• CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level.

      El Mouali, Youssef; Gaviria-Cantin, Tania; Sánchez-Romero, María Antonia; Gibert, Marta; Westermann, Alexander J; Vogel, Jörg; Balsalobre, Carlos; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-01-01)
      Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3'UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3'UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3'UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.
    • Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein.

      Gerovac, Milan; El Mouali, Youssef; Kuper, Jochen; Kisker, Caroline; Barquist, Lars; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Cold Spring Habor Laboratory Press and RNA Society, 2020-07-09)
      RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs.
    • Global RNA interactome of Salmonella discovers a 5' UTR sponge for the MicF small RNA that connects membrane permeability to transport capacity.

      Matera, Gianluca; Altuvia, Yael; Gerovac, Milan; El Mouali, Youssef; Margalit, Hanah; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier (Cell Press), 2022-01-20)
      The envelope of Gram-negative bacteria is a vital barrier that must balance protection and nutrient uptake. Small RNAs are crucial regulators of the envelope composition and function. Here, using RIL-seq to capture the Hfq-mediated RNA-RNA interactome in Salmonella enterica, we discover envelope-related riboregulators, including OppX. We show that OppX acts as an RNA sponge of MicF sRNA, a prototypical porin repressor. OppX originates from the 5' UTR of oppABCDF, encoding the major inner-membrane oligopeptide transporter, and sequesters MicF's seed region to derepress the synthesis of the porin OmpF. Intriguingly, OppX operates as a true sponge, storing MicF in an inactive complex without affecting its levels or stability. Conservation of the opp-OppX-MicF-ompF axis in related bacteria suggests that it serves an important mechanism, adjusting envelope porosity to specific transport capacity. These data also highlight the resource value of this Salmonella RNA interactome, which will aid in unraveling RNA-centric regulation in enteric pathogens.
    • In vivo targets of Salmonella FinO include a FinP-like small RNA controlling copy number of a cohabitating plasmid.

      El Mouali, Youssef; Gerovac, Milan; Mineikaitė, Raminta; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxford Academic, 2021-05-03)
      FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.