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 *

Riboswitches are ligand-binding elements contained within the 5' untranslated regions of bacterial transcripts, which generally regulate expression of downstream open reading frames. Here, we show that in Listeria monocytogenes, a riboswitch that binds vitamin B12 controls expression of a noncoding regulatory RNA, Rli55. Rli55, in turn, controls expression of the eut genes, whose products enable ethanolamine utilization and require B12 as a cofactor. Defects in ethanolamine utilization, or in its regulation by Rli55, significantly attenuate Listeria virulence in mice. Rli55 functions by sequestering the two-component response regulator EutV by means of a EutV-binding site contained within the RNA. Thus, Rli55 is a riboswitch-regulated member of the small group of regulatory RNAs that function by sequestering a protein and reveals a distinctive mechanism of signal integration in bacterial gene regulation.