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Discovery and characterization of a fourth class of guanidine riboswitches.

ABSTRACT: Riboswitches are RNAs that specifically sense a small molecule and regulate genes accordingly. The recent discovery of guanidine-binding riboswitches revealed the biological significance of this compound, and uncovered genes related to its biology. For example, certain sugE genes encode guanidine exporters and are activated by the riboswitches to reduce toxic levels of guanidine in the cell. In order to study guanidine biology and riboswitches, we applied a bioinformatics strategy for discovering additional guanidine riboswitches by searching for new candidate motifs associated with sugE genes. Based on in vitro and in vivo experiments, we determined that one of our six best candidates is a new structural class of guanidine riboswitches. The expression of a genetic reporter was induced 80-fold in response to addition of 5 mM guanidine in Staphylococcus aureus. This new class, called the guanidine-IV riboswitch, reveals additional guanidine-associated protein domains that are extremely rarely or never associated with previously established guanidine riboswitches. Among these protein domains are two transporter families that are structurally distinct from SugE, and could represent novel types of guanidine exporters. These results establish a new metabolite-binding RNA, further validate a bioinformatics method for finding riboswitches and suggest substrate specificities for as-yet uncharacterized transporter proteins.

SUBMITTER: Lenkeit F

PROVIDER: S-EPMC7736828 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Discovery and characterization of a fourth class of guanidine riboswitches.

Lenkeit Felina F Eckert Iris I Hartig Jörg S JS Weinberg Zasha Z

Nucleic acids research 20201201 22

Riboswitches are RNAs that specifically sense a small molecule and regulate genes accordingly. The recent discovery of guanidine-binding riboswitches revealed the biological significance of this compound, and uncovered genes related to its biology. For example, certain sugE genes encode guanidine exporters and are activated by the riboswitches to reduce toxic levels of guanidine in the cell. In order to study guanidine biology and riboswitches, we applied a bioinformatics strategy for discoverin ...[more]

PMID: 33237283

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Project description:Riboswitches are messenger RNA (mRNA) domains that regulate gene function in response to the intracellular concentration of a variety of metabolites and second messengers. They control essential genes in many pathogenic bacteria, thus representing an inviting new class of biomolecular target for the development of antibiotics and chemical-biological tools. In this Account, we briefly review the discovery of riboswitches in the first years of the 21st century and their ensuing characterization over the past decade. We then discuss the progress achieved so far in using riboswitches as a focus for drug discovery, considering both the value of past serendipity and the particular challenges that confront current researchers. Five mechanisms of gene regulation have been determined for riboswitches. Most bacterial riboswitches modulate either transcription termination or translation initiation in response to ligand binding. All known examples of eukaryotic riboswitches, and some bacterial riboswitches, control gene expression by alternative splicing. The glmS riboswitch, which is widespread in Gram-positive bacteria, is a catalytic RNA activated by ligand binding: its self-cleavage destabilizes the mRNA of which it is part. Finally, one example of a trans-acting riboswitch is known. Three-dimensional structures have been determined for representatives of 13 structurally distinct riboswitch classes, providing atomic-level insight into their mechanisms of ligand recognition. While cellular and viral RNAs have attracted widespread interest as potential drug targets, riboswitches show special promise due to the diversity of small-molecule recognition strategies that are on display in their ligand-binding pockets. Moreover, riboswitches have evolved to recognize small-molecule ligands, which is unique among known structured RNA domains. Structural and biochemical advances in the study of riboswitches provide an impetus for the development of methods for the discovery of novel riboswitch activators and inhibitors. Recent rational drug design efforts focused on select riboswitch classes have yielded a small number of candidate antibiotic compounds, including one active in a mouse model of Staphylococcus aureus infection. The development of high-throughput methods suitable for riboswitch-specific drug discovery is ongoing. A fragment-based screening approach employing equilibrium dialysis that may be generically useful has demonstrated early success. Riboswitch-mediated gene regulation is widely employed by bacteria; however, only the thiamine pyrophosphate-responsive riboswitch has thus far been found in eukaryotes. Thus, riboswitches are particularly attractive as targets for antibacterials. Indeed, antimicrobials with previously unknown mechanisms have been found to function by binding riboswitches and causing aberrant gene expression.

Project description:BACKGROUNDOver the past 75 years, human pathogens have acquired antibiotic resistance genes (ARGs), often from environmental bacteria. Integrons play a major role in the acquisition of antibiotic resistance genes. We therefore hypothesized that focused exploration of integron gene cassettes from microbial communities could be an efficient way to find novel mobile resistance genes. DNA from polluted Indian river sediments were amplified using three sets of primers targeting class 1 integrons and sequenced by long- and short-read technologies to maintain both accuracy and context.RESULTSUp to 89% of identified open reading frames encode known resistance genes, or variations thereof (>?1000). We identified putative novel ARGs to aminoglycosides, beta-lactams, trimethoprim, rifampicin, and chloramphenicol, including several novel OXA variants, providing reduced susceptibility to carbapenems. One dihydropteroate synthase gene, with less than 34% amino acid identity to the three known mobile sulfonamide resistance genes (sul1-3), provided complete resistance when expressed in Escherichia coli. The mobilized gene, here named sul4, is the first mobile sulfonamide resistance gene discovered since 2003. Analyses of adjacent DNA suggest that sul4 has been decontextualized from a set of chromosomal genes involved in folate synthesis in its original host, likely within the phylum Chloroflexi. The presence of an insertion sequence common region element could provide mobility to the entire integron. Screening of 6489 metagenomic datasets revealed that sul4 is already widespread in seven countries across Asia and Europe.CONCLUSIONSOur findings show that exploring integrons from environmental communities with a history of antibiotic exposure can provide an efficient way to find novel, mobile resistance genes. The mobilization of a fourth sulfonamide resistance gene is likely to provide expanded opportunities for sulfonamide resistance to spread, with potential impacts on both human and animal health.

Dataset Information

Discovery and characterization of a fourth class of guanidine riboswitches.

Publications

Discovery and characterization of a fourth class of guanidine riboswitches.

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