The sRNA LhrC5 and Hfq post-transcriptionally regulate ferritin expression in Listeria monocytogenes

The RNA chaperone Hfq facilitates base-pairing interactions between small regulatory RNAs (sRNAs) and their target mRNAs in Gram-negative bacteria. In Gram-positive species, however, its analogous matchmaker function remains poorly defined, with the notable exception of the Hfq-dependent sRNA LhrA in the pathogen Listeria monocytogenes. This bacterium also encodes the LhrC family of homologous sRNAs, which typically repress gene expression by base-pairing near ribosome-binding sites of target mRNAs. Although LhrC1-5 bind Hfq, their previously characterized regulatory functions are Hfq-independent. Here, we focus on LhrC5, which is uniquely encoded within the operon of ferritin (Fri), an iron-storage protein critical for virulence and stress adaptation. We used a combination of in vitro and in vivo methods including co-immunoprecipitation, northern blot, EMSA, RNA structure probing, in vitro translation, Hfq overexpression, and RNA stability analysis. The fri gene is transcribed from one σB-dependent and two σA-dependent promoters. We demonstrate that Hfq specifically promotes the binding of LhrC5-but not the homologous LhrC4-to the ribosome-binding site of the σA1-derived fri mRNA, resulting in reduced translation in vitro. No interaction between LhrC5 and transcripts originating from the σA2 or σB promoters was detected. Consistent with this specificity, cellular analyses show that LhrC5, but not LhrC1-4, selectively destabilizes the σA1-derived fri mRNA under osmotic stress conditions, thereby fine-tuning its expression. However, Hfq was not required for this LhrC5-dependent regulation of fri mRNA stability under physiological conditions. Notably, Hfq overexpression during σA1-to-σB switching of the fri promoter leads to accelerated decay of both σA1- and σB-derived fri mRNAs, consistent with the involvement of Hfq in post-transcriptional downregulation of fri expression. Collectively, this study identifies fri as a new regulatory target of the LhrC sRNA family member LhrC5 and reveals a previously unrecognized role of Hfq in LhrC5-dependent control. These findings uncover additional complexity in riboregulation in this pathogen, with potential implications for iron homeostasis.