RESUMEN
Phage-encoded endolysins have emerged as a potential substitute to conventional antibiotics due to their exceptional benefits including host specificity, rapid host killing, least risk of resistance. In addition to their antibacterial potency and biofilm eradication properties, endolysins are reported to exhibit synergism with other antimicrobial agents. In this study, the synergistic potency of endolysins was dissected with antimicrobial peptides to enhance their therapeutic effectiveness. Recombinantly expressed and purified bacteriophage endolysin [T7 endolysin (T7L); and T4 endolysin (T4L)] proteins have been used to evaluate the broad-spectrum antibacterial efficacy using different bacterial strains. Antibacterial/biofilm eradication studies were performed in combination with different antimicrobial peptides (AMPs) such as colistin, nisin, and polymyxin B (PMB) to assess the endolysin's antimicrobial efficacy and their synergy with AMPs. In combination with T7L, polymyxin B and colistin effectively eradicated the biofilm of Pseudomonas aeruginosa and exhibited a synergistic effect. Further, a combination of T4L and nisin displayed a synergistic effect against Staphylococcus aureus biofilms. In summary, the obtained results endorse the theme of combinational therapy consisting of endolysins and AMPs as an effective remedy against the drug-resistant bacterial biofilms that are a serious concern in healthcare settings.
Asunto(s)
Antibacterianos , Péptidos Antimicrobianos , Biopelículas , Sinergismo Farmacológico , Endopeptidasas , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa , Staphylococcus aureus , Biopelículas/efectos de los fármacos , Endopeptidasas/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Pseudomonas aeruginosa/efectos de los fármacos , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/química , Nisina/farmacología , Nisina/química , Polimixina B/farmacología , Bacteriófagos , Colistina/farmacología , Bacteriófago T4/efectos de los fármacos , Bacteriófago T4/fisiología , Bacteriófago T7/efectos de los fármacos , Bacteriófago T7/genéticaRESUMEN
Covering: up to the end of 2015While Nature continues to deliver a myriad of potent and structurally diverse biologically active small molecules, the cellular targets and modes of action of these natural products are rarely identified, significantly hindering their development as new chemotherapeutic agents. This article provides an introductory tutorial on the use of T7 phage display as a tool to rapidly identify the cellular targets of natural products and is aimed specifically at natural products chemists who may have only limited experience in molecular biology. A brief overview of T7 phage display is provided, including its strengths, weaknesses, and the type of problems that can and cannot be tackled with this technology. Affinity probe construction is reviewed, including linker design and natural product derivatisation strategies. A detailed description of the T7 phage biopanning procedure is provided, with valuable tips for optimising each step in the process, as well as advice for identifying and avoiding the most commonly encountered challenges and pitfalls along the way. Finally, a brief discussion is provided on techniques for validating the cellular targets identified using T7 phage display.
Asunto(s)
Bacteriófago T7/efectos de los fármacos , Productos Biológicos , Bacteriófago T7/metabolismo , Productos Biológicos/química , Productos Biológicos/farmacología , Humanos , Estructura MolecularRESUMEN
We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity.
Asunto(s)
Bacteriófago T7/genética , Didesoxinucleótidos/farmacología , Escherichia coli/enzimología , Inhibidores de la Síntesis del Ácido Nucleico , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Nucleótidos de Timina/farmacología , Bacteriófago T7/efectos de los fármacos , Bacteriófago T7/enzimología , Bacteriófago T7/crecimiento & desarrollo , ADN Viral/biosíntesis , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Didesoxinucleótidos/metabolismo , Escherichia coli/virología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Fosforilación , Pirimidina Fosforilasas/genética , Pirimidina Fosforilasas/metabolismo , Eliminación de Secuencia , Timidina/metabolismo , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Nucleótidos de Timina/metabolismo , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Disinfecting pathogenic contaminated water rapidly and effectively on sites is one of the critical challenges at point-of-use (POU) situations. Currently available technologies are still suffering from irreversible depletion of disinfectants, generation of toxic by-products, and potential biofouling problems. Herein, we developed a chlorine rechargeable biocidal nanofibrous membrane, poly(acrylonitrile-co-5-methyl-5-(4'-vinylphenyl)imidazolidine-2,4-dione) (P(AN-VAPH)), via a combination of a free radical copolymerization reaction and electrospun technology. The copolymer exhibits good electrospinnability and desirable mechanical properties. Also, the 5-methyl-5-(4'-vinylphenyl)imidazolidine-2,4-dione (VAPH) moieties containing unique hydantoin structures are able to be chlorinated and converted to halamine structures, enabling the P(AN-VAPH) nanofibrous membrane with rapid and durable biocidal activity. The chlorinated P(AN-VAPH) nanofibrous membranes showed intriguing features of unique 3D morphological structures with large specific surface area, good mechanical performance, rechargeable chlorination capacity (>5000 ppm), long-term durability, and desirable biocidal activity against both bacteria and viruses (>99.9999% within 2 min of contact). With these attributes, the chlorinated P(AN-VAPH) membranes demonstrated promising disinfecting efficiency against concentrated bacteria-contaminated water during direct filtration applications with superior killing capacity and high flowing flux (5000 L m-2 h-1).
Asunto(s)
Antibacterianos/farmacología , Antivirales/farmacología , Desinfectantes/farmacología , Hidantoínas/farmacología , Membranas Artificiales , Nanofibras/química , Resinas Acrílicas/síntesis química , Resinas Acrílicas/farmacología , Antibacterianos/síntesis química , Antivirales/síntesis química , Bacteriófago T7/efectos de los fármacos , Desinfectantes/síntesis química , Desinfección/instrumentación , Escherichia coli/efectos de los fármacos , Filtración/instrumentación , Hidantoínas/síntesis química , Listeria/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Polivinilos/síntesis química , Polivinilos/farmacología , Purificación del Agua/instrumentaciónRESUMEN
Viruses are notoriously capable of evolving resistance to drugs. However, if the endpoint of resistance evolution is only partial escape, a feasible strategy should be to stack drugs, so the combined effect of partial inhibition by several drugs results in net inhibition. Assessing the feasibility of this approach requires quantitative data on viral fitness before and after evolution of resistance to a drug, as done here with bacteriophage T7. An inhibitory gene expressed from a phage promoter aborts wild-type T7 infections. The effect is so severe that the phage population declines when exposed to the inhibitor but expands a billion-fold per hour in its absence. In prior work, T7 evolved modest resistance to this inhibitor, an expected result. Given the nature of the inhibitor, that it used the phage's own promoter to target the phage's destruction, we anticipated that resistance evolution would be limited as the phage may need to evolve a new regulatory system, with simultaneous changes in its RNA polymerase (RNAP) and many of its promoters to fully escape inhibition. We show here that further adaptation of the partially resistant phage led to complete resistance. Resistance evolution was due to three mutations in the RNAP gene and two other genes; unexpectedly, no changes were observed in promoters. Consideration of other mechanisms of T7 inhibition leaves hope that permanent inhibition of viral growth with drugs can in principle be achieved.
Asunto(s)
Antivirales/metabolismo , Bacteriófago T7/efectos de los fármacos , Bacteriófago T7/genética , Farmacorresistencia Viral/genética , Evolución Molecular , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Bacterias/virología , Bacteriófago T7/fisiología , Expresión Génica , Plásmidos/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
During the development of antibacterial and antiviral materials for personal protective equipment (PPE), daylight active functional polymeric materials containing vitamin K compounds (VKs) and impacts of polymer structures to the functions were investigated. As examples, hydrophobic polyacrylonitrile (PAN) and hydrophilic poly(vinyl alcohol-co-ethylene) (PVA-co-PE) polymers were directly blended with three VK compounds and electrospun into VK-containing nanofibrous membranes (VNFMs). The prepared VNFMs exhibited robust photoactivity in generating reactive oxygen species (ROS) under both daylight (D65, 300-800 nm) and ultraviolet A (UVA, 365 nm) irradiation, resulting in high antimicrobial and antiviral efficiency (>99.9%) within a short exposure time (<90 min). Interestingly, the PVA-co-PE/VK3 VNFM showed higher ROS production rates and better biocidal functions than those of the PAN/VK3 VNFM under the same photoirradiation conditions, indicating that PVA-co-PE is a better matrix polymer material for these functions. Moreover, the prepared PVA-co-PE/VK3 VNFM maintains its powerful microbicidal function even after five times of repeated exposures to bacteria and viruses, showing the stability and reusability of the antimicrobial materials. The fabrication of photoinduced antimicrobial VNFMs may provide new insights into the development of non-toxic and reusable photoinduced antimicrobial materials that could be applied in personal protective equipment with improved biological protections.
Asunto(s)
Antibacterianos/farmacología , Antivirales/farmacología , Nanopartículas/química , Equipo de Protección Personal , Rayos Ultravioleta , Vitamina K/farmacología , Antibacterianos/química , Antivirales/química , Bacteriófago T7/efectos de los fármacos , Escherichia coli O157/efectos de los fármacos , Listeria/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Tamaño de la Partícula , Teoría Cuántica , Propiedades de Superficie , Vitamina K/análogos & derivados , Vitamina K/químicaRESUMEN
Synthesis of oligoribonucleotide primers for lagging-strand DNA synthesis in the DNA replication system of bacteriophage T7 is catalyzed by the primase domain of the gene 4 helicase-primase. The primase consists of a zinc-binding domain (ZBD) and an RNA polymerase (RPD) domain. The ZBD is responsible for recognition of a specific sequence in the ssDNA template whereas catalytic activity resides in the RPD. The ZBD contains a zinc ion coordinated with four cysteine residues. We have examined the ligation state of the zinc ion by X-ray absorption spectroscopy and biochemical analysis of genetically altered primases. The ZBD of primase engaged in catalysis exhibits considerable asymmetry in coordination to zinc, as evidenced by a gradual increase in electron density of the zinc together with elongation of the zinc-sulfur bonds. Both wild-type primase and primase reconstituted from purified ZBD and RPD have a similar electronic change in the level of the zinc ion as well as the configuration of the ZBD. Single amino acid replacements in the ZBD (H33A and C36S) result in the loss of both zinc binding and its structural integrity. Thus the zinc in the ZBD may act as a charge modulation indicator for the surrounding sulfur atoms necessary for recognition of specific DNA sequences.
Asunto(s)
Bacteriófago T7/enzimología , ADN Primasa/química , ADN Primasa/metabolismo , ADN Viral/metabolismo , Zinc/metabolismo , Sustitución de Aminoácidos/efectos de los fármacos , Sustitución de Aminoácidos/genética , Bacteriófago T7/efectos de los fármacos , Biocatálisis/efectos de los fármacos , Cloruros/farmacología , Cristalografía por Rayos X , Cisteína/metabolismo , Estabilidad de Enzimas/efectos de los fármacos , Histidina/metabolismo , Ligandos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oligorribonucleótidos/biosíntesis , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Moldes Genéticos , Espectroscopía de Absorción de Rayos X , Compuestos de Zinc/farmacologíaRESUMEN
The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to remove non-enveloped viruses from aqueous media. Here we tested the virus inactivation capability of meso-Tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4-N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be efficient photosensitizers of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding.
Asunto(s)
ADN/metabolismo , Fotoquímica/métodos , Porfirinas/farmacología , Porfirinas/efectos de la radiación , Inactivación de Virus , Bacteriófago T7/efectos de los fármacos , Bacteriófago T7/efectos de la radiación , Cationes/efectos de la radiación , ADN/efectos de los fármacos , ADN/efectos de la radiación , Fármacos Fotosensibilizantes , Porfirinas/metabolismoAsunto(s)
Bacteriófago T7/química , Endotoxinas/aislamiento & purificación , Ultrafiltración/métodos , Bacteriófago T7/efectos de los fármacos , Bacteriófago T7/genética , Ácido Desoxicólico/química , Ácido Desoxicólico/farmacología , Detergentes/química , Detergentes/farmacología , Endotoxinas/químicaRESUMEN
Ultraviolet A (UVA) light (315-400 nm) is ubiquitously found in our environment and constitutes about 95% of the total solar UV; all UVC and most UVB being absorbed by the stratospheric ozone layer. Compared with UVB and C, UVA does not show any direct effect on biological systems. Indirect effects of UVA, however, have been recognised overwhelmingly and this includes photosensitization of biological and non-biological compounds and production of free radicals many of which include oxygen and are hence known as reactive oxygen species or ROS. Several types of free radicals have been identified although their impacts on various macro- and micro-biomolecules are yet to be fully elucidated. beta-Phenylpyruvic acid is ubiquitously found in eukaryotic cells as a metabolite of phenylalanine, which is subsequently converted to phenyllactate and/or to 2-hydroxyphenylacetate and mandelate. In patients suffering from phenylketonuria the hydroxylation of phenylalanine to tyrosine is defective due to lack of phenylalanine hydroxylase. These result in accumulation and excretion of this compound in the urine. Here we present evidence that photolysis of beta-phenylpyruvic acid by a skin tanning lamp, emitting 99% UVA (315-400 nm) and 1% UVB (290-315 nm) generates carboxyl radicals (CO(2)(*)) and also possibly causes direct electron transfer (or type 1) reactions. Electron paramagnetic resonance was used to detect the free radicals. To determine the biological effects of this photolytic reaction, T7 was exposed to these photolytic reactive agents and found to lead to high levels of phage inactivation. Damage to DNA and/or components such as tail fibre proteins may be involved in T7 inactivation. In addition, our unpublished data suggest that certain phenylketonuria cell lines are more sensitive to PPA+NUV, lending importance to photolytic studies of this agent.
Asunto(s)
Daño del ADN , Radicales Libres/síntesis química , Ácidos Fenilpirúvicos/efectos de la radiación , Especies Reactivas de Oxígeno/síntesis química , Rayos Ultravioleta , Bacteriófago T7/efectos de los fármacos , Ensayo Cometa , Espectroscopía de Resonancia por Spin del Electrón , FotólisisRESUMEN
We investigated the efficiency and the mechanism of action of a tetraphenyl porphyrin derivative in its photoreaction with T7 phage as surrogate of non-enveloped DNA viruses. TPFP was able to sensitize the photoinactivation of T7 phage in spite of the lack of its binding to the nucleoprotein complex. The efficiency of TPFP photosensitization was limited by the aggregation and by the photobleaching of porphyrin molecules. Addition of sodium azide or 1,3-dimethyl-2-thiourea (DMTU) to the reaction mixture moderated T7 inactivation, however, neither of them inhibited T7 inactivation completely. This result suggests that both Type I and Type II reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated nucleoprotein complex. Polymerase chain reaction (PCR) also failed to demonstrate any DNA damage. Circular dichroism (CD) spectra of photosensitized nucleoprotein complex indicated changes in the secondary structure of both the DNA and proteins. We suggest that damages in the protein capsid and/or loosening of protein-DNA interaction can be responsible for the photodynamic inactivation of T7 phage. The alterations in DNA secondary structure might be the result of photochemical damage in phage capsid proteins.
Asunto(s)
Bacteriófago T7/efectos de los fármacos , Virus ADN/efectos de los fármacos , Galactósidos/farmacología , Fotoquimioterapia , Porfirinas/farmacología , Dicroismo Circular , Daño del ADN , Reacción en Cadena de la PolimerasaRESUMEN
We study here the effect on phage T7 RNA polymerase activity of lac repressor bound downstream of the T7 promoter. When repressor binds in vitro at an operator centered at +13 or +15 with respect to transcription start, it does not prevent initiation, though the transcript yield is reduced. However, the processivity of the polymerase is depressed and transcript extension is blocked at positions +4 and +6, respectively. These results indicate that repressor and polymerase do not simply exclude each other from the promoter. Rather, they would come into steric conflict and compete for establishment or retention of interactions with the same segment of DNA, without this leading to the immediate displacement of either polymerase or repressor. The resulting destabilization of the transcription complex would depress both initiation rate and enzyme processivity. In contrast to the above results, little reduction in runoff transcription is observed when operator is centered at +47. The decreased sensitivity of polymerase to repressor bound at +47 versus +13 or +15 is likely to be due to the higher stability of the elongation complex during the transcription of downstream regions in comparison with the first transcribed nucleotides. We also show that under conditions of leaky repression and with operator centered at +13, a mutant T7 RNA polymerase showing normal promoter affinity but a slower elongation rate is more sensitive to repression than the wild-type enzyme, both in vitro and in vivo. In vitro, this higher sensitivity is largely due to a reduced ability of the mutant to overcome the elongation block at position +4. The parallel between the in vitro and in vivo data suggests that in vivo the repressor also does not prevent polymerase from binding to promoter, but interferes with subsequent steps in initiation and transcript extension, in this case presumably largely extension beyond +4.
Asunto(s)
Proteínas Bacterianas/farmacología , Bacteriófago T7/enzimología , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Proteínas de Escherichia coli , Proteínas Represoras/farmacología , Proteínas Bacterianas/metabolismo , Bacteriófago T7/efectos de los fármacos , Bacteriófago T7/genética , Secuencia de Bases , Sitios de Unión , ADN Viral/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virología , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Represoras Lac , Datos de Secuencia Molecular , Regiones Operadoras Genéticas , Mutación Puntual , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas ViralesRESUMEN
We investigated the efficiency and the mechanism of action of two--one symmetrically and one asymmetrically substituted--glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 phage as a model of nucleoprotein (NP) complexes. A correlation was found between the dark inactivation of T7 and the binding of porphyrins determined by fluorescence spectroscopy. Both types of porphyrin sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different. There was no correlation between the dark binding and the photosensitizing efficacy of the two derivatives. Inactivation was moderated by 1,3-diphenylisobenzofuran and 1,3-dimethyl-2-thiourea; however, neither of them inhibited T7 inactivation completely. This result suggests that both Type-I and Type-II reactions play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated NP complex. Polymerase chain reaction analysis of a 555 bp segment of gene 1 and a 3826 bp segment of genes 3 and 4 failed to demonstrate any DNA damage.
Asunto(s)
Bacteriófago T7/efectos de los fármacos , Bacteriófago T7/efectos de la radiación , Luz , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Escherichia coli/virología , Fármacos Fotosensibilizantes/química , Porfirinas/química , Espectrometría de FluorescenciaRESUMEN
This study investigates the importance of DNA damage in viral inactivation by phenothiazines and light. Phenothiazines, including methylene blue (MB), toluidine blue and azure B are of particular interest because of their ability to bind to nucleic acids in vitro. Initial studies employing phages T7, MS2 and PM2 indicated that both DNA and RNA phages as well as enveloped and nonenveloped phages can be inactivated by phenothiazine photosensitization. PM2, which contains a lipid-protein bilayer and supercoiled DNA, was used for the mechanistic studies to model blood-borne viruses. Viral DNA damage was assessed following treatment of phage to known levels of viral inactivation by extracting the DNA and analyzing for both direct and piperidine-catalyzed strand cleavage by gel electrophoresis. DNA strand cleavage was found to be both sensitizer concentration and light dose dependent. Both viral inactivation and DNA damage were found to be oxygen-dependent events. In parallel experiments, strand cleavage of isolated PM2 DNA treated with MB and light was also found to be oxygen dependent, in contrast to some previous reports. Transfection studies, which measure the infectivity of the extracted viral DNA, indicated that DNA from MB-treated phage was just as capable of generating progeny virus as the untreated controls. It was therefore concluded that the observed DNA damage is not correlated with loss of phage infectivity.
Asunto(s)
Bacteriófagos/efectos de la radiación , Daño del ADN , Bacteriófago T7/efectos de los fármacos , Bacteriófago T7/efectos de la radiación , Bacteriófagos/efectos de los fármacos , Sangre/microbiología , ADN Viral/efectos de los fármacos , ADN Viral/efectos de la radiación , Humanos , Técnicas In Vitro , Levivirus/efectos de los fármacos , Levivirus/efectos de la radiación , Azul de Metileno/farmacología , Fármacos Fotosensibilizantes/farmacologíaRESUMEN
Bispsoralen derivatives possessing two psoralens and one piperazine molecule, 1,4-bis[n'-(8-psoralenoxy) alkyl] piperazine (Bis[PsCn]PIP, n = 4, 6, 8), show high water solubility, efficient intercalation into DNA and good photocrosslinking efficiency of DNA. Bis(PsC4)PIP shows high lethality on bacteriophage T7 and can effectively inhibit the amplification of DNA by stopping the polymerase chain reactions in a short period of irradiation time.
Asunto(s)
Furocumarinas/química , Furocumarinas/farmacología , Fármacos Sensibilizantes a Radiaciones/química , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Bacteriófago T7/efectos de los fármacos , Bovinos , Reactivos de Enlaces Cruzados/síntesis química , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/farmacología , ADN/efectos de los fármacos , ADN/metabolismo , Furocumarinas/síntesis química , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Fotobiología , Fotoquímica , Piperazinas/síntesis química , Piperazinas/química , Piperazinas/farmacología , Fármacos Sensibilizantes a Radiaciones/síntesis químicaRESUMEN
The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter. Upon induction with L-arabinose, recombinant cells produced 100-fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol. This protein yield accounts for 30% of total cell protein content. In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20-30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome. In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using a two-stage feeding strategy to compartmentalize cell growth and protein synthesis. In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase. Subsequently, glycerol was fed to the culture broth and L-arabinose was augmented to induce protein production when cells entered the late log growth phase. As a result, a carbamoylase yield corresponding to 5525 units was obtained, which amounts to a 337-fold increase over that achieved on a shake-flask scale. Taken together, these results illustrate the practical usefulness of T7 system under control of the araBAD promoter for heterologous protein production.
Asunto(s)
Amidohidrolasas/biosíntesis , Bacteriófago T7/genética , Escherichia coli/enzimología , Escherichia coli/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Amidohidrolasas/análisis , Arabinosa/genética , Arabinosa/farmacología , Bacteriófago T7/efectos de los fármacos , Proteínas Portadoras , Recuento de Células , Cromosomas Bacterianos/genética , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/clasificación , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli , Estudios de Factibilidad , Fermentación , Modelos Moleculares , Rhizobium/enzimología , Rhizobium/genética , Sensibilidad y Especificidad , Proteínas ViralesRESUMEN
The discovery of new metal-selective complexing agents may be facilitated by applying an in vitro selection strategy. Such a strategy was recently devised to identify and enrich populations of bacteriophage that rely on Mg(II)-, Zn(II)-, or Au(III)-selective stabilization for survival in the presence of denaturing urea. The potential for extension of the strategy to other metal ions is investigated here. The kinetics of phage inactivation in 5 M urea was measured for a spectrum of metals. At a concentration of 1 mM, Mg(II), Ca(II), Co(II), and Ni(II) were found to be the most stabilizing, followed by Cd(II), Cu(II), and Zn(II), respectively. K(I) had virtually no effect. In contrast, Al(III) and Au(III) significantly destabilized the phage, even at concentrations of 0.25 mM.
Asunto(s)
Bacteriófago T7/efectos de los fármacos , Metales/farmacología , Aluminio/farmacología , Cationes/farmacología , Oro/farmacología , Cinética , Urea/farmacologíaRESUMEN
Treatment of bacteriophage T7 with methyl methanesulfonate perturbed phage-specific genetic expression in both repair-proficient and repair-deficient Escherichia coli cells. In wild-type cells (AB1157), the time course of protein synthesis was slowed down but an entire complement of phage proteins was synthesized. In cells (BK2114, tag-) unable to repair 3-methyladenine, the toxic lesion produced by methyl methanesulfonate, alkylated phage produced only early (class I) proteins. These results suggested that late transcription was inhibited in infected tag- cells. These cells were shown to contain a significant amount of active T7 RNA polymerase, a class I protein. Thus, the cause of inhibition appeared to be the inability of T7 RNA polymerase to use unrepaired DNA as template. In vitro transcription assays with alkylated T7 DNA as template supported this proposal. T7 RNA polymerase proved to be very sensitive to the presence of alkylation lesions. In addition, the phage enzyme was much more sensitive to these lesions than was its bacterial counterpart, E. coli RNA polymerase. These results suggest that 3-methyladenine exerts its toxic action, in the T7 system, at the level of transcription by T7 RNA polymerase. To further characterize the reduced activity of the T7 enzyme, an in vitro transcription assay using linearized plasmid DNA with one T7 promoter was devised. Gel electrophoresis revealed that only one transcript of well-defined length was synthesized by T7 RNA polymerase on this template. Alkylation of the template did not alter the size of the transcript produced. Simultaneous measurement of chain initiation and chain elongation confirmed this result by showing that both steps were reduced to the same extent by alkylation of template DNA. Thus T7 RNA polymerase does not appear to be blocked by 3-methyladenine. Rather the lesion must hinder translocation of T7 RNA polymerase along the DNA template during chain elongation.
Asunto(s)
Adenina/análogos & derivados , Bacteriófago T7/efectos de los fármacos , ADN Glicosilasas , Adenina/farmacología , Alquilación , Bacteriófago T7/genética , Reparación del ADN , ADN Bacteriano/metabolismo , ADN Viral/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Metilmetanosulfonato/farmacología , N-Glicosil Hidrolasas/metabolismo , Plásmidos , ARN Viral/metabolismo , Especificidad por Sustrato , Moldes Genéticos , Transcripción Genética , Proteínas Virales/biosíntesisRESUMEN
We have examined protein synthesis directed by bacteriophage T7 which had been alkylated with methyl methanesulfonate so as to produce apurinic sites in its DNA in vivo. Both repair-proficient and repair-deficient (xth nfo mutant) strains of Escherichia coli served as host cells. In repair-proficient cells, all three classes of phage proteins were synthesized, although with significant delays. In mutant cells, only class I proteins were produced and their synthesis was delayed and reduced, demonstrating a perturbation of protein synthesis and providing the first in vivo indication that transcription is inhibited by abasic sites. However, the proposed effects of abasic sites on transcription appear to be weaker than those on replication.
Asunto(s)
Ácido Apurínico/metabolismo , Bacteriófago T7/genética , Daño del ADN , Proteínas de Escherichia coli , Regulación Viral de la Expresión Génica/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Proteínas Virales/biosíntesis , Alquilación , Autorradiografía , Bacteriófago T7/efectos de los fármacos , Reparación del ADN , ADN Viral/efectos de los fármacos , ADN Viral/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ARN Polimerasas Dirigidas por ADN/biosíntesis , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/deficiencia , Escherichia coli/enzimología , Exodesoxirribonucleasas/deficiencia , Metilmetanosulfonato/toxicidad , Mutagénesis , Factores de Tiempo , Replicación Viral/efectos de los fármacosRESUMEN
Psoralens react photochemically with DNA to form interstrand crosslinks as well as two types of monoadduct (furan-side and pyrone-side adducts). To investigate the relative roles of these adducts in toxicity, we have studied the interaction of 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) with bacteriophage T7. These two derivatives differ in the fraction of pyrone-side monoadducts formed, TMP producing very small amounts of this type of adduct. The results show similar phage survival for the two psoralen analogs at equivalent numbers of crosslinks per DNA molecule. However, the survival fraction of treated phage is significantly lower than the fraction of noncrosslinked DNA molecules. Phage survival decreases after secondary irradiation which is used to transform monoadducts into crosslinks, but this decrease is not due solely to crosslinks; at doses beyond that required to transform all crosslinkable monoadducts into crosslinks, phage survival continues to decrease, pointing to the production of other genotoxic lesions during secondary irradiation. These results indicate that, although crosslinks can kill phage T7, as shown by the secondary irradiation results, they are not sufficient in number to explain the psoralen toxicity after primary irradiation. Therefore monoadducts, both furan-side and pyrone-side types, must in large part be responsible for phage inactivation.