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Experimental Discovery of Small Rnas in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism

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Experimental Discovery of Small Rnas in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism. Chantal Bohn, Candice Rigoulay, Svetlana Chabelskaya, Cynthia Sharma, Antonin Marchais, Patricia Skorski, Elise Borezée-Durant, Romain Barbet, Eric Jacquet, Annick Jacq, et al. To cite this version: Chantal Bohn, Candice Rigoulay, Svetlana Chabelskaya, Cynthia Sharma, Antonin Marchais, et al.. Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism.. Nucleic Acids Research, Oxford University Press, 2010, 38 (19), pp.6620-36. 10.1093/nar/gkq462. hal-00534360 HAL Id: hal-00534360 https://hal.archives-ouvertes.fr/hal-00534360 Submitted on 9 Apr 2013 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. 6620–6636 Nucleic Acids Research, 2010, Vol. 38, No. 19 Published online 28 May 2010 doi:10.1093/nar/gkq462 Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism Chantal Bohn1, Candice Rigoulay1, Svetlana Chabelskaya2, Cynthia M. Sharma3, Antonin Marchais1, Patricia Skorski1, Elise Boreze´ e-Durant4, Romain Barbet5, Eric Jacquet5, Annick Jacq1, Daniel Gautheret1, Brice Felden2, Jo¨ rg Vogel3 and Philippe Bouloc1,* 1Institut de Ge´ ne´ tique et Microbiologie, CNRS/UMR 8621, IFR115, Centre scientifique d’Orsay, Universite´ Paris-Sud, baˆ timent 400, 91405 Orsay Cedex, 2Universite´ de Rennes I, Inserm U835, UPRES EA2311, Biochimie Pharmaceutique, 2 avenue du Prof. Le´ on Bernard 35043 Rennes, France, 3Max Planck Institute for Infection Biology, Charite´ platz 1, 10117 Berlin, Germany, 4Institut Micalis, UMR1319, Institut National de la 5 Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex and Centre de recherche de Gif, Downloaded from Institut de Chimie des Substances Naturelles, UPR2301 CNRS, IMAGIF, qPCR-Platform, 91198 Gif sur Yvette Cedex, France Received January 31, 2010; Revised April 5, 2010; Accepted May 11, 2010 http://nar.oxfordjournals.org/ ABSTRACT and the folate-dependent one-carbon metabolism. Using an experimental approach, we investigated As we observed that RsaE accumulates transiently the RNome of the pathogen Staphylococcus in late exponential growth, we propose that RsaE aureus to identify 30 small RNAs (sRNAs) including functions to ensure a coordinate downregulation 14 that are newly confirmed. Among the latter, 10 of the central metabolism when carbon sources are encoded in intergenic regions, three are become scarce. generated by premature transcription termination by guest on April 9, 2013 associated with riboswitch activities, and one is ex- INTRODUCTION pressed from the complementary strand of a Regulatory RNAs in prokaryotes are frequently referred transposase gene. The expression of four sRNAs in- to as small RNA (sRNAs). The sRNAs are typically creases during the transition from exponential to between 50 and 300 nts in length. Most sRNAs with stationary phase. We focused our study on RsaE, identified regulatory functions pair with mRNAs to an sRNA that is highly conserved in the bacillales modulate their translation, stability or transcription (1). order and is deleterious when over-expressed. We The sRNAs encoded opposite to mRNA genes are show that RsaE interacts in vitro with the 50 region of referred to as ‘cis-encoded sRNAs, since they often opp3A mRNA, encoding an ABC transporter compo- regulate the associated mRNA expressed from the com- nent, to prevent formation of the ribosomal initiation plementary coding strand by extended perfect base-pairing (2). In contrast, sRNAs expressed from intergenic regions complex. A previous report showed that RsaE (IGRs) often act on target mRNAs transcribed from gen- opp3B opp3A targets which is co-transcribed with . etically unlinked regions. Base-pairing between these Thus, our results identify an unusual case of trans-encoded sRNAs and mRNA targets is usually im- riboregulation where the same sRNA controls an perfect and limited to short sequences. In many bacteria, operon mRNA by targeting two of its cistrons. A including the Gram-negative bacterium Escherichia coli, combination of biocomputational and transcription- the activity of trans-encoded sRNAs requires the al analyses revealed a remarkably coordinated RNA-chaperone Hfq (3). However, the Gram-positive RsaE-dependent downregulation of numerous bacterium Staphylococcus aureus might not require Hfq metabolic enzymes involved in the citrate cycle for sRNA activity since an hfq deletion mutant of this *To whom correspondence should be addressed. Tel: +33 1 69 15 70 16; Fax: +33 1 69 15 66 78; Email: [email protected] ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2010, Vol. 38, No. 19 6621 organism has no discernable phenotype (4). A third class downregulates multiple enzymes of two major metabolic of sRNAs can exert a regulatory function by directly inter- pathways, likely to help S. aureus prepare for the entry acting with proteins (5–7). Lastly, some mRNAs possess into stationary phase. structured 50 untranslated regions (UTR) that, under specific growth conditions, can adopt alternative conform- ations to affect gene expression of the downstream genes MATERIALS AND METHODS in cis. Binding of molecules such as metabolites or tRNAs Bacterial strains, plasmids and growth conditions to these so-called riboswitches induces RNA structure changes of the transcribed UTR, causing premature tran- The RNomics approach was performed on strain N315 scriptional termination or translational inhibition (8). (27). Transcriptome analysis was performed on strain Some riboswitches are known to produce sRNAs (9), RN6390 (40), a laboratory derivative of the NCTC8325 which might independently function on trans-encoded clinical strain (41) that was shown to be virulent in differ- target mRNAs by base-pairing mechanisms (10). ent animal models (42,43). Note that for simplicity, we Several methods have been used to identify sRNAs, only used the N315 gene name nomenclature. including computational predictions, direct labeling, Engineered plasmids were constructed in E. coli TG1, DNA microarrays and shotgun sequencing (11,12). and transferred to RN4220 (rsbU, agr), which is trans- Escherichia coli and Salmonella each encode an estimated formable with exogenous DNA (44), and subsequently 100 sRNAs (11). These respond to diverse environmental to RN6390. factors such as glucose availability, iron limitation, Staphylococcus aureus strains were routinely grown in Downloaded from envelope stress, or transition to anaerobiosis, to facilitate BHI medium with aeration at 37C. Escherichia coli TG1 adjustments, respectively, in carbon metabolism (13,14), used for cloning experiments was grown in LB broth at of levels of iron-requiring enzymes (15–17), amounts of 37C. Chloramphenicol was added to media as needed at metabolic transporters (18,19) or quantities of enzymes the following concentrations: 5 mg/ml for S. aureus and required for anaerobic growth (20,21). Other regulatory 10 mg/ml for E. coli. To determine the effect of sRNA in- functions of sRNAs include quorum sensing and virulence duction, staphylococcal cultures were prepared as follows: http://nar.oxfordjournals.org/ (22–26). 10-fold serial dilutions of exponential cultures were Staphylococcus aureus is a human commensal of skin spotted on BHI plates containing or not 1 mM anhydrote- and nares and an opportunistic pathogen responsible for tracycline (aTc) and incubated at 37C for 24 h. a wide variety of human infections including superficial pAT12 contains the repA, repC, tetR genes and the skin and wound infections to deep abscesses (endocarditis xyl/tetO promoter (45). The pAT12 derivative containing and meningitis), septicemia or toxin-associated syndromes rsaA, rsaH, rsaE or expressing RNA sequences antisense (27). It is a leading cause of nosocomial and community of RsaA, RsaH and RsaE (named pAT12-RsaA, acquired diseases and many strains acquire antibiotic re- pAT12-RsaH, pAT12-RsaE, pAT12-ASRsaA, pAT12- by guest on April 9, 2013 sistance (28). Staphylococcus aureus virulence gene expres- ASRsaH and pAT12-ASRsaE, respectively) were con- sion is regulated by the coordinated action of numerous structed as follows: DNA sequences of interest were regulators. One of the best characterized regulators is an amplified from N315 genomic DNA by PCR, using sRNA called RNAIII (514 nt), which modulates virulence primers indicated in Supplementary Data S1. The result- gene expression (29). Its 30 domains can mediate transla- ing products were digested by EcoRI and NotI, ligated to tional repression of S. aureus virulence genes directly by EcoRI-NotI-treated pAT12. Expression of the cloned se- interacting with spa mRNA, which encodes the
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