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Expression of Iron, the Salmochelin Siderophore Receptor, Requires Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues Roberto Balbontín, Nicolás Villagra, Maria Pardos de la Gándara, Guido Mora, Nara Figueroa-Bossi, Lionello Bossi To cite this version: Roberto Balbontín, Nicolás Villagra, Maria Pardos de la Gándara, Guido Mora, Nara Figueroa- Bossi, et al.. Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues. Molecular Microbiology, Wiley, 2016, 100 (1), pp.139 - 155. 10.1111/mmi.13307. hal-01412678 HAL Id: hal-01412678 https://hal.archives-ouvertes.fr/hal-01412678 Submitted on 8 Dec 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues Roberto Balbontın,1† Nicolas Villagra,2 to the polychromatic landscape of sRNA-mediated Maria Pardos de la Gandara, 1‡ Guido Mora,2 regulation. Nara Figueroa-Bossi1* and Lionello Bossi1 1Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Universite Paris-Sud, Universite Paris- Introduction Saclay, 91198 Gif-sur-Yvette, France. Rewiring of gene expression networks in response to 2Laboratorio de Patogenesis Molecular y chemical or physical cues is central to the ability of bac- Antimicrobianos, Facultad de Medicina, Universidad terial pathogens to adapt to environmental changes. Andres Bello, Echaurren 183, Santiago, Chile. Adaptive responses can be finely tuned through the interplay of transcriptional and post-transcriptional regu- lators (Mandin and Guillier, 2013). Small non-coding Summary RNAs (sRNAs) are key components of this regulatory circuitry (Gottesman and Storz, 2011). In gram-negative The iroN gene of Salmonella enterica and uropa- bacteria, a large family of sRNAs act by binding to chap- thogenic Escherichia coli encodes the outer mem- eron protein Hfq and establishing short, often imperfect, brane receptor of Fe31-bound salmochelin, a base-pair interactions with target messenger RNAs siderophore tailored to evade capture by the host’s (mRNAs). Hfq protects unpaired sRNAs against degra- immune system. The iroN gene is under negative dation by RNases and it is thought to facilitate mRNA control of the Fur repressor and transcribed under recognition (De Lay et al., 2013; Vogel and Luisi, 2011). iron limiting conditions. We show here that tran- The majority of Hfq binding sRNAs are negative regula- scriptional de-repression is not sufficient to allow tors. Most members of this class target sequences iroN expression, as this also requires activation by within ribosome binding sites where formation of the either of two partially homologous small RNAs RNA duplex obstructs the entry of the 30S ribosomal (sRNAs), RyhB1 and RyhB2. The two sRNAs target subunit, thereby inhibiting translation initiation. Transla- the same sequence segment approximately in the tional repression is generally accompanied by the desta- middle of the 94-nucleotide 50 untranslated region bilization of both mRNA and sRNA (Masse et al., 2003). (UTR) of iroN mRNA. Several lines of evidence On one hand, loss of ribosomal shielding renders the suggest that base pair interaction stimulates iroN mRNA susceptible to internal attack by RNase E (Drey- mRNA translation. Activation does not result from fus, 2009). Conversely, the ability of Hfq to bind RNase the disruption of a secondary structure masking E (Morita et al., 2005) can promote the active recruit- the ribosome binding site; rather it involves ment of this enzyme near the site of the sRNA-mRNA sequences at the 50 end of iroN 50 UTR. In vitro association (Ikeda et al., 2011) or even stimulate its ‘toeprint’ assays revealed that this upstream site activity at a distant site (Prevost et al., 2011). Although binds the 30S ribosomal subunit provided that a downstream event in all above pathways (Morita et al., RyhB1 is paired with the mRNA. Altogether, our 2006), the RNase E step is important to make the data suggest that RyhB1, and to lesser extent sRNA action stoichiometric and irreversible. Finally, RyhB2, activate iroN mRNA translation by promot- sRNA-mediated silencing can also proceed through ing entry of the ribosome at an upstream ‘standby’ instructed mRNA decay as opposed to translational inhi- site. These findings add yet an additional nuance bition. In the regulation of Salmonella ompD gene, the sRNA MicC pairs with internal sequences in the mRNA and guides RNase E to cleave the mRNA at a position adjacent to the pairing site (Bandyra et al., 2012). Small RNAs can also activate gene expression, typically by stimulating translation initiation. The paradigmatic 1 example is represented by the rpoS mRNA whose 50 UTR way originates from the horizontal acquisition of the iroA folds into a secondary structure that sequesters the locus, which comprises the iroBCDE operon and the Shine-Dalgarno (SD) motif causing the translation initia- adjacent, convergently oriented iroN gene (Baumler€ tion step to be intrinsically inefficient. Any of three different et al., 1998). The iroBCDE operon encodes functions sRNAs, can counter this effect by establishing an alterna- involved in salmochelin synthesis and export; IroN is the tive pairing configuration that frees the ribosome-binding outer membrane receptor through which ferric salmo- site thus activating translation (McCullen et al., 2010; chelin is internalized (Baumler€ et al., 1998; Hantke Soper et al., 2010). More recently, a number of alternative et al., 2003). The relevance of this pathway in pathoge- sRNA-mediated activating mechanisms have emerged nicity was highlighted by the finding that the iroBCDE (reviewed in Papenfort and Vanderpool, 2015). One sys- iroN locus confers a competitive advantage to Salmo- tem features a direct competition between Hfq, acting as a nella in colonizing the inflamed intestine of wild-type translational repressor, and the sRNA, which upon pairing mice but not of mice deficient in lipocalin-2 production with the target sequence redirects Hfq to a different site (Raffatellu et al., 2009). Once delivered to the periplasm (Salvail et al., 2013). Other mechanisms are independent through the respective receptors, Fe31-enterobactin and of translation and rely on the sRNA preventing or slowing Fe31-salmochelin are chaperoned by FepB to the down degradation of the mRNA (Papenfort et al., 2013; FepDGC transporter and translocated to the cytoplasm Frohlich et al., 2013). where they are degraded by the Fes and IroD hydro- The data described in this paper point to the exis- lases, respectively (Lin et al., 2005; Zhu et al., 2005; tence of a further variation in the RNA-dependent acti- Crouch et al., 2008). The iron released from hydrolyzed vation theme, whereby the sRNA stimulates translation siderophores is transferred to bacterial proteins and of the target gene by facilitating ribosomal recruitment at ligands. At some point along these processes, some an upstream ‘standby’ site. This mechanism was uncov- reduced iron is produced and maintained in a soluble ered during a study of the regulation of iron uptake in form. The size of this labile Fe21 pool is tightly regulated Salmonella. to limit the harmful effects of Fenton chemistry. Fe21 To acquire iron from the environment, most free-living levels are controlled homeostatically through modulation microorganisms synthesize and secrete a class of of iron uptake and allocation. organic molecules named siderophores, which capture In Escherichia coli, iron homeostasis is achieved ferric ion from organic or inorganic sources (reviewed in through the interplay of two global regulators: the Fur Crosa and Walsh, 2002; Fischbach et al., 2006; Wan- protein and RyhB small RNA. Fur is a Fe21-binding dersman and Delepelaire, 2004). The iron-siderophore polypeptide that, upon undergoing Fe21-dependent complex is then imported into the cell through high affin- dimerization, binds specific DNA sequences (Fur boxes) ity receptors (Braun and Braun, 2002; Braun and in the promoter regions of iron uptake genes and Hantke, 2011). Enterobactin, the archetypal siderophore represses transcription (Carpenter et al., 2009; Hantke, of enterobacterial species like Escherichia coli and Sal- 2001). A decline in Fe21 levels shifts the equilibrium monella enterica, is synthesized through a pathway that towards Fe21-free Fur monomers, resulting in the pro- branches off from the biosynthetic pathway of aromatic gressive relief of repression. Included in the Fur regulon amino acids and is carried out by the products of the is the gene for RyhB sRNA. Accumulating under iron- ent genes. Enterobactin export involves the functions of limiting conditions, RyhB downregulates the synthesis of EntS (Furrer et al., 2002) and TolC (Bleuel et al., 2005) a number of non-essential iron-utilization and iron- while the import of its iron-bound form occurs through storage proteins (including superoxide dismutase SodB the high affinity, TonB-gated FepA
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