No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus

BACKGROUND: The RNA-binding protein Hfq is involved in stress and virulence of several pathogens, probably due to its role as mediator in small RNA (sRNA)-mRNA interactions. In this study, we investigate the function of Hfq in the Gram-positive pathogen Staphylococcus aureus, by constructing hfq null mutant derivatives. RESULTS: We report that unexpectedly, in S. aureus, Hfq does not seem to play a crucial role in stress response, RNAIII or spa mRNA quantity and exoprotein expression, as tested in three virulent genetic backgrounds. Moreover, a global analysis of the RN6390 hfq mutant, which tests ~ 2000 phenotypes, supports our results concerning the non-implication of Hfq in stress response, and shows that Hfq is also not involved in resistance to several chemical agents and antibiotics and does not seem to be implicated in metabolic pathways. CONCLUSION: Our data suggest that although sRNA-mRNA interactions in S. aureus are decisive for gene expression regulation, they do not require the RNA-chaperone protein Hfq. These interactions possibly require an RNA-chaperone protein other than Hfq, which remains to be found

Whole genome mapping of 5' RNA ends in bacteria by tagged sequencing : A comprehensive view in Enterococcus faecalis

Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first comprehensive view of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5'RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites and 352 processing sites in E. faecalis. A blind motif search retrieved canonical features of SigA- and SigN-dependent promoters preceding TSSs mapped. We discovered 95 novel putative regulatory RNAs, small- and antisense RNAs, and 72 transcriptional antisense organisations. Presented data constitute a significant insight into bacterial RNA landscapes and a step towards the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner

Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism

Using an experimental approach, we investigated the RNome of the pathogen Staphylococcus aureus to identify 30 small RNAs (sRNAs) including 14 that are newly confirmed. Among the latter, 10 are encoded in intergenic regions, three are generated by premature transcription termination associated with riboswitch activities, and one is expressed from the complementary strand of a transposase gene. The expression of four sRNAs increases during the transition from exponential to stationary phase. We focused our study on RsaE, an sRNA that is highly conserved in the bacillales order and is deleterious when over-expressed. We show that RsaE interacts in vitro with the 5′ region of opp3A mRNA, encoding an ABC transporter component, to prevent formation of the ribosomal initiation complex. A previous report showed that RsaE targets opp3B which is co-transcribed with opp3A. Thus, our results identify an unusual case of riboregulation where the same sRNA controls an operon mRNA by targeting two of its cistrons. A combination of biocomputational and transcriptional analyses revealed a remarkably coordinated RsaE-dependent downregulation of numerous metabolic enzymes involved in the citrate cycle and the folate-dependent one-carbon metabolism. As we observed that RsaE accumulates transiently in late exponential growth, we propose that RsaE functions to ensure a coordinate downregulation of the central metabolism when carbon sources become scarce

Evidence for the Role of Horizontal Transfer in Generating pVT1, a Large Mosaic Conjugative Plasmid from the Clam Pathogen, Vibrio tapetis

The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600T reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae

The Staphylococcus aureus RNome and Its Commitment to Virulence

Staphylococcus aureus is a major human pathogen causing a wide spectrum of nosocomial and community-associated infections with high morbidity and mortality. S. aureus generates a large number of virulence factors whose timing and expression levels are precisely tuned by regulatory proteins and RNAs. The aptitude of bacteria to use RNAs to rapidly modify gene expression, including virulence factors in response to stress or environmental changes, and to survive in a host is an evolving concept. Here, we focus on the recently inventoried S. aureus regulatory RNAs, with emphasis on those with identified functions, two of which are directly involved in pathogenicity

Global Analysis of Extracytoplasmic Stress Signaling in Escherichia coli

The Bae, Cpx, Psp, Rcs, and σE pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signal-inducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response

Déblocage de ribosomes et étiquetages de polypeptides par trans-traduction chez Escherichia coli

L ARNtm et SmpB permettent la libération des ribosomes lorsque la traduction est défectueuse. Les polypeptides dont la biosynthèse est bloquée sont en même temps étiquetés, ce qui induit leur élimination par des protéases. Ce mécanisme est nommé trans-traduction. SmpB et l ARNtm sont ubiquitaires chez les eubactéries. Nous montrons que les SmpB de plusieurs espèces bactériennes peuvent étiqueter des polypeptides avec l ARNtm d E. coli in vivo, ce qui illustre la conservation fonctionnelle du système de trans-traduction. Nous montrons aussi que la trans-traduction n est pas affectée in vivo à des concentrations sublétales aminoglycosides néomycine B ou paromomycine. Elle améliore même la survie d E. coli en présence de ces antibiotiques et d érythromycine, probablement en limitant l accumulation de polypeptides anormaux et la séquestration des ribosomes.Par ailleurs, le gène codant pour l ARNtm a été génétiquement modifié pour identifier des polypeptides préférentiellement étiquetés in vivo chez E. coli. Nous montrons que des protéines complètes peuvent être étiquetées par l ARNtm, si la terminaison de la traduction de leur messager est défectueuse. Enfin, nous montrons que le messager secM est trans-traduit, car il est clivé à un site de blocage traductionnel précédemment caractérisé. Nous proposons qu il existe une activité endoribonucléasique encore inconnue, qui serait associée au ribosome et activée en réponse à un blocage traductionnel. Elle agirait en concertation avec la trans-traduction. Elle pourrait intervenir dans certaines voies de régulation cellulaire ou limiter certains évènements de recodage.tmRNA and SmpB can rescue ribosomes stalled on a messenger during translation. Nascent polypeptides are tagged, which induces their degradation by proteases. This mechanism is called trans-translation. First, we show that SmpB from multiple bacterial species can tag polypeptides in vivo, together with E. coli tmRNA. This result illustrates the remarquable functionnal conservation of the trans-translation mechanism. Second, we show that trans-translation is not affected in vivo by sublethal concentrations of the aminoglycosides neomycin B and paromomycin. Moreover, trans-translation conferes a growth advantage in the presence of these antibiotics and erythromycin, probably by limiting accumulation of abnormal polypeptides and stalled ribosomes. Third, the gene encoding tmRNA was genetically modified, in order to identify polypeptides that are preferentially tagged in vivo by trans-translation in E. coli. We show that complete proteins can also be tagged by tmRNA, if translation termination of their messengers is poorly efficient. Finally, we show that secM messenger is trans-translated, because it is cleaved at a site of ribosome pausing during translation elongation. We propose that their is an endonucleolytic activity associated with the ribosome, which is induced in response to ribosome stalling. In cooperation with trans-translation, it could participate in some regulation pathways or could limit some recoding events during translation.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Regulatory RNAs in bacteria: From identification to function

International audienceEditorial: The discovery that RNA functions extend beyond their roles as messengers of DNA information and key components of the translation machinery has shaken up the RNA field..

Les acides ribonucléiques régulateurs du staphylocoque doré et leurs rôles dans la virulence

National audienc