RESUMEN
Singlet oxygen generation has long been considered the key feature that allows genetically encoded fluorescent tags to produce polymeric contrast agents for electron microscopy. Optimization of the singlet oxygen sensitization quantum yield has not included the effects of electron-rich monomers on the sensitizer's photocycle. We report that at monomer concentrations employed for staining, quenching by electron transfer is the primary deactivation pathway for photoexcitations. A simple photochemical model including contributions from both processes reproduces the observed reaction rates and indicates that most of the product is driven by pathways that involve electron transfer with monomersânot by the sensitization of singlet oxygen. Realizing the importance of these competing reaction pathways offers a new paradigm to guide the development of genetically encodable tags and suggests opportunities to expand the materials scope and growth conditions for polymeric contrast agents (e.g., biocompatible monomers, O2 poor environments).
Asunto(s)
Medios de Contraste , Polimerizacion , Transporte de Electrón , Medios de Contraste/química , Oxígeno Singlete/química , Flavoproteínas/química , Flavoproteínas/metabolismo , Fármacos Fotosensibilizantes/química , Procesos FotoquímicosRESUMEN
RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Gram-positive and Gram-negative bacteria. Suitable HBC-Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30-tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.
Asunto(s)
Aptámeros de Nucleótidos , ARN , Escherichia coli/genética , Antibacterianos , Bacterias Gramnegativas/genética , Bacterias Grampositivas , Salmonella typhimurium/genética , Aptámeros de Nucleótidos/genéticaRESUMEN
In this study, we describe the first real-time live cell assay for compound accumulation and permeability in both Gram positive and Gram negative bacteria. The assay utilizes a novel fluorogenic tagging strategy that permits direct visualization of compound accumulation dynamics in the cytoplasm of live cells, unobscured by washing or other processing steps. Quantitative differences could be reproducibly measured by flow cytometry at compound concentrations below the limit of detection for MS-based approaches. We establish the fluorogenic assay in E. coli and B. subtilis and compare the intracellular accumulation of two antibiotics, ciprofloxacin and ampicillin, with related pharmacophores in these bacteria.
Asunto(s)
Antibacterianos , Escherichia coli , Antibacterianos/farmacología , Bacterias Gramnegativas , Bacterias Grampositivas , Permeabilidad , Pruebas de Sensibilidad MicrobianaRESUMEN
Engineered RNAs have applications in diverse fields from biomedical to environmental. In many cases, the folding of the RNA is critical to its function. Here we describe a strategy to improve the response time of a riboswitch-based fluorescent biosensor. Systematic mutagenesis was performed to either make transpose or transition base pair mutants or introduce orthogonal base pairs. Both natural and unnatural base pair mutants were found to improve the biosensor response time without compromising fold turn-on or ligand affinity. These strategies can be transferred to improve the performance of other RNA-based tools.
Asunto(s)
Técnicas Biosensibles , Riboswitch , Emparejamiento Base , Tiempo de Reacción , Mutación , ARNRESUMEN
Cyclic-di-AMP (CDA) is a signaling molecule that controls various cellular functions including antibiotic tolerance and osmoregulation in Staphylococcus aureus (S. aureus). In this study, we developed a novel biosensor (bsuO P6-4) for in vivo detection of CDA in S. aureus. The fluorescent biosensor is based on a natural CDA riboswitch from Bacillus subtilis connected at its P6 stem to the dye-binding aptamer Spinach. Our study showed that bsuO P6-4 could detect a wide concentration range of CDA in both laboratory and clinical strains, making it suitable for use in both basic and clinical research applications.
RESUMEN
Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s-1 and 0.35 s-1, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Técnicas Biosensibles/métodos , Escherichia coli/genética , Colorantes Fluorescentes , Cinética , ARN/genéticaRESUMEN
Bacterial second messengers are important for regulating diverse bacterial lifestyles. Cyclic di-GMP (c-di-GMP) is produced by diguanylate cyclase enzymes, named GGDEF proteins, which are widespread across bacteria. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been described in some bacteria, which produce both c-di-GMP and a more recently identified bacterial second messenger, 3',3'-cyclic-GMP-AMP (cGAMP). One of these proteins was found in the predatory Bdellovibrio bacteriovorus, Bd0367. The bd0367 GGDEF gene deletion strain was found to enter prey cells, but was incapable of leaving exhausted prey remnants via gliding motility on a solid surface once predator cell division was complete. However, it was unclear which signal regulated this process. We show that cGAMP signalling is active within B. bacteriovorus and that, in addition to producing c-di-GMP and some c-di-AMP, Bd0367 is a primary producer of cGAMP in vivo. Site-directed mutagenesis of serine 214 to an aspartate rendered Bd0367 into primarily a c-di-GMP synthase. B. bacteriovorus strain bd0367S214D phenocopies the bd0367 deletion strain by being unable to glide on a solid surface, leading to an inability of new progeny to exit from prey cells post-replication. Thus, this process is regulated by cGAMP. Deletion of bd0367 was also found to be incompatible with wild-type flagellar biogenesis, as a result of an acquired mutation in flagellin chaperone gene homologue fliS, implicating c-di-GMP in regulation of swimming motility. Thus the single Bd0367 enzyme produces two secondary messengers by action of the same GGDEF domain, the first reported example of a synthase that regulates multiple second messengers in vivo. Unlike roles of these signalling molecules in other bacteria, these signal to two separate motility systems, gliding and flagellar, which are essential for completion of the bacterial predation cycle and prey exit by B. bacteriovorus.
Asunto(s)
Bdellovibrio bacteriovorus , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bdellovibrio bacteriovorus/genética , Bdellovibrio bacteriovorus/metabolismo , Nucleótidos Cíclicos/metabolismoRESUMEN
The Hypr cGAMP signaling pathway was discovered via the function of the riboswitch. In this study, we show the development of a method for affinity capture followed by sequencing to identify non-coding RNA regions that bind nucleotide signals such as cGAMP. The RNAseq of affinity-captured cGAMP riboswitches from the Geobacter sulfurreducens transcriptome highlights general challenges that remain for this technique. Furthermore, by applying riboswitch reporters in vivo, we identify new growth conditions and transposon mutations that affect cGAMP levels in G. sulfurreducens. This work reveals an extensive regulatory network and supports a second functional cGAMP synthase gene in G. sulfurreducens. The activity of the second synthase was validated using riboswitch-based fluorescent biosensors, and is the first known example of an active enzyme with a variant GGDDF motif.
Asunto(s)
Proteínas Bacterianas/metabolismo , Geobacter/metabolismo , Mutación , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/metabolismo , Riboswitch , Transcriptoma , Proteínas Bacterianas/genética , Geobacter/genética , Nucleotidiltransferasas/genética , Transducción de SeñalRESUMEN
The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a profluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. In this updated chapter, we have added procedures on using biosensors in flow cytometry to detect exogenously added compounds. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.
Asunto(s)
Aptámeros de Nucleótidos/genética , Técnicas Biosensibles/métodos , Citometría de Flujo/métodos , Colorantes Fluorescentes/análisis , Microscopía Intravital/métodos , Microscopía Fluorescente/métodos , ARN de Transferencia/genética , ARN/genética , Riboswitch/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Compuestos de Bencilo , Clonación Molecular/métodos , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imidazolinas , Isopropil Tiogalactósido/farmacología , Conformación de Ácido Nucleico , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , PlásmidosRESUMEN
Cell-based sensors are useful for many synthetic biology applications, including regulatory circuits, metabolic engineering, and diagnostics. While considerable research efforts have been made toward recognizing new target ligands and increasing sensitivity, the analysis and optimization of turn-on kinetics is often neglected. For example, to our knowledge there has been no systematic study that compared the performance of a riboswitch-based biosensor versus reporter for the same ligand. In this study, we show the development of RNA-based fluorescent (RBF) biosensors for guanidine, a common chaotropic agent that is a precursor to both fertilizer and explosive compounds. Guanidine is cell permeable and nontoxic to E. coli at millimolar concentrations, which in contrast to prior studies enabled direct activation of the riboswitch-based biosensor and corresponding reporter with ligand addition to cells. Our results reveal that the biosensors activate fluorescence in the cell within 4 min of guanidine treatment, which is at least 15 times faster than a reporter derived from the same riboswitch, and this rapid sensing activity is maintained for up to 1.6 weeks. Together, this study describes the design of two new biosensor topologies and showcases the advantages of RBF biosensors for monitoring dynamic processes in cell biology, biotechnology, and synthetic biology.
Asunto(s)
Técnicas Biosensibles/métodos , Guanidina/análisis , Riboswitch , Escherichia coli/genética , Escherichia coli/metabolismo , Guanidina/metabolismo , Cinética , Ingeniería Metabólica , ARN/química , ARN/metabolismoRESUMEN
Synthetic biology has potential spaceflight applications yet few if any studies have attempted to translate Earth-based synthetic biology tools into spaceflight. An exogenously inducible biological circuit for protein production in Arabidopsis thaliana, pX7-AtPDSi (Guo et al. 2003), was flown to ISS and functionally investigated. Seedlings were grown in a custom built 1.25 U plant greenhouse. Images recorded during the experiment show that leaves of pX7-AtPDSi seedlings photobleached as designed while wild type Col-0 leaves did not, which reveals that the synthetic circuit led to protein production during spaceflight. Polymerase chain reaction analysis post-flight also confirms that the Cre/LoxP (recombination system) portions of the circuit were functional in spaceflight. The subcomponents of the biological circuit, estrogen-responsive transcription factor XVE, Cre/LoxP DNA recombination system, and RNAi post-transcriptional gene silencing system now have flight heritage and can be incorporated in future designs for space applications. To facilitate future plant studies in space, the full payload design and manufacturing files are made available.
Asunto(s)
Arabidopsis/metabolismo , Vuelo Espacial , Biología Sintética/métodos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Estradiol , Integrasas , Hojas de la Planta/genética , Hojas de la Planta/efectos de la radiación , Plantas Modificadas Genéticamente , Interferencia de ARN , ARN de Planta , Receptores de Estrógenos/genética , Factores de TranscripciónRESUMEN
Bacteria contain a diverse set of RNAs to provide tight regulation of gene expression in response to environmental stimuli. Bacterial small RNAs (sRNAs) work in conjunction with protein cofactors to bind complementary mRNA sequences in the cell, leading to up- or downregulation of protein synthesis. In vivo imaging of sRNAs can aid in understanding their spatiotemporal dynamics in real time, which inspires new ways to manipulate these systems for a variety of applications including synthetic biology and therapeutics. Current methods for sRNA imaging are quite limited in vivo and do not provide real-time information about fluctuations in sRNA levels. Herein, we describe our efforts toward the development of an RNA-based fluorescent biosensor for bacterial sRNA both in vitro and in vivo. We validated these sensors for three different bacterial sRNAs in Escherichia coli and demonstrated that the designs provide a bright, sequence-specific signal output in response to exogenous and endogenous RNA targets.
Asunto(s)
Técnicas Biosensibles/métodos , Escherichia coli/genética , Colorantes Fluorescentes/química , ARN Bacteriano/análisis , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Sitios de Unión , Proteínas de Escherichia coli/genética , Citometría de Flujo , Proteína de Factor 1 del Huésped/genética , Hibridación de Ácido Nucleico , ARN Bacteriano/química , Espectrometría de FluorescenciaRESUMEN
Chemiluminescent biosensors have been developed and broadly applied to mammalian cell systems for studying intracellular signaling networks. For bacteria, biosensors have largely relied on fluorescence-based systems for quantitating signaling molecules, but these designs can encounter issues in complex environments due to their reliance on external illumination. In order to circumvent these issues, we designed the first ratiometric chemiluminescent biosensors for studying a key bacterial second messenger, cyclic di-GMP. We have shown recently that these biosensors function both in vitro and in vivo for detecting changes in cyclic di-GMP levels. In this chapter, we present a practical and broadly applicable method for high-throughput quantitation of cyclic di-GMP in bacterial cell extracts using the high affinity biosensor tVYN-TmΔ that could serve as the "Bradford assay" equivalent for this bacterial signaling molecule.
Asunto(s)
Técnicas Biosensibles , Vibrio cholerae , Animales , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas , GMP Cíclico , Regulación Bacteriana de la Expresión Génica , Sistemas de Mensajero Secundario , Transducción de Señal , Vibrio cholerae/metabolismoRESUMEN
Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an "atomic mutagenesis" strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.
Asunto(s)
GMP Cíclico/inmunología , Interferones/inmunología , Nucleótidos Cíclicos/inmunología , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Inmunidad Innata , Interferones/química , Interferones/genética , Nucleótidos Cíclicos/química , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Second messenger signaling networks allow cells to sense and adapt to changing environmental conditions. In bacteria, the nearly ubiquitous second messenger molecule cyclic di-GMP coordinates diverse processes such as motility, biofilm formation, and virulence. In bacterial pathogens, these signaling networks allow the bacteria to survive changing environmental conditions that are experienced during infection of a mammalian host. While studies have examined the effects of cyclic di-GMP levels on virulence in these pathogens, it has not been possible to visualize cyclic di-GMP levels in real time during the stages of host infection. Toward this goal, we generate the first ratiometric, chemiluminescent biosensor scaffold that selectively responds to c-di-GMP. By engineering the biosensor scaffold, a suite of Venus-YcgR-NLuc (VYN) biosensors is generated that provide extremely high sensitivity (KD < 300 pM) and large changes in the bioluminescence resonance energy transfer (BRET) signal (up to 109%). As a proof-of-concept that VYN biosensors can image cyclic di-GMP in tissues, we show that the VYN biosensors function in the context of a tissue phantom model, with only â¼103-104 biosensor-expressing E. coli cells required for the measurement. Furthermore, we utilize the biosensor in vitro to assess changes in cyclic di-GMP in V. cholerae grown with different inputs found in the host environment. The VYN sensors developed here can serve as robust in vitro diagnostic tools for high throughput screening, as well as genetically encodable tools for monitoring the dynamics of c-di-GMP in live cells, and lay the groundwork for live cell imaging of c-di-GMP dynamics in bacteria within tissues and other complex environments.
Asunto(s)
Proteínas Bacterianas/metabolismo , Técnicas Biosensibles/métodos , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Luciferasas/metabolismo , Proteínas Luminiscentes/metabolismo , Transducción de Señal/fisiología , Proteínas Bacterianas/genética , GMP Cíclico/análisis , GMP Cíclico/metabolismo , Transferencia de Energía , Escherichia coli , Proteínas de Escherichia coli/genética , Límite de Detección , Luciferasas/genética , Sustancias Luminiscentes/química , Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/genética , Prueba de Estudio Conceptual , Unión Proteica , Ingeniería de Proteínas , Vibrio choleraeRESUMEN
Genetically encodable fluorescent biosensors provide spatiotemporal information on their target analytes in a label-free manner, which has enabled the study of cell biology and signaling in living cells. Over the past three decades, fueled by the development of a wide palette of fluorescent proteins, protein-based fluorescent biosensors against a broad array of targets have been developed. Recently, with the development of fluorogenic RNA aptamer-dye pairs that function in live cells, RNA-based fluorescent (RBF) biosensors have emerged as a complementary class of biosensors. Here we review the current state-of-the-art for fluorogenic RNA aptamers and RBF biosensors for imaging small molecules and RNAs, and highlight some emerging opportunities.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Colorantes Fluorescentes , Proteínas , ARN/genéticaRESUMEN
3',3'-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.
Asunto(s)
Proteínas Bacterianas/metabolismo , Myxococcus xanthus/enzimología , Nucleótidos Cíclicos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismoAsunto(s)
Desoxirribonucleasas/metabolismo , Inmunidad Innata , Nucleótidos Cíclicos/inmunología , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/metabolismo , Virus Vaccinia/metabolismo , Animales , Cristalografía por Rayos X , Citosol/metabolismo , ADN Viral/inmunología , ADN Viral/metabolismo , Desoxirribonucleasas/química , Humanos , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/inmunología , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismo , Virus Vaccinia/inmunología , Vacunas ViralesRESUMEN
A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3',3'-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 Å-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Geobacter/enzimología , Geobacter/metabolismo , Metales/metabolismo , Nucleótidos Cíclicos/metabolismo , Transducción de Señal , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Cinética , Modelos Teóricos , Oxidación-Reducción , Conformación ProteicaRESUMEN
In bacteria and archaea, small RNAs (sRNAs) regulate complex networks through antisense interactions with target mRNAs in trans, and riboswitches regulate gene expression in cis based on the ability to bind small-molecule ligands. Although our understanding and characterization of these two important regulatory RNA classes is far from complete, these RNA-based mechanisms have proven useful for a wide variety of synthetic biology applications. Besides classic and contemporary applications in the realm of metabolic engineering and orthogonal gene control, this review also covers newer applications of regulatory RNAs as biosensors, logic gates, and tools to determine RNA-RNA interactions. A separate section focuses on critical insights gained and challenges posed by fundamental studies of sRNAs and riboswitches that should aid future development of synthetic regulatory RNAs.