appears to be part of a novel molecular process responsible for targeting the Cry and Cyt proteins to their cytoplasmic compartments. This paper describes several previously unknown features of the Bti organelle, representing a first step to understanding the biology of a unique process of sorting and packaging of proteins into PBs. The insights from this research suggest a potential for future applications in nanotechnology.
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Thurincin H, a bacteriocin produced by Bacillus thuringiensis, exhibits antibacterial activity against Gram-positive and Gram-negative bacteria. While much is known about its expression and antimicrobial spectrum, its hemolytic property has yet to be established. In this study, thurincin H was produced in a plasmid-free acrystalliferous strain of B. thuringiensis (Bt Cry-B) that naturally lacked antimicrobial and hemolytic activities. When grown in Tryptic Soy Broth (TSB), the bacteriocin's maximal production in Bt Cry-B harboring the thurincin H genetic cluster (Bt Cry-B/pThur) was observed at 24 h. Thurincin H was purified as a sole peptide of ~5 kDa using three purification steps, i.e., salt precipitation, ultrafiltration, and gel filtration chromatography. The bacteriocin showed inhibitory activity against B. cereus (5631 U), Bt Cry-B (8827 U), E. faecium wild type (11,197 U), and E. faecium ATCC 19,434 (6950 U), but not against Bt Cry-B/pThurH and Bt Cry-B/pThurHΔThnA. In addition, a minimum inhibitory concentration (MIC) of 5.0 µg/mL against B. cereus 183 was observed. In silico predictions suggested that thuricin H lacks hemolytic activity, which was validated in vitro using 4 × the MIC, i.e., 20 µg/ml. Our data lay a foundation for the potential safe use of thurincin H as an antibacterial peptide for medical use, in food products, and for expression in probiotic bacteria.
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Bacillus thuringiensis , Bacteriocinas , Antibacterianos/química , Bacillus thuringiensis/genética , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Bactérias Gram-Positivas , Bactérias Gram-Negativas , Bacteriocinas/genética , Bacteriocinas/farmacologiaRESUMO
Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.
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Bacillus thuringiensis , Nanopartículas , Animais , Proteínas de Bactérias/toxicidade , Endotoxinas , Proteínas Hemolisinas/toxicidade , Larva , Controle de MosquitosRESUMO
AIMS: The objective of this study was to evaluate the inhibitory activity of compounds secreted by bacteria isolated from a hydrogen-producing bioreactor to understand how these microorganisms interact in this community. METHODS AND RESULTS: In vitro inhibitory assays were performed using samples secreted by bacteria subject to different treatments to determine if their inhibitory effect was due to organic acids, non-proteinaceous compounds or bacteriocin-like inhibitory substances (BLIS). Bacterial isolated were suppressed 43%, 30% and 27% by neutralized, precipitated and non-neutralized cell-free supernatants, respectively. Non-hydrogen producers (non-H2 P) lactic acid bacteria (LAB) (Lactobacillus plantarum LB1, Lactobacillus pentosus LB7, Pediococcus acidilactici LB4) and hydrogen producers (H2 P) LAB (Enterococcus faecium F) were inhibited by the production of organic acids, non-proteinaceous compounds and BLIS. Meanwhile, the obligate anaerobe H2 P (Clostridium beijerinckii B) inhibited by the production of non-proteinaceous compounds and BLIS. The presence of BLIS was confirmed when proteolytic enzymes affected the inhibitory activity of secreted proteins in values ranging from 20% to 42%. The BLIS produced by L. plantarum LB1, P. acidilactici LB4, L. pentosus LB7 and E. faecium F showed molecular masses of ~11, 25, 20 and 11 kDa, respectively. CONCLUSIONS: It was demonstrated antagonistic interactions between Lactobacillus-Enterococcus and Pediococcus-Enterococcus species, generated by the secretion of organic acids, non-proteinaceous compounds and BLIS. SIGNIFICANCE AND IMPACT OF THE STUDY: We report the interactions between LAB isolated from hydrogen-producing bioreactors. These interactions might impact the dynamics of the microbial population during hydrogen generation. Our work lays a foundation for strategies that allow controlling bacteria that can affect hydrogen production.
Assuntos
Bacteriocinas , Enterococcus faecium , Lactobacillales , Bacteriocinas/metabolismo , Bacteriocinas/farmacologia , Reatores Biológicos , Enterococcus/metabolismo , Fermentação , Hidrogênio/metabolismo , Lactobacillales/metabolismo , Pediococcus/metabolismo , Triticum/metabolismoRESUMO
Ascoviruses are large dsDNA viruses characterized by the extraordinary changes they induce in cellular pathogenesis and architecture whereby after nuclear lysis and extensive hypertrophy, each cell is cleaved into numerous vesicles for virion reproduction. However, the level of viral replication and transcription in vesicles compared to other host tissues remains uncertain. Therefore, we applied RNA-Sequencing to compare the temporal transcriptome of Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV) at 7, 14, and 21 days post-infection (dpi). We found most transcription occurred in viral vesicles, not in initial tissues infected, a remarkably novel reproduction mechanism compared to all other viruses and most other intracellular pathogens. Specifically, the highest level of viral gene expression occurred in hemolymph, for TnAV at 7 dpi, and SfAV at 14 dpi. Moreover, we found that host immune genes were partially down-regulated in hemolymph, where most viral replication occurred in highly dense accumulations of vesicles.
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Ascoviridae/genética , Hemolinfa/virologia , Transcriptoma/genética , Tropismo/genética , Animais , Vírus de DNA/genética , DNA Viral/genética , Genoma Viral/genética , Fases de Leitura Aberta/genética , Reprodução/genética , Análise de Sequência de DNA/métodos , Spodoptera/genética , Vírion/genética , Replicação Viral/genéticaRESUMO
Agaves are a group of succulent plants that thrive in arid or semiarid environments. Indeed, genes associated with their resilience are a potential resource for genetic engineering of other agronomically important crops grown in adverse climates. Agave is mainly used for the production of distilled (spirits) and non-distilled alcoholic beverages, including tequila, mezcal, bacanora, raicilla, and pulque, all of which have special connections to Mexican history and culture, and contribute to the Mexican economy. In recent years, there has been growing interest to maximize the use of agave plant materials for other purposes, as the bulk of their biomass pre- and post-production is wasted. In traditional practice, during the passage from fields to factories, only agave cores are used, and the leaves and bagasse are not always harnessed. To place this in perspective, during the period from 2010 to 2019, 2674.7 × 106 L of tequila was produced in Mexico, which required 9 607 400 tons of agave cores. This generated approximately the same amount of leaves and 3 842 960 tons of bagasse. The economic base of agave plants can be expanded if expended biomass could be transformed into products that are useful for applications in food, forage, ensilage, agriculture, medicine, energy, environment, textiles, cosmetics, and esthetics. This review focuses on the current utility of agave plants, as well as our perspective for future studies and uses of this formidable plant. © 2020 Society of Chemical Industry.
Assuntos
Agave/química , Resíduos/análise , Bebidas Alcoólicas/análise , Celulose/análise , Fermentação , México , Energia RenovávelRESUMO
The objective of this study was to determine whether seeds of Brassica oleracea var. italica (i.e. broccoli, an edible plant) produce defensins that inhibit phytopathogenic fungi and pathogenic bacteria of clinical significance. Crude extracts obtained from broccoli seeds were fractioned by molecular exclusion techniques and analyzed by liquid chromatography-high-resolution mass spectrometry. Two peptides were identified, BraDef1 (10.68 kDa) and BraDef2 (9.9 kDa), which were categorized as Class I defensins based on (a) their primary structure, (b) the presence of four putative cysteine disulfide bridges, and (c) molecular modeling predictions. BraDef1 and BraDef2 show identities of, respectively, 98 and 71%, and 67 and 85%, with defensins from Brassica napus and Arabidopsis thaliana. BraDef (BraDef1 + BraDef2) disrupted membranes of Colletotrichum gloeosporioides and Alternaria alternata and also reduced hyphal growth of C. gloeosporioides by ~ 56% after 120 h of incubation. Pathogenic bacteria (Bacillus cereus 183, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and Vibrio parahaemolitycus) were susceptible to BraDef, but probiotic bacteria such as Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus casei were not inhibited. To our knowledge, this is the first report of defensins present in seeds of B. oleracea var. italica (i.e. edible broccoli). Our findings suggest an applied value for BraDef1/BraDef2 in controlling phytopathogenic fungi and pathogenic bacteria of clinical significance.
Assuntos
Anti-Infecciosos/farmacologia , Brassica/química , Defensinas/farmacologia , Sequência de Aminoácidos , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Bactérias/efeitos dos fármacos , Cromatografia Líquida , Defensinas/química , Defensinas/isolamento & purificação , Fungos/efeitos dos fármacos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Extratos Vegetais/química , Sementes/químicaRESUMO
Ascoviruses are large, enveloped DNA viruses that induce remarkable changes in cellular architecture during which the cell is partitioned into numerous vesicles for viral replication. Previous studies have shown that these vesicles arise from a process resembling apoptosis yet which differs after nuclear lysis in that mitochondria are not degraded but are modified by the virus, changing in size, shape, and motility. Moreover, infection does not provoke an obvious innate immune response. Thus, we used in vivo RNA sequencing to determine whether infection by the Spodoptera frugiperda ascovirus 1a (SfAV-1a) modified expression of host mitochondrial, cytoskeletal, and innate immunity genes. We show that transcripts from many mitochondrial genes were similar to those from uninfected controls, whereas others increased slightly during vesicle formation, including those for ATP6, ATP8 synthase, and NADH dehydrogenase subunits, supporting electron microscopy (EM) data that these organelles were conserved for virus replication. Transcripts from 58 of 106 cytoskeletal genes studied increased or decreased more than 2-fold postinfection. More than half coded for mitochondrial motor proteins. Similar increases occurred for innate immunity transcripts and their negative regulators, including those for Toll, melanization, and phagocytosis pathways. However, those for many antimicrobial peptides, such as moricin, increased more than 20-fold. In addition, transcripts for gloverin-3, spod_x_tox, Hdd23, and lebocin, also antimicrobial, increased more than 20-fold. Interestingly, a phenoloxidase inhibitor transcript increased 12-fold, apparently to interfere with melanization. SfAV-1a destroys most fat body cells by 7 days postinfection, so innate immunity gene transcripts apparently occur in remaining cells in this tissue and possibly other major tissues, namely, epidermis and tracheal matrix.IMPORTANCE Ascoviruses are large DNA viruses that infect insects, inducing a cellular pathology that resembles apoptosis but which differs by causing enormous cellular hypertrophy followed by cleavage of the cell into numerous viral vesicles for replication. Previous EM studies suggest that mitochondria are important for vesicle formation. Transcriptome analyses of Spodoptera frugiperda larvae infected with SfAV-1a showed that mitochondrial transcripts were similar to those from uninfected controls or increased slightly during vesicle formation, especially for ATP6, ATP8 synthase, and NADH dehydrogenase subunits. This pattern resembles that for chronic disease-inducing viruses, which conserve mitochondria, differing markedly from viruses causing short-term viral diseases, which degrade mitochondrial DNA. Though mitochondrial transcript increases were low, our results demonstrate that SfAV-1a alters host mitochondrial expression more than any other virus. Regarding innate immunity, although SfAV-1a destroys most fat body cells, certain immunity genes were highly upregulated (greater than 20-fold), suggesting that these transcripts may originate from other tissues.
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Ascoviridae/genética , Mitocôndrias/genética , Replicação Viral/genética , Animais , Ascoviridae/metabolismo , Perfilação da Expressão Gênica , Imunidade Inata/genética , Larva/virologia , Mitocôndrias/metabolismo , Análise de Sequência de RNA , Spodoptera/genética , Spodoptera/metabolismo , Transcriptoma , Proteínas Virais/genética , Replicação Viral/fisiologiaRESUMO
Streptomyces spp. are Gram-positive bacteria well-known for their ability to produce antibiotics and other metabolites, but few studies on bacteriocins produced by these bacteria have been reported. We tested eight Streptomyces strains against different pathogenic bacteria, and selected S. griseus, S. nigrescens, S. bottroprensis, and S. violaceoruber for further study based on their inhibitory effects against bacteria, including human pathogens. S. bottropensis reached its highest activity at 312 h and was higher than the activities of S. violaceoruber and S. nigrescens. The best condition for bacteriocin precipitation was using diammonium sulfate at 50% saturation. Bacteriocins were susceptible to proteinase treatments and stable at high temperature (up to 100 °C). The highest inhibitory activities were observed between pH 5 and 6. Cross-activity assays indicated that each Streptomyces strain produced different bacteriocins. When preparations of S. griseus and S. nigrescens were subjected to SDS-PAGE, bands of inhibition were observed in the gel overlay assay at a position corresponding to ~ 2 and 3 kDa, respectively, suggesting that both strains are potential sources for novel bacteriocins.
Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Streptomyces/metabolismo , Sulfato de Amônio/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Estabilidade Proteica , Especificidade da Espécie , Streptomyces/classificação , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , TemperaturaRESUMO
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
The most important bioinsecticide used worldwide is Bacillus thuringiensis and its hallmark is a rich variety of insecticidal Cry protein, many of which have been genetically engineered for expression in transgenic crops. Over the past 20 years, the discovery of other insecticidal proteins and metabolites synthesized by B. thuringiensis, including chitinases, antimicrobial peptides, vegetative insecticidal proteins (VIP), and siderophores, has expanded the applied value of this bacterium for use as an antibacterial, fungicidal, and nematicidal resource. These properties allow us to view B. thuringiensis not only as an entity for the production of a particular metabolite, but also as a multifaceted microbial factory. In particular, chitinases of B. thuringiensis are secreted enzymes that hydrolyze chitin, an abundant molecule in the biosphere, second only to cellulose. The observation that chitinases increase the insecticidal activity of Cry proteins has stimulated further study of these enzymes produced by B. thuringiensis. Here, we provide a review of a subset of our knowledge of B. thuringiensis chitinases as it relates to their phylogenetic relationships, regulation of expression, biotechnological potential for controlling entomopathogens, fungi, and nematodes, and their use in generating chitin-derived oligosaccharides (ChOGs) that possess antibacterial activities against a number of clinically significant bacterial pathogens. Recent advances in the structural organization of these enzymes are also discussed, as are our perspective for future studies.
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Eukaryotic dsDNA viruses use small basic protamine-like proteins or histones, typically <15 kDa, to condense and encapsidate their genomic (g)DNAs during virogenesis. Ascoviruses are large dsDNA (~100â»200 kbp) viruses that are pathogenic to lepidopteran larvae. Little is known about the molecular basis for condensation and encapsidation of their gDNAs. Previous proteomic analysis showed that Spodoptera frugiperda ascovirus (SfAV-1a) virions contain a large unique DNA-binding protein (P64; 64 kDa, pI = 12.2) with a novel architecture proposed to condense its gDNA. Here we used physical, biochemical, and transmission electron microscopy techniques to demonstrate that P64's basic C-terminal domain condenses SfAV-1a gDNA. Moreover, we demonstrate that only P64 homologs in other ascovirus virions are unique in stably binding DNA. As similar protein families or subfamilies were not identified in extensive database searches, our collective data suggest that ascovirus P64 homologs comprise a novel family of atypical large viral gDNA condensing proteins.
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OBJECTIVES: To develop a recombinant strain of Bacillus thuringiensis that synthesizes two bacteriocins that enhance the antibacterial potency of the strain and that could have applied clinical and industrial value. RESULTS: We cloned the thurincin H cluster into the pHT3101 vector by assembling two genetic cassettes harboring genes for the synthesis, modification, immunity and transport of thurincin H. This construct was used to transform a thurincin H-sensitive strain of B. thuringiensis that synthesizes the kenyacin 404 to generate the recombinant Btk 404/pThurH which was immune to thurincin H and produces bacteriocins of approximately 3 kDa. A significant increase in the inhibitory activity, respectively, ~ 40 and 300%, was observed when compared with parental Btm 269 and Btk 404. Btk 404/pThurH showed increased activity against ten Gram-positive bacteria, including B. cereus, Listeria monocytogenes and B. pseudomycoides, and the Gram-negative bacterium, Sphingobacterium cabi. However, an antagonistic effect against Vibrio parahaemolyticus, relative to native strains, was observed. CONCLUSIONS: We have generated a recombinant strain of B. thuringiensis that co-synthesizes two bacteriocins (kenyacin 404, thurincin H) with improved inhibitory activity, when compared with parental strains. To our knowledge, this is the first study that shows that B. thuringiensis could be manipulated to produce two bacteriocins, one being of heterologous origin, that enhance the antibacterial activity of the recombinant strain.
Assuntos
Antibacterianos/biossíntese , Bacillus thuringiensis/química , Bacteriocinas/biossíntese , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Clonagem Molecular , Escherichia coli/genética , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
The structural gene that encodes thurincin H, a bacteriocin produced by Bacillus thuringiensis, is harboured in a genetic cluster (thnP, E, D, R, A1, A2, A3, B, T, I) that controls its synthesis, modification, secretion and autoimmunity. The specific genes in the cassette that confer immunity in B. thuringiensis to thurincin H are unknown. To identify these immunity determinants, we generated constructs that were used to transform a natural thurincin H-sensitive B. thuringiensis strain (i.e. Btk 404), and resistance or susceptibility to the bacteriocin in resultant recombinants was evaluated. When Btk 404/pHT3101-ThnARDEP and Btk 404/pHT3101-ThnABTI were exposed to thurincin H, immunity was demonstrated by the former only, indicating that ThnI does not play a role in resistance to the bacteriocin as previously proposed. Furthermore, we generated different sub-cassettes under the control of divergent promoters pThnR and pThur of the thurincin H locus, and pChi, and using the green fluorescent protein gene as reporter, which demonstrated that all promoters were recognised by ThnR, except pChi. We show for the first time that the small operon composed of thnR, thnD and thnE is required for immunity of B. thuringiensis to thurincin H, and thnI is not necessary for this response.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Bacillus thuringiensis/imunologia , Bacteriocinas/imunologia , Transportadores de Cassetes de Ligação de ATP/genética , Autoimunidade , Bacillus thuringiensis/efeitos dos fármacos , Bacillus thuringiensis/genética , Bacteriocinas/genética , Bacteriocinas/farmacologia , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Família Multigênica , Óperon , Regiões Promotoras GenéticasRESUMO
Two mosquitocidal bacteria, Bacillus thuringiensis subsp. israelensis (Bti) and Lysinibacillus sphaericus (Ls) are the active ingredients of commercial larvicides used widely to control vector mosquitoes. Bti's efficacy is due to synergistic interactions among four proteins, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, whereas Ls's activity is caused by Bin, a heterodimer consisting of BinA, the toxin, and BinB, a midgut-binding protein. Cyt1Aa is lipophilic and synergizes Bti Cry proteins by increasing midgut binding. We fused Bti's Cyt1Aa to Ls's BinA yielding a broad-spectrum chimeric protein highly mosquitocidal to important vector species including Anopheles gambiae, Culex quinquefasciatus, and Aedes aegypti, the latter an important Zika and Dengue virus vector insensitive to Ls Bin. Aside from its vector control potential, our bioassay data, in contrast to numerous other reports, provide strong evidence that BinA does not require conformational interactions with BinB or microvillar membrane lipids to bind to its intracellular target and kill mosquitoes.
Assuntos
Inseticidas/química , Inseticidas/farmacologia , Mosquitos Vetores/efeitos dos fármacos , Proteínas/química , Proteínas/farmacologia , Proteínas Recombinantes de Fusão , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacologia , BioensaioRESUMO
The biopesticide used most effectively to control mosquito and blackfly vectors of human diseases worldwide is Bacillus thuringiensis subsp. israelensis. The high efficacy of this bacterium is due to synergistic interactions among four protein entomotoxins assembled individually into a single parasporal body (PB) during sporulation. Cyt1Aa, the primary synergist, is the most abundant toxin, comprising approximately 55% of the PB's mass. The other proteins are Cry11Aa at â¼35%, and Cry4Aa and Cry4Ba, which together account for the remaining â¼10%. The molecular genetic basis for the comparatively large amount of Cyt1Aa synthesized is unknown. Here, in addition to the known strong BtI (σE) and BtII (σK) promoters, we demonstrate a third promoter (BtIII) that has high identity to the σE promoter of Bacillus subtilis, contributes to the large amount of Cyt1Aa synthesized. We also show that a cyt1Aa-BtIII construct was not functional in a σE-deficient strain of B. subtilis. Comparison of transcription levels and protein profiles for recombinant strains containing different combinations of BtI, BtII and BtIII, or each promoter alone, showed that BtIII is active throughout sporulation. We further demonstrate that a stable stem-loop in the 3'-untranslated region (3'-UTR, predicted ΔG=-27.6) contributes to the high level of Cyt1Aa synthesized.