Highlights•Human STING variants respond poorly to cGAMP but still cytosolic DNA•DNA sensing through cGAS leads production of a noncanonical 2′-5′ linked cGAMP•STING robustly and not canonical cGAMPSummaryThe presence foreign DNA in the cytosol mammalian cells elicits potent antiviral interferon response. Recently, was proposed induce synthesis cyclic GMP-AMP (cGAMP) upon binding an enzyme called synthase (cGAS). activates response by downstream receptor STING. Here, we identify natural human...

Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety pathogenic responses in bacteria, but tools for monitoring their regulation vivo lacking. We have designed RNA-based fluorescent biosensors cyclic di-GMP and AMP-GMP by fusing the Spinach aptamer to variants natural GEMM-I riboswitch. In live cell imaging experiments, these demonstrate fluorescence turn-on response dinucleotides, they were used confirm production enzyme DncV.

Riboswitches are structured RNAs typically located in the 5' untranslated regions of bacterial mRNAs that bind metabolites and control gene expression. Most riboswitches sense one metabolite function as simple genetic switches. However, we found region Bacillus clausii metE messenger RNA includes two respond to S-adenosylmethionine coenzyme B12. This tandem arrangement yields a composite system functions two-input Boolean NOR logic gate. These findings discovery additional riboswitch...

Significance Bacteria are hidden forces of nature. For example, Geobacter bacteria play important roles in geochemistry by reducing metals the environment. Scientists also exploring application these toward toxic metal remediation and as “living batteries” that can generate electricity from biowaste. However, there is limited understanding signaling pathways regulate this extracellular metal-reducing activity. Here we have discovered sulfurreducens use riboswitch sensors for a molecule...

Cyclic di-AMP (cdiA) is a second messenger predicted to be widespread in Gram-positive bacteria, some Gram-negative and Archaea. In the human pathogen Listeria monocytogenes, cdiA an essential molecule that regulates metabolic function cell wall homeostasis, decreased levels of result increased antibiotic susceptibility. We have generated fluorescent biosensors for through fusion Spinach2 aptamer ligand-binding domains riboswitches. The biosensor was used visualize intracellular live L....

High-throughput enzyme activity screens are essential for target characterization and drug development, but few assays employ techniques or reagents that applicable to both in vitro live cell settings. Here, we present a class of selective sensitive fluorescent biosensors S-adenosyl-l-homocysteine (SAH) provide direct "mix go" assay methyltransferases (MTases), an includes several cancer therapeutic targets. Our riboswitch-based required alternate inverted fusion design strategy, retained...

Bacteria occupy a diverse set of environmental niches with differing oxygen availability. Anaerobic environments such as mammalian digestive tracts and industrial reactors harbor an abundance both obligate facultative anaerobes, many which play significant roles in human health biomanufacturing. Studying bacterial function under partial or fully anaerobic conditions, however, is challenging given the paucity suitable live-cell imaging tools. Here, we introduce series RNA-based fluorescent...

A novel family of riboswitches, called SAM-IV, is the fourth distinct set mRNA elements to be reported that regulate gene expression via direct sensing S -adenosylmethionine (SAM or AdoMet). SAM-IV riboswitches share conserved nucleotide positions with previously described SAM-I despite rearranged structures and family-specific identities. Sequence analysis molecular recognition experiments suggest similar ligand binding sites, but have different scaffolds. Our findings support view RNA has...

Cyclic dinucleotides are second messengers that target the adaptor STING and stimulate innate immune response in mammals. Besides protein receptors, there bacterial riboswitches selectively recognize cyclic dinucleotides. We recently discovered a natural riboswitch targets 3′,3′-cGAMP, which is distinguished from endogenous mammalian signal 2′,3′-cGAMP by its backbone connectivity. Here, we report on structures of aptamer domain 3′,3′-cGAMP Geobacter c-di-GMP bound states. The adopts tuning...

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger modulates the key bacterial lifestyle transition from motile sessile biofilm-forming state. Since then, ubiquity of genes encoding proteins in genomes has established dominance cdiG signaling bacteria. However, observation proteobacteria encode large number proteins, nearing 1% coding sequences some cases, raises question why bacteria need so many...

Expanding DNA sequence databases and improving methods for comparative analysis are being exploited to identify numerous noncoding RNA elements including riboswitches. Ligands many riboswitch classes usually can be inferred based on the genomic contexts of representative RNAs, complex formation or genetic regulation subsequently demonstrated experimentally. However, there several candidate riboswitches which ligands have not been identified. In this report, we discuss three most compelling...

The vitamin folate is required for methionine homeostasis in all organisms. In addition to its role protein synthesis, the precursor S-adenosyl-methionine (SAM), which used myriad cellular methylation reactions, including histone reactions. Here, we demonstrate that and deficiency led reduced of lysine 4 H3 (H3K4) Saccharomyces cerevisiae. effect nutritional on H3K79 was less pronounced, but exacerbated S. cerevisiae carrying a hypomorphic allele Dot1, enzyme responsible methylation. This...

Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility biofilm formation. To interrogate di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging have been developed. However, need external illumination precludes use these tools bacteria their natural including deep tissues whole...

Nearly all representatives of experimentally validated riboswitch classes in bacteria control the expression genes for transport or synthesis key metabolic compounds. Recent findings have revealed that some riboswitches also regulate involved physiological changes, virulence, and stress responses. Many novel RNA motifs are being identified by using bioinformatics algorithms search conserved sequence structural features located intergenic regions. Some these RNAs likely to function as...

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated bacterial cyclic di-GMP signaling. This system provides rare opportunity study how proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, Hypr in Geobacter sulfurreducens, specifically regulates GMP-AMP (3',3'-cGAMP) levels vivo stimulate gene expression metal reduction separate from electricity production....

Cyclic di-GMP (c-di-GMP) is a second messenger that important in regulating bacterial physiology and behavior, including motility virulence. Many questions remain about the role regulation of this signaling molecule, but current methods detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage natural, high affinity receptor c-di-GMP, Vc2 riboswitch aptamer, to develop sensitive rapid electrophoretic mobility shift assay...

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 pathways for inferred display a narrow distribution based upon characterized synthases, DncV Hypr GGDEFs. Here, we report that ubiquitous second messenger AMP (cAMP) an activator GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, show inhibited directly by di-GMP, which provides evidence cross-regulation between different CDN...

Second messenger signaling networks allow cells to sense and adapt changing environmental conditions. In bacteria, the nearly ubiquitous second molecule cyclic di-GMP coordinates diverse processes such as motility, biofilm formation, virulence. bacterial pathogens, these bacteria survive conditions that are experienced during infection of a mammalian host. While studies have examined effects levels on virulence in it has not been possible visualize real time stages host infection. Toward...