In gram-positive bacteria, it is estimated that 2-5% of all genes are regulated by riboswitches and 5’ UTR elements that exert their effect through the control of premature transcriptional termination. The real extent of this regulatory strategy was estimated to be much broader, but there was so far no method for genome wide discovery of regulated termination. Here we developed a novel deep-sequencing platform that directly measures the in-vivo activity of every regulator in the genome under physiological conditions, and so enables high-throughput screening for unique regulator types. Using our approach, we discovered that antibiotic resistance genes across many pathogenic bacteria are regulated via premature transcriptional termination. In the absence of antibiotics, transcription of these genes is aborted prematurely; but in the presence of antibiotics, a specific structure in the 5’ UTR is modified to allow productive transcription. We further show that several of these regulators function through a unique ribosome mediated attenuation mechanism. Our results suggest a major, previously unappreciated, role for regulated termination in bacterial antibiotic resistance, with potential clinical implications.
