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Streptomyces are renowned for their prolific production of specialized metabolites with applications in medicine and agriculture. These multicellular bacteria present a sophisticated developmental cycle and play a key role in soil ecology. Little is known about the impact of Streptomyces phage on bacterial physiology. In this study, we investigated the conditions governing the expression and production of "Samy", a prophage found in Streptomyces ambofaciens ATCC 23877. This siphoprophage is produced simultaneously with the activation of other mobile genetic elements. Remarkably, the presence and production of Samy increases bacterial dispersal under in vitro stress conditions. Altogether, this study unveiled a new property of a bacteriophage infection in the context of multicellular aggregate dynamics.
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Profagos , Streptomyces , Streptomyces/virología , Streptomyces/fisiología , Streptomyces/genética , Profagos/genética , Profagos/fisiología , Activación Viral/genéticaRESUMEN
Microbial specialized metabolite biosynthetic gene clusters (SMBGCs) are a formidable source of natural products of pharmaceutical interest. With the multiplication of genomic data available, very efficient bioinformatic tools for automatic SMBGC detection have been developed. Nevertheless, most of these tools identify SMBGCs based on sequence similarity with enzymes typically involved in specialised metabolism and thus may miss SMBGCs coding for undercharacterised enzymes. Here we present Synteruptor (https://bioi2.i2bc.paris-saclay.fr/synteruptor), a program that identifies genomic islands, known to be enriched in SMBGCs, in the genomes of closely related species. With this tool, we identified a SMBGC in the genome of Streptomyces ambofaciens ATCC23877, undetected by antiSMASH versions prior to antiSMASH 5, and experimentally demonstrated that it directs the biosynthesis of two metabolites, one of which was identified as sphydrofuran. Synteruptor is also a valuable resource for the delineation of individual SMBGCs within antiSMASH regions that may encompass multiple clusters, and for refining the boundaries of these SMBGCs.
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Bacterial chromosomes are large molecules that need to be highly compacted to fit inside the cells. Chromosome compaction must facilitate and maintain key biological processes such as gene expression and DNA transactions (replication, recombination, repair, and segregation). Chromosome and chromatin 3D-organization in bacteria has been a puzzle for decades. Chromosome conformation capture coupled to deep sequencing (Hi-C) in combination with other "omics" approaches has allowed dissection of the structural layers that shape bacterial chromosome organization, from DNA topology to global chromosome architecture. Here we review the latest findings using Hi-C and discuss the main features of bacterial genome folding.
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Streptomyces are prolific producers of specialized metabolites with applications in medicine and agriculture. Remarkably, these bacteria possess a large linear chromosome that is genetically compartmentalized: core genes are grouped in the central part, while the ends are populated by poorly conserved genes including antibiotic biosynthetic gene clusters. The genome is highly unstable and exhibits distinct evolutionary rates along the chromosome. Recent chromosome conformation capture (3C) and comparative genomics studies have shed new light on the interplay between genome dynamics in space and time. Here, we review insights that illustrate how the balance between chance (random genome variations) and necessity (structural and functional constraints) may have led to the emergence of spatial structuring of the Streptomyces chromosome.
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Streptomyces are prolific producers of specialized metabolites with applications in medicine and agriculture. These bacteria possess a large linear chromosome genetically compartmentalized: core genes are grouped in the central part, while terminal regions are populated by poorly conserved genes. In exponentially growing cells, chromosome conformation capture unveiled sharp boundaries formed by ribosomal RNA (rrn) operons that segment the chromosome into multiple domains. Here we further explore the link between the genetic distribution of rrn operons and Streptomyces genetic compartmentalization. A large panel of genomes of species representative of the genus diversity revealed that rrn operons and core genes form a central skeleton, the former being identifiable from their core gene environment. We implemented a new nomenclature for Streptomyces genomes and trace their rrn-based evolutionary history. Remarkably, rrn operons are close to pericentric inversions. Moreover, the central compartment delimited by rrn operons has a very dense, nearly invariant core gene content. Finally, this compartment harbors genes with the highest expression levels, regardless of gene persistence and distance to the origin of replication. Our results highlight that rrn operons are structural boundaries of a central functional compartment prone to transcription in Streptomyces.
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Streptomyces , Streptomyces/genética , Operón de ARNr , Cromosomas Bacterianos/genética , ARN Ribosómico/genéticaRESUMEN
The production of specialized metabolites by Streptomyces bacteria is usually temporally regulated. This regulation is complex and frequently involves both global and pathway-specific mechanisms. Streptomyces ambofaciens ATCC23877 produces several specialized metabolites, including spiramycins, stambomycins, kinamycins and congocidine. The production of the first three molecules has been shown to be controlled by one or several cluster-situated transcriptional regulators. However, nothing is known regarding the regulation of congocidine biosynthesis. Congocidine (netropsin) belongs to the family of pyrrolamide metabolites, which also includes distamycin and anthelvencins. Most pyrrolamides bind into the minor groove of DNA, specifically in A/T-rich regions, which gives them numerous biological activities, such as antimicrobial and antitumoral activities. We previously reported the characterization of the pyrrolamide biosynthetic gene clusters of congocidine (cgc) in S. ambofaciens ATCC23877, distamycin (dst) in Streptomyces netropsis DSM40846, and anthelvencins (ant) in Streptomyces venezuelae ATCC14583. The three gene clusters contain a gene encoding a putative transcriptional regulator, cgc1, dst1, and ant1, respectively. Cgc1, Dst1, and Ant1 present a high percentage of amino acid sequence similarity. We demonstrate here that Cgc1, an atypical orphan response regulator, activates the transcription of all cgc genes in the stationary phase of S. ambofaciens growth. We also show that the cgc cluster is constituted of eight main transcriptional units. Finally, we show that congocidine induces the expression of the transcriptional regulator Cgc1 and of the operon containing the resistance genes (cgc20 and cgc21, coding for an ABC transporter), and propose a model for the transcriptional regulation of the cgc gene cluster. IMPORTANCE Understanding the mechanisms of regulation of specialized metabolite production can have important implications both at the level of specialized metabolism study (expression of silent gene clusters) and at the biotechnological level (increase of the production of a metabolite of interest). We report here a study on the regulation of the biosynthesis of a metabolite from the pyrrolamide family, congocidine. We show that congocidine biosynthesis and resistance are controlled by Cgc1, a cluster-situated regulator. As the gene clusters directing the biosynthesis of the pyrrolamides distamycin and anthelvencin encode a homolog of Cgc1, our findings may be relevant for the biosynthesis of other pyrrolamides. In addition, our results reveal a new type of feed-forward induction mechanism, in which congocidine induces its own biosynthesis through the induction of the transcription of cgc1.
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Regulación Bacteriana de la Expresión Génica , Netropsina , Streptomyces , Distamicinas , Genes Bacterianos , Familia de Multigenes , Netropsina/biosíntesis , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
Bacteria of the genus Streptomyces are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in Streptomyces ambofaciens correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather 'open' to a 'closed' conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of S. ambofaciens is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.
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Antibacterianos/metabolismo , Cromosomas Bacterianos , Streptomyces/genética , Streptomyces/metabolismo , Estructuras Cromosómicas , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Familia de Multigenes , TranscriptomaRESUMEN
BACKGROUND: The CRISPR/Cas (clustered regularly interspaced short palindromic repeat and CRISPR-associated nucleases) based technologies have revolutionized genome engineering. While their use for prokaryotic genome editing is expanding, some limitations remain such as possible off-target effects and design constraints. These are compounded when performing systematic genome editing at distinct loci or when targeting repeated sequences (e.g. multicopy genes or mobile genetic elements). To overcome these limitations, we designed an approach using the same sgRNA and CRISPR-Cas9 system to independently perform gene editing at different loci. RESULTS: We developed a two-step procedure based on the introduction by homologous recombination of 'bait' DNA at the vicinity of a gene copy of interest before inducing CRISPR-Cas9 activity. The introduction of a genetic tool encoding a CRISPR-Cas9 complex targeting this 'bait' DNA induces a double strand break near the copy of interest. Its repair by homologous recombination can lead either to reversion or gene copy-specific editing. The relative frequencies of these events are linked to the impact of gene editing on cell fitness. In our study, we used this technology to successfully delete the native copies of two xenogeneic silencers lsr2 paralogs in Streptomyces ambofaciens. We observed that one of these paralogs is a candidate-essential gene since its native locus can be deleted only in the presence of an extra copy. CONCLUSION: By targeting 'bait' DNA, we designed a 'generic' CRISPR-Cas9 toolkit that can be used to edit different loci. The differential action of this CRISPR-Cas9 system is exclusively based on the specific recombination between regions surrounding the gene copy of interest. This approach is suitable to edit multicopy genes. One such particular example corresponds to the mutagenesis of candidate-essential genes that requires the presence of an extra copy of the gene before gene disruption. This opens new insights to explore gene essentiality in bacteria and to limit off-target effects during systematic CRISPR-Cas9 based approaches.
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Sistemas CRISPR-Cas , Edición Génica/métodos , ARN Guía de Kinetoplastida/genética , Proteínas Bacterianas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , Recombinación Homóloga , Streptomyces/genéticaRESUMEN
JC virus (JCV), a ubiquitous human polyomavirus, can cause fatal progressive multifocal leukoencephalopathy (PML) in immune compromised patients. The viral genome is composed of two conserved coding regions separated by a highly variable non-coding control region (NCCR). We analyzed the NCCR sequence from 10 PML JCV strains and found new mutations. Remarkably, the NCCR f section was mutated in most cases. We therefore explored the importance of this section in JCV expression in renal (HEK293H) and glioblastoma (U-87MG) cell lines, by adapting the emerging technology of DNA minicircles. Using bidirectional fluorescent reporters, we revealed that impaired NCCR-driven late expression in glioblastoma cells was restored by a short deletion overlapping e and f sections. This study evidenced a relevant link between JCV NCCR polymorphism and cell-type dependent expression. The use of DNA minicircles opens new insights for monitoring the impact of NCCR variation.
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Regulación Viral de la Expresión Génica , Virus JC/genética , Leucoencefalopatía Multifocal Progresiva/virología , Infecciones por Polyomavirus/virología , Línea Celular Tumoral , Genoma Viral , Células HEK293 , Humanos , Mutación , Polimorfismo Genético , Regiones no TraducidasAsunto(s)
Antineoplásicos/aislamiento & purificación , Reactores Biológicos/microbiología , Escherichia coli , Metotrexato/aislamiento & purificación , Purificación del Agua/métodos , Animales , Antineoplásicos/farmacocinética , Biodegradación Ambiental , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Ingeniería Metabólica/métodos , Metotrexato/farmacocinética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Organismos Modificados Genéticamente , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Aguas Residuales/química , Aguas Residuales/microbiología , Contaminantes Químicos del Agua/aislamiento & purificación , Contaminantes Químicos del Agua/farmacocinética , Purificación del Agua/instrumentación , gamma-Glutamil Hidrolasa/genética , gamma-Glutamil Hidrolasa/metabolismoRESUMEN
In enteropathogenic Escherichia coli (EPEC), the locus of enterocyte effacement (LEE) encodes a type 3 secretion system (T3SS) essential for pathogenesis. This pathogenicity island comprises five major operons (LEE1 to LEE5), with the LEE5 operon encoding T3SS effectors involved in the intimate adherence of bacteria to enterocytes. The first operon, LEE1, encodes Ler (LEE-encoded regulator), an H-NS (nucleoid structuring protein) paralog that alleviates the LEE H-NS silencing. We observed that the LEE5 and LEE1 promoters present a bimodal expression pattern, depending on environmental stimuli. One key regulator of bimodal LEE1 and LEE5 expression is ler expression, which fluctuates in response to different growth conditions. Under conditions in vitro considered to be equivalent to nonoptimal conditions for virulence, the opposing regulatory effects of H-NS and Ler can lead to the emergence of two bacterial subpopulations. H-NS and Ler share nucleation binding sites in the LEE5 promoter region, but H-NS binding results in local DNA structural modifications distinct from those generated through Ler binding, at least in vitro Thus, we show how two nucleoid-binding proteins can contribute to the epigenetic regulation of bacterial virulence and lead to opposing bacterial fates. This finding implicates for the first time bacterial-chromatin structural proteins in the bimodal regulation of gene expression.IMPORTANCE Gene expression stochasticity is an emerging phenomenon in microbiology. In certain contexts, gene expression stochasticity can shape bacterial epigenetic regulation. In enteropathogenic Escherichia coli (EPEC), the interplay between H-NS (a nucleoid structuring protein) and Ler (an H-NS paralog) is required for bimodal LEE5 and LEE1 expression, leading to the emergence of two bacterial subpopulations (with low and high states of expression). The two proteins share mutual nucleation binding sites in the LEE5 promoter region. In vitro, the binding of H-NS to the LEE5 promoter results in local structural modifications of DNA distinct from those generated through Ler binding. Furthermore, ler expression is a key parameter modulating the variability of the proportions of bacterial subpopulations. Accordingly, modulating the production of Ler into a nonpathogenic E. coli strain reproduces the bimodal expression of LEE5 Finally, this study illustrates how two nucleoid-binding proteins can reshape the epigenetic regulation of bacterial virulence.
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Cromatina/genética , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Islas Genómicas/genética , Fosfoproteínas/genética , Proteínas Bacterianas/genética , Cromatina/química , Epigénesis Genética , Operón , Regiones Promotoras Genéticas , Factores de Transcripción/genética , VirulenciaRESUMEN
Measuring gene expression at the single cell and single molecule level has recently made possible the quantitative measurement of stochasticity of gene expression. This enables identification of the probable sources and roles of noise. Gene expression noise can result in bacterial population heterogeneity, offering specific advantages for fitness and survival in various environments. This trait is therefore selected during the evolution of the species, and is consequently regulated by a specific genetic network architecture. Examples exist in stress-response mechanisms, as well as in infection and pathogenicity strategies, pointing to advantages for multicellularity of bacterial populations.
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Bacterias/genética , Epigénesis Genética , Expresión Génica , Redes Reguladoras de Genes , Ambiente , Modelos Genéticos , Fenotipo , Procesos EstocásticosRESUMEN
Integration of HIV-1 linear DNA into host chromatin is required for high levels of viral expression, and constitutes a key therapeutic target. Unintegrated viral DNA (uDNA) can support only limited transcription but may contribute to viral propagation, persistence and/or treatment escape under specific situations. The molecular mechanisms involved in the differential expression of HIV uDNA vs integrated genome (iDNA) remain to be elucidated. Here, we demonstrate, for the first time, that the expression of HIV uDNA is mainly supported by 1-LTR circles, and regulated in the opposite way, relatively to iDNA, following NF-κB pathway modulation. Upon treatment activating the NF-κB pathway, NF-κB p65 and AP-1 (cFos/cJun) binding to HIV LTR iDNA correlates with increased iDNA expression, while uDNA expression decreases. On the contrary, inhibition of the NF-κB pathway promotes the expression of circular uDNA, and correlates with Bcl-3 and AP-1 binding to its LTR region. Finally, this study identifies NF-κB subunits and Bcl-3 as transcription factors binding the HIV promoter differently depending on viral genome topology, and opens new insights on the potential roles of episomal genomes during the HIV-1 latency and persistence.
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Regulación Viral de la Expresión Génica , Genoma Viral , VIH-1/genética , FN-kappa B/metabolismo , Transducción de Señal/genética , Transcripción Genética , Integración Viral/genética , Línea Celular , ADN Circular/genética , ADN Viral/genética , Duplicado del Terminal Largo de VIH/genética , Humanos , Ácidos Nucleicos/metabolismo , Unión Proteica , ARN Viral/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genéticaRESUMEN
Photodynamic therapy (PDT) leads to cell death by using a combination of a photosensitizer and an external light source for the production of lethal doses of reactive oxygen species (ROS). Since a major limitation of PDT is the poor penetration of UV-visible light in tissues, there is a strong need for organic compounds whose activation is compatible with near-infrared excitation. Triphenylamines (TPAs) are fluorescent compounds, recently shown to efficiently trigger cell death upon visible light irradiation (458 nm), however outside the so-called optical/therapeutic window. Here, we report that TPAs target cytosolic organelles of living cells, mainly mitochondria, triggering a fast apoptosis upon two-photon excitation, thanks to their large two-photon absorption cross-sections in the 760-860 nm range. Direct ROS imaging in the cell context upon multiphoton excitation of TPA and three-color flow cytometric analysis showing phosphatidylserine externalization indicate that TPA photoactivation is primarily related to the mitochondrial apoptotic pathway via ROS production, although significant differences in the time courses of cell death-related events were observed, depending on the compound. TPAs represent a new class of water-soluble organic photosensitizers compatible with direct two-photon excitation, enabling simultaneous multiphoton fluorescence imaging of cell death since a concomitant subcellular TPA re-distribution occurs in apoptotic cells.
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Compuestos de Anilina/metabolismo , Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Imagen Óptica/métodos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Muerte Celular , Citometría de Flujo , Células HeLa , Humanos , Luz , Especies Reactivas de Oxígeno/análisisRESUMEN
Xenotropic Murine Leukemia Virus-related Virus (XMRV) is a new gammaretrovirus generated by genetic recombination between two murine endogenous retroviruses, PreXMRV1 and PreXMRV2, during passaging of human prostate cancer xenografts in laboratory mice. XMRV is representative of an early founder virus that jumps species from mouse to human cell lines. Relatively little information is available concerning the XMRV integrase (IN), an enzyme that catalyzes a key stage in the retroviral cycle, and whose sequence is conserved among replication competent retroviruses emerging from recombination between the murine endogenous PreXMRV-1 and PreXMRV-2 genomes. Previous studies have shown that IN inhibitors efficiently block XMRV multiplication in cells. We thus aimed at characterizing the biochemical properties and sensitivity of the XMRV IN to the raltegravir, dolutegravir, 118-D-24 and elvitegravir inhibitors in vitro. We report for the first time the purification and enzymatic characterization of recombinant XMRV IN. This IN, produced in Escherichia coli and purified under native conditions, is optimally active over a pH range of 7-8.5, in the presence of Mg(2+) (15 mM and 30 mM for 3'-processing and strand transfer, respectively) and is poorly sensitive to the addition of dithiothreitol. Raltegravir was shown to be a very potent inhibitor (IC50 â¼ 30 nM) whereas dolutegravir and elvitegravir were less effective (IC50 â¼ 230 nM and 650 nM, respectively). The 118-D-24 drug had no impact on XMRV IN activity. Interestingly, the substrate specificity of XMRV IN seems to be less marked compared to HIV-1 IN since XMRV IN is able to process various donor substrates that share little homology. Finally, our analysis revealed some original properties of the XMRV IN such as its relatively low sequence specificity.
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Inhibidores de Integrasa/farmacología , Integrasas/química , Integrasas/metabolismo , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/enzimología , Secuencia de Aminoácidos , Ditiotreitol/farmacología , Integrasa de VIH/química , Compuestos Heterocíclicos con 3 Anillos/farmacología , Concentración de Iones de Hidrógeno , Integrasas/genética , Integrasas/aislamiento & purificación , Datos de Secuencia Molecular , Oxazinas , Piperazinas , Piridonas , Pirrolidinonas/farmacología , Quinolonas/farmacología , Raltegravir Potásico , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Xenotropic Murine leukemia virus-Related Virus (XMRV) directly arose from genetic recombinations between two endogenous murine retroviruses that occurred during human xenografts in laboratory mice. Studies on XMRV could thus bring clues on how a new retrovirus could circumvent barrier species. We observed that XMRV exhibits a weak promoter activity in human cells, similar to the transcription level of a Tat-defective HIV-1. Despite this low fitness, XMRV can efficiently propagate through the huge accumulation of viral copies (≈40 copies per cell) that compensates for the low expression level of individual proviruses. We further demonstrate that there is an inverse relationship between the maximum number of viral copies per infected cell and the level of viral expression, which is explained by viral envelope interference mechanisms. Low viral expression compensation by viral copy accumulation through delayed interference could a priori contribute to the propagation of others viruses following species jumps.
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Gammaretrovirus/fisiología , Expresión Génica , Provirus/fisiología , Transcripción Genética , Replicación Viral , Animales , Línea Celular , Gammaretrovirus/genética , Humanos , Ratones , Regiones Promotoras Genéticas , Provirus/genética , Transducción GenéticaRESUMEN
Ku, a cellular complex required for human cell survival and involved in double strand break DNA repair and multiple other cellular processes, may modulate retroviral multiplication, although the precise mechanism through which it acts is still controversial. Recently, Ku was identified as a possible anti-human immunodeficiency virus type 1 (HIV-1) target in human cells, in two global approaches. Here we investigated the role of Ku on the HIV-1 replication cycle by analyzing the expression level of a panel of non-replicative lentiviral vectors expressing the green fluorescent protein in human colorectal carcinoma HCT 116 cells, stably or transiently depleted of Ku. We found that in this cellular model the depletion of Ku did not affect the efficiency of (pre-)integrative steps but decreased the early HIV-1 expression by acting at the transcriptional level. This negative effect was specific of the HIV-1 promoter, required the obligatory step of viral DNA integration and was reversed by transient depletion of p53. We also provided evidence on a direct binding of Ku to HIV-1 LTR in transduced cells. Ku not only promotes the early transcription from the HIV-1 promoter, but also limits the constitution of viral latency. Moreover, in the presence of a normal level of Ku, HIV-1 expression was gradually lost over time, likely due to the counter-selection of HIV-1-expressing cells. On the contrary, the reactivation of transgene expression from HIV-1 by means of trichostatin A- or tumor necrosis factor α-administration was enhanced under condition of Ku haplodepletion, suggesting a phenomenon of provirus latency. These observations plead in favor of the hypothesis that Ku has an impact on HIV-1 expression and latency at early- and mid-time after integration.
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ADN Helicasas/metabolismo , VIH-1/fisiología , ADN Helicasas/genética , Células HCT116 , Humanos , Autoantígeno Ku , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Integración Viral/fisiología , Latencia del Virus/fisiologíaRESUMEN
Gene transfer for research or gene therapy requires the design of vectors that allow for adequate and safe transgene expression. Current methods to modulate the safety and expression profile of retroviral vectors can involve the insertion of insulators or scaffold/matrix-attachment regions in self-inactivating long terminal repeats (SIN-LTRs). Here, we generated a set of lentiviral vectors (with internal CMV or PGK promoter) in which we inserted (at the level of SIN-LTRs) sequences of avian (i.e., chicken hypersensitive site-4, cHS4), human (i.e., putative insulator and desert sequence), or bacterial origin. We characterized them with respect to viral titer, integration, transduction efficiency and transgene expression levels, in both integrase-proficient and -deficient contexts. We found that the cHS4 insulator enhanced transgene expression by a factor of 1.5 only when cloned in the antisense orientation. On the other hand, cHS4 in the sense orientation as well as all other inserts decreased transgene expression. This attenuation phenomenon persisted over long periods of time and did not correspond to extinction or variegation. Decreased transgene expression was associated with lower mRNA levels, yet RNA stability was not affected. Insertions within the SIN-LTRs may negatively affect transgene transcription in a direct fashion through topological rearrangements. The lentiviral vectors that we generated constitute valuable genetic tools for manipulating the level of transgene expression. Moreover, this study demonstrates that SIN-LTR inserts can decrease transgene expression, a phenomenon that might be overcome by modifying insert orientation, thereby highlighting the importance of careful vector design for gene therapy.
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Región de Flanqueo 3'/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/genética , Lentivirus/genética , Secuencias Repetidas Terminales/genética , Transgenes/genética , Animales , Pollos/genética , Vectores Genéticos/biosíntesis , Células HeLa , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The Bae, Cpx, Psp, Rcs, and sigma(E) pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signal-inducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response.
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Citoplasma/genética , Escherichia coli/genética , Perfilación de la Expresión Génica , Transducción de Señal/genética , Estrés Fisiológico/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Regulón/genéticaRESUMEN
DjlA is an inner membrane cochaperone belonging to the DnaJ family, which has been shown to be involved in Legionella sp. pathogenesis. In this study, we explored the role of this protein in the physiology and virulence of Vibrio tapetis, the etiological agent of brown ring disease (BRD) in Manila clam (Ruditapes philippinarum). Analysis of the djlA locus in V. tapetis revealed a putative organization in an operon with a downstream gene that we designated duf924(Vt), which encodes a conserved protein with an unknown function and has homologues in bacteria and eukaryotes. djlA mutants displayed a reduced growth rate and showed an important loss of cytotoxic activity against R. philippinarum hemocytes in vitro, which could be restored by extrachromosomal expression of wild-type djlA(Vt) but not duf924(Vt). These results are in keeping with the potential importance of DjlA for bacterial pathogenicity and open new perspectives for understanding the mechanism of action of this protein in the novel V. tapetis-R. philippinarum interaction model.