Rli51 Attenuates Transcription of the Listeria Pathogenicity Island 1 Gene mpl and Functions as a Trans-Acting sRNA in Intracellular Bacteria

<i>Listeria</i> pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen <i>Listeria monocytogenes</i>. Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding <i>mpl</i>. So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a <i>cis</i>- and as a <i>trans</i>-acting sRNA. Under nutrient-rich conditions, <i>rli51-mpl</i> transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, <i>rli51</i> transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into <i>mpl</i>. Comparative intracellular bacterial transcriptomics in <i>rli51</i>-null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a <i>trans</i>-acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended <i>mpl</i> transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.