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

How bacteria switch between tracks Bacterial riboswitches prevent the formation of full-length messenger RNA, and hence proteins, via transcriptional termination in response to metabolites. However, identifying riboswitches within the genome has previously required comparative analysis, which may miss species- and environmentally specific responses. Dar et al. developed a method called term-seq to document all riboswitches in a bacterial genome, as well as their metabolite counterparts (see the Perspective by Sommer and Suess). The method revealed a role for pathogenic bacterial riboswitches in antibiotic resistance. Thus, transcription may be one way pathogens fend off antibiotic attack. Science, this issue p. 10.1126/science.aad9822; see also p. 144 A powerful technique is described for discovering riboswitches and attenuators that respond to previously unknown ligands. [Also see Perspective by Sommer and Suess] INTRODUCTION Riboswitches and attenuators are cis-regulatory RNA elements (ribo-regulators), which in most cases control bacterial gene expression via ligand-mediated, premature transcription termination. Depending on the presence or absence of the specific ligand, the formation of a transcription terminator upstream of the gene causes transcription to abort prematurely, generating short unproductive transcripts. In response to changes in the metabolite concentrations, the structure of the ribo-regulator is altered, destabilizing the terminator and allowing read-through into the gene, thus resulting in expression of the full-length mRNA. These ribo-regulators play central roles in bacterial physiology and virulence and have been used for synthetic biology applications as well as recognized as therapeutic targets for antibiotics. RATIONALE Despite the importance of riboswitches and attenuators, there is currently no experimental high-throughput method for the discovery of such ribo-regulators across bacterial genomes. Furthermore, given a metabolite or ligand of interest, until now there was no efficient experimental approach to identify natural riboswitches or attenuators that sense and respond to it. RESULTS We developed term-seq, a method that enables quantitative mapping of all exposed RNA 3′ ends in bacteria and allows unbiased, genome-wide identification of genes that are regulated by premature transcription termination. This method quantitatively measures the in vivo activities of all expressed ribo-regulators in a given genome simultaneously and under physiological conditions, thus enabling high-throughput discovery of ribo-regulators that respond to a metabolite of interest. Application of term-seq to the model bacteria Bacillus subtilis, Listeria monocytogenes, and Enterococcus faecalis detected the vast majority of known riboswitches as well as multiple previously unidentified regulators that function via conditional termination. We demonstrate the utility of our approach by screening for ribo-regulators that specifically respond to small antibiotic molecules. We found that numerous antibiotics resistance genes, in both pathogenic bacteria and in the human microbiome, are regulated via termination-based ribo-regulators that allow read-through when the antibiotic is present in the cell. Focusing on lmo0919, one of the antibiotic-regulated genes we detected in Listeria monocytogenes, revealed that this locus confers specific resistance to the translation-inhibiting antibiotic lincomycin. In the absence of the antibiotic, transcription is terminated prematurely by the ribo-regulator. However, upon exposure to lincomycin, drug-inhibited ribosomes stall over a conserved three-amino-acid upstream open reading frame found within the ribo-regulator, thus triggering a conformational change in the transcriptional terminator and inducing the expression of the full-length mRNA that encodes the resistance gene. CONCLUSION These results describe a high-throughput method for ribo-regulator discovery in either bacterial monocultures or complex bacterial communities such as the human microbiome. Furthermore, they reveal a broad role for conditional termination in regulating multiple classes of antibiotic resistance genes in the human microbiome and provide a general tool for discovering riboswitches and attenuators that respond to specific metabolites of choice. Genome-wide discovery of antibiotic responsive ribo-regulation in bacteria. (A) Direct mapping of RNA 3′ ends reveals gene regulation by conditional termination in a genome-wide manner. Differential sequencing of monoculture or complex bacterial communities under metabolite-rich and -poor conditions (metabolite in this case being an antibiotic) detects ribo-regulators that specifically respond to the metabolite. (B) Mechanism of regulation of the Listeria monocytogenes lmo0919 antibiotic responsive ribo-regulator. 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 ribo-regulators 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 previously unknown ligands.