THE LISTERIA MONOCYTOGENES ncRNA RLI60 IS A BCAAS SENSOR THAT REGULATES BCAAS BIOSYNTHESIS AND VIRULENCE GENE EXPRESSION VIA A MECHANISM OF RIBOSOME MEDIATED ATTENUATION

Listeria monocytogenes ncRNA rli60 is a ~200 bp element specific to Listeria, located upstream to the branch chained amino acids (BCAAs) biosynthesis operon (the ilv operon). rli60 was identified in several transcriptome analyses, however its function was not investigated. In this work we found that during growth at low concentrations of BCAAs, rli60 is co-transcribed with the ilv operon, while under rich media conditions the transcription is terminated immediately downstream the rli60 gene, suggesting it might function as a cis-regulatory element. Further genetic characterization has demonstrated that rli60 negatively regulates the ilv operon under intermediate levels of BCAAs, while under high BCAAs levels the global regulator CodY fulfills this function. Moreover, under very low BCAAs levels rli60 was still able to negatively regulate the ilv operon (to some extant) while CodY was shown to function as an activator. Interestingly, we identified a short peptide encoded within rli60, exhibiting multiple BCAAs codons which resembled regulatory codons/peptides of a ribosome-mediated attenuation mechanism. Under such a mechanism, a leader peptide is fully formed when BCAAs are available, leading to the formation of a terminator structure that terminates transcription upstream the ilv genes. However, under BCAAs starvation, the ribosome is stalled at the BCAAs codons resulting in the formation of an anti-terminator structure that allows transcription of the downstream ilv genes. Preliminary data indicates that this is the mechanism used by L. monocytogenes to fine-tune the expression of the ilv operon under varying concentrations of BCAAs. Finally, as low BCAAs concentration signals L. monocytogenes of its intracellular location, we find that rli60 also affects the expression of virulence genes via modulation of CodY activity as a repressor.