Term-seq reveals abundant ribo-regulation of antibiotics resistance in bacteria

Abstract

Riboswitches and attenuators are cis-regulatory RNA elements, most of which control bacterial gene expression via metabolite-mediated, premature transcription termination. We developed an unbiased experimental approach for genome-wide discovery of such ribo-regulators in bacteria. We also devised an experimental platform that quantitatively measures the in-vivo activity of all such regulators in parallel, and enables rapid screening for ribo-regulators that respond to metabolites of choice. Using this approach we detected numerous antibiotic-responsive riboregulators that control antibiotic resistance genes in pathogens and in the human microbiome. Studying one such regulator in Listeria monocytogenes revealed an attenuation mechanism mediated by antibiotic-stalled ribosomes. Our results expose broad roles for conditional termination in regulating antibiotic resistance, and provide a tool for discovering riboswitches and attenuators that respond to novel ligands.Riboswitches and attenuators are 5'UTR-residing, cis-regulatory RNA elements (riboregulators) that tune gene expression in bacteria by sensing key metabolites, amino acids, nucleotides and ions (1-6). These RNA elements can regulate the expression of the downstream gene either at the transcription or the translation level. When riboswitches and attenuators control transcription they usually generate a condition-specific, regulated transcriptional terminator, such that termination results in a prematurely aborted transcript whereas read-through generates a full length, productive mRNA (5) (Fig. 1A). In the case of riboswitches, the 5'UTR RNA sensor differentially folds to form a terminator or an antiterminator in the presence or absence of a regulating metabolite, respectively; in attenuators, the formation of a transcriptional terminator is mediated by the rate of translation of an upstream ORF (uORF), as exemplified in the Trp operon (4). Regulation by conditional termination controls key processes in bacteria including core metabolism (7,8), motility (9) biofilm formation (9, 10), and virulence (11,12). Riboswitches enable optimization of metabolite production in bacterial expression systems (13,14), are readily *

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1riboswitches inE.coli

“…If the respective metabolite is essential for life, this will lead to a growth stop and/or death of the bacterial cell. Several studies have demonstrated that riboswitches are indeed druggable [6–8,16,47–49]. The most prominent investigation employed a phenotypic screen and identified ribocil that acts as a structurally distinct mimic of the natural ligand flavin mononucleotide to repress ribB gene expression and inhibit cell growth [7].…”

“…Riboswitches have emerged as possible targets for the development of alternative antimicrobial approaches [1–8]. They are typically located in the 5ʹ noncoding regions of bacterial mRNA and are able to bind specific metabolites to their aptamers with very high selectivity [9–11].…”

RNA Regulators: Formidable Modulators of Yersinia Virulence

“…A bioinformatics analysis predicted a few conserved riboswitches (Moco, FMN, cobalamin, yybP-ykoY element), the characterized Mg 2+ -responsive riboswitch mgtA [39], and 17 additional riboswitch-like elements, indicating that the Yersinia riboswitch-repertoire still remains undiscovered. Recently, a powerful unbiased high-throughput approach for global discovery of riboswitches and attenuators (Term-seq) has been described [40]. The application of the Term-seq approach to Yersinia grown in different growth media or host environments will not only allow a rapid screening for riboregulators which respond to metabolites of choice, it will also enable the discovery of yet unknown virulence-related riboswitches.…”

How the Study of Listeria Monocytogenes has Led to New Concepts in Biology

“…The first noncoding RNAs (ncRNAs) in L. monocytogenes were identified by coimmunoprecipitation with Hfq, a small RNA-binding protein required for the activity of small regulatory RNAs in prokaryotes [21] and by an in silico-based approaches [22]. Since then, the use of high-density tiling arrays and RNA deep sequencing provided a picture of the whole L. monocytogenes transcriptome in multiple conditions, allowing the discovery of many regulatory RNAs [23][24][25][26][27][28] and the annotation of hundreds of regulatory RNAs in L.…”

“…The vast majority of antibiotics affecting translation have RNA binding properties and it has been speculated that RNA-based regulation could be involved in antibiotic resistance genes expression. Recently, several antibiotics resistance genes regulated via termination-based ribo-regulators were identified in L. monocytogenes [24]. Indeed, the discovery of ribo-regulators that specifically respond to antibiotics occurred by applying term-seq, a method enabling quantitative mapping of all exposed RNA 3' ends, to L. monocytogenes bacteria grown in the presence or absence of antibiotic, such as lincomycin.…”