Cyclic diguanylate (c-di-GMP) is a second messenger of bacteria and its detection is an important issue in basic and applied microbiology. As c-di-GMP riboswitch ligand-binding domains (aptamer domains) capture c-di-GMP with high affinity and selectivity, they are promising platforms for the development of RNA-based c-di-GMP sensors. We analyzed two previously reported c-di-GMP sensor RNAs derived from the Vc2 riboswitch. We also designed and tested their variants, some of which showed improved properties as RNA-based c-di-GMP sensors.
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Recognition of cyclic-di-GMP by a riboswitch conducts translational repression through masking the ribosome-binding site distant from the aptamer domain.
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Department of Chemistry, Graduate School of Science and Engineering, University of Toyama, Toyama, Japan.
The riboswitch is a class of RNA-based gene regulatory machinery that is dependent on recognition of its target ligand by RNA tertiary structures. Ligand recognition is achieved by the aptamer domain, and ligand-dependent structural changes of the expression platform then usually mediate termination of transcription or translational initiation. Ligand-dependent structural changes of the aptamer domain and expression platform have been reported for several riboswitches with short (<40 nucleotides) expression platforms.
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Department of Chemistry, University of California, Berkeley, 94720, USA.
Second-generation RNA-based fluorescent biosensors have been developed that enable flow cytometry experiments to monitor the population dynamics of c-di-GMP signaling in live bacteria. These experiments are high-throughput, provide information at the single-cell level, and can be performed on cells grown in complex media and/or under anaerobic conditions. Here, we describe flow cytometry methods for three applications: (1) high-throughput screening for diguanylate cyclase activity, (2) analyzing c-di-GMP levels under anaerobic conditions, and (3) monitoring cell population dynamics of c-di-GMP levels upon environmental changes.
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Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France; Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France; Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France. Electronic address:
Clostridium difficile (CD)-associated diarrhoea is currently the most prevalent nosocomial diarrhoea worldwide. Many characteristics of CD pathogenicity remain poorly understood. Recent data strongly indicate the importance of an RNA network for the control of gene expression in CD.
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J Biosci Bioeng
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Department of Chemistry, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan. Electronic address:
Cyclic diguanylate (c-di-GMP) is a second messenger of bacteria and its detection is an important issue in basic and applied microbiology. As c-di-GMP riboswitch ligand-binding domains (aptamer domains) capture c-di-GMP with high affinity and selectivity, they are promising platforms for the development of RNA-based c-di-GMP sensors. We analyzed two previously reported c-di-GMP sensor RNAs derived from the Vc2 riboswitch.
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