RESUMEN
Small regulatory RNA (sRNAs) are key mediators of posttranscriptional gene control in bacteria. Assisted by RNA-binding proteins, a single sRNA often modulates the expression of dozens of genes, and thus sRNAs frequently adopt central roles in regulatory networks. Posttranscriptional regulation by sRNAs comes with several unique features that cannot be achieved by transcriptional regulators. However, for optimal network performance, transcriptional and posttranscriptional control mechanisms typically go hand-in-hand. This view is reflected by the ever-growing class of mixed network motifs involving sRNAs and transcription factors, which are ubiquitous in biology and whose regulatory properties we are beginning to understand. In addition, sRNA activity can be antagonized by base-pairing with sponge RNAs, adding yet another layer of complexity to these networks. In this article, we summarize the regulatory concepts underlying sRNA-mediated gene control in bacteria and discuss how sRNAs shape the output of a network, focusing on several key examples.
Asunto(s)
ARN Bacteriano , ARN Pequeño no Traducido , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Regulón , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Regulación Bacteriana de la Expresión Génica , Bacterias/genética , Bacterias/metabolismo , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/metabolismoRESUMEN
Base pairing RNAs modulate gene expression in all studied organisms. In many bacteria, the base pairing between most small regulatory RNAs (sRNAs) and their targets is mediated by the Hfq RNA chaperone. However, recent studies have shown FinO-domain proteins also bind sRNAs. To examine the global contribution of the FinO-domain ProQ protein in Escherichia coli, we carried out RIL-seq to identify RNA pairs bound to this protein. The RNA-RNA interactome for ProQ contains hundreds of pairs. Intriguingly, a significant fraction of the ProQ-bound RNA pairs are also found associated with Hfq, indicating overlapping, complementary, or competing roles for the two proteins. Characterization of one novel RNA pair bound by both chaperones revealed that while Hfq is required for RNA sponge-mediated downregulation of the sRNA, ProQ can inhibit this regulation. Overall, our results uncover increased complexity in RNA regulatory networks involving RNA chaperone proteins, RNases, sRNAs, and mRNAs.
Asunto(s)
ADN Bacteriano/genética , Proteínas de Escherichia coli/genética , Proteína de Factor 1 del Huésped/genética , ARN Bacteriano/genética , Proteínas de Unión al ARN/genética , Emparejamiento Base/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Chaperonas Moleculares/genética , Dominios Proteicos/genética , ARN Mensajero/genética , ARN Pequeño no Traducido/genéticaRESUMEN
The RNA chaperone Hfq plays important regulatory roles in many bacteria by facilitating the base pairing between small RNAs (sRNAs) and their cognate mRNA targets. In the gram-negative opportunistic pathogen Pseudomonas aeruginosa, over a hundred putative sRNAs have been identified but for most, their regulatory targets remained unknown. Using RIL-seq with Hfq in P. aeruginosa, we identified the mRNA targets for dozens of previously known and unknown sRNAs. Strikingly, hundreds of the RNA-RNA interactions we discovered involved PhrS. This sRNA was thought to mediate its effects by pairing with a single target mRNA and regulating the abundance of the transcription regulator MvfR required for the synthesis of the quorum sensing signal PQS. We present evidence that PhrS controls many transcripts by pairing with them directly and employs a two-tiered mechanism for governing PQS synthesis that involves control of an additional transcription regulator called AntR. Our findings in P. aeruginosa expand the repertoire of targets for previously known sRNAs, reveal potential regulatory targets for previously unknown sRNAs, and suggest that PhrS may be a keystone sRNA with the ability to pair with an unusually large number of transcripts in this organism.
Asunto(s)
Pseudomonas aeruginosa , ARN Pequeño no Traducido , Pseudomonas aeruginosa/genética , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , ARN Mensajero/genética , Bacterias/genética , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/genéticaRESUMEN
Small RNAs (sRNAs) associated with the RNA chaperon protein Hfq are key posttranscriptional regulators of gene expression in bacteria. Deciphering the sRNA-target interactome is an essential step toward understanding the roles of sRNAs in the cellular networks. We developed a broadly applicable methodology termed RIL-seq (RNA interaction by ligation and sequencing), which integrates experimental and computational tools for in vivo transcriptome-wide identification of interactions involving Hfq-associated sRNAs. By applying this methodology to Escherichia coli we discovered an extensive network of interactions involving RNA pairs showing sequence complementarity. We expand the ensemble of targets for known sRNAs, uncover additional Hfq-bound sRNAs encoded in various genomic regions along with their trans encoded targets, and provide insights into binding and possible cycling of RNAs on Hfq. Comparison of the sRNA interactome under various conditions has revealed changes in the sRNA repertoire as well as substantial re-wiring of the network between conditions.
Asunto(s)
Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Proteína de Factor 1 del Huésped/genética , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Emparejamiento Base , Sitios de Unión , Mapeo Cromosómico , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Proteína de Factor 1 del Huésped/metabolismo , Motivos de Nucleótidos , Unión Proteica , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/metabolismoRESUMEN
It is now established that base-pairing regulatory RNAs are key players in post-transcriptional regulatory networks where they affect the translation and/or stability of their target RNAs. In many cases, the base-pairing between two RNAs is facilitated by an RNA-binding protein (RBP) that serves as an RNA chaperone. Recent advances in sequencing methods have revealed the RNA populations bound by the RBPs, yielding insights valuable into regulatory networks. Further analyses of these networks can improve our understanding of the roles played by RBPs in the regulation of gene expression by regulatory RNAs, especially when multiple RBPs are involved. For example, using an RNA sequencing-based methodology that captures RNA-RNA pairs on RBP, an interplay between two RBPs in bacteria that compete on the same RNA-RNA pair was revealed. In this case, one protein promotes negative regulation of the target RNA, while the second protein can block this regulation. In this mini-review, I outline the exciting future directions that can be taken to deepen our understanding of the roles played by RBPs in post-transcriptional regulation, and discuss how the different sequencing methods can assist in deciphering the relationships among RBPs, and between the RBPs and the RNAs they bind. Having a more detailed picture of the RBPs-RNAs network will elucidate how bacteria can have nuanced control of gene expression, critical for survival in the varied environments in which bacteria live.
Asunto(s)
Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , ARN/genéticaRESUMEN
The occurrence of pharmaceuticals, including antibacterial compounds, in the environment has been acknowledged as an emerging and troubling issue in environmental safety; their usage is constantly on the rise, and their effects on the environment are only partially understood. Such compounds can accumulate, contaminate the ecosystem, and contribute to the spreading of antibiotic resistance among bacteria, hindering human health. Bioluminescent Escherichia coli reporter strains, engineered to detect antibiotic compounds by fusing the promoter of the global regulator soxS to the Photorhabdus luminescens luxCDABE cassette, were further modified by altering their membrane permeability and efflux capabilities. This was accomplished by introducing several mutations in the efflux system (ΔemrE, ΔacrB, and ΔtolC) and by overexpressing OmpF, a porin located in the outer membrane that allows passive diffusion of molecules. Combinations of these alterations had a cumulative effect in lowering the detection threshold of several antibiotics, in some of the cases to concentrations reported from pharmaceutical-polluted environments.
Asunto(s)
Antibacterianos/análisis , Fenómenos Fisiológicos Bacterianos , Técnicas Biosensibles/métodos , Permeabilidad de la Membrana Celular , Contaminantes Ambientales/análisis , Escherichia coli/efectos de los fármacos , Antibacterianos/farmacología , Transporte Biológico , Contaminantes Ambientales/farmacología , Escherichia coli/enzimología , Escherichia coli/genética , Eliminación de Gen , Expresión Génica , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Photorhabdus/enzimología , Photorhabdus/genética , Sensibilidad y EspecificidadRESUMEN
Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here, we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3´ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.
Asunto(s)
Proteínas de Escherichia coli , ARN Pequeño no Traducido , Proteínas de Escherichia coli/metabolismo , ARN Pequeño no Traducido/metabolismo , ARN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/genética , Flagelos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/genéticaRESUMEN
Small regulatory RNAs (sRNAs) acting in concert with the RNA chaperone Hfq are prevalent in many bacteria and typically act by base-pairing with multiple target transcripts. In the human pathogen Vibrio cholerae, sRNAs play roles in various processes including antibiotic tolerance, competence, and quorum sensing (QS). Here, we use RIL-seq (RNA-interaction-by-ligation-and-sequencing) to identify Hfq-interacting sRNAs and their targets in V. cholerae. We find hundreds of sRNA-mRNA interactions, as well as RNA duplexes formed between two sRNA regulators. Further analysis of these duplexes identifies an RNA sponge, termed QrrX, that base-pairs with and inactivates the Qrr1-4 sRNAs, which are known to modulate the QS pathway. Transcription of qrrX is activated by QrrT, a previously uncharacterized LysR-type transcriptional regulator. Our results indicate that QrrX and QrrT are required for rapid conversion from individual to community behaviours in V. cholerae.
Asunto(s)
Vibrio cholerae , Humanos , Vibrio cholerae/genética , ARNRESUMEN
Recent advances in the convergence of the biological, chemical, physical, and engineering sciences have opened new avenues of research into the interfacing of diverse biological moieties with inanimate platforms. A main aspect of this field, the integration of live cells with micro-machined platforms for high throughput and bio-sensing applications, is the subject of the present review. These unique hybrid systems are configured in a manner that ensures positioning of the cells in designated patterns, and enables cellular viability maintenance, and monitoring of cellular functionality. Here we review both animate and inanimate surface properties and how they affect cellular attachment, describe relevant modifications of both types of surfaces, list technologies for platform engineering and for cell deposition in the desired configurations, and discuss the influence of various deposition and immobilization methods on the viability and performance of the immobilized cells.
Asunto(s)
Bacterias/aislamiento & purificación , Técnicas Biosensibles/métodos , Análisis por Micromatrices/métodos , Técnicas Biosensibles/instrumentación , Adhesión Celular , Supervivencia Celular , Células Inmovilizadas/citología , Análisis por Micromatrices/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Propiedades de SuperficieRESUMEN
We describe a flow-through biosensor for online continuous water toxicity monitoring. At the heart of the device are disposable modular biochips incorporating agar-immobilized bioluminescent recombinant reporter bacteria, the responses of which are probed by single-photon avalanche diode detectors. To demonstrate the biosensor capabilities, we equipped it with biochips harboring both inducible and constitutive reporter strains and exposed it to a continuous water flow for up to 10 days. During these periods we challenged the biosensor with 2-h pulses of water spiked with model compounds representing different classes of potential water pollutants, as well as with a sample of industrial wastewater. The biosensor reporter panel detected all simulated contamination events within 0.5-2.5 h, and its response was indicative of the nature of the contaminating chemicals. We believe that a biosensor of the proposed design can be integrated into future water safety and security networks, as part of an early warning system against accidental or intentional water pollution by toxic chemicals.
Asunto(s)
Bacterias/metabolismo , Técnicas Biosensibles/instrumentación , Monitoreo del Ambiente/instrumentación , Mediciones Luminiscentes/instrumentación , Análisis por Micromatrices/instrumentación , Sistemas en Línea/instrumentación , Contaminantes Químicos del Agua/toxicidad , Antimonio/análisis , Antimonio/toxicidad , Arsénico/análisis , Arsénico/toxicidad , Genes Reporteros , Residuos Industriales/análisis , Factores de Tiempo , Eliminación de Residuos LíquidosRESUMEN
Small RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. To enable transcriptome-wide mapping of bacterial sRNA-target pairs, we developed RIL-seq (RNA interaction by ligation and sequencing). RIL-seq is an experimental-computational methodology for capturing sRNA-target interactions in vivo that takes advantage of the mutual binding of the sRNA and target RNA molecules to the RNA chaperone protein Hfq. The experimental part of the protocol involves co-immunoprecipitation of Hfq and bound RNAs, ligation of RNAs, library preparation and sequencing. The computational pipeline maps the sequenced fragments to the genome, reveals chimeric fragments (fragments comprising two ligated independent fragments) and determines statistically significant overrepresented chimeric fragments as interacting RNAs. The statistical filter is aimed at reducing the number of spurious interactions resulting from ligation of random neighboring RNA fragments, thus increasing the reliability of the determined sRNA-target pairs. A major advantage of RIL-seq is that it does not require overexpression of sRNAs; instead, it simultaneously captures the in vivo targets of all sRNAs in the native state of the cell. Application of RIL-seq to bacteria grown under different conditions provides distinctive snapshots of the sRNA interactome and sheds light on the dynamics and rewiring of the post-transcriptional regulatory network. As RIL-seq needs no prior information about the sRNA and target sequences, it can identify novel sRNAs, along with their targets. It can be adapted to detect protein-mediated RNA-RNA interactions in any bacterium with a sequenced genome. The experimental part of the RIL-seq protocol takes 7-9 d and the computational analysis takes â¼2 d.
Asunto(s)
Mapeo Cromosómico/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Análisis de Secuencia de ARN/métodos , Transcriptoma/genética , Genoma Bacteriano , GenómicaRESUMEN
Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in incorporating multiple reporter cells into a single platform; the technology now allows the simultaneous scrutiny of a large collection of sensor strains. We review current aspects in cell array technology with emphasis on microbial sensor arrays. We consider various techniques for patterning live cells on solid surfaces, describe different array-based applications and devices, and highlight recent efforts for live cell storage. We review mathematical approaches for deciphering the data emanating from bioreporter collections, and discuss the future of single cell arrays. Innovative technologies for cell patterning, preservation and interpretation are continuously being developed; when they all mature, cell arrays may become an efficient analytical tool, in a scope resembling that of DNA microarray biochips.
Asunto(s)
Técnicas Biosensibles/métodos , Análisis de Matrices Tisulares/métodos , BacteriasRESUMEN
The ever-growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy-based non-antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.
Asunto(s)
Antibacterianos/análisis , Antibacterianos/farmacología , Técnicas Biosensibles/métodos , Contaminantes Ambientales/análisis , Contaminantes Ambientales/farmacología , Escherichia coli/efectos de los fármacos , Antibacterianos/clasificación , Contaminantes Ambientales/clasificación , Escherichia coli/genética , Genes Reporteros , Humanos , Luciferasas/análisis , Luciferasas/genética , Organismos Modificados Genéticamente/genéticaRESUMEN
The last decade has witnessed a significant increase in interest in whole-cell biosensors for diverse applications, as well as a rapid and continuous expansion of array technologies. The combination of these two disciplines has yielded the notion of whole-cell array biosensors. We present a potential manifestation of this idea by describing the printing of a whole-cell bacterial bioreporters array. Exploiting natural bacterial tendency to adhere to positively charged abiotic surfaces, we describe immobilization and patterning of bacterial "spots" in the nanolitre volume range by a non-contact robotic printer. We show that the printed Escherichia coli-based sensor bacteria are immobilized on the surface, and retain their viability and biosensing activity for at least 2 months when kept at 4 °C. Immobilization efficiency was improved by manipulating the bacterial genetics (overproducing curli protein), the growth and the printing media (osmotic stress and osmoprotectants) and by a chemical modification of the inanimate surface (self-assembled layers of 3-aminopropyl-triethoxysilane). We suggest that the methodology presented herein may be applicable to the manufacturing of whole-cell sensor arrays for diverse high throughput applications.