RIBOSOME-MEDIATED CONTROL OF BRANCH-CHAINED AMINO ACIDS BIOSYNTHESIS AS A REGULATORY MECHANISM OF VIRULENCE GENE EXPRESSION AND SEMI-AUXOTROPHY

Listeria monocytogenes is an intracellular human pathogen that is also able to grow as a saprophyte. The transition between these two lifestyles is regulated by multiple environmental signals, including the branch-chained amino acids (BCAAs) that are sensed by the transcriptional regulator CodY. L. monocytogenes` rli60 is a ~200 bp small non-coding RNA specific to Listeria, located upstream the branch chained amino acids biosynthesis operon, the ilv operon. rli60 was identified in several transcriptome analyses, however its function was not investigated. In this work we found that in bacteria growing in media with low concentrations of BCAAs, rli60 is co-transcribed with the ilv operon from a single promoter, while in media rich in BCAAs the transcription of this operon is prematurely terminated, suggesting it functions as a cis-regulatory RNA element. Here we show that the regulation of the ilv operon is hierarchal and dependent on the level of BCAAs available. Under high BCAAs levels CodY represses the ilv operon, including rli60, however upon a further drop in BCAAs levels, rli60 becomes the main repressor of the ilv operon. We identified a short coding sequence within rli60 which exhibits multiple BCAAs codons and showed that it functions as a leader peptide of ribosome-mediated transcriptional attenuator. This attenuator forms a RNA structure that prematurely terminates the transcription of ilv genes under rich BCAAs conditions and an anti-terminator structure that promotes ilv transcription under BCAAs starvation. We find this mechanism to tightly regulate the expression of the ilv operon under changing BCAAs levels. We further show that rli60 is responsible for L. monocytogenes semi-auxotrophy for BCAAs, and that it serves as a mechanism to regulate virulence gene expression.